Nano Pat Ology

N A N O PAT H O L O G Y
THE HEALTH IMPACT OF

NANOPARTICLES
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ANTONIETTA M GATTI
University of Modena & Reggio Emilia, Italy
STEFANO MONTANARI
Laboratory Nanodiagnostics, Italy
N A N O PAT H O L O G Y
THE HEALTH IMPACT OF
NANOPARTICLES
Published by
Pan Stanford Publishing Pte. Ltd.
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British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.
NANOPATHOLOGY
The Health Impact of Nanoparticles
Copyright © 2008 by Pan Stanford Publishing Pte. Ltd.
All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means,
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ISBN-13 978-981-4241-00-7
ISBN-10 981-4241-00-8
Printed in Singapore.
Rhaimie - Nanopathology.pmd 1 10/3/2007, 5:24 PM

This book is dedicated to those who are neither young nor
old enough to know everything, but know, as we do,
that they don’t know anything.
Acknowledgements
All this research would not have come into fruition if it had not been
financed by the European Project NANOPATHOLOGY. Word of thanks
to the independence of the referees and the European Commission for its
farsightedness. It was due to this initiative that people could now be
properly treated and the cause of their contamination could be identified
and eradicated.
Prof. James Kirkpatrick, Prof. William Bonfield, Prof. Peter Revell
and Dr. Diana Boraschi were the first to understand what we meant and
to support and encourage us.
We are indebted to Dr. Andrea Gambarelli, Dr. Roberta Salvatori,
Dr. Federico Capitani, Dr. Daniela Tossini, Dr. Gianluca Sighinolfi for
their technical help.
Special thanks to Miss Lavinia Nitu, our efficient and patient
secretary.
vii
Preface
Nanotechnologies can represent a real innovation for human society

and life. The possibility of “bottom-up” construction makes man look
like God, but the wise man knows that every progress can have a
negative side and too often, when he realizes that, disasters have already
occurred.
The primary raison d’être of this book is to help society avoid the
repeat of the mistakes made by the Curies and their followers when they
discovered radioactivity and started, on the wings of enthusiasm, to use it
on people affected by a number of diseases, and did that without being
able to anticipate the harmful side effects of those therapies.
A new trend has spread all over Europe, meetings after meetings
dedicated to radiotherapy were organized, and people have even started
to wear necklaces with beads of Cobalt core.
It was only a few decades later, after having paid a high price
in terms of deaths, that the side effects of radioactivity became
evident, but now we can use radioactive materials in a safe way daily,
taking advantage from this phenomenon and the technologies it had
generated.
The new frontier opened by nanotechnology especially in medicine
looks extremely exciting. In the future we might see nanodevices
equipped with nanomotors inserted in the blood vessels and driven to
areas damaged by an infarction to destroy the thrombus or the atheroma
and restore circulation, or toward the pneumothorax area to seal the
lesion. Or even devices that act like guardians to check the onset of
inflammations in precancerous areas.
All these may look like a dream, but, it is a dream fast becoming a
reality and, before, it becomes real, it is crucial that we verify how
organisms, tissues, cells react to the presence of nanoparticles, i.e.
foreign bodies whose behaviour is still largely unknown.
ix
x Nanopathology
Recent European research projects (Nanosafe1, Nanosafe2,

Nanoderm, Nanopathology) have explored the possible risks of
nanoparticles on human health, and their results are controversial. Some
assert the safety of nanoparticles through in-vitro tests, others are more
doubtful and less optimistic, while a few scientists have already
presented clinical evidences of the presence of nanoparticles in
pathological tissues [A.M. Gatti, 2005]. Besides other pieces of evidence,
unintentionally released, nanosized particles were found in soldiers who
served in former Yugoslavia during the Balkans War (1993-97). It is
widely known that the explosion of Depleted Uranium bombs can
develop a temperature exceeding 3000°C [Annual Report, 1978]. The
magnitude of this combustion is capable of vaporizing everything. As
soon as the vaporized materials cool down, nanosized particles are
created and are scattered in the environment. The inhalation or ingestion
of those mainly metallic particles by humans and animals can bring
about pathological effects [Nemmar A. et al., 2002]. Warfare is not the
only one to be blamed for the formation of nanoparticles as a pollutant.
Car engines, industry, incineration and high-temperature procedures in
general are just a few examples of particulate pollutants producers. It is
easy to conclude that in more than one instance, the environment is
already contaminated by nanosized particles.
It has been proved that 100-nm-sized particles when inhaled can
bypass the lung barrier in 60 seconds and reach the liver in 60 minutes.
It may not be possible to eliminate nanoparticles from the
environment, but, awareness of their possible adverse effects on human
health is important. More research are needed to determine the safest
procedures to handle them.
This book intends to help society change its mind set as in the words
of John Steinbeck, Nobel Prize Winner, 1964,
“The ability to think differently today than yesterday is

what separates the wise from the stubborn.”
Preface xi
Bibliography
Annual Technical report of the Air Force Armament Laboratory. (1978). Armament
development and test Center, Eglin Air Force Base, Florida, USA. Project n°
06CD0101 (From October 1977 to October 1978). Gatti, A.M. Handbook (2005).
Risk assessment of micro and nanoparticles and the human health, Handbook of
nanostructured biomaterials and their applications. Ed. by American Scientific
Publisher USA, cap. 12, pp. 347-369.
Nemmar A., Hoet P.H.M., Vanquickenborne B., Dinsdale D., Thomeer M., Hoylaerts
M.F., Vanbilloen H., Mortelmans L., Nemery B. (2002). Passage of inhaled
particles in to the blood circulation in humans, Circulation, 105 (4), pp. 411-417.
Authors
Antonietta M Gatti
University of Modena & Reggio Emilia, Italy
Stefano Montanari
Laboratory Nanodiagnostics, Italy
Contents
Acknowledgements vii
Preface ix
1. How the Whole Thing Began or the Logic Path Towards a

Discovery 1
1.1 Introduction...................................................................................... 6
1.2 Bibliography .................................................................................... 10
2. In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 11

2.1 Introduction...................................................................................... 11
2.2 Nanoparticles and medical devices .................................................. 13
2.2.1 Dentistry ................................................................................ 13
2.2.2 Orthopaedics.......................................................................... 14
2.2.3 Nanostructured surfaces ........................................................ 16
2.2.4 Drug delivery......................................................................... 17
2.2.5 Nanomedicine........................................................................ 19
2.3 The results of the nanopathology project ......................................... 22
2.3.1 In-vivo experiments ............................................................... 28
2.4 Bibliography .................................................................................... 33
3. Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System,

Vessels, Sperm 39
3.1 Introduction...................................................................................... 39
3.2 Lung ................................................................................................. 42
3.3 Blood................................................................................................ 70
3.4 Liver................................................................................................. 80
3.5 Kidney and adrenal gland ................................................................ 95
3.6 Digestive system .............................................................................. 102
3.7 Vessels ............................................................................................. 114
3.8 Sperm ............................................................................................... 115
3.9 A few considerations on reproduction ............................................. 121
3.10 Bibliography .................................................................................... 131
4. Six “Detective Stories” 135

4.1 The 1st case....................................................................................... 135
4.2 The 2nd case...................................................................................... 142
4.3 The 3rd case ...................................................................................... 145
4.4 The 4th case ...................................................................................... 148
xiii
xiv Nanopathology
4.5 The 5th case ...................................................................................... 152

4.6 The 6th case ...................................................................................... 155
5. War and Nanoparticles 161

5.1 Civilians living around a firing ground ............................................ 198
5.2 A few reflections.............................................................................. 199
5.3 Bibliography .................................................................................... 200
6. Nanoparticles in the Environment and Working Places 203

6.1 Introduction...................................................................................... 203
6.2 Chimneysweeps: a historical case .................................................... 205
6.3 Welding metals ................................................................................ 207
6.4 Toner................................................................................................ 210
6.5 The particulate active filter or FAP.................................................. 212
6.6 The environment around a foundry.................................................. 215
6.7 The environment around a power plant............................................ 220
6.8 The case of a ship............................................................................. 227
6.9 Incinerators ...................................................................................... 228
6.10 Tobacco smoke ................................................................................ 231
6.11 Bibliography .................................................................................... 237
7. Nanoparticles in Food, Cosmetics and Other Products 239

7.1 Bibliography .................................................................................... 249
8. New York 9/11 251

8.1 Bibliography .................................................................................... 263
9. The Future and Prevention Criteria 265

9.1 The state of the art............................................................................ 265
9.2 What is next?.................................................................................... 267
9.3 The future......................................................................................... 280
9.4 A few reflections.............................................................................. 281
9.5 Bibliography .................................................................................... 284
Appendix 287
Index 291
Chapter 1
How the Whole Thing Began or the

Logic Path Towards a Discovery
___________
%
ack in 1990, the hospital of Monza, a branch of the University of
Milan - Italy, sent to the Laboratory of Biomaterials of the
University of Modena a vena cava filter (Montanari, 2000) which
had two broken legs and that had been explanted surgically from a 62-
year-old female patient (Emanuelli, et al., 1995) (Gatti, et al., 2006). The
surgeon who had first implanted and then removed the filter just wanted
to know why the device had failed and solving his problem was not
particularly hard, as it was due to a caudal migration with the two legs
trapped in a collateral vein. The filter moved downward, while the two
legs could not follow the movement, broke. One of the consequences was
that the fracture surfaces remained exposed to the blood and interacted
with its components. What remained of the filter and the broken legs was
observed under a Scanning Electron Microscope equipped with an X-ray
microprobe (Energy Dispersive Spectroscope or EDS).
This analysis [A highly energetic electron beam is aimed at the
sample, and that yields a number of by-products, among which X-rays.
Each chemical element has characteristic energies/wavelengths which
can be detected using a solid state energy dispersive spectrometer
detector] (See Appendix) on this surface, where the legs had broken,
revealed a relatively high concentration of Chlorine, Silicon, Phosphorus
and Magnesium; all elements that did not belong either to the filter’s
alloy - an AISI 316L stainless steel, whose composition is Chromium
18%, Nickel 14%, Molybdenum 3%, Carbon below 0.03% and Iron to
balance - or to the blood, at least not in such quantities. At that time, we
could not find any reasonable explanation to what we had seen.
1
2 Nanopathology
A couple of years later, the same hospital sent another broken vena
cava filter to the Laboratory. This time the device was a temporary one
(Bovyn, et al., 1997) removed from a 58-year-old male 23 days after
having been implanted. Also that filter had failed, having lost one of its
legs which was then retrieved from the patient’s right inferior interlobar
renal vein by means of a transvenous noose catheter. The scanning-
electron-microscope observation of the fracture showed a few inorganic
deposits that could not belong to the product’s composition. As the EDS
analysis proved that the filter’s alloy was composed of Cobalt,
Chromium, Nickel, Molybdenum, Silicon and Iron (alloy called Phynox),
while Calcium and Aluminium were found in one of the deposits,
Chlorine, Silicon, Potassium, Calcium, Sulphur, Aluminium, Sodium,
Titanium and Magnesium in another, and single particles of Aluminium
and Calcium-Sulphur were also detected. Those deposits had obviously
formed in vivo, since the specimen had been carefully washed in a
Potassium oxide solution, and cleaned in a Hydrogen peroxide ultrasonic
bath, a treatment that destroys biofilm and leaves only insoluble
precipitates strictly adhered to the surface. Some of the materials
detected can be found as trace elements in ionic form or bound to organic
molecules in the human organism, but what we saw was particles and the
quantities we were confronted with were comparatively high. Again, no
satisfactory explanation could be offered then to that phenomenon.
In December 1997 a 62-year-old patient (Ballestri, et al., 2001)
(Gatti, et al., 2002) was admitted to the University hospital of Modena,
where the Laboratory of Biomaterials was then located. He was affected
by acute renal failure, hepato-splenomegaly and a mild haemolytic
anaemia. In addition to that, for the last eight years he had suffered from
a slight increase of the body temperature, a phenomenon that manifested
itself in the late afternoon and remitted to a normal temperature in a
matter of a few hours, without recurring to any medication. That fever
followed periodic cycles, each of which lasted a few months. Among the
symptoms the patient suffered from there was also a constant tiredness.
Blood and urine culture had been consistently negative and, despite
numerous hospitalizations, no aetiology could be determined for such
collection of symptoms.
How the Whole Thing Began or the Logic Path Towards a Discovery 3
So, a percutaneous echo-assisted biopsy was performed in the liver

and the lower pole of the left kidney, revealing non-caseating
granulomatous areas with Langhans-type giant cells in the kidney, while
the liver showed a mild fibrosis of the portal tracts filled with
mononuclear infiltrates and with the presence of epithelioid giant cells.
Scattered polarized light-blue particles either in the kidney and the liver
were shown by polarized-light microscopy observation, often inside
giant cells located in granulomatous areas, suggesting a foreign-body
multi-system granulomatosis. No yeasts or alcohol-acid resistant bacteria
could be identified. Clinical and immunological tests excluded
tuberculosis, mycotic infection, protozoa infestation, granulomatous
vasculitis or sarcoidosis, and the patient’s history was negative for
exposure to chemicals, drug abuse or arteriographic procedures.
That was indeed a very hard problem to solve through the traditional
means of investigation. So, the samples were given to our Laboratory
where we studied the debris by means of a scanning electron microscope
(SEM XL40 by Philips) and an environmental scanning electron
microscope (ESEM XL50 by Fei Company) equipped with an EDS. Both
in the liver and kidney specimens, we could identify solid particles
whose electronic density was higher than the biological background’s.
Their size ranged 6-20 microns in the liver and was smaller than 6
microns in the kidney, and the EDS elemental analyses, identical in both
types of specimens, revealed that they were made of Aluminium, Silicon,
Oxygen, Sodium, and Potassium or Barium, a composition that
suggested that of a ceramic material (and was compatible with feldspars,
non-fibrous silicates that are regular components of porcelain). In order
to understand the origin of such debris, a specific anamnestic study of the
patient was carried out, looking for an internal source of these debris like
prostheses, implant, etc.
What we learned from the patient, was that nine years before, two
ceramic bridges had been placed in his left upper and right lower dental
arches. That caused immediately a marked discomfort, an uncontrolled
lachrymation of the left eye, and a homolateral earache that could not be
solved by the antibiotic treatments the patient had undergone. In addition
to that, the patient became a bruxer.
4 Nanopathology
The prostheses had been milled a little later to try and solve the
problem of malocclusion and bruxism, so leaving a highly worn
prosthetic surface.
So, we started to suspect that the source of the debris found in the
patient’s tissues could be those prostheses and had both removed. They
were cross-sectioned and subjected to an Rx microanalysis that showed
the same elemental composition as the foreign bodies discovered in the
tissues. Ceramic particles smaller or as large as 40 microns were found
in the patient’s stools before the prostheses were removed, but none
one month after. So the patient underwent a 6-month therapy of
methylprednisolone and that induced the remission of the fever along
with the reversal of hepato-splenomegaly. Inflammation, haemolysis
and cholestasis disappeared, while the renal function recovered. After
therapy, no steroids were administered for another six month and
the clinical picture worsened. A recourse to methylprednisolone and
cyclophosphamide, an immunosuppressive therapy, lead to a complete
remission. Liver and kidney biopsies performed three years after the first
hospitalization showed a marked reduction of the granulomatosis.
Now, the question was: how could that debris, whose origin was hard
to call into question, have reached the liver and the kidneys? In our
opinion, the hypothesis that those particles had been absorbed by the
gastrointestinal system looked the likeliest, but no literature existed to
support what we suspected. Debris of different sizes had been detected in
the stools, the same kind of foreign bodies had been found in the liver
and the kidneys, but those in the kidneys were smaller than those in the
liver. That could suggest that the particles, undoubtedly present in the
bowels, which were absolutely healthy in our patient, had passed through
the intestinal mucosa and had been cleared by the liver before entering
the general circulation and, from there, the kidneys.
Such passage was not described in the then current handbooks of
Physiology and sounded hard to believe.
Professor Peter Revell of the Free Royal Hospital of London gave us
then four bioptic samples of liver granulomatosis whose origin had been
declared to be unknown, but viruses, bacteria and parasites had been
ruled out as possible causes. In three of them we found evident traces of
surprisingly enough but undoubtedly environmental dust, while in one

we detected particles of a metal that had been used for therapeutic
purposes. Gold nanoparticles had been injected in the patient’s knee joint
to treat an arthrosis and it was that Gold that we found in the liver tissue.
That made us understand that solid environmental pollution could be
suspected as being able to penetrate the body and settle in the liver. How,
it was far from clear, but the most reasonable ways of entry seemed us to
be the digestive system – for which we had already a piece of evidence
with the case of the dental prosthesis - and, perhaps - but it was just a
guess - the respiratory system.
In the meantime, we had received more samples both from our
University Hospital and the Istituto Tumori (Cancer Institute) of Milan –
Italy, and they regarded cases of colon cancer and Crohn’s disease. In all
instances, inorganic dust was present in considerable concentrations and
variety. In one case we could even detect 15 different chemical elemental
compositions in the same cluster in less than 1 cm2 of tissue.
Thanks to our experience in caval filtration and the new availability
of filters that could be explanted even after a long time, we started to
take into consideration the blood clots that were regularly found stuck to
the device when it was removed from the patient and the thrombi that the
device had trapped. If inorganic dust, especially composed of chemical
elements that should not be present in the organism, had been detected,
that would have meant, without any possible doubt, that that dust had an
exogenous origin. As a matter of fact, as will be illustrated in detail later
in this book, all samples showed that presence.
Finding once unexpected inorganic particulate had become a daily
experience, but we could say very little or nothing at all about its way of
entrance.
So, we started to look at some pathological lung sample we got from
our University and the University of Siena (Italy), and among them there
were also some cases of pneumothorax. Then, we studied cases of cystic
fibrosis, sarcoidosis and of different forms of lung cancer.
Inorganic micro- and nanodust was to be found with a great
frequency.
We decided, then, to present a project to the European Community, a
project we called “Nanopathology”, by that neologism we had created
6 Nanopathology
meaning the pathologies due to micro- and nanodust, and by that project
intending to study if what we had observed for a few years had a
scientific basis.
1.1 Introduction
Man has only recently landed on what to him was a new planet: Nano.
But Nature has always been there. Just to give one example, Nature uses
a “nanocode” called genome to preserve all the information about the
species each individual, be it a man, a virus or any other living being,
belongs to, and that code is extremely complex, sophisticated and
efficient. Genome, in its turn, gives all the necessary instructions, many
of which we are not aware of, to build the (nanosized) proteins necessary
to live, and does that at a nanoscale.
We are accustomed to interacting with objects the size we can handle
and/or see, and have also been accustomed for a relatively long time to
investigating and trying to understand how atoms and molecules behave;
but Nano is something different from both worlds. Nanoscale systems
are too big to be considered molecules, but definitely too small to be
understood in terms of macro, and attempting to extrapolate their
behaviour starting either from the smaller or from the larger would lead
us astray. Neither quantum nor classic physics can be fully applied.
Of this sort of “mesoworld” we know very little, but what has
immediately become visible is that the properties of those objects are
extremely interesting from a scientific point of view and, in addition to
that, offer many prospective possibilities to be exploited to our benefit.
Right for that latter reason, an enormous throng of scientists, technicians,
industrialists and financiers flung themselves, and are still doing with
growing enthusiasm and hopes, headlong into that field, the way pioneers
did in the past when a new land was discovered or gold was
unexpectedly found somewhere.
More than a couple of decades ago, in 1986, Kim Eric Drexler, an
American engineer, published Engines of Creation, a book describing
nanomachines capable of reproducing both themselves and virtually any
material object, extending life duration, curing diseases, in short,
working all sorts of wonders and, at the same time, reducing pollution.
Many scientists ridiculed such outlook, and nevertheless the book
exerted a strong influence on many people. And many people means
good business. Now, after more than twenty years have elapsed, we are
still far from what Drexler we do not know how rightfully anticipated,
but it is a matter of fact that nanotechnology is finding more and more
applications and the limit seems to be imagination.
According to Forbes, (web ref. 1) now the most common applications
of nanotechnology are in sports goods, ski wax, tennis racquets and
tennis balls being the top three, but some medical applications are
already in use and many more are being attempted, cosmetics,
photocatalists and computer chips are already available and what are
called “smart” surfaces, by that meaning hydrophobic, self-cleaning,
anti-bacteria, anti-mould treatments are best sellers. Just as a brief
observation, the adjective “smart” sounds very à-la Drexler.
So, after having landed in what if not a promised is at least a
promising land, it would be fool not to advance and explore it with the
purpose of exploiting it. But like any unknown territory, also this can
hide unexpected dangers and, for that reason, using prudence and
exercising some patience may look advisable. Who carried out
exhaustive studies about the impact those technology and, in particular,
handling nanodust may have on human and animal organisms? How can
we know how long those entities will take to interact with environment
and organisms, if ever they will interact in a perceptible way? Is there
any long enough experience in that field? No satisfactory answer can be
given to those questions.
What we can do is extrapolate.
Micro- and, much more rarely, nano-scale particles have always been
produced by a number of natural sources: belching volcanoes, forest
fires, rock erosion, and airborne desert and beach sand, but the greatest
quantity of such dust, particularly the smaller, comes from man. Much of
it is the undesired by-product of combustion, a process that has started to
be used on a large, industrial scale only a couple of centuries ago, i.e.
roughly 1/10,000 of the time man has spent on the Earth. Generating heat
at temperatures higher than that of burning wood has always been
difficult and, when the concept of selling value was introduced,
8 Nanopathology
expensive. For those reasons, materials such as, for example, glass have
been rather precious for a long time. But when we started to exploit fossil
combustibles, heat has grown cheaper and cheaper and easy to come by,
and now very high temperatures can be attained without any difficulty
and at a low cost. All that creates dust, and, as a rule of thumb, the higher
the temperature, the tinier the dust.
So, foundries, cement plants, incinerators and internal-combustion
engines, among an indefinite number of other sources, produce particles,
and their quantity is constantly on the increase, particularly as regards
nanoparticles, since industrial filters are not efficient enough to trap them
and the increase in temperature makes microparticles rarer and
nanoparticles more common.
Particulate matter is released in the atmosphere and behaves in a way
that is very similar, or all but identical in the case of nanosize, to gas.
Some of those particles, especially the small ones, tend to form clusters
and, for that reason, making a clear distinction between micro and nano
is not always possible and often not even meaningful. However, in any
case the structure of a nanoparticle remains the same.
The dust we deal with is inorganic, most of the times crystalline,
insoluble in water or other common, natural solvents, and non
biodegradable. Being so small, pressure and thermal gradients and wind
carry it virtually everywhere, and when it eventually falls to the ground,
a gentle breath of wind can lift it up again, thus restarting the cycle. That
way, those particles can travel very long distances and stay with us
forever.
Besides, when we look at those inorganic particles from the pollution
point of view, we must not forget that they are often the carriers of
organic pollutants like, for example and among many others, dioxins,
furans or polycyclic aromatic hydrocarbons. And we must also remember
that in a number of environmental conditions, even inside biological
tissues, those particles tend to coalesce and, for that reason,
differentiating micro and nanoparticles is not always possible and in
some instances could be meaningless. Another important point is that
spherical particles generated by combustion, especially the larger ones,
are often hollow and very fragile, so, when they break, they create
fragments which are obviously smaller and, as a consequence,

microparticles can create nanoparticles.
From the compositional point of view, the nanoparticles we deal with
are very often alloys fortuitously created by the fact that the chemical
elements that make them up were present in the materials burnt and
combined to form an alloy that in most cases is not to be found in any
metallurgy handbook. So, their elemental chemical composition can be a
telltale indication that allows to identify the source.
But there are also cases when those particles come from wear and
friction of machines or industrial working, and heat is not involved in
their formation. In that case, their composition is the same as that of the
original material.
An important issue regards mass and volume. If, for the sake of
simplification, we assume that particles are spherical (which is often the
case with the particles we deal with, when created at high temperature),
we can easily calculate their volume and their mass, which depends,
obviously, on the matter that object is made of. Let’s suppose that we
have a sphere whose diameter is 10 µm, i.e. that of a coarse particle. In
that case, its volume is 4πr3/3 = 523.3 µm3. Now, let us suppose that we
wish to make spheres with a diameter of 2.5 µm, i.e. ¼ of the one of the
former ball. In that case, the volume of each sphere will be 8.0593 mµ3,
i.e.64 times as small. That means that, using the same amount of matter
necessary to make a particle whose diameter is 10 µm, we can create 64
particles whose diameter is 4 times as small. And, continuing, we can
create 1,000 particles with a diameter of 1 µm or 1,000,000 with a
diameter of 0.1 µm. This is particularly important if we look at this
simple geometric fact with the nanopathologist’s eye, since the same
amount of matter, if burnt at different temperatures, can give origin to
particles whose effects on the organism is very different [see also
Chapter 6 – Introduction]
On that dust we collected some experience and thanks to that we can
try and extrapolate about nanotechnological dust and its biological
behaviour.
Small particles are classified in a number of different ways, according
to the classifier and his discipline. Physicists, chemists and biologists do
not seem to be always agreed. We are not much interested in academic
10 Nanopathology
classifications and we do not really know where micro ends and nano
starts in the organism. It is highly probable that a threshold exists below
which natural, physiological barriers are ineffective and even one below
which cells do not oppose any resistance to the entrance of foreign
bodies. Our studies do not allow us to quantify that barrier nor to say if
and, in case, to what extent that threshold is influenced by size, shape,
surface/volume ratio, chemistry, state of aggregation or other factors.
Ours is a work in progress.
1.2 Bibliography
Ballestri, M., Baraldi, A., Gatti, A. M., Furci, L., Bagni, A., Loria, P., Rapanà, R. M.,
Carulli, N. and Albertazzi, A., (2001), Liver and Kidney Foreign Bodies
Granulomatosis in a Patient with Malocclusion, Bruxism and Worn Dental
Prosthesis – Gastroenterology,121:1234-38
Bovyn, G., Gory, P., Reynaud, P. and Ricco, J. B., (1997), The Tempofilter: A
multicenter study of a new temporary caval filter implantable for up to six weeks –
Ann Vasc Surg;11:520-25
Emanuelli, G., Gatti, A. M., Cigada, A. and Brunella, M. F., (1995), Physico-chemical
observations on a failed Greenfield vena cava filter – J Cardiovasc Surg;36:121-5
Gatti, A. M. and Montanari, S., (2006), Retrieval Analysis of Clinical Explanted Vena
Cava Filters – J Biomed Mat Res Part B: Appl Biomater 77B:307-314
Gatti, A. M., Ballestri, M. and Bagni, A., (2002), Granulomatosis associated to porcelain
wear debris – American Journal of Dentistry, Vol. 15, No. 6
Montanari, S., (2000), Malattia tromboembolica e filtri cavali - Ed. C. Rabbia, G.
Emanuelli – 90-140 Minerva Medica – Turin
Ref.web 1
[http://www.forbes.com/investmentnewsletters/2005/01/12/cz_jw_0112soapbox.html]
Chapter 2
In-vitro and in-vivo Biological Behaviour of

Micro and Nanoparticles
___________
2.1 Introduction
7
he 21st century opened with a revolution due to emerging
technologies called nanotechnologies, which are growing
exponentially and even if we know that they must have a physical
limit, their outlook seems to have no end.
“Nano” is a word of Greek origin meaning “very small”, and
nanotechnologies are characterized by smaller and smaller technologies,
i.e. they deal with items ranging in magnitude between 10-9 and 10-8 m.
The first definition was given by Norio Taniguchi, of the Tokyo Science
University, in 1971: “Nano-technology is the production technology to
get the extra high accuracy and ultrafine dimensions, i.e. the preciseness
and fineness of the order of 1 nm (10-9 m in length)”. This definition has
been updated by the Europäische Akademie in 2003:
“Nanotechnology deals with functional systems based on the use of
subunits with specific size-dependent properties of the individual sub-
units,” in order to include not only the production of nanoparticles, but
also the production of systems or processes based on the highest possible
miniaturization.
Nanotechnologies refer to the technological field concerning the
controlled manufacturing of functional nanosystems or the creation of
nanostructures, which results in the production of entities with at least
one dimension of 100 nm-length scale.
11
12 Nanopathology
To give an idea of the smallness of these things, we can compare

them with some biological objects (see Fig. 2.1). A human hair can range
between 10 and 60 microns in width, human red cells have a diameter of
5-7 microns, while, at the cell order, ATPsynthase measures 10 nm and a
DNA molecule ranges between 0.5 nm and 2 nm.
Fig. 2.1 Scale of dimensions.
The synthesis of such small entities, never seen in the world, as the
ones we include in the definition of nano and their (sometimes
involuntary) dissemination can represent a new pollution and new stimuli
for the animal and human organisms strongly influencing their health.
Nobody knows what the impact will be of these new technologies on
our society and what side effects are to be expected. It is a novel situation
and it is imperative to carry out researches to investigate if and how
nanoparticles can represent a possible risk to the environment and who
lives in that environment.
But our aim is to investigate:
- what is considered “nano” for the human body, namely,
- what is its reaction against a discrete (not continuous) stimulus,
- what is the threshold size below which tissues or cells do not react
in a known way since the sensors do not “see” or recognize the stimulus.
Nanopathology will try to give an answer.
In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 13
Applications in the medical field are already possible and a brief

overview of the most promising ones is given below.
2.2 Nanoparticles and medical devices
Applications of nanotechnologies are being experimented in a number of

specific fields of medical interest, but dentistry, by the creation of
nanocomposites for dental restorations, is the discipline where they are
already commonly available.
2.2.1 Dentistry
At present, dental restorations employ amalgams, composites, and Gold

or ceramic inlays.
There is a real need to dispose of mouldable, more resistant materials,
especially for posterior teeth where mastication load is particularly high,
capable of replacing amalgam, long suspected to be toxic. Though there
are no final scientific demonstrations to support the thesis of its toxicity,
a few diseases, and some lethal ones at that, have been associated to
the use of amalgams, in particular, amiotrophic lateral sclerosis and
Alzheimer’s disease. Amalgam is, in fact, the most common, and
inescapable for its carriers, source of human exposure to Mercury, and a
chronic low-level exposure to Mercury should be considered as a factor
in Alzheimer’s disease or in other diseases of the central nervous system,
including multiple sclerosis and Parkinson’s disease (Wenstrup et al.,
1990) (Saxe et al., 1999).
Some dental materials such as composites, dental filling resins
employing nanoparticles made, for instance, of Zirconia (Zirconium
oxide), are already available on the market, but other, more sophisticated,
materials are being developed. Organo-functionalized nanospheres,
containing X-ray absorbing elements such as Zirconium, combined with
liquid crystal monomers produce, in fact, composites with minimal
polymerization shrinkage and improve mechanical properties (Mitra et
al., 2003), (Mayer et al., 1998), (Williams et al., 2003).
14 Nanopathology
The improvement of these properties is only partial, since the wear

due to mastication cycles is not decreased and the risk of wear debris
ingestion continues (Ballestri et al., 2001).
Until a few years ago, this event did not cause any alarm, since it was
believed that that debris was gotten rid of with the faeces like all the
non degradable, small things people, and babies in particular, ingest
involuntarily. Unfortunately, it is not so.
It is growing clearer and clearer that biocompatible materials can
lose their biocompatibilty when they pass from a bulk form to a
fragmented one. The nanosized form is responsible for until-now
hardly suspected direct interactions with single cells, and those
interactions can induce unknown, unexpected reactions (Peters et al.,
2004), (Lucarelli, et al 2004). Toothpastes with either Silver or Gold or
hydroxypatite nanoparticles and used as nanofillers are already on
the market, as well as the bristles of some toothbrushes coated with
nanoSilver (see Chapter 7).
2.2.2 Orthopaedics
Besides dental implantology, another field of application of

nanotechnology is orthopaedics.
Orthopaedic devices, especially hip and knee joint prostheses,
present two main problems that show over time: wear of the matching
surfaces in the joint and loosening of the stem in the femoral channel.
The former causes the failure of the device because of the granulomatous
reaction induced by the wear debris, the loss of the three-dimensional
movement due to the loss of sphericity of the head, and, finally,
the rupture of the acetabulum with the ensuing migration of the
head.
To overcome these problems, new nanocomposite-based, wear-
resistant and self-lubricating PVD (plasma vapour deposition) coatings
(Stueber et al. 2002) are being studied. The wear of the hip joint
prostheses and the health problem due to the production of wear debris
induce researchers to develop new coatings more resistant to wear and,
possibly, self-lubricating. A possible solution can be the development

of a metastable hard matrix incorporating homogenously distributed
nanoclusters of MoS2. The use of cross-linked polyethylene does not
seem to solve the problems, also because wear produces sub-micronic
particles that can trigger a tissue reaction and be widely disseminated in
the tissues because of their ultrafine size (Urban et al., 2000), (Revell
et al., 1997).
The creation of nanosized-morphology surfaces enhances
biocompatibility and is supposed to ameliorate the interaction between
the device and specific cellular components. In biomaterial research, it
has been found that even though a bulk material may be well tolerated by
the body, finely divided particles of the same material can often lead to
severe, even carcinogenic, complications. Differences in particle size
influence histological reactions and cytokine production. Macrophage
response to particulate debris appears, for example, to be dependent on
particle size, composition, and dose as given by surface area ratio.
Several studies have also been carried out to determine the relationship
between cell and particle size with respect to cytotoxicity. (Goodman
et al., 1990), (Shanbagh et al., 1994), (Tamura, 2003).
Carbon nanofibres have been proposed as a possible, new
orthopaedic implant material because of their unique mechanical,
electrical and cytocompatibility properties. These fibres have a size close
to hydroxyapatite (Calcium phosphate) crystals and collagen fibres
found in the bone. Moreover, “in vitro” studies provided evidence that
nanophase Carbon fibres enhance osteoblast function over conventional
Carbon fibres and current orthopaedic implant materials (such as
titanium). To determine the influence of Carbon nanofibre wear debris on
osteoblast viability, direct contact toxicity studies were performed. The
results proved that nanophase Carbon fibres were less detrimental to
osteoblast viability than larger-diameter, conventional Carbon fibres. In
other words, they provide evidence of the promise of nanophase
materials in improving orthopaedic implant efficiency (Price et al.,
2004).
16 Nanopathology
2.2.3 Nanostructured surfaces
Implantable medical devices are bound to employ biocompatible

materials, i.e. materials that must be not only accepted by the biological
environment, but also promote an appropriate and favourable biological
interaction. The basic concept is that biological activity depends on the
structure of the matching matter and, at the same time, much of the
matter properties depends on size (Dobson et al., 1999).
So the biocompatibility of a material must have an “appropriate” cell
adhesion, that is preceded by a specific protein adsorption from the
extracellular matrix. The adsorption of a specific monolayer of proteins
will be of the utmost importance for the success of an implant. In fact,
once the proper protein has been adsorbed by the implanted surface, the
appropriate cells need to adhere and proliferate. The target of achieving
proper cell adhesion and proliferation is every biomaterial engineer’s
challenge. (Whiteside et al., 1991).
There are many cellular processes which are triggered by the type of
protein adsorbed, its conformation and its biological activity. If the
presence of a certain protein is requested to guarantee a “proper”
interaction of an implant with the biological environment, it may be
possible to manipulate the implant surface in order to induce in advance
that situation. The so-called “biomimetic” surfaces base their activity in
the human body upon this concept and, typically, biomimetics aims at
copying nature’s way of organizing and ordering complex molecules at
surface, to inhibit non-specific surface reactions and, finally, to create
“smart surfaces” that undergo rapid shifts in properties with small
external change (Castner et al., 2002).
The ability to design such a system is greatly supported by
biotechnologies and nanotechnologies. Examples of that are the
molecular self–assembly as a strategy for nanofabrication that involves
designing molecules and supramolecular entities so that shape-
complementarity causes them to aggregate into desired structures. Just as
an example, the Self-Assembly Monolayers (SAMs) are considerably
important in developing arterial prostheses, where it is absolutely
essential that the interior of the walls gets quickly coated by endothelial
cells. The efforts to attach heparin molecules to the surface of vascular
catheters in order to avoid blood clotting was not particularly successful,

especially because thrombosis cannot be reduced to a simple surface
problem, but the new strategy of SAMs can certainly improve and
stabilize these surfaces, and help to achieve better results. (Zhang et al.,
2002).
Nanotechnologies can create functionalised nanosurfaces attaching
polipetides or dendrimers (repeatedly branched molecules) that can
increase the biocompatibility of the implantable devices. Suitable
candidates for dendrimers synthesis and study include monomers
currently used for the preparation of medical-grade linear polymers,
and natural metabolites. For example, monomers from synthetic
polymers such as polyethylene-glycol, polytrimethylene-carbonate and
natural amino-acids represent classes of monomers suitable for use.
Incorporation of one or more of these building blocks in a dendritic
structure provides new opportunities to create well-defined polymers for
tissue engineering applications. The versatility of dendrimer technology
provides also unlimited possibilities in the synthesis of novel
nanomaterials and unprecedented control over these systems (Eppel
et al., 2002), (Bellingham et al., 2003), (Carnahan et al., 2001), (Balogh
et al., 1999).
2.2.4 Drug delivery
Other emerging and especially promising applications are those in the

drug-delivery field and gene therapy. Those are made possible by the
versatility of nanotechnologies and, particularly, by the extremely
reduced size of those devices, equal to that of their targets. In the targeted
delivery of drugs there are attempts to use dendrimers because drug
molecules can be loaded both in the interior of the dendrimers and
attached to the surface groups in order to control the rate of drug’s
release into the body. ( Kilts, 2003), (Orive et al., 2003), (Buxton, 2003).
One of the most promising application of nanoparticles is their use in
pharmaceuticals as a drug-releasing support for neural diseases, capable
of negotiating the blood-brain barrier. The existence of this barrier is
in fact a severe limitation for the delivery of potentially useful drugs
such as cytostatics and central nervous system in-vitro active agents.
18 Nanopathology
Nevertheless, it has been demonstrated that different drugs bound to

nanoparticles can be transported across the blood-brain barrier and
achieve pharmacological effects in the brain (i.e. brain tumour treatment)
(Li et al., 2003), (Lockman et al., 2002), (Koziara et al., 2003), (Rantioa
et al., 2004), (Schroeder et al., 1998).
Nanosized materials can be the substrate where viruses or DNA
molecules can be encapsulated or sorted. The interaction of these
biomolecules with the nanoparticle, nanotube or nanosized surface can
serve to detect specific proteins or viruses and can guarantee that the
molecules are carried to the specific target. The integration of biological
molecules with Carbon nanotubes (i.e. fullerene molecules with a
cylindrical or toroidal shape) has potential applications in gene- and
drug-delivery technology and enables the use of hybrid systems like
biomolecular sensors. In addition, peptide functionalized nanoparticles
were created as a new class of therapeutic and diagnostic agents with the
purpose of specifically recognizing and binding the characteristic
molecular signature of cancer and other pathological conditions. (Salem
et al, 2003), (Zheng et al, 2003), (Sando et al,2003), (Chakrabarti et al.,
2003), (Lehmann-Horn et al., 2003), (Ai et al. 2003), (Gao et al., 2003).
But, besides its visible advantages, the possibility to interact with the
smallest components of the human body makes nanoparticles potentially
dangerous, and verifying the results of this interaction is an awkward
matter (Pereira et al., 1999), (Saebo, 2004).
Many researchers and organizations (web ref. 1), (Tran et al., 2005),
(web ref. 2), (Greenpeace Environment Trust Report, 2003), (US EPA
Report, 2005), (SCHENIR Report, 2005), (web ref. 3), (Proceedings of
St. Gallen, 2006), (web ref. 4), (web ref. 5), (web ref. 6) are busy
discussing the possible risks related to these new technologies and the
possibility to manipulate in such a small-scale way the cell response.
They can actually enter the caveolar openings in cell membranes and
the perturbations produced can cause the death of the cell. Nanoparticles
have also a high surface reactivity and can interact with enzymes or
proteins, disturbing biological processes and causing impairments to
structural or metabolic processes. (Schler et al., 2003), (Colvin, 2003),
(Warheit, 2004), (Borm et al., 2004), (Brook et al., 2004), (Maynard et
al., 2004), (Hett, 2004).
2.2.5 Nanomedicine
Nanomedicine (Editorial, 2003), (web ref. 7), (web ref. 8) is a new field
of research that deals with all the applications of nanoparticles or
nanodevices for medical uses.
It refers to highly specific medical intervention at the molecular scale
that involves the use of engineered nanodevices and nanostructures to
monitor, repair, construct and control the human biological system. The
most elementary of nanomedical devices will be used in the diagnosis of
illness. A more advanced use of nanotechnology might involve, for
example, implanted devices to dispense drugs or hormones as needed in
people with chronic imbalance or deficiency states. Lastly, the most
advanced nanomedicine involves the use of nano-robots as miniature
surgeon. Such machines might repair damaged cells or even get inside
cells and replace or assist damaged intracellular structures (heart
defibrillators and pacemakers are likely to be replaced by more
sophisticated devices that can act on the individual cells). And in a
possibly not so far future, nanomachines might replicate themselves or
correct genetic deficiencies by replacing DNA molecules.
Nanospheres are already employed in humans, though only
experimentally, in diagnostics. Inhalable nanoparticles can sense, for
example, the local ventilatory status, releasing drugs at appropriate local
dosage in response to physiological stimuli and lipid-based nanoparticles
are being considered for site-specific delivery of antifungal therapy
to the pulmonary epithelium. Moreover, it was observed that highly
lymphotrophic super-paramagnetic nanoparticles (monocrystalline Iron
oxide) can easily gain access to lymph-nodes by means of interstitial-
lymphatic transport in patients affected by prostate cancer. Their
presence in lymph-nodes can be detected by MRI (Magnetic Resonance
Imaging) and their concentration can indicate a metastasis (Harishingani
et al., 2003), (Babes et al., 1999).
Scientists have also shown that Iron oxide nanoparticles as small as a
virus can outline not only brain tumours under MRI, but also other
cerebral lesions caused by multiple sclerosis, stroke and neurological
disorders, that may otherwise go unnoticed. Iron oxide nanoparticles can
20 Nanopathology
be delivered across the blood-brain barrier and stay in the brain lesions at
least for days. For this reason, Iron oxide has some advantages over
Gadolinium, which must be administered just before surgery and which
doesn’t enter cells. Advances in magnetic resonance imaging using Iron
oxide are showing to be particularly promising to improve diagnoses also
in liver and neck tumours (Mack et al., 2002), (Schultz et al., 1999).
The same nanoparticles can be used also for cancer therapy. Once
delivered in the tumour they are involved in a field of radiofrequency
waves that induce a heating of the Iron particles and of the cells
that produces the death of the cells and the necrosis of the tumour.
(Brigge et al., 2002), (Fahlvik et al., 1990), (Boomemain et al., 2001),
(Hogemann et al., 2001), (Moghimi et al., 2001), (Allport et al., 2001),
(Wunderbaldinger, 2001), (Wilhelm et al., 2002), (Moore et al., 2001).
This new possible therapy is under evaluation with in-vitro and
in-vivo experiments. The given references can be the base for a
deepening of the matter.
From our point of view, we look with a critical eye such an invasive
diagnosis/therapy that releases intentionally not biodegradable particles
inside the organism, particles which are inevitably sensed as foreign
bodies. The reaction against that unusual, unexpected invasion is not the
one produced against, for example, foreign proteins: we do not have
specific enzymes, specific antibodies and there is no anaphylactic shock.
Those particles represent something unknown to the human body, but
when they are inside the blood circulation they are immediately sensed
and interact with the blood’s components and they do it physically,
chemically and biologically. This interaction is the key-point of
modifications that can have systemic effects, not immediately the way it
happens with foreign proteins, but after a more or less long while.
Nanoparticles have a size that is roughly the same as the tri-dimensional
size of proteins. The attachment of a nanoparticle to a protein (for
physical adhesion, for electrostatic attraction, etc.) can cause a stretching
of chemical bonds, a stress of the protein’s morphology, and the
denaturation of the proteinic structure, causing the non recognition of the
compound as something rightfully belonging to the organism. That can
be the foundation of an immunological disease.
But what is very critical, is the fact that after the imaging or the
therapy have been performed, these foreign bodies cannot be disposed of,
remain inside the body as foreign particulate matter, and their long- term
effects are mostly unknown. Something about those effects is described
in the following paragraph.
Also the application of quantum dots in Medicine may be seen as a
possible cause of unknown effects.
Quantum dots (QDs) are semiconductive crystals that do not exist in
nature, with dimensions ranging from 1 to 10 nm, and that can be
fluorescent. They can emit light throughout the visible spectrum from the
infrared to the ultraviolet. What makes our dots unique is that their
luminescence can be tuned to any wavelength over a broad spectral range
and be stable under ambient conditions (Bruchez Jr M et al., 1998),
(Bruchez Jr M, 2005). (web ref. 9).
Examples of QDs are Cadmium selenide, Lead selenide, and Lead
Sulphur. Cadmium selenide can be the core of a QD with an outer shell
of Zinc.
Their peculiarity is the fluorescence emitted after having been
excited. That fluorescence is more perceptible and more prolonged
as to that of the normal fluorescent agents used in Medicine
(fluorescin, etc.)
They can be combined with antibodies in an easy way and can
be driven inside a cell. Their fluorescence can be detected and be
used to verify its state. Studies to use them in vivo seem to demonstrate
their capacity to be attracted by tumour cells and show their borders
in a body.
But the main problem that remains beyond the technological beauty
of these items is “what is the fate of them after use inside the body?”
They can enter easily in the cells but they are inorganic matter masked by
organic one.
22 Nanopathology
2.3 The results of the nanopathology project
In 2001, we coined a new word: “nanopathology”, meaning by that the

collection of pathologies due to micro- and nanoparticles. At that time,
this was a void concept, since a comparatively small number of
pathologies were recognized worldwide as triggered by particulate
matter. Silica particles and asbestos fibres are recognized to be able to
induce lung diseases as silicosis and asbestosis. Inhalation of dust
containing particles of quartz or fibres of a silicate is known to trigger
lethal pathologies of the lungs. It is a well-known fact that the inhalation
of mine dust or of cigarette smoke are risk factors for the onset of lung
cancer. But a possible correlation with nanoscaled particulate matter was
completely unknown.
In 2001, submicronic matter was recognized to be dispersed in the
environment, though very few studies existed, though none of them was
exhaustive, on its behaviour, but nothing had been done for nanosized
particles.
No evidence identified nanodust as responsible for pathologies. Just
micronic and, in very few instances, submicronic particles were
considered to be the cause of pathologies (e.g. pneumoconioses).
Now that nanotechnologies are a rapidly growing discipline, society
is scared by possible side effects of the production, manipulation and use
of nanoparticles, and nanosafety has become by necessity a priority.
Many researchers are investigating the toxicity of nanoparticles towards
cells, while our project has already gone further by investigating their in-
vivo interaction and the impact they have to humans.
Among the most abundant air pollutants in urban areas is particulate
matter with a mean diameter ≤10 µm (also called PM10 by
environmental toxicologists, defined as particulate matter with an
aerodynamic size of 10 µm or smaller). Over the years, it has become
clear that particles with a very low size (especially ≤100 nm) are more
significant health-wise than larger particles, since they have been shown
to be capable of inducing far more severe effects. (Oberdörster, 2001).
The surface/size-ratio increases exponentially with the decreasing of
particle sizes, leading to enhanced surface reactivity. This enhanced
surface reactivity might lead to greater biological activity per given mass,
compared to larger particles, which, in turn, might have effects, for
example, on the internalization of particles into tissues, cells and
organelles, or on the induction of oxidative stress (Oberdörster et al.,
2005). By definition, particles with a size below 100 nm are called nano-
scaled particles (short: nanoparticles) or ultrafine particles by
toxicologists.
Because of the minute size of nanoparticles, the internalization into
the body’s tissues appears to be extremely easy. This was shown by
experiments in human volunteers with radioactive-labelled Carbon
nanoparticles (i.e. `Technegaz`) that were shown to pass rapidly into the
systemic circulation after inhalation. Radioactivity could already be
detected in the blood one minute after inhalation (Nemmar A. et al.,
2002). Furthermore, animal studies revealed that inhaled nanoparticles
were relocated into the liver (Oberdörster G. et al., 2002) and the brain
(Oberdörster G. et al., 2004). Thus, nanoparticles seem to be able to
circumvent the tight blood-brain-barrier and possibly cross the blood-
placenta barrier (Reichrtova E. et al., 1998), (Kaiglova A. et al., 2001).
Moreover, it has been suggested, and we offered further evidence, that
nanoparticles are involved in thrombus formation (Nemmar A. et al.
2002), (Gatti A.M. et al., 2004) and today we know that particulate air
pollution is associated with enhanced mortality from respiratory and
cardiovascular diseases (Pope C.A., 2000).
As the sources of internalized nanoparticles (food, air, etc.) and the
location of particle detection are generally far apart, a distribution via the
blood stream must have occurred. Thus, endothelial cells, which line the
inner surface of blood vessels, will have direct contact with the particles.
Those cells are important in inflammation mechanisms and wound
healing. Upon pro-inflammatory stimulation of the endothelium,
adhesion molecules are expressed on the cell surface, thus mediating
leukocyte attachment (e.g. E-selectin and intercellular adhesion mole-
cule-1/ICAM-1). Besides, endothelial cells can release cytokines, such as
interleukin-8 (IL-8, a key factor in neutrophil chemotaxis). Thus, these
features contribute to the pro-inflammatory endothelial phenotype that
permits the transmigration of leukocytes from the blood into the
perivascular space (Cook-Mills J.M. et al., 2005).
24 Nanopathology
The activation of IL-8, E-selectin and ICAM-1 is regulated by the

same transcription factors NF-κB (nuclear factor-κB) and AP-1 (acti-
vator protein-1) (Montgomery K.F. et al., 1991), (Roebuck K.A., et al.,
1995), (Mukaida N. et al., 1994).
The in-vitro tests we carried out within the scope of the European
project considered the interaction of 5 different engineered nanoparticles:
Titanium oxide: titania (TiO2), Silicon oxide: silica (SiO2),
Polyvynilchoride (PVC), Cobalt and Nickel with cells (human
endothelial, gut epithelial, liver epithelial and monocytic cells) and
organs (in-vivo tests with rats). Those nanoparticles were examined with
respect to the cellular internalisation and their influence on the cell
viability, proliferative activity, and the pro-inflammatory endothelial
phenotype. Titanium oxide (titania) was selected since it is employed in
many beauty and sun-screen creams and in wall paints, Silicon oxide for
its use in chewing gum and coatings, PVC because it is used in a bulk
form for medical devices, Cobalt because of its toxicity and Nickel
because it is allergenic1.
One of the main characteristics of nanoparticles is their clustering,
namely their capability to form microsized aggregates that provoke their
sinking in the medium. So, a real interaction with cells is not allowed if
proper procedures to disperse them in the medium are not adopted. Some
authors claim good non-cytotoxic results, but they did not verify if a real
cell-nano interaction was set-up.
The nanoparticles mentioned were added to macrophages/monocytes,
gut and liver epithelial cell culture medium and the cytotoxicity assays
verified the cell survival, the production of inflammatory and defence
mediators as the cytokine production, and the modulation of TLR
expression. These are parameters that can give an idea if the presence of
nanoparticles weakened the “healthy” state of the cells .
The cytotoxicity studies carried out by Dr. D. Boraschi (CNR Pisa –
Italy) showed that Cobalt nanoparticles induced a lesser expression of
1
SiO2 and TiO2 particles were produced by flame spray pyrolysis. The size range of SiO2
was 4-40 nm with 14 nm mean particle size; of TiO2 20-160 nm (mean size 70 nm),
of Co particles (Sigma) 50-200 nm (mean size 120 nm), of Ni was about 62 nm
(Nanoamor), of PVC (polyvinylcloride without phthalate) 100 nm.
TLR4, TLR7, TLR8, TLR5 TLR10 and CD14. The other materials have
different behaviours (Lucarelli M. et al., 2004).
The tests verified that:
1- Nano-particles do not affect cell survival or proliferation. Only Co
nano-particles are toxic at >100 mg/106 cells
2- Co nano-particles could have an M2 biasing inhibiting effect of
TLR4/CD14-mediated responses. It was noted a down-regulation
of TLR4 and CD14 expression, inhibition of reactivity to LPS in
macrophages.
3- Nano-particles, in particular ZrO2, induce increase expression of viral
TLR. There is a possible impact on cell reactivity to viral infections
4- SiO2 nano-particles have M1 biasing effect on macrophages. There is
the induction of M1 markers in naive cells, amplification in M1 cells
5- Nano-particles, in particular TiO2, amplify IL-18 activity (role in
initiation of autoimmunity?). That means that there is a differential
expression of IL-18 regulatory molecules in macrophages and
hepatocytes.
So there is not an evident and significant cytotoxic effects at subtoxic
concentration, but an impairment of cellular viability was observed at
different levels (i.e. decrease of cell number, protein expression of the
proliferation marker Ki67, and the metabolic activity).
According to studies carried out by J. Kirkpatrick (University of
Mainz – Germany) a pro-inflammatory effect in HDMEC (Human
Dermal Microvascular Endothelial Cells) occurred after exposure to
SiO2, Co, and Ni particles and was apparent by an enhanced release of
IL-8. Only higher particle concentrations (25 and 50 µg/ml) induced this
increase in IL-8 release. The E-selectin protein expression was enhanced
by high amounts of Co-particles whereas Ni-particles induced no protein
expression of E-selectin. In contrast to the particles, divalent Co and Ni
ions induced the expression of all pro-inflammatory markers tested (i.e.
IL-8, E-selectin, ICAM-1) (Peters K. et al., 2004).
That means that the normal cell defences are weakened in presence of
nanosized foreign bodies
In order to evaluate and quantify of pro- and anti-angiogenic
characteristics of the nanoparticles we selected another model system
26 Nanopathology
that leads to the formation of in-vitro capillaries within a three-dimen-

sional extracellular matrix (fibrin and type I collagen).
The addition of the different particles (TiO2, SiO2, Ni, Co, PVC) into
the three-dimensional matrix revealed that only the presence of Co
particles led to changes in the in-vitro capillary phenotype. A number of
abortive sprouts were formed and the developing sprouts were not so
pronounced as in the positive control. However, cell viability appeared
unaffected. Software-supported quantification of these Co-particle-
induced changes revealed a significant reduction in the degree of
angiogenesis in vitro.
Also the tests employing the co-cultivation of endothelial cells
together with the monocytic cell line U937 in the angiogenesis model
system verified the hypothesis according to which the addition of PMA-
stimulated U937 cells to the culture could lead to a distinct decrease of
the length of in-vitro-capillaries and also to a reduction of endothelial
cell number.
In detail the tests with endothelial cells verified:
a- The nanoparticles investigated do not affect cell survival or
proliferation. Only Co nanoparticles are toxic at >100 mg/106 cells. They
inhibit the expression of LPS receptors TLR4 and CD14 and induce a
down-regulation of mRNA expression for TLR4 and CD14 (the two
receptor chains that recognise bacterial lipopolysaccharide and activate
macrophage defence functions).
An interesting result is that Co nanoparticles impair macrophages
activation by bacterial LPS, in fact a negligible cytokine production
was detected. These results mean that in presence of these metallic
nanoparticles the macrophages become unable to mount appropriate
defences to bacterial challenge. That means that danger exists of
increased susceptibility to infections. These very interesting results were
not obtained with Ni nanoparticles since, due to their forming micrometric
aggregates and sinking in the medium, they did not interact with the cells.
b- The Cobalt nanoparticles with endothelial cells showed an
angiogenic behaviour.
Our study has shown that human endothelial cells possess a large
capability to internalize nanoparticles. All nanoparticles tested were
taken up by the endothelial cells and to a major extent into vacuoles.
The Cobalt nanoparticles showed an angiogenic behaviour, that can

trigger a carcinogenic reaction.
The results induced to put forward the hypothesis that the observed
pro-inflammatory activation after Co particle exposure may be attributed
to a release of divalent Co ions by the particles [Cobalt nanoparticles
inside the cell corrode and release Cobalt ions], since the exposure of en-
dothelial cells to these ions leads to the impaired endothelial viability and
pro-inflammatory stimulation. This contrasts with the effects of the Ni
particles. Here, the suggestion of a Ni ion release by the particles
resulting in an induced pro-inflammatory stimulation is not congruent
with the pro-inflammatory effects induced by the respective ions, since
Ni ions induced both an increase in the release of IL-8 and the protein
expression of endothelial cells adhesion molecules (i.e. E-selectin and
ICAM-1), whereas Ni particles induced only an increased IL-8 release
and the expression of adhesion molecules was not initiated. This
indicates an activation mechanism for the Ni-particles that deviates from
the Ni-ion-induced activation that is shown to occur via a cooperation of
the above-mentioned transcription factors NF-κB and AP-1. Since
oxidative stress is also a relevant aspect in the mechanisms of (Ni-)
particulate matter-induced effects (Koziara et. al., 2003) this mechanism
of differential activation of pro-inflammatory gene promoters might
play a role. Thus, it can be suggested that the Ni-ion release by the
nanoparticles remains under the critical limit for pro-inflammatory
activation but further Ni nanoparticle induced effects (possibly oxidative
stress) are responsible for the enhanced IL-8 release. Also the tests with
the co-cultures (macrophage-endothelial cells) indicate a deep interaction
of Co nanoparticles with the cells. The addition of PMA-stimulated
U937 cells to the culture led to a distinct decrease in the length of in-
vitro capillaries and also to a reduction of endothelial cell number. In
addition to that, the number of U937 colonies within the 3-dimensional
extracellular matrix rose.
These in-vitro results indicate some mechanisms of actions of
nanoparticles with cells and explain the in-vivo results and the clinical
evidence.
28 Nanopathology
2.3.1 In-vivo experiments
The 5 different materials mentioned above (Cobalt, Nickel, silica, titania,

PVC) were implanted in rats in two forms (bulk samples as discs, and
nanoparticles).
That experiment was performed in order to verify if the size of these
debris is an important factor in triggering pathological reaction.
Each rat (see Fig. 2.3.1) was implanted in the dorsal muscle with two
similar implants: an amount of 5 cc of nanoparticles and a 10-mm
diameter, 0.8 mm height disc of the same chemical composition. For
each group of materials, 2 rats were sacrificed after 6 months (short
term), and 3 rats were sacrificed after 12 months (long term). In addition,
5 control rats were implanted with a reference material. Two of them
were sacrificed after 6 months and 3 of them were sacrificed after 12
months. Five rats were fed by gavage with a mixture of nanoparticles
(50% Ni and 50% ZrO2 with two different ranges) (about 0.030 – 60 mg
by day): 2 were sampled after 6 months and 3 were sampled after 12
months, in order to see if the small-size particles can negotiate the bowel
barrier and contribute to the pathogenesis of some diseases. The animals
were sacrificed after 6 and 12 months.
After the short- and long-term implantation, samples were explanted
and the gross necropsy of the animals performed. All samples were
formalin-fixed and paraffin-embedded, microtome-sliced and Hematoxylin-
Eosin-stained. The sections were analyzed histopathologically and under
ESEM in order to verify the interface of the bulk and nanosized materials
after the interaction.
Our animal studies verified that the metallic nanoparticles were
cancerogenic, while the bulk material induced just a fibrotic capsule with
a granulomatosic reaction.
Fig. 2.3.1 Cobalt and Nickel nanoparticles induced rhabdomyosarcoma in the rats’ back.
The metallic disks induced only fibrotic capsules. (Courtesy of Biomatech-France)
The other materials (PVC, silica, and titania) developed only a

fibrotic capsule or a granulomatous tissue. The reason could be the
agglomeration of the nanoparticles that transforms them to micrometric
debris. In this case, the body reacts to the size of particulate matter, as the
materials are chemically inert.
A summary of macroscopic and microscopic findings are presented in
Tab. 2.3.1.
30 Nanopathology
Tab. 2.3.1 Synopsis of the histological findings.
Material 6-month period 12-month period

TiO2: NP Granuloma Granuloma
TiO2: B Inflammation Inflammation
SiO2: NP Inflammation Inflammation
SiO2: B Inflammation Inflammation
Granuloma + fibroblastic
PVC: NP Granuloma
proliferation
PVC: B Inflammation Inflammation
Co: NP Preneoplasia Neoplasia*
Inflammation +
Co: B Preneoplasia*
granuloma
Ni: NP Neoplasia #
Ni:B Neoplasia #
Note: B= bulk, NP= Nanoparticles; * Animals were euthanized after 8 months. # No

analysis - animals were euthanized after 6 months.
TiO2 – In detail: a thin, fibrous, moderately capillarized pocket

surrounding the bulk material implanted subcutaneously was observed in
all cases, but no granulomas were present.
SiO2 – In detail: in all cases, an adherent thin fibrous pocket
surrounding the bulk material implanted subcutaneously could be seen,
like what happened with TiO2, without any evidence of local intolerance.
The inflammatory infiltrate consisted mainly of lymphocytes and
monocytes, but not of mast cells.
At the site of the intramuscular implantation, neither particles nor
macroscopic abnormality could be seen. Histological examination of the
specimens with implanted particles revealed inflammatory infiltrates
which were localised subcutaneously and intramuscularly. An increased
numbers of mast cells, typically localized perivascularly, could be
detected in most cases.
PVC – Also in all these cases, a thin fibrous pocket around the bulk
material implanted subcutaneously was observed, without any visible,
local lesion. And also in these cases, the bulk material itself did not
reveal anything abnormal. Histological evaluation of implanted PVC
bulk showed discrete chronic inflammatory infiltrates. No particles could

be observed.
The sites implanted intramuscularly with nanoparticles did not show
anything macroscopically remarkable. In 8 cases out of 10, particles were
easily visible in the subcutaneous tissue. On histology, all cases of
implanted PVC nanoparticles revealed intramuscular foreign body-type
granuloma. Macrophages and multinucleated foreign-body giant cells
could be seen around amorphous, unstained polygonal material that did
not exhibit birefringence under polarized light. However, the particles
revealed intense cherry-red colour by staining with the Oil Red-reaction.
A slight perigranulomatous fibrosis was also observed. An interesting
find is the observation of foci of fibroblastic proliferation in three
animals. The cells were spindle-shaped and showed some nuclear
pleomorphism. On immuno-histochemistry, they showed an enhanced
nuclear expression of PCNA (Proliferating Cell Nuclear Antigen).
Cobalt – Clinical examination revealed the formation of nodules in
three animals occurred within the 6-month observation period. Then, all
remaining animals implanted with Co developed nodules at the sites
where nanoparticles had been implanted. One animal of the 12-month
observation group died 8 months after implantation. So, for ethical
reasons because of the development of these handicapping tumors, all
remaining animals were sacrificed at 8 months. In three out of the four
cases belonging to the 6-month group and in six cases of the 8-month
group, nodules developed at the site where nanoparticles had been
implanted.
Through histological observation of the specimens with implanted
nanoparticles, malignant, intramuscular, mesenchymal tumors at the
original implantation site was demonstrated in one out of four animals of
the 6-month observation period and in five out of six cases of the 8-
month period. Particles were observed in three of six animals belonging
to the 8-month group.
No such tumors were not observed around the bulk material, where
a discrete inflammatory infiltrate composed of mononuclear cells and
lymphocytes was present. In the subcutaneous area, a discrete fibrosis
had developed, but not granulomas.
32 Nanopathology
In two of the four animals of the 6-month observation period (at the
nanoparticulate site) and in two of the six animals of the 8-month
observation period, a capsule with fibroblastic proliferations was visible.
The cells showed an increased pleomorphism of the nuclei and mitotic
rate. In addition, they displayed strong expression of the proliferation
marker PCNA. These lesions are classified as pre-neoplasia.
Grouped together, these results show the presence of a malignant
mesenchymal tumor in five of the six cases of implanted Co nanoparticle
sites and two further cases (one around bulk material and one at the
nanoparticulate site) with pre-neoplasia.
Nickel – Three of the animals from the Ni-implanted group died
respectively 4, 4.5 and 5.5 months after implantation. Because of such a
high mortality rate and the development of tumors, we decided to
sacrifice all the remaining animals belonging to that group at 6 months.
Both subcutaneous and intramuscular implantation sites had developed
visible nodules in all cases and in all cases necrosis was detected inside
the nodule associated with deposits and exudate seen in the implanted
subcutaneous pocket.
Both Ni bulk and nanoparticles of implanted Ni showed a malignant
mesenchymal tumor surrounded by a fibrous capsule localized
subcutaneously and intramuscularly. In general, abundant areas of central
necrosis, dystrophic calcification and ossification were also observed. In
contrast with that, tumors in tissue specimens implanted with bulk
material showed a central cystic component, suggestive of the implant
site. In addition to that, necrosis looked less important, and ossification
was more rarely seen. Particles could not be observed histologically.
The findings indicate a malignant mesenchymal tumor, which is
classified on the basis of the morphology and the immuno-histochemical
marker profile as rhabdomyosarcoma.
The results obtained, though preliminary and in need to be confirmed
by further experiments, show that size is a decisive factor in determining
the compatibility of a material with the organism. As to materials, metals
seem to be much more reactive in a pathogenic way then ceramics and
polymers. These results must be reminded for all the following chapters.
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Chapter 3
Clinical Cases:
Lung, Blood, Liver, Kidney, Digestive System,
Vessels, Sperm
___________
3.1 Introduction
7
his chapter contains a number of clinical cases studied on bioptic or
autoptic samples. No homogeneity or statistical meaning is to be
expected or looked for here, as this is just the start of a collection of
cases that will hopefully keep coming and being added to the ones
illustrated here. Anyhow, what is described in this part of the book
represents answers given to patients, or their relatives or doctors when
the patients had died, regarding the exposure those patients had
undergone. In many instances, we had no advice to give as to a possible
treatment, but when somebody knows he is dying, knowing why can be
important to him. In any case, the experience we are collecting can be
useful to prevent the repetition of similar cases or to find an explanation
to cryptogenic diseases.
It was hard, when not impossible at all, to guess how particles
actually behave once they have entered the organism. So, one of the
problems was to select the organs that make up a favourite mark for
those particles, if such a thing as favourite organs exists.
Inflammatory pathologies of unknown origin had already proved to
be a very interesting source of knowledge, but in our then limited
experience we had seen that dust might possibly be blamed for other
forms of adverse reactions like, for example, the formation of blood
39
40 Nanopathology
thrombi or - but in that case the thing was far more complicated - for
some neurological diseases or pathologies whose classification is
controversial like chronic fatigue.
We studied more than 600 aggregate cases of vein thrombosis,
chronic fatigue, lymphomas, solid cancers of various organs, etc.
Reference tissues were obtained from cadavers of young, presumably
healthy, subjects, died in car accidents.
All the pathological specimens we had the chance to examine showed
the presence of inorganic, non biocompatible and non biodegradable
debris, ranging 10 nm - 10 microns. Some of those debris (10-100 nm)
were also photographed inside cellular nuclei.
The observation and study of the pathological samples we came
across and are described here showed a few interesting data. In the cases
of biological reactions typical of some diseases like cryptogenic
granulomatosis, sarcoidosis and Crohn’s disease, foreign bodies were
always present inside the granulomatous tissue. In some cases, the
chemical composition of the particulate detected made it possible to
identify the kind of exposure the patient had undergone. Micro and
nanoparticles were always present in cancerous tissues, but it must be
emphasized that this constant presence was due to a pre-selection of the
cases we observed. As a matter of fact, in a number of patients suffering
from cancer we had the chance to check outside the scope of this project,
no particles were found. This is only natural, as predisposing and causing
factors exist, such as genetics, radiations, exposure to organic solvents
and many other pollutants. In the cases investigated, mainly concerning
primary cancers, the inorganic foreign bodies were concentrated at the
interface between cancer and healthy tissue. This specific location, never
discovered before, seems to be meaningful in understanding the
mechanism regulating the onset of cancerogenicity. This specific
location could also explain why in some cases we can’t find anything:
The planar section we analyzed was part of a bulk, tri-dimensional
sample and it is likely that the interface was not included in that
particular section.
Once, we could not use the tissue harvested from one of the “healthy”
cadavers we used as reference, as it contained Calcium carbonate. It
turned out that that boy had been drug addicted and the mineral was used
Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System, Vessels, Sperm 41
to blend the drug. After that, considering the difficulties that carrying out
an anamnestic study implies with relatives or friends about the health
state of somebody who has died, our attention was addressed to people
we had reasons enough to consider “non contaminated”.
After the 3-year project sponsored by the European Community we
carried out, the reference samples, i.e. “the standards”, are represented by
the internal organs of feti of induced abortions. Their tissues are “clean”,
since we believed they had not been exposed to environmental pollution.
That was an extreme choice to obtain reference samples with “zero
contamination”, but that choice reserved some surprise.
In fact, the study mentioned above involved the verification of animal
and human malformed feti. In a few instances, dust was detected in their
tissues, and that showed that nano-contamination can be shared between
mother and child through the umbilical circulation.
Fig. 3.1 Schematic view of the entrance ways of particles in the organism.
42 Nanopathology
Fig. 3.1 serves to understand the paragraphs that follow, as it

describes graphically how particles, be they inhaled or ingested or, as a
matter of fact, introduced in any other way into the organism, reach any
tissue. The lung is the first structure to be interested, followed by the
blood, the liver and the kidney. After that, the digestive system has been
treated, but the contaminations found in the liver and the kidney could
have originated from inhalation as well as from ingestion. Sperm closes
the chapter. We did not describe anything regarding the brain, as we
could examine only two cases, in both of which particulate matter could
be seen.
All the images below are electron-microscope picture of pathological
tissues. They are presented together with their relative chemical EDS
spectrum (see Appendix). All spectra show the peaks of Carbon and
Oxygen that belong to the biological substrate, to the Carbon support
where the specimen is laid, but may as well be part of the spot aimed at,
which is in all cases an inorganic particle. We cannot be certain if
Carbon and Oxygen are real components of the particle or just an
artifact. For that reason, Carbon and Oxygen are not listed in the
elements detected.
3.2 Lung
We may say that we inaugurate our life with an inspiration of air. In fact,
neither our entire body nor any of the cells that make it up could survive
without Oxygen, since much of our metabolism is based upon a series of
oxidations.
That is the reason why the inhalation of air, a blend of gases where
Oxygen represents about one fifth, is necessary. But the air we breath is
not so pure as we would desire it to be. Among many other pollutants,
it contains all the by-products of the combustion processes related to
fires, heating, and industrial and automotive processes. So it contains
gases like Carbon dioxide and Carbon monoxide, furans, dioxins,
hydrocarbons, etc. And it contains dust which for size and chemical
composition is different from the natural one.
Medical Geology is a new discipline of investigation that studies the

impact of natural dust on human health. (Centeno, 2005).
Who, for instance, lives close to a volcano has a higher probability to
suffer from lung diseases, since, even if that volcano is not active, the
erosion of lava streams by rain, winds and temperature changes pollutes
the environment in a constant way. But the industrial and/or combustive
processes release dusts with chemical compositions that have never been
present on this planet and, therefore, are completely unknown to the
human body and its metabolism.
Because of its morphology and size, inhaled dust can interfere
physically with the passage of Oxygen through the alveoli, since it
creates a hard-to-negotiate barrier to the exchanges of the gases (Oxygen
against Carbon dioxide). But dust, perceived as a foreign body in the
alveoli, can also create a different local problem consisting in an
inflammatory reaction which can degenerate.
For those very well-known reasons, many environment-protection
agencies, among which that of the European Community, set limits to the
concentration of particulate pollution in the atmosphere. Nowadays those
limits are based on the mass of PM10 in a fixed volume, but a more
scientific determination, like number of particles evaluated according to
the reciprocal of their size is certainly hoped for. (Armaroli, N. et al.,
2003), (L.M. Brown et al., 2000), (J. Ruuskanen et al., 2001), (S. Ebelt
et al. 2001), (R.M. Harrison et al., 2000). The European Community is
preparing a new regulation based on PM2.5, unfortunately still
considering just mass.
For these reasons, we started to investigate samples of lungs affected
from pathologies with different levels of seriousness. Tab. 3.2.1 reports
the list of the pathologies investigated and all the images that follow are
related to these cases.
As can be seen, there are some cases seemingly not related in a direct
way to lung pathologies. For instance: Hodgkin’s and non-Hodgkin’s
lymphoma, skin pesudolymphoma, Wegener’s granulomatosis,
nephrocalcinosis, and Gulf War Syndrome. All these pathologies are
multi-organ-diseases and we analyzed many samples coming from the
same subject, included the lung, even if that was not the organ that
looked mainly interested by the pathology.
44 Nanopathology
We selected the particles shown according to peculiarity of

morphology, size or chemistry.
The lungs, as well as the mouth, the stomach or the colon have a
surface that has been exposed for a long time to pollution, so we find
much dust that remained trapped for a certain period of time. In the case
of a half-mm2 sample of the mouth mucosa we found as many as 600
sub-micronic particles of 10 different chemical compositions.
Tab. 3.2.1 List of the investigated samples of the lung.
Pathology No.
Fibrosis 8
Silicosis 1
Granulomatosis 3
Wegener’s granulomatosis 1
Pulmonary Emphysema 1
Sarcoidosis 3
Gulf War Syndrome 2
Pneumothorax 14
Pleural Coniosis 1
Nephrocalcinosis 1
Racemose ossification 1
Hodgkin’s Lymphoma 1
Non-Hodgkin’s Lymphoma 1
Skin Pseudo-lymphoma 1
Pleural Mesothelioma 32
Cancer 17
Not diagnosed 4
Reference 20
Total 112
First of all, a basic concept must be clear: most gross particles remain
on the inner surface of the alveoli, while the smaller ones, and the more
so the smaller they are, can negotiate that anatomical barrier and enter
the bloody stream.
Fig. 3.2.1a shows the surface of a lung alveolus with large particles of
surely combustive origin. That can be inferred by their high porosity,
something typical of combustion processes. Fig. 3.2.1b, instead, shows
the section of a blood vessel with red cells in its lumen stuck together,
and white particles with a size smaller than 2 µm. They look whiter than
the biologic matter around them because of their atomic density, higher
than the biological surroundings’. In this context they can express their
possible non-biocompatibility by a thrombogenic effect and thus induce
the formation of a thrombus. Pulmonary thromboembolism may be the
consequence of that phenomenon in the venous circulation, while stroke
and infarction may be what the phenomenon triggers in the arteries.
What we documented in the blood vessels suggests also another
possible effect. Particles are much harder than any blood components
and of the tissue making up the vessels. They are transported by the flow,
so they can hit and “scratch” the endothelium, causing a vasculitis. As a
matter of fact, many of the pathologies investigated present also that
particular symptom.
Fig. 3.2.1a – Fig. 3.2.1b ESEM images of 2 types of particles. The former is deposited on
the surface of an alveolus, while the latter is trapped inside a blood vessel.1
Much literature exists describing an increase of lethal episodes in

cardiopaths in relation with an increase of PM2.5 in the atmosphere, and
the fact becomes easy to explain in the light of our observations (Courter
1
This photo was published in Handbook of Nanostructured Biomaterials and Their
Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and
nanoparticles and the human health”, cap. 12, 347-369 ed American Scientific Publisher
USA 2005.
46 Nanopathology
L. A. et al., 2007), (Schwarze, P. E. et al., 2006), (Newby, J. A. et al.,

2006).
For a long time, silica and silicates particles have been known as the
responsible to a form of pneumoconiosis called silicosis, while asbestos
has been blamed as the cause of mesothelioma (asbestos can be
considered nanoparticle, since one of its dimensions is in the nanosize).
Pleural Malignant Mesothelioma, a malignant tumour that spreads
from the parietal pleura in the chest cavity and sometimes involves the
lung, is a kind of tumour strictly correlated with inflammatory reactions
and is due to environmental contamination.
Though not the only one, asbestos exposure is the most common risk
factor (involving more or less 80 percent of mesothelioma patients), but
other promoting factors are known, including chronic lung infections,
tuberculous pleuritis, radiation and exposure to the simian virus 40
(SV40) or other mineral fibres.
Of that highly malignant tumour very little is known to date about its
molecular and metabolic features and awareness is very recently growing
about the relevance of cell-to-cell signalling involved in the modulation
of its cell growth and differentiation, e.g. the presence of NOX and other
signal molecules (Soini et al., 2000) recently found. Of course it is
impossible to see gaseous compounds like NOx, COx, SOx, etc., so only
their effects are detectable, like the production of cytokines or others
compounds of the cell down to gene expression.
Our studies started from a pragmatic vision: if particulate matter
originating from environmental pollution is to be blamed for some
human diseases, we must find it where the biological tissue shows its
main lesions.
Fig. 3.2.2 shows asbestos fibres (a silicatic compound containing,
according to their species, Calcium, Silicon, Magnesium and Iron) inside
a pathological human lung tissue affected by mesothelioma. Around a
fibre, formations called “pearls of asbestos” can be seen. Those are
precipitates of Iron protein induced by the chemical reaction of the
silicatic fibre with the extracellular matrix, rich in Iron. The periodicity
of those pearls is due to the diffusion of ions that rules the phenomenon.
This aspect is well known among biomaterialists expert of bioactive
glasses (a compound of Silicon-Sodium-Calcium and Phosphorus),
materials commonly used for bone defect repair. (Gatti, et al, 1998) In
fact, these materials are not inert in the human organism, but degrade
releasing ions that create a concentration gradient activating diffusive
processes. In this specific case, the glass releases immediately Sodium
ions and there is a diffusion from the core toward the surface of the glass
of the Phosphorus and Calcium ions, leaving a matrix rich in Silicon (a
Silicon hydrated gel). The surface grows now rich in Calcium and
Phosphorus that precipitate as Calcium phosphate, the matrix for the
colonization of the osteocytes. This way, new bone is rapidly generated.
Fig. 3.2.2 ESEM Images of asbestos fibres with its chemical composition, found inside
a pleural mesothelioma. “Pearls of asbestos” (arrow) are visible(marker 5 µm), (marker
10 µm).2
Pleural mesothelioma has been described in literature for a long time

as related to the inhalation of asbestos nanoparticles with their peculiar
form of needles. But it also a fact that some patients who developed that
2
This photo was published in Handbook of Nanostructured Biomaterials and Their
Applications, Volume 2, Gatti A.M., Montanari S. “Risk assessment of micro and
nanoparticles and the human health”, cap. 12, 347-369, ed American Scientific Publisher
USA 2005.
48 Nanopathology
disease were never exposed to that pollutant. It is a well-known

fact that there is a correlation between environmental pollution and
some diseases, so we looked for environmental contaminants in the
pathological lung tissues.
Fig. 3.2.3 shows another example of asbestos fibres embedded in the
lung tissue. Iron protein pearls are clearly visible.
Fig. 3.2.3 Asbestos fibre in a pleural mesothelioma. (marker 10 µm).
Asbestos is evident in the latter picture, with the typical pearls of Iron
protein. It is important to observe the pointed shape of asbestos crystals,
a shape that makes penetration into tissue easier as compared to other,
bulkier shapes.
Racemose ossification, the disease related to Fig. 3.2.4 is a, rare,
diffuse pulmonary ossification of unknown origin in which mature bone
is found in the pulmonary parenchyma. It affects in general middle-aged
males and is asymptomatic. In the vast majority of cases, the condition is
discovered incidentally at autopsy. The arrow in Fig. 3.2.4 shows a
particle of Iron embedded in the new “bone”. (Wells et al. 1943), (Fried
et al. 1992) (Ikeda et al. 1998), (Trejo et al. 2002).
Fig. 3.2.4 Image of a racemose ossification. The arrow indicates a particle entrapped in
the part of lung that modified its chemical content into a Calcium-phosphate (marker
50 µm).
Fig. 3.2.5 shows a case of round atelectasis (Stark, 1982) (Menzies

et al., 1987). That disease is a rare lung pseudotumour that in most cases
is associated with asbestos-related pleural disease but can result from
a variety of other chronic pleural diseases like, for example, an acute
pleuropulmonary illness caused by Legionella pneumophila. The disease
presents often as an asymptomatic radiographic finding associated with
chronic pleural disease, usually related to asbestos exposure. In the case
we studied, we found silicatic particles containing Silicon-Aluminium-
Magnesium-Iron-Sodium-Phosphorus-Sulphur-Chlorine-Potassium-
Calcium-Titanium-Iron inside the round-shaped whiter areas. (Stark P.,
1982), (Menzies R et al., 1987).
50 Nanopathology
Fig. 3.2.5 Image of a lung sample affected by round atelectasis. Inside the lung fibrotic
tissue with chronic inflammation we found “round” areas full of silicatic nanoparticles.
Like all sarcoidoses, lung sarcoidosis is characterized by the presence

of small granulomas. In the case of the lung, granulomas can appear on
the walls of the alveoli or on those of the bronchioles. Its origin is still
unknown, but most scientists think it is caused by a disorder of the
immune system (Heffner DK, 2007). The case to which Fig. 3.2.6 refers
shows Mercury-Sulphur nanoparticles in the reaction cutaneous
lymphoid infiltrate. Granulomas can contain foreign bodies. In this case
the nanoparticles detected are chemically toxic.
Fig. 3.2.6 Lung sarcoidosis. Inside a non necrotizing dermal granulomas, chemically toxic
nanoparticles of Mercury-Sulphur-Silicon-Chlorine were found. (marker 5 µm).
Fig. 3.2.7 concerns a case affected from tubercolo-silicosis. At optical

microscopic observation, the lung parenchyma showed silicotic nodules
with concentric bands of hyalinized collagen surrounded by fibroblasts
and histiocytes with a blackish pigment .
We report images of wide lung calcific areas (white dots). Their
chemical analyses reveal that that is not a normal calcification but the
biological tissue turned into Calcium phosphate. This tissue is hard, not
elastic and can compromise the elasticity of the lung and of breathing.
Fig. 3.2.7 Low-magnification image of tubercolo-silicosis with wide Calcium phosphate

areas (marker 300 µm).
Fig. 3.2.8 High-magnification image of tubercolo-silicosis. At such magnification, Silicon-

Titanium-Aluminium-Iron particles become visible. (marker 50 µm).
52 Nanopathology
At higher magnification (Fig. 3.2.8), it can be seen that the white dots
are micro and nanoparticles of a compound made of Titanium-Silicon-
Aluminium-Iron-Magnesium-Sodium-Chorine-Potassium-Calcium-
Sulphur-Phosphorus. It is hard to tell the origin of that pollution, but
wide areas of the lung are interested by these foreign bodies. The lung
tissue is a composite material with altered morphology and physical
properties.
Fig 3.2.9 Image of a squamocellular carcinoma sample containing Tungsten–based particles

(marker 20 µm).
The patient whom the biopsy relative to a squamocellular carcinoma

(Fig. 3.2.9 and Fig. 3.2.10) was taken from worked at the milling and
rough-hewing of metal bushings for oil-pressure pumps where mineral
oils derived from hydrocarbons were used. Tungsten-Aluminium-Iron-
Titanium nanoparticles were present in his pathological tissue. At
histopathological observation, the squamocellular carcinoma was hardly
differentiated, and differentiation areas were in an adeno-squamous
direction. Wide necrotic foci and marked atypias were also present.
Fig. 3.2.10 Squamous cellular carcinoma with Titanium-Silicon-Aluminium-Phosphorus-

Chlorine-Potassium-Iron particles (marker 20 µm).
Fig. 3.2.11 Iron nanoparticles found in the lung of a soldier suffering from Gulf War
Syndrome (marker 5 µm).
54 Nanopathology
Fig. 3.2.12 Solitary Gold-Silver-Copper particle found in the lung of a soldier suffering
from Gulf War Syndrome (marker 10 µm).
The images of Fig. 3.2.11 and 3.2.12 show nanoparticulate matter of

different compositions in the lung tissue of a veteran from the First Gulf
War, suffering from the so-called “Gulf War Syndrome”
The samples come from an officer active in the Canadian Intelligence
who died in 1999 as a victim of the so-called Gulf War, during which he
served as a mine clearer. Natural Uranium (90%) and depleted Uranium
(9-10%) were found in his urine by another laboratory when the patient
was still alive. No such element, though, was found in particulate form in
our investigations. In the samples studied we found nanoparticles of
Titanium compounds and of Iron, along with particulate matter of Silver
and precious alloys like Gold-Copper. Finding such small particles inside
that patient’s lungs was strange indeed, as he had lived most of his life
lived in the Canadian countryside, and had never worked in a
nanotechnology laboratory where particles that size are common. He
grew ill after a 6-month mission in Iraq and, in fact, had been exposed to
the war pollution created by bombing. (See Chapter 5: War and
nanoparticles)
In another case, this time of a patient affected from the Gitelman’s
syndrome, we found many different foreign bodies in the same sample.
Gitelman’s syndrome is a rare, often asymptomatic, autosomal-
recessive disorder affecting the renal tubules, causing them to pass
Sodium, Magnesium, Potassium and Chloride into the urine. The patient,
suffering from chronic interstitial pneumonia with allergic fibrosing

alveolitis, had been in touch with depleted Uranium. Inactivating
mutation in the SLC12A3 gene was found in the patient, which is
compatible with a diagnosis of Gitelman’s syndrome. (O’Shaughnessy
KM et al., 2004), (Naesens M. et al., 2004).
Nanoparticles of a rather complex composition are visible in the
tissue. The different chemical composition of the particles found
indicates an origin from different sources.
Fig. 3.2.13 Low-magnification image of a lung tissue affected by Gitelman’s Syndrome.

Arrows show Silicon-based pollution (marker 300 µm).
The first image (Fig. 3.2.13) shows a common silicate particle

(normal dust) and the second one (Fig. 3.2.14) is a round-shaped particle
of a compound of Cerium-Lanthanum-Neodymium. In our experience it
can have been originated by smoking, as in some tobacco leaves we
found the presence of this strange environmental, weakly radioactive
composition. The third image (Fig. 3.2.15) is related to the presence
of another unusual compound containing Copper-Chlorine-Silicon-
Aluminium-Sulphur-Phosphorus-Iron.
56 Nanopathology
Fig. 3.2.14 Images of a single Cerium-Neodymium-Iron particle in a lung sample affected

by Gitelman’s Syndrome (marker 20 µm).
Fig. 3.2.15 Images of a lung sample affected by Gitelman’s Syndrome. Note the nano-scale
Chlorine-Copper-Silicon-Sulphur particulate matter (marker 2 µm).
The case shown in Fig. 3.2.16 concerns a 42-year-old woman who

died of malignant pleural mesothelioma without having been exposed to
asbestos. In fact, no such mineral could be found in her pathological
samples, but we detected Iron nanoparticles and fibres of Aluminium-
Silicon-Iron, whose long-shaped morphology is similar to asbestos. The
only anamnestic detail of this patient is that she underwent a five-year
chemotherapy treatment because of a breast cancer, before falling ill of
mesothelioma (Melato M et al., 2001). Another case of the same disease
(Fig. 3.2.17) shows the presence of Iron micro and nanoparticulate.
Fig. 3.2.16 Images of malignant pleural mesothelioma’s sample. A fibre of Aluminium

silicate is clearly visible (marker 20 µm).
Fig. 3.2.17 Images of malignant pleural mesothelioma’s sample with Iron micro and
nanoparticles. (marker 10 µm).
58 Nanopathology
Fig. 3.2.18 Iron-based particles in the sample of a patient with a multi-visceral

granulomatosis (marker 100 µm).
Fig. 3.2.18 concerns the case of a patient for whom no clear diagnosis
was issued. He suffered from a multi-visceral granulomatosis and what
we found in the lung and spleen samples were wide areas where Iron-
Silicon-Aluminium-Magnesium-Sulphur-Phosphorus were present. The
presence of Silicon, Aluminium and Magnesium with Iron induces to
think that Iron is not endogenous but comes from outside the body.
The case of Fig. 3.2.19 is about a young lady of 22 who suffered from
talcosis and pneumothorax. The histological study revealed the presence
of blebs, fibrosis, lymphocytic phlogosis and giant cells with particles.
The particles we detected were composed mainly of Copper (Fig. 3.2.20)
and of Silicon-Magnesium (talc).
Fig. 3.2.19 Talc particles contained cells in a pneumothorax (talcosis) (marker 10 µm).
Fig. 3.2.20 Solitary Copper particle in a pneumothorax area (marker 10 µm).
The round-shaped Copper particle shows that the patient had been
exposed to a toxic material coming from a combustion occurred at about
1,100°C, since the melting temperature of Copper is 1,083°C. That
element has been known for a long time to be toxic both in vitro and in
vivo causing tissue necrosis. If the particles are located outside of the
lung, in touch with the pleura, they can induce a necrosis, a rupture and
the onset of a pneumothorax.
60 Nanopathology
Fig. 3.2.21 Case of pneumotorax with the presence of a Titanium needle (marker 10 µm).
Fig. 3.2.22 Case of pneumotorax with the presence of macrophages full of Iron-Silicon-
Aluminium-Calcium particles (marker 20 µm).
In a 65-year-old workman a pneumoconiosis was diagnosed. The

histology showed blebs, fibrosis, lymphocytic inflammation and giant
cells with particles we showed to be composed of Iron-Silicon-Sulphur-
Calcium-Aluminium (Fig. 3.2.22). The pricking needles of Titanium
(Fig. 3.2.21) can be responsible of the pneumotorax, but the damage
created by the Iron particles may have also contributed.
Fig. 3.2.23 Cells full of Iron-Silicon-Aluminium-Phosphorus-Sulphur-Calcium-Titanium-

Chromium particles surrounding a pneumothorax area (marker 20 µm).
Fig. 3.2.24 Particular of Fig. 3.2.24 (marker 10 µm).
A further case of pneumothorax, this time of a 42-year-old solderer

came to our observation. Coniotic materials in giant cells (at different
magnifications) were easily visible, composed of Iron-Silicon-Titanium-
Calcium-Aluminium-Phosphorus-Sulphur-Chromium (Fig. 3.2.23 and
Fig. 3.2.24). White, round shaped particles are clearly visible inside the
cells. This evidence is compatible with a working-place exposure.
62 Nanopathology
Fig. 3.2.25 Area around pneumothorax. Many silicatic particles are visible (marker
20 µm).
Fig. 3.2.26 Particular of the lung sample around the area of the pneumothorax above.
Cerium-Iron particles are contained in cells (marker 10 µm).
A businessman of 42 suffered from a pneumothorax. Blebs with

fibrosis, chronic lymphocytic infiltration and giant cells containing
nanoparticles whose main component was Silicon were present. (Fig.
3.2.25). Also in this case, in the sample of the lung lesion, the tissue was
full of micro and nanosized foreign bodies. Rather peculiar is the
probably weakly radioactive debris containing Cerium-Iron-Aluminium-
Silicon-Phosphorus and Sulphur (Fig. 3.2.26).
Fig. 3.2.27 Image of a lung sample in the area of the pneumothorax. Silicon-Aluminium
based particles are visible (marker 100 µm).
Fig. 3.2.28 Particular of Fig. 3.2.27. At higher magnification, numerous needle-shaped

Silicon-Aluminium based particles can be seen (marker 10 µm).
A case similar to the one illustrated in Fig. 3.2.25 is shown in

Fig. 3.2.27. In this instance the patient was a 71-year-old workman. The
coniotic nodule and the giant cells contained particles whose principal
component was a composition of Silicon-Aluminium-Potassium-
Titanium-Iron-Magnesium-Sodium-Phosphorus-Sulphur, i.e. a ceramic
dust. Fig. 3.2.28 regards the same patient as above, but in this portion
of the lung the particles had the same composition but a different
morphology.
64 Nanopathology
Fig. 3.2.29 Cluster of Silver nanoparticles in a lung sample affected by allergic pneumonia
(marker 5 µm).
The picture above (Fig. 3.2.29) shows a case of chronic, allergic

pneumonia of exogenous origin with lung interstitial fibrosis and
secondary leiomyomatosis, a rare, benign tumour that generally develops
in the uterus, but can affect organs with smooth muscle cells. In this case,
clusters of Silver nanoparticles were clearly visible in the pathological
tissue.
Fig. 3.2.30 Unusual morphology of pulmonary cells (adenocarcinoma) containing Iron-

Sulphur-based particles (marker 50 µm).
Fig. 3.2.31 Same sample as the one of Fig. 3.2.29. Normal red cells are inside the ellipse,
while lighter red cells are indicated by arrows (marker 20 µm).
Fig. 3.2.32 Same sample as the one of Fig. 3.2.29 and Fig. 3.2.30. What look like Iron
phosphate precipitates on a needle-shaped Iron particle (marker 10 µm).
Figs. 3.2.30-31 and 32 show different types of particles found in a

small lung sample affected by adenocarcinoma. The first image show
round-shaped organic bodies with Iron nanoparticles inside. The second
one shows metallic particles (Chromium-Iron-Nickel) embedded in red
cells (lighter in colour and indicated by arrows), while the third one is an
Iron needle with endogenous Iron-phosphatic crystals. A surgical metal
clip was detected at histology. It could be responsible for the debris
found.
A few cases of pleural mesothelioma are shown below.
66 Nanopathology
Fig. 3.2.33 Images of Iron-Sulphur and Iron-Zinc-Sulphur-Silicon-Phosphorus

nanoparticles of in a lung tissue affected by mesothelioma (markers 10 µm and 20 µm
respectively).
In the same case of mesothelioma, two different types of

nanoparticles were detected. One was made of Iron-Sulphur, while the
other was composed of Iron, Zinc, Silicon, Sulphur and Phosphorus
(Fig. 3.2.33).
Among the particles found in the case illustrated in Fig. 3.2.34 and
Fig. 3.2.35, we focused our attention to some debris with unusual
compositions. The former contained Uranium-Phosphorus-Potassium
particles, (a radioactive composition) the latter contained Tungsten,
Tantalum, Niobium, Phosphorus and Sulphur.
Fig. 3.2.34 Pleural mesothelioma with particles containing Uranium (marker 50 µm).
Fig. 3.2.35 Lung tissue affected by mesothelioma with Tungsten-Tantalum nanoparticles

(marker 20 µm).
68 Nanopathology
Fig. 3.2.36 The same sample of a tissue affected by mesothelioma are shown at different
magnifications (marker 200, 50 and 5 µm respectively).
This case of mesothelioma is rather peculiar. In fact, macrophages

were found in the tissue full of small, round-shaped particles of
Aluminium-Magnesium. Fig. 3.2.36 shows part of the tissue at
increasing magnifications. The second image shows a blood vessel
surrounded by whiter cells. They are full of sub-micronic globular
entities containing Aluminium.
The patient had worked for a long time in a mechanic industry and he
was exposed to vapours exhaled by what is called “Aluminothermy
process”. Also this case confirms that what was found inside is the
expression of the environmental exposure the patient underwent. The
inorganic matter we inhale can remain trapped in the lung at different
tissue levels. In the case of needle-shaped particles, once inside the lung,
because of the respiratory acts, they are pushed toward the more external
part of the organ, i.e. the pleura. When they are there, they have the time
to express their toxicity and induce a tissue or cell reaction. The
biocompatibility of a material is also a function of time: a short time
interaction is less detrimental then a long term contact.
But in the analysis of the possible damage due to environmental
pollution we need to take into account other phenomena that were
observed by the School of Leuven (Belgium).
The main work is due to Dr. Nemmar who had some 100 nm-sized
Technetium-radiolabelled Carbon nanoparticles inhaled by 5 volunteers.
The experiment showed that those particles negotiate the lung barrier in
60 seconds and their dissemination in internal organs, the liver in this
particular case was the organ observed, occurs in 60 minutes. (Nemmar
et al., 2002)
That simple experiment simulated what probably happens every day.
It is well-known that 100nm-sized particles are released, of course
unintentionally, by car engines, industrial fumes, incinerators, etc. and
the paper mentioned offers an explanation to what we find in many
tissues other than hepatic (Tonomori et al., 2004). The next paragraphs
show that contamination can be very deep, and no organ is free from
danger. It is only a matter of size, chemical composition, affinity for
some function of the organ and chemistry of the particle.
70 Nanopathology
3.3 Blood
The presence of the inorganic particles found in the internal organs was
explained putting forward the hypothesis that they are carried by the
blood circulation.
In order to demonstrate this presence inside the blood, we wanted to
take pictures of circulating particles, but also of possible problems
induced by their interaction with the blood components. To this aim, we
tackled the problem from three different points of view.
Firstly we looked for inorganic debris in the blood of clinically
healthy people living in polluted areas.
Then, we checked thrombi formed in-vivo, either in people at risk of
pulmonary thromboembolism and in patients who had a myocardial
infarction. In the former case, we analyzed venous clots trapped by caval
filters, and in the latter the thrombi removed from the coronary arteries
by means of a new procedure making use of a thrombectomy equipment.
And finally we analyzed samples of patients suffering from vasculitis,
a cryptogenic inflammation of the blood vessels.
The list of Tab. 3.3.1 shows the pathologies considered.
Fig. 3.3.1 shows the image of red cells smeared on an acetate sheet.
Small particles are visible, but it is hard to tell if they are immersed in the
plasma, attached to the cell or embedded inside it.
Tab. 3.3.1 List of the pathologies analysed.
Pathologies No.
Thromboembolic disease 65
Aneurism 1
Idiopathic Thrombocytosis 1
Diabetes 1
Parassitosis 2
Cerebral angioma 1
Hemangioma - Angiolipoma 1
non-Hodgkin’s Lymphoma 1
Lymphadenopathy 2
Not diagnosed cases 6
Reference samples 2
Total 84
This analysis itself is simple to perform and particles are easy to

detect, but that is not enough to allow us to say that the presence of
foreign bodies means that a pathology is starting.
More interesting proved the study of thrombi formed in-vivo, since
particles found inside them can mean that they acted as nucleating
agents, or their non biocompatibility triggered the coagulation cascade.
Fig. 3.3.1 Image of an Antimony–Cobalt nanoparticle and red cells (marker 5 µm).
72 Nanopathology
In order to demonstrate the interaction particle-blood in vivo, we took

patients treated with caval filters. Those are medical devices that are
implanted in the inferior vena cava either as a preventive means before
surgery in patients who were deemed as being at risk of developing a
deep-vein thrombosis (DVT) with a consequent pulmonary embolism
(PE), or to prevent PE episodes in potentially relapsing patients. In those
cases, a metallic structure is inserted in the inferior vena cava and left
there to capture the thrombi formed in the lower limbs and pelvis.
Fig. 3.3.2 Image of a vena cava filter by ALN (France).
The analyses of explanted temporary vena cava filters (ALN Implants

Chirurgicaux, - France) (Fig. 3.3.2) offered the possibility to analyse the
thrombus entrapped, without influencing the system. Those filters,
usually implanted in the lumen of the inferior vena cava, are the most
common mechanical device for the prevention of pulmonary thrombo-
embolism and their use is growing more and more widespread, especially
in the USA.
The observation of the filters explanted allowed to analyze the small
thrombi captured in the human venous circulation. (Gatti, A. M. et al.,
2006) (Montanari S., 2000). The vena-cava filters examined are
composed of nine AISI-316L stainless steel, 0.3-mm diameter, wires
gathered together inside an ogival capsule made of the same material,

from where they branch out, forming a conical skeleton. Each of the
shorter 6 of the 9 prongs that make up the device has a distal hook that
penetrates the venous wall and serves to keep the filter anchored in place,
while the longer three are meant as stabilizers to keep the device lined up
with the vascular axis, and have no hooks. One of the long prongs has a
small ring at its distal end. The overall height of the device is 5 cm and,
being it elastic, its diameter adjusts itself to the section of the vessel up to
32 mm.
In two different studies, we analyzed respectively 14 and 20
temporary filters explanted from patients who had had this device
implanted for different reasons and for different periods (from 18 to 384
days). The main indications were:
1. Anti-thrombosis prophylaxis in bone fractures or implant of hip
joint prosthesis or cancer;
2. Deep Vein Thrombosis (DVT);
3. Protection in loco-regional thrombolysis treatment.
The particles found in the thrombi trapped in the filters were
characterized with a size ranging from 10 down to 0.1 micron and for a
wide variety of chemical compositions.
Some of the compositions found are listed below. The first element
considered is that expressed in the spectrum with the highest peak:
- Si;
- Si; Mg;
- Cr, Fe, Si, S, Na;
- S, O, Ba ,Na;
- Fe, Sb, S, P, Si, Na;
- Sb, S;
- W, O, S, P, Fe;
- Bi ,S, Cl, Si, Na Ca;
- Pb, Si, Al ,Cr, Fe, Mg, Ca;
- Ag, Si, Mg, S, Bi, Cl, O, Ca, Cu;
- Co, S, Ag, Ca, Cl, P, W, Al, Zn, Fe;
- S, Ba, Ca, Cl, Na;
- Fe, Cr, O, S, Ca, Cl, Cu;
- S, Cl, Ca, Bi, P, Na, Mg;
74 Nanopathology
- Ag, O, S, Ca, P, Si, Mg, Ni;

- Au, Cl, S, O, Ca, Ag, Na, Si, Mg, Cu, Fe.
It is interesting to see how all the thrombi come to our observation
had foreign bodies trapped.
One case was particularly interesting from the nanopathological point
of view, as the thrombus contained micro- and nano-sized foreign bodies
composed of as many as 6 different chemical compositions (Co-W, Al-
Si, Si, Fe, Ag-S, Fe-Cr-Ni). Fig. 3.3.3 and 3.3.4 show particles of Barium
Sulphate and Silver dispersed in the thrombus. (Gatti, A. M. et al., 2006)
Fig. 3.3.3 Image of red cells in a thrombus trapping nanoparticles of Barium-Sulphate

(marker 10 µm).
Fig. 3.3.4 Image of a particle composed of Silver and Calcium found in a thrombus (marker
10 µm).
Fig. 3.3.5 Sulphur-Barium-Sodium-Aluminium-Silicon-Phosphorus-Calcium-Iron particles

in an fibrotic tissue adherent to a caval filter (marker 10 µm).
The presence and quantity of thrombotic material found in the filters

did not seem to depend on the time the device had stayed implanted in
the organism, but on the extent of contamination of the blood, i.e. how
many particles per volume unit were present. That means that the higher
the patient’s exposure was, the greater was the concentration in the blood
and the higher was their probability to induce a thrombotic reaction. It
has been demonstrated that foreign bodies the size of 100 nanometres,
once inhaled, can leave the alveoli and enter the blood circulation in 60
seconds. (Nemmar et al. 2002). In Fig. 3.3.5 Sulphur-Barium-Sodium-
Aluminium-Silicon-Phosphorus-Calcium-Iron nanoparticles are shown
trapped in the fibrotic tissue adherent to a caval filter.
76 Nanopathology
Fig. 3.3.6 Cluster of nanoparticles composed of Titanium-Sulphur–Barium-Calcium-

Chlorine (marker 10 µm).
Fig. 3.3.6 and Fig. 3.3.7 show metallic debris found in thrombi
trapped by two caval filters. The former is a cluster of Titanium-
Sulphur–Barium-Calcium-Chlorine nanodebris, while the latter is a
single Iron-Chromium-Nickel needle-shaped particle.
Fig. 3.3.7 Image of stainless-steel debris found inside a thrombus (marker 20 µm).
In the cases we had the chance to observe, particles much larger than
100-nanometer were detected in the blood. Whether they are the cause of
the thromboses or they are just mechanically trapped by the blood during
its pathologic coagulation may be discussed, but it is not unreasonable to
suppose that a foreign body can be thrombogenic, especially, as has
always been the case in this study, when those foreign bodies are neither
biodegradable nor biocompatible, and being not biocompatible means, in
the vast majority of cases, being also thrombogenic.
Recently it was demonstrated that when the environmental
concentration of PM2.5 increases, the mortality rate of cardiopaths, or
patients suffering from cardiovascular diseases increases as well. The
images shown can confirm these data.
The size and composition of the debris found is rather typical of
industrial pollution, in particular of high-temperature processes involving
inorganic materials, such as occurs in a foundry, in a cement plant, in an
incinerator or in engine fumes.
All patients were considered to be at risk of developing a deep-vein
thrombosis (DVT) or, in some cases, were already diagnosed to be
affected by that disease. This is one of reasons that make us suspect that
the thrombi detected or, at least, part of them, embolized from the DVT
foci. Some of them, though, could have been originated by inorganic
particulate circulating in the blood and become actually thrombogenic
once the organism has been stimulated by particular, well-known,
conditions like, for example, surgery, bone fractures, cancer or a
relatively long stay in bed.
A number of the many pulmonary embolism episodes observed
worldwide are classified as idiopathic, since no focus can be identified
nor an explanation regarding their origin is possible according to the so-
called Virchow’s Triad, that states that pathological clotting of the blood
in the vessels occurs when the blood flow is somehow obstructed or
slowed down, when the vascular endothelium is damaged or when the
chemistry of the blood is disturbed or incompetent. In many instances, it
is a combination of the three factors that is responsible for the
thrombosis, as one single factor may not always be enough.
The presence of particulate matter in the blood, added to the Triad as
a fourth factor, may explain in part or totally the not infrequent cases
78 Nanopathology
when none of those classical three causes exist, yet a pulmonary

thromboembolism occurs.
That particulate air pollutants are responsible for or, at least,
have some form of relationship with the onset of cardiovascular and
pulmonary diseases has been amply demonstrated (Peters A. et al.,
1997), (Samet J. M. et al., 2000) and is supported by clinical evidence,
though much remains still to be explained as to the patho-mechanisms
they follow. At present, for instance, we are not in a position to say if the
blood reacts to the presence of clusters of nanoparticles and of isolated
microparticles in a different way.
Also analyzing the material that occluded a coronary vessel we found
foreign bodies.
Myocardial infarction is caused by an acute obstruction of the
coronary lumen and in many cases that obstruction is caused by a
thrombus or, at least, contains thrombotic material together with
atheromatous plaque. A few years ago, a mechanical technique aimed at
aspiring that material grew popular in interventional cardiology. (Jun-
ichi K. et al., 2002), (Andrew, M. W. et al., 2006). We had the chance to
investigate on a sample taken from a myocardial infarction thanks to the
technique of mechanical coronary thrombectomy and found micro and
nanoparticulate in that thrombus as well.
Fig. 3.3.8 and the ones that follow (3.3.9 and 3.3.10) show inorganic
particulate inside coronary thrombi. Such unexpected finding may rouse
Fig. 3.3.8 Coronary thrombus with an agglomeration of many nanoparticles with one
microparticle of a Titanium-Bismuth-Sulphur-Phosphorus compound (marker 10 µm).
the suspicions that those foreign bodies can have triggered the
pathological coagulation. This could explain why a higher cardiogenic
mortality is reported when PM2.5 concentration in the atmosphere grows
higher (Pope, C. A. et al., 2006), (Pope, C. A. et al., 2006).
Fig. 3.3.9 Small Platinum-Tungsten-Copper-Sodium debris found in a coronary thrombus

(marker 10 µm).
Fig. 3.3.10 Cluster of nanoparticles composed of Antimony-Cobalt Sulphur-Sodium

embedded in a fibrin reticulum (marker 10 µm).
80 Nanopathology
3.4 Liver
Granulomatous hepatitis is a lesion found in a wide spectrum of liver

diseases (Denk, H. et al., 1994). TBC and sarcoidosis infection have been
most frequently incriminated, while other causes are represented by viral
infections, immunological disorders, drug-induced injuries, Crohn’s
disease and foreign-body reaction (Anderson, D. S. et al., 1998).
A minor percentage of cases, about 10%, remains of unknown
etiology and is indicated as GLUS (Granulomatous Lesions of Unknown
Significance), (Brinker, H., 1990)
From the histological point of view, such lesions are usually non
caseating, with a few multinucleate giant cells and some surrounding
chronic inflammatory infiltrate. Medical literature analyses local effects
of wear from joint replacements, identifying the debris as the main result
of in situ degradation of implants (Jacobs, J. J. et al., 1998). In addition
to obvious local effects, particulate debris created by an implanted device
has been reported as present in lymph nodes (Albores-Saavedra, J. et al.,
1994), (Bos, I. et al., 1990) and in distant organs, but literature on the
subject is rather scanty (Urban, R. M. et al., 2000).
As a general rule, the larger particles thus generated cause a fibrous
or giant-cell reaction, while the smaller ones prompt a macrophage
phagocytosis reaction. Chapter 2 demonstrates that nanoparticles behave
in a different way and do not induce “normal” inflammatory reactions.
Our first case of liver granulomatosis is the one described in Chapter
1. In this later series, we analyzed a group of patients with phlogosis
(13 cases) and “granulomatous phlogosis” (resulted from liver biopsies)
(29 cases), since they are related to tissue inflammations also caused by
foreign bodies . The relevant data are shown in Tab. 3.4.1.
In order to have a reference, not shown in the table, five specimens of
liver taken from presumably healthy young people dead in car accidents
were observed in the same way under ESEM together with ten more
samples from feti. Those specimens were in all cases free from any
particulate contamination.
Tab. 3.4.1 List of the pathologies investigated with the relevant number of cases.
Pathology No.
Phlogosis 13
Fibrosis 2
Granulomatosis 29
Steatosis 7
Hepatitis 6
Siderosis 3
Haemorrhage 3
Wilson’s disease 1
Cancer 28
Metastasis 17
Gulf War Syndrome 1
Nephrocalcinosis 1
Not diagnosed cases 23
Reference samples 15
Total 154
All these cases were checked by a histopathologist. Just three of the

cases chosen showed the presence of foreign material inside the
granulomas, whose nature remained undetermined to him. In any case,
he ruled out any infectious origin according to the results obtained with
Pas, Grocott and Zihl-Nielsen reactions.
Once again, the observations under ESEM proved essential in the
identification of materials responsible for granulomatous reactions, as the
presence of micro- and nano-debris in all samples except the references
was consistently found. The particles detected were different in size (in
general <20 µm) and in chemistry. As all the cases selected are
meaningful in demonstrating the hypothesis mentioned above, some of
them are discussed singularly because of their clinical history. Their
anamnestic records confirmed the exposure we found in the tissue.
Case No. 1: 61-year old female with a clinical history of rheumatoid
arthritis; increase of GOT, GPT and gamma-GT. Use of corticosteroids
and Gold salts injected directly in her joints. Histologically, the liver
82 Nanopathology
biopsy showed a mild chronic phlogosis and a small granuloma in the

proximity of a portal tract, with black material inside.
At ESEM analysis, particles trapped in a degenerated tissue
containing Gold nanoparticles were identified. (Fig. 3.4.1). The oddity of
such finding got us to analyse more in detail different areas of the same
specimen. The EDS spectrum (Fig. 3.4.2) showed that Gold alone was
not accompanied by any other element, the spherical, homogenous
morphology of the particles as small as 50-nm excluded a dental origin.
The study of the clinical record revealed that years before falling ill of
liver granulomatosis, the patient had suffered from a knee arthrosis and
had been subjected to a colloidal Gold therapy. We could not find any
other reasonable explanation to the presence of that form of pure Gold in
the liver, where it had behaved like a foreign body, but a migration from
the joints, where it had been injected.
Fig. 3.4.1 Gold nanoparticles in a liver granulomatosis (marker 50 µm).

Fig. 3.4.2 Cluster of Gold nanoparticles in a liver granuloma (marker 2 µm).
Case No. 2: A 25-year-old female was affected by a hepatic

echinococcosis. At ESEM observation, small debris of Barium-sulphate
could be detected in the section. The patient’s clinical file revealed that
she suffered from digestive disorders, which suggested the possibility
that she had undergone a gastroscopy with BaSO4 used as contrast
medium. The fact was confirmed by the patient herself who also reported
an allergic reaction from that chemical.
Case No. 3: A 63-year-old male suffered from indefinite symptoms,
and a constant feeling of fatigue. Liver biopsy showed the presence of a
lympho-monocytic infiltrate, areas of macrosteatosis and an epithelioid
granuloma lined with a lympho-monocitic halo. At ESEM, degenerated
areas of the liver showed numerous particles of Calcium-sulphate
(gypsum) and fillo-silicate (Fig. 3.4.3). Such chemistry suggested the
possibility that the patient lived or worked in a particularly polluted
environment. As a matter of fact, the clinical record reported that he was
a mason.
84 Nanopathology
Fig. 3.4.3 Gypsum and fillo-silicate micro and nanoparticles in an epithelioid granuloma.3
Case No. 4: A 65-year-old African male was operated on for a spleen

cancer. Surgery revealed that his liver was compromised and a biopsy of
that organ was taken, which showed a large granuloma containing
necrotic tissue. The ESEM observation in that tissue revealed the
presence small spherical Calcium-phosphate particles with a narrow
distribution of diameters (2-7µm) (Fig. 3.4.4). The hypothesis we
advanced, a hypothesis never confirmed because of the patient’s death
and the consequent stop of all investigations, was that those findings
were of biological origin, perhaps a parasite.
3
This photo was published in Biomateriali, vol 23, issue 11, Gatti A.M., Rivasi F.
“Biocompatibility of micro- and nanoparticles Part I in liver and kidney”, June 2002,
p. 2381-2387.
Fig. 3.4.4 400-µm granuloma with fibrous capsule (low-magnification). (marker 200 µm).
Fig. 3.4.5 Same sample as above. Higher magnification shows Calcium phosphate
nanospherules (marker 5 µm).
The same morphology, size range and composition as those seen in

this case was found in a number of other biological tissues (Fig. 3.4.5).
Still today we do not understand if the reaction is a sort of unusual
calcification (why spheres? Why Sodium and Magnesium?) or it is due
to some other mechanism.
Case No. 5: The only symptom of a 27-year-old male with a history
of drug abuse was fever. Liver echography showed multiple nodules of a
diameter never exceeding 2 cm. The liver samples we had the chance to
86 Nanopathology
check showed the presence of foreign bodies with different chemical

composition (silicates). Such finding was probably due to the powder
used to mix the drug.
Case No. 6: The liver biopsy of a 62-year-old male with a clinical
history of peritoneal dialysis showed granulomas in correspondence with
the glissonian membrane and focal hepatic fibrosis. The spectrum of the
debris seen in the pathological tissue reveals the presence of Carbon,
Oxygen, Silicon, Sulphur, Calcium and Titanium. Such debris might be
correlated with a possible pollution of the dialysis solutions or to the
pharmaceutics taken in a chronic way.
Diagnostics uses either light transmission microscopy with a
maximum of up to 400 magnifications or molecular biology, that
analyses the sequence of the aminoacidic bases, whose size is
nanometric. Between this two terms there is a wide interval spanning
about 2-3 orders of magnitude where information about particular
pathologies, not otherwise explained, may be lost. And such information
may be important or even essential to understand the problem.
As mentioned above, no less than 10% of all cases of liver
granulomatosis remains unexplained as to their origin, and that brought
us to face this problem under the different point of view of our technique.
The use of a novel technique, a technique typical of physicists,
represented by the use of Environmental Scanning Electron Microscopy
(ESEM) that allows the analysis of samples under different gradients of
pressure, without any physical and chemical pre-treatment demonstrated
that within this gap evidence can be found essential to issue a correct
diagnosis.
In fact, just as an example, the case of the patient with a fever and
renal failure of unexplained aetiology described in Chapter 1, whose
renal and liver biopsies were subjected to ESEM examination, showed a
spectrum of elemental composition compatible with the porcelain his
dental prosthesis was made of. This could explain the aetiology of his
granulomatous hepatic lesions and was fundamental to set an efficacious
therapy.
In the same way, the X-ray microanalyses carried out on the
particulate matter of case No. 3, gave information on the particles’
chemistry and these data could be related, at least in part, to the clinical,
environmental and employment history of that patients. A likely
identification of the cause of the disease is certainly useful for the
therapy, and, which is of extreme importance, to set prevention
measures.
On the basis of these observations, we can assume that defining “non
toxic” what are just chemically inert (or hardly reactive) particles is
erroneous. Below a critical size, still hard to establish, particles, whatever
they are made of, are not biocompatible and may interact with biologic
tissues just because of their presence, and they may be responsible of
chronic inflammatory reactions that can lead to more severe forms of
disease. Nano-scaled particles can induce other unknown phenomena not
yet described in books or papers.
This first part concerning the pathologies of the liver has been
dedicated to cases of granulomatosis because such reaction by tissues
against foreign bodies has been often described in literature. Local
granulomatosis around the head of hip joint prostheses due to the wear
debris of the matching surfaces (head against acetabulum) is a far from
rare complication in orthopaedics. So, there is no problem in accepting
the existence of a correlation between presence of foreign bodies and
biological reaction. More difficult to accept are the following images.
They show the presence of dust that induced more severe reactions, like
cancer.
For a better understanding of the subject, we are presenting a genetic
disease involving the metabolism of endogenous Iron, in order to show
the difference between a genetic and an acquired disease. The genetic
disease we will deal with is siderosis.
Siderosis is a pathological build up of Iron in the organism. The lungs
are the most interested organs, but the liver and the urinary system can be
also affected. An Iron overload is known to impair the immune response
of the liver, and cause hepatic fibrosis and cirrhosis. There are different
opinions about the relative risk of developing hepato-cellular carcinoma
in patients with siderosis as compared with patients with hepatic fibrosis
and cirrhosis and the mechanism of liver carcinogenesis in genetic
hemochromatosis is still to be discovered. The main association with
genetic hemochromatosis is with a defect in gene HFE which helps
88 Nanopathology
regulate the amount of Iron absorbed from food. In the case below
(Fig. 3.4.6), precipitates of Iron in the form of numerous nanoparticles
are clearly visible inside the cytoplasmatic area. The picture shows some
nuclei surrounded by Iron precipitates (whiter).
Fig. 3.4.6 Image of a liver affected by siderosis. Globular Iron precipitates in the cytoplasm
can be seen (marker 20 µm).
In two cases of liver cancer, we detected particles inside the nucleus

of viable, “healthy” cells (Fig. 3.4.7). The mechanism of entry of those
particles is still to be explained.
Fig. 3.4.7 ESEM image of a liver cancerous tissue with a viable cell containing
nanoparticles in the nucleus (marker 10 µm).
Fig. 3.4.8 Silicon-Aluminium-Phosphorus-Sulphur-Iron particles in a non clearly diagnosed

liver pathology (marker 20 µm).
This sample of Fig. 3.4.8 was taken from a cylinder of hepatic tissue
that showed a partially changed architecture. Portal spaces looked
slightly dilated, with inflammatory infiltrate. No steatosis, no Iron or
biliary accumulations were visible. Kupfer cells were not evident.
Some hepatocytes looked glycogenated. Foreign-body giant cells
with a marked lymphocyte halo were present at one of the edge of
the cylinder. Silicon-Aluminium-Phosphorus-Sulphur-Iron particles were
disseminated inside the area and could be detected in the tissue.
Fig. 3.4.9 Chlorine-Potassium-Sulphur-Nickel crystal in an apparently empty cavity

(marker 20 µm).
90 Nanopathology
Fig. 3.4.9 is a cylinder of liver tissue with a preserved lobular

architecture. A small quantity of inflammatory, lymphocyte infiltrate is
visible in the portal spaces. No Iron or bile, but some small Iron
accumulation were present. Kupfer cells were not evident. At an edge of
the sample, some giant cells mingled with a thick lymphocyte infiltrate
were visible. At ultrastructural observation, the sample revealed the
presence of Chlorine-Potassium-Sulphur-Nickel particles. The edged
morphology defines it as a crystal. The void space around it may mean
that it is toxic.
Fig. 3.4.10 Two images of a neoplastic sample of the liver cancer where Sulphur-based
debris are visible (marker 10 µm).
The images of Fig. 3.4.10 show a wide dissemination of Sulphur-

Phosphorus-Barium-Calcium-Potassium-Iron-Sodium-Copper micro and
nanoparticles inside the hepatic tissue. The same chemistry was
homogeneously found throughout all the sample. The patient suffered
from a colon adeno-carcinoma, but his liver was already interested by
metastases. The portal space was larger and infiltrated by lymphocytes
and the metastatic tissue was interested by fibrosis, phlogosis and
formation of ductal-like structures. The particles of the bottom image of
Fig. 3.4.10 are attached to the surface of red cells.
Another pathology of unknown origin is lung fibrosis. Among the
cases examined, in one of them we found Lead-Iron nanoparticles in the
lung and in the liver. Fig. 3.4.11 presents a preserved liver architecture
with a lymphocyte infiltration, without neoplasm, and without Iron
accumulation. No mycetes were present, but chemically toxic foreign
bodies of Lead and Iron at nano-scale level.
Fig. 3.4.11 The image shows a hepatic tissue affected by a mild fibrosis. Lead-Iron-based
nanoparticles can be easily seen (marker 20 µm).
After a cholecystectomy, the patient developed a diabetes and later a

pancreas neoplasm. The liver presented a fibrosis, where we found the
toxic Lead-Iron-based nanoparticles.
92 Nanopathology
Fig. 3.4.12 Zinc-Sulphur-Phosphorus particles in a liver. The same particles were found in
the lung cancer of the patient (marker 20 µm).
Fig. 3.4.13 Liver cancer sample with two Zinc nanoparticles inside a cell nucleus (marker
5 µm).
Figs. 3.4.12 and 3.4.13 show a particular of the liver with small
steatosic modifications. Zinc nanoparticles are clearly visible inside a
cell nucleus. Fig. 3.4.13 shows that there is no efficient physiological
barrier at cellular level for nano-scaled particles.
Fig. 3.4.14 The image shows a particular of a neoplastic tissue of a cholangiocarcinoma full
of Barium-Sulphate micro and nanodebris (marker 20 µm).
Fig. 3.4.15 Same hepatic tissue as that in Fig. 3.4.14, interested by cancer with round-
shaped particles of a Magnesium-Aluminium-Barium compound (marker 20 µm).
Fig. 3.4.14 and Fig. 3.4.15 are about the same case of
cholangiocarcinoma. A particle inside a cell nucleus is visible in Fig.
3.4.15. Particles gather and accumulate in preferential locations and
finding those is essential to investigations. Another important point is the
influence locations could have.
94 Nanopathology
Fig. 3.4.16 Liver with metastases from colon adenocarcinoma (marker 50 µm).
Fig. 3.4.16 shows a cancerous metastasis characterized by the

formation of fibrosis and gland-like structures. The sample is full of
nanoparticles composed of Barium-Sulphur-Iron. We did not have the
possibility to check if the same particulate was present in what was
called primary cancer located in the colon.
Fig. 3.4.17 Image of a small particle of a Aluminium-Phosphorus-Chlorine-Sulphur-

Calcium-Iron-Lead-Strontium compound (marker 50 µm).
Fig. 3.4.17 shows a small particle of a strange Aluminium-Phosphorus-

Barium-Chlorine-Sulphur-Calcium-Iron-Lead-Strontium compound found
in the liver of a patient affected by the so-called Gulf-War syndrome.
That alloy does not exist in any metallurgy handbook. Every time we
find such strange chemical compositions, not having any application, we
have reasons to believe they are created by uncontrolled combustions:
Outside the chimney of an incineration plant or during the explosion of a
weapon against a target or when an airplane crashes or explodes inside a
building as happened in New York in 2001, a matter vaporization occurs
with the creation of a new pollution whose composition changes from
point to point. So, a coupling of elements is obtained that are not
matched in any intentionally made alloy.
3.5 Kidney and adrenal gland
From a certain point of view, the kidney tissue behaves like a mechanical
filter, the way other organs, the liver, for example, do. The difference
between kidney and liver seen as mechanical filters is that the former can
trap tinier objects.
Also for this organ we analyzed samples from subjects suffering from
different pathologies, as reported in Tab. 3.5.1.
Tab. 3.5.1 List of the renal diseases examined.
Pathology No.
Granulomatosis 2
Glomerulo-nephritis 1
Gulf War Syndrome 3
Neu-Laxova Syndrome 2
Cancer 2
Nephrocalcinosis 1
Kidney failure (IRA) 1
Reference samples 3
Total 16
96 Nanopathology
The first case concerns a granulomatose reaction. Granuloma is a

typical reaction of animal tissues to the presence of a foreign body that
can be either organic (bacteria, parasites, etc) or inorganic (dust, etc).
Sometimes, a fibrotic capsule is created in the attempt to isolate the
exogenous inorganic presence from the biological context. Fig. 3.5.1
shows this type of reaction. When the foreign body is isolated that way,
it can stay there for years without inducing particularly severe symptoms.
An example may be the debris of a bullet that can remain sequestered in
the tissues.
Fig. 3.5.1 Fibrous capsule in a kidney grown around a silicate particle (marker 20 µm).
The image above shows the presence of a microsized debris of a

silicate, i.e. a ceramic material, in the kidney. That debris can be “regular
household” dust, but can as well be a material coming from sources like,
for instance, ingested food. Chapter 1 showed the presence of dental
prosthetic debris in a granuloma of the liver and the kidneys with the
larger particles in the liver and the smaller in the kidneys. So, finding
silicate debris present in the composition of chewing gums in those
organs should not be surprising. As a matter of fact, chewing gums sold
on the Italian market contain silicates under particular form, as the
producer claims they can remove food remains from between the teeth
when no water is available to brush them. During mastication, the gum
matrix releases this particulate matter which gets swallowed and, thanks
to its small size, can pass through the bowel barrier and be filtered by
this tissue.
Fig. 3.5.2 with two spectra is about a patient suffering from acute
renal failure (ARF), i.e. a loss of kidney function occurring very rapidly.
The immediate result is a retention of urea and creatinine and non-
Nitrogenous waste products that are normally excreted by the kidney.
According to the severity and duration of the renal dysfunction, this
accumulation is accompanied by a number of metabolic disturbances,
such as metabolic acidosis, hyperkalaemia, changes in body fluid
balance, and adverse effects on many other organ systems. The condition
can be characterized by oliguria or anuria. The patient died of multiorgan
failure after sessions of plasmapheresis and continuous arterovenous
filtration. The histologic aspect was that of a tubular necrosis. Slight
lymph-monocyte infiltrate in the interstitium
98 Nanopathology
Fig. 3.5.2 Silicate and Tin foreign body in a renal tissue (marker 50 µm).
Fig. 3.5.3 Right adrenal gland with adenoma containing Iodine-Copper-Sulphur particles
(marker 20 µm).
The patient to what Fig. 3.5.3 refers was a career soldier in the
coastguard corps who had been on duty in Albania 2000-2001. In 2003 a
Crohn’s syndrome was diagnosed, from adrenal-gland adenoma. In this
case of Crohn’s syndrome, strange particles of Iodine-Copper-Sulphur
were found. Copper is a toxic metal, but no literature could be found
about the biocompatibility of this material in the adrenal gland.
Fig. 3.5.4 Chromium-Iron nanoparticles in a kidney, most probably a stainless steel (marker
5 µm).
100 Nanopathology
Fig. 3.5.5 Image of a particular of globular calcification in a kidney (marker 100 µm and
50 µm respectively).
Fig. 3.5.4 and Fig. 3.5.5 refer to a patient died of multi-organ

failure. Fig. 3.5.4. shows the presence of what looks like stainless steel
nanoparticles, while Fig. 3.5.5 shows globular entities, in fact Calcium-
Phosphate precipitates, formed inside the kidney. The calcification
occurred modifies the physical properties of this tissue, like, for instance
its elasticity. As a consequence of this irreversible phenomenon, the
tissue grew harder and lost much of its functionality.
Figs. 3.5.6a and 3.5.6b and spectrum. The images show a calcification (Calcium-
Phosphorus-Magnesium-Chlorine-Sodium) formed by single spherules (markers 200 µm
and 5 µm respectively).
The patient the sample of Fig. 3.5.6 comes from suffered from a
multi-organ failure with no clear diagnosis about its aetiology. He
presented many different particles disseminated in all organs, but in the
kidneys we found these Calcium-phosphate particles with a narrow range
of size distribution (1–10 micron). The difference with the previous case
is not only in the morphology but also in the composition. The spheres
are also composed of Sodium and Magnesium. The case looks like that
of the liver granuloma of Fig. 3.4.4, full of spherules very similar to
these.
Even after consulting one of the greatest experts of Calcium-
phosphates, Dr. Rachel LeGeros of the University of New York, we
cannot tell if it has an organic or an inorganic origin, nor if it is
endogenous or exogenous.
102 Nanopathology
The series of images shown demonstrates that environmental

pollution can be trapped also in the kidneys. It is possible that the
smallest nanoparticles can escape through the mesh of this filter,
go through the renal tubules, the ureters and the bladder and are
drained through the urethra. Our studies on urine could not confirm
any possible hypothesis, since the salt crystallization masks any other
presence.
3.6 Digestive system
Our studies on the digestive system showed that every inorganic, non
biodegradable, particulate matter can remain sequestered at different
levels along the pathway between the mouth and the anus.
This particulate pollution can be intentionally or unintentionally
contained in the food, or in drugs as excipient, or can derive from the
wear debris of dental materials. Part remains stuck in the oral mucosa
and cases of “Burning Mouth Disease”, ameloblastoma, cancer examined
confirmed the local entrapment.
We present an example of a very small bioptic sample taken from the
oral mucosa of a subject affected by a Burning Mouth Disease (Fig.
3.6.1). The patient was cured with tranquilizers and antidepressant drugs.
In the one-by-one millimetre specimen we detected more than 600
particles, the debris from a dental prosthesis were found. During
restoration works, the dentist drilled the old Gold-Platinum alloy
prosthetic material without the protection of a dam and polluted the
whole mouth cavity, but specially the mucosa, with the drilling debris.
The mucosa responded with an inflammatory, highly disseminated,
reaction.
Fig. 3.6.1 Oral mucosa (marker 500 µm).
Our study of stomach cancer is just at an initial stage.

The few cases, but particularly the too few samples we had available
do not allow to put forward any hypothesis. Another important point as
to the stomach cases is that we have never had the possibility to select in
the explanted cancerous mass the interface between cancer and healthy
tissue. From our point of view, that is the most significant position where
inorganic pollution concentrates. The only meaningful oddity we came
across was the finding of titania (Titanium dioxide) nanoparticles in a
cancer tissue of a stomach of a 75-year old patient. Titania nanoparticles
are something that is being employed more and more frequently in
sunscreen creams and in paints as an application of nanotechnology, but
whose use is rather recent. So, it is strange to find high-technology
material in the stomach of an old person. A possible, though not
demonstrated, explanation is that he may have eaten them mixed with
some food. That and all the following images witness that those particles
can remain entrapped inside the tissues and are not entirely eliminated
through defecation.
More than 50% of the 400 samples of food examined show a
contamination coming either from environmental sources or from the
wear of the machines used to work it (See Chapter 7).
104 Nanopathology
In the case of pathologies of the most distal tract of the digestive

system, we analyzed different levels of seriousness of disease (Tab.
3.6.1): from mildly inflamed tissues to very severe illnesses like cancer.
Tab. 3.6.1 List of the pathologies examined.
Pathology No.
Phlogosis 2
Crohn’s disease 3
Ulcerous rectocolitis 2
Granulomatosis 2
Cancer 30
Mesothelioma 1
Metastasis from hepatic carcinoma 1
Reference samples 3
Total 45
One of the cases of Crohn’s disease we examined is particularly

interesting. A 27-year-old patient had taken a antistaminic tablets since
the age of 7. In his inflamed tissue we found silicate particles which,
according to the drug’s producer, were part of the excipient used in those
tablets.
The next image (Fig. 3.6.2) gives an idea of what our colon is: a
receptor of everything is introduced through the mouth. In this case, a
large, curled debris of what is probably Teflon is adherent to the mucosa
wall. The polymeric material is easily identified because of the presence
of Fluorine in the Carbon-Oxygen spectrum. Observing the morphology
of the debris, a suggestive hypothesis can be proposed: When food is
cooked in an anti-adherent pan with a Teflon coating and is stirred or
turned with a non appropriate tool like a knife or fork, the possibility
exists of scratching the surface with the ensuing detachment of the
Teflon layer with its typical folds.
The deposition of such large debris on the mucosa surface can lead to
the following consequences: They behave as mechanical barriers
obstructing the adsorption of nutritive components. They are foreign
bodies that the tissue tries to eliminate with spasms (probably), pain,
inflammatory reaction, granuloma or fibrotic capsule formation, etc. But
what may look strange is that also this macroscopic debris was not
eliminated with defecation.
The images below witness the presence of inorganic foreign bodies in
the digestive tract wall. In a case we found 14 chemically different
particles in a 2-mm square sample. In this situation it is particularly
difficult to find a correlation between the particles, their origin and the
disease. That is likely to be the result of multiple “insults”, all concurring
to trigger a reaction.
Fig. 3.6.2 Bulk sample of a colon mucosa where a folded Teflon debris (arrow) is deposited.
106 Nanopathology
Fig. 3.6.3 Lead-Chlorine-Aluminium nanoparticles in a patient suffering from Crohn’s

disease (marker 10 µm).
The sample illustrated in Fig. 3.6.3, containing Lead-Chlorine-

Aluminium nanoparticles, comes from a patient affected by Crohn’s
disease, a pathology characterized by granulomatous reactions that,
in the case shown, were treated with Asacol (a non steroidal anti-
inflammatory drug). Inside the tissue we found also toxic particles
containing Lead.
Fig. 3.6.4 Colon microvilli with Aluminium-Copper micro and nanoparticles (marker
100 µm).
In the colon cancer shown in Fig. 3.6.4 we found, among other

particles, Aluminium-Copper micro and nanoparticulate. In many
pathologic instances, the colon contains particles presenting different
shape, size and composition. It is likely that the reaction is mainly due to
the presence of foreign bodies without a particular influence due to their
chemistry.
Fig. 3.6.5 Probably zirconia nanoparticles in a cancerous tissue (marker 10 µm).
Fig. 3.6.6 Tin-Calcium-Silicon, Aluminium-Magnesium-Sodium-Phosphorus-Sulphur-Iron

particles sequestered in a colon reaction tissue (marker 100 µm).
108 Nanopathology
The images of Figs. 3.6.5 and 3.6.6 present another case of colon
cancer with particles of what is probably zirconia (Zirconium dioxide)
and a strange compound whose complex composition can be seen in the
spectrum attached. It is difficult to guess both the origin and the
biological properties of these particles.
Fig. 3.6.7 A radioactive micro-sized particle in a peritoneal mesothelioma (marker 20 µm).
Fig. 3.6.8 Cluster of Lead-Silicon-Titanium-Chromium-Iron-Aluminium-Magnesium

nanoparticles (marker 10 µm).
Fig. 3.6.9 Round-shaped Silicon-Silver-based particle (marker 5 µm).
The images of Figs. 3.6.7, 3.6.8 and 3.6.9 show different samples
coming from a case of peritoneal mesothelioma. We analyzed more than
that 10 samples from different areas compromised by the disease and
there we found numerous different micro and nanosized particles. What
we found most frequently were stainless steel particles (non shown), but
we could also identify toxic Lead particles (Fig. 3.6.8), Silver-Silicon
particles and some ceramic radioactive material like that shown in Fig.
3.6.7. That material contained Titanium, Zirconium, Cerium but also
Uranium, Thorium, Niobium and Yttrium. Industrial ceramic material
could be the origin of such particulate.
Fig. 3.6.10 Particle aggregate of a salt in a sigma adenocarcinoma.

110 Nanopathology
Beside unusual chemical compositions detected in the intestine, we

found also common products. In this case (Fig. 3.6.10) a Sodium-
Chlorine particle is shown. The presence of a familiar composition like
that is intriguing, because those are the elements composing soluble salts.
So we wondered why such a compound could be present in the intestinal
tissue in an evidently insoluble form. In an medium like urine, salts like
Sodium chloride or Sodium chlorate solidify only during the sample
preparation and at that time they crystallize forming dendrites. In this
case we suppose they were already aggregated in an insoluble way
before fixation of the sample. We are not in a position to tell if such a
particle, with its composition and its being insoluble, therefore not a
source of ions, is biocompatible.
Fig. 3.6.11 Particular of a colon mucosa affected by cancer with sub-micronic stainless steel
particles (marker 10 µm).4
4
This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of
micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.
Fig. 3.6.12 Higher magnification of Fig. 3.6.11.
The case Fig. 3.6.11 and Fig. 3.6.12 refers to is rather singular as
the patient, suffering from a colon cancer, was a 19-year-old girl. Her
pathologic specimens were full of stainless steel particulate matter.
Fig. 3.6.13 Cluster of Silver-Molybdenum nanoparticles in a cancerous colon mucosa.
The previous images are referred to colon cancer samples. In the

same specimen we found metals like Silver-Molybdenum (Fig. 3.6.13) or
Silver alone (Fig. 3.6.14). If we were somewhat surprised at finding
those metals in the colon, the analysis of some food containing Silver
(see Chapter 7) offered us a possible explanation for those presences.
112 Nanopathology
Fig. 3.6.14 Cluster of Silver nanoparticles deposited on the colon mucosa in a cancer case.
Red cells are clearly visible at the right, lower corner.
A case of an amateur long-distance runner is shown below (Fig.

3.6.15). During his sports activity, a period of about 20 years, the subject
had taken very large quantities of food integrators and stimulant drugs
without any medical supervision. At the age of 50 a colon cancer was
diagnosed. In this case, we were not in a position to blame either diet or
drugs for his disease, since, because of the variety of substances
introduced, many of which escaped from the subject’s memory, no
traceability study was possible.
(a) (b)
Figs. 3.6.15a and 3.6.15b5 Silver nanoparticles inside the colon mucosa (marker 20 and
2 µm).
5
This photo was published in Biomateriali vol.25, A.M. Gatti “Biocompatibility of
micro- and nano-particles in the colon (part II)”, 385-392, Feb 2004.
Fig. 3.6.16 Silicon–Magnesium particle, i.e. talc, trapped in a colon cancer.
The colon cancer of Fig. 3.6.16 contained talc particles. That mineral
is generally ingested only as excipient in tablets.
The samples above show particulate matter in a wide variety of
morphologies, sizes and chemical compositions. Today’s world has
grown particularly complex and taking into account all the variables that
make it up and can interfere with living organisms is all but impossible.
If we limit ourselves to food and consider it under the nanopathological
point of view, we have to consider that:
a- an overwhelming majority of primary products grow in polluted
environments;
b- cereals and flours are generally stored in silos, many of which are
old and release debris that is inevitably added to food;
c- manufacturing adds further contaminants like the wear debris of
crushing and grinding tools;
d- polluted ingredients can be added to clean ones, thus giving origin
to a contaminated final product;
e - animals fed with polluted food can retain contamination, and that
contamination is transferred to man when he eats those animals which
are never checked under this point of view.
114 Nanopathology
3.7 Vessels
As demonstrated, environmental pollution can contaminate the blood, but

also the vessel structure can be interested by those entities. Those
pollutants are mainly metallic or ceramic and they are obviously harder
than the red and white cells, the platelets, and the intima, the inner lining
of the blood vessels. Because of that, when carried by the blood flow,
they can graze those structures and damage them, for example scratch the
vessel walls. A vessel damaged that way might be more prone to start
thrombosis or host atheromasic deposits or calcifications. We saw that
the particles carried by the blood can cross the vessel’s wall, perhaps
pushed by the blood pressure or through other mechanisms we still do
not know. During that passage, they can be trapped by the wall. It is only
reasonable to accept the fact that they can interact with that tissue.
Fig. 3.7.1 shows a silicatic particle that, probably because of its
arrow-shaped morphology, scraped the endothelium and remained stuck
in the vascular wall. Fig. 3.7.2 represents another example of entrapment
of debris.
Fig. 3.7.1 Silicatic particle in a vessel wall.

Fig. 3.7.2 Silver-Chlorine particle trapped in an inflammatory aourtic aneurism case.
In Chapter 4, the case of a girl affected by vasculitis will be discussed

extensively where Tungsten carbide nanoparticles were found. It is
certain that they were present in the blood where they induced the
disease, but also extravasated, i.e. they passed through the vessel wall
and went into the muscles where they caused a different reaction.
3.8 Sperm
Our first analysis of a sperm sample was made as a consequence of a

telephone call from a mother, whose son had died after his return from a
peace-keeping mission in the Balkans in 2001. Her son had decided to
join the Italian mission after the end of the Balkan war, had left perfectly
healthy and after a three-month mission had come home ill. His
conditions were underestimated and misdiagnosed by the doctors who
took care of him and he died of a Hodgkin’s lymphoma. Before
undergoing chemotherapy, he donated his sperm in order to use it for a
future fertilization in the hope he could recover from his disease.
After his death, his parents were informed that the sperm should have
been destroyed, but his mother asked to get it and gave it to us to be
examined.
That first analyses gave important results (see Tab. 3.8.1), since we
found foreign bodies there. At the beginning, since it was our first match
with sperm, finding foreign bodies in a fluid we believed to be protected
116 Nanopathology
enough surprised us, but, continuing our experience, we noticed that the
fact is far from uncommon.
Tab. 3.8.1 List of the sperm samples analyzed with the relevant patient’s pathologies.
Pathology No.
Non-Hodgkin’s Lymphoma 3
Lymphoma 1
Testicle cancer 1
Gulf War Syndrome 3
Blood disorder 2
Non diagnosed cases 2
Reference samples 5
Total 18
The following images show some examples of the debris and their
chemical compositions. The study involved mainly the sperm of veterans
from Iraq and former Yugoslavia (see Chapter 5) given to us because of
the fear of contaminations suspected be responsible for the children
malformation sometimes observed among American veterans. At
present, the epidemiological studies tend to deny any correlation among
the “working site exposure” of the fathers and these pathologies, but it is
true that the protocols of the US Army suggest to avoid procreation
within a year after the mission or longer than that.
Another case dealt with a soldier who had some health problems after
having returned from the Balkans and asked to have his sperm analyzed
and there we found metallic particles of different compositions, mainly
of Tungsten-Cobalt-Copper-Titanium-Sodium-Aluminium-Phosphorus-
Sulphur-Chlorine-Potassium-Calcium. The round-shape of most particles
was the obvious consequence of fortuitous combustions, a fact confirmed
by the great quantity of different elements present in the composition.
Fig. 3.8.1 Tungsten-Cobalt-Copper-Titanium nanoparticle in a sperm sample. (marker

20 µm).
The soldier, to whom Fig. 3.8.1 refers, and his wife wished to have a
child, but were afraid something wrong could happen as a consequence
of the pollution we had detected and, they asked for our help. So, with
the help of two young doctors, we tried a sort of decontamination,
separating the spermatozoa from the plasma, and we saw that the
particles remained in the plasma. Having seen that, we suggested to recur
to artificial fertilization with the “clean” spermatozoa and the result was
a couple of healthy twins.
Fig. 3.8.2 A viable spermatozoon is close to a Lead-Chromium particle (marker 20 µm).

118 Nanopathology
Fig. 3.8.2 shows a viable spermatozoon close to two Lead-Chromium

(two toxic elements) particles. The obvious questions are: “What is the
fate of this spermatozoon in contact with a toxic material? What is the
fate of this particle when the sperm is donated during a sexual
intercourse? What will the answer be of the vaginal mucosa and the
uterus when a toxic particle remains in close contact with them? Is
Burning Semen Disease (see Chapter 5) the response for this presence?
All that is something really new, very little has been done in that field
and we do not have the answers. We can formulate only hypotheses, and
those hypotheses may show the way the research should follow.
Another case we came across and illustrated in Fig. 3.8.3 is that of a
soldier whose sperm was polluted by clusters of nanoparticles composed
mainly of Lead and Iron.
Fig. 3.8.3 Cluster of Lead-Chromium-Iron-Titanium-Silicon-Aluminium-Magnesium-

Sodium-Chlorine-Calcium nanoparticles (marker 10 µm).
Fig. 3.8.4 Image of an Iron particle together with a viable spermatozoon (marker 20 µm).
Both pictures (Fig. 3.8.4 and Fig. 3.8.5) show foreign bodies in a drop
of sperm from two different subjects.
Fig. 3.8.5 Image of a spherical zirconia particle in the sperm (marker 20 µm).
120 Nanopathology
Fig. 3.8.6 Cluster of Lead-Chromium-Chlorine nanoparticles (marker 20 µm).
Fig. 3.8.7 Image of a viable spermatozoon with a spherical Antimony-Cobalt-Sulphur-

Sodium nanoparticle (marker 20 µm).
Figs. 3.8.6 and Fig. 3.8.7 come from the same patient and show
viable spermatozoa in contact with toxic foreign bodies. This finding
interested us for two reasons: the first because the so-called physiological
barriers prove to be inefficient against such form of assault, the second
because it may represent a key to understand some medical mysteries of
our age. For instance, the so-called “industrial sterility” that affects some
workers in certain industries could be explained if we accept that the
contamination in some working places, represented by a toxic pollution,
reaches the testicles, pollutes the sperm and causes the death of
spermatozoa or reduces their vitality. This kind of contamination could
be verified also in the female partners suffering from Burning Semen
Disease.
In the case of contaminated sperm, the simplest prevention measure
that can be adopted to avoid possible problems to the female partner is
the use of a condom
3.9 A few considerations on reproduction
We leave in an age whose sexual customs are somewhat looser than in

the past, and meeting occasional partners without knowing anything
about their health is common enough. Knowing that we do not know
what we should and that ignorance may lead to grievous consequences
should induce to adopt more prudent approaches.
What we found in the sperm has other important consequences.
What happens if an egg is fertilized by contaminated sperm? What
happens to a fertilized egg that develops in a medium where particles of
toxic material are present?
All those questions remain without direct answers, but we had a
chance to observe some of the possible consequences. When we
analyzed malformed feti (both human and animal), we discovered
something that had escaped consideration until that moment.
We received two malformed lambs whose mothers had been grazing
close to an interforce military firing ground in Sardinia, and samples of
the internal organs of three feti affected by Neu-Laxova syndrome from
Malta. The peculiarity of the latter was that they were born, along with
122 Nanopathology
three more, in a period of twenty moths, something really surprising,

since that syndrome is extremely rare and Malta counts no more than
400,000 inhabitants.
The animal cases presented two different macroscopic kinds of
malformations: the first lamb had no developed brain nor eyes, in whose
place sat its ears. The second had the extrusion of the intestine. Both
survived just few hours.
The three cases examined out of six feti affected by Neu-Laxova
syndrome were sent to us by the University of Malta for the search of
possible genotoxic material. Despite its extreme rarity, six cases were
reported in Malta, a country with a population of 400,000 inhabitants, in
the space of just 18 months.
Neu-Laxova syndrome is a rare, incompatible with life, syndrome of
unknown origin characterized by ichthyosis (scaly skin), intrauterine
growth retardation, microcephaly, short neck, central nervous system
abnormalities, hypoplastic or atelectasia of the lungs, deformities of the
limbs, oedema, polyhydramnios (an abnormally high level of amniotic
fluid), and short umbilical cord.
As a matter of fact, inorganic particulate matter was clearly visible in
all samples we examined.
At the same time, we analyzed some internal organs of fetus from
induced abortions and in all cases they appeared free from any
particulate contamination.
Fig. 3.9.1 Morphology of four different tissues: brain, thymus gland, spleen and kidney of
a fetus whose delivery was artificially induced (markers: A = 20 µm, B = 50 µm, C =
100 µm, D = 100 µm).
Fig. 3.9.1 shows different tissues belonging to a healthy fetus from an

induced abortion. The tissues appear free of any foreign body. They are
composed of only Carbon and Oxygen. (Hydrogen and Nitrogen are part
of the composition of the natural tissues, but it is impossible to see them
with our equipment).
All the images that follow regard samples taken from malformed feti.
Surprisingly enough, since those beings had never lived in the
environment we all share, particulate of exogenous origin could be
detected in their tissues. The compositions of some of those particles
were particularly strange and, in a few cases, certainly toxic.
124 Nanopathology
Fig. 3.9.2 Antimony particle inside the baby’s liver affected by Neu-Laxova syndrome
(marker 20 µm).
Fig. 3.9.3 Strontium-Sulphur-Iron-Aluminium particle inside a baby’s liver affected by

Neu-Laxova syndrome (marker 20 µm).
Fig. 3.9.2 and Fig. 3.9.3 regard the same case.

Fig. 3.9.4 Lead-Bismuth particle surrounded by red cells in a liver sample of a case of
Neu-Laxova syndrome (marker 10 µm).
Fig. 3.9.5 Antimony particle inside a baby’s kidney affected by Neu-Laxova syndrome.
The subject is the same as the one of Fig. 3.9.4 (marker 10 µm).
Fig. 3.9.4 and Fig. 3.9.5 belong to the same subject. In the three cases
we had the chance to examine, the particles found showed different
compositions but Antimony was always present.
126 Nanopathology
Fig. 3.9.6 Zinc particle in a Neu-Laxova’s syndrome liver (marker 10 µm).
Fig. 3.9.7 Antimony particles inside a baby’s kidney affected by Neu-Laxova syndrome
(marker 20 µm).
The pictures above (Figs. 3.9.6 and 3.9.7) belong to the same case.
All the three cases of malformed feti showed the presence of micro
and nanoparticles inside the liver and the kidneys. The spectral analyses
of all the particles found in the small samples received revealed that they
are contaminated with metallic foreign bodies, sometimes toxic, like the
compounds of Antimony, Strontium or Lead. This presence is really
peculiar, if we consider that feti are never directly exposed to the
environment and its pollution. According to what we found, those
specimens present different levels of pollution as far as concentration is
concerned, but they all share the presence of Antimony compounds. A
study aimed at searching the origin of this contaminant investigating

parents’ life and environment they lived in is in progress.
Fig. 3.9.8 Iron-Zirconium-Silicon-Aluminium-Magnesium-Zinc-Copper-Chromium-Calcium-

Antimony particle in the liver of a malformed fetus (marker 10 µm).
Fig. 3.9.9 Lung tissue from a malformed fetus. Clustered Nickel-Chromium nanoparticulate
is present (marker 20 µm).
128 Nanopathology
Among the oddities found in the lung pathologic tissues, we want to

show also an indirect exposure. The direct exposure to pollution regarded
a pregnant woman, but that pollution moved to the fetus through the
placental blood circulation. The pulmonary tissue of a 20-week fetus is
shown in Fig. 3.9.8 and Fig. 3.9.9. The artificial abortion was due to the
discovery of a 2-cm vertebral fission. Clusters of micro and nanoparticles
were visible in the lung tissue, composed of Nickel-Chromium. This
debris is chemically toxic. The particle shown in Fig. 3.9.8 is rather
large, about the same size of a red cell, and its composition is very
complex. Also in the case of this fetus, Antimony was present.
The next is not a human case, but a veterinary one (Fig. 3.9.10 and
Fig. 3.9.11). It regards the malformed fetus of a lamb died immediately
after delivery. Its mother grazed in the close vicinity of a firing ground in
Sardinia where grass was contaminated by the dust coming from the
explosions consequent to military experiments and drills.
Fig. 3.9.10 Antimony-Sulphur particle found in a malformed lamb’s brain (marker 10 µm).
Also in the cases of the malformed lambs we found a wide

dissemination of nanoparticles in all its organs, including the brain and
testicles. As in the human cases, Antimony was a constant presence.
Fig. 3.9.11 Antimony-Cobalt-Sulphur nanoparticle in the liver tissue of the malformed lamb
of Fig. 3.9.10 (marker 20 µm).
It was somewhat odd to find recurrent chemical compositions shared

by either the human feti of Malta and the animal ones in Sardinia. But
Antimony, the element in question, could also be found in combination
with Cobalt in the sperm of a soldier who worked in a firing ground in
Sardinia and in a Canadian soldier who served during the First Gulf War.
This series of coincidences urged us to start a project on fetal
malformation which is in progress.
130 Nanopathology
Tab. 3.9.1 Flow-chart of nanoparticle dissemination with the entrance points.
Note: The number 1 in the insets means a primary “door” of entrance of the particles
inside the human body.
All the images shown in the previous pages suggest a particular

dissemination of the nanoparticles once inhaled or ingested in the human
body. Their persistence in the tissues can induce possible pathological
reactions.
The Tab. 3.9.1 is an attempt to summarize the achieved experience.
Further studies are necessary to confirm the findings.
3.10 Bibliography
Albores-Saavedra, J., Vitch, F., Delgado, R., Wiley, E. and Hagler, H., (1994). Sinus
histiocytosis of pelvic lymph nodes after hip replacement. A histogenic
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Chapter 4
Six “Detective Stories”

___________
7
he technique we developed allowed us to set-up a new investigative
method to assess the patient’s actual exposure to micro and
nanopollution.
The identification of the particle chemistry found in the pathological
tissues, along with their size and shape, induced us to investigate on the
patient’s life and look for possible sources of that particular
contamination, the real possibilities of his exposure and how that
pollution contamination may have occurred. In some cases we saw that
the exposure continued to be possible, so, some suggestions were given
to the patient to avoid it.
That kind of work had us discover unexpected ways of contamination
and, quite often, unknown ways of dissemination of the particles inside
the human body.
Six of such cases are briefly described below.
The 1st Case
The first case is related to a 25-year old non smoking girl.

She was admitted to the hospital because of vascular problems
affecting her hands and an altered electrical conduction of her peripheral
nerves.
The first diagnosis she received was sclerodermia, but later, after
the neuro-electrical test results, a diagnosis of ischemic miopathy was
advanced. The steroid therapy undertaken proved to be ineffective.
135
136 Nanopathology
A biopsy of the left-leg muscles (Fig. 4.1) was performed and specific
tests on the fresh tissue revealed an endothelial vasculitis, diagnosis
confirmed by Harvard University researchers.
Another histological section was sent to us to be processed and
analyzed with our technique. What we saw among other particulate were
submicronic particles of Carbon-Tungsten outside a blood vessel.
Fig. 4.1 Image of Carbon-Tungsten nanoparticles inside the muscle biopsy (marker 20 µm).
The way we do in similar cases, because of the presence of such

unusual foreign bodies which we took as a sort of marker, we started to
investigate on the patient’s occupational life that had taken place all
along in a small hairdresser’s shop.
We checked a great variety of the products used there to see if
Tungsten was present somewhere, but no trace of that metal was to be
found. So, we analyzed the dust from the plaster of the shop premises,
the indoor particulate pollution in the air, the dust inside the hairdryers
(the electrical resistances of the hairdryers can be made of Tungsten) and
the smoke of the cigarettes smoked by the only smoker who worked
there. Again, no trace of Tungsten was to be found.
Special attention was dedicated to parents’ story, also considering the
fact that the patient’s father had died of a lung cancer. That diagnosis had
been issued by observing bronchial mucus smeared on a glass slide, and
we look for it in the hospital’s archive. Had we found Tungsten there, we
could have identified a pollutant shared by both father and daughter and
that would have put us on an easier track.
Six “Detective Stories” 137
It must be said that biological samples on glass are not ideal for our
analyses, since glass issues signals that can interfere with those issued by
the particles.
In that particular specimen we found a few metallic particles, but no
Tungsten. So, that hypothesis of domestic pollution had to be ruled out
and we had to restart the investigation, which we did in the patient’s
house.
The indoor particulate contamination in the air was analyzed together
with the wall plaster, the cigarettes smoked by her mother and the food
more frequently eaten by the patient. But not even there any trace of
Tungsten was found.
We had to find a source of Tungsten if we wanted to solve the case.
That metal or its carbide (Carbon-Tungsten particles were what we had
found in the girl’s biopsies) are rather common in metallurgical
industries, but the patient did not seem to be likely to be or to have been
involved in such a contamination, as she had lived all her life in a flat
downtown, far from industrial sites.
In the course of our many interviews, the girl told us that two years
ago she had had a bleeding from the endometrium which did not respond
to any pharmaceutical treatment and that was solved surgically. Before
the operation, she had also suffered from two bleeding episodes from the
anus, and in both cases she was treated at the Emergency Room of the
local Hospital.
During the surgical operation, a small biological sample of
endometrium (Fig. 4.2) was taken and processed for the usual
histological tests. We analyzed the archived sample and found small
particles of Iron-Chromium-Nickel (stainless steel) there. The presence
of very small particles ranging from 0.5 to 8 µm in such an anatomical
site seemed very strange. Their presence could have been the cause of
the bleeding, since those are bodies foreign to the human organism and,
in contact with the mucosa, can trigger an inflammatory reaction and, in
some instances, a localized necrosis. The inflammation can also reach
deeper into the tissues. That could explain the strange episodes of
bleeding from the anus which could depend on an acute inflammation of
the internal tissue in close vicinity of the vagina or the uterus.
138 Nanopathology
Fig. 4.2 Image of a sample of endometrium with stainless steel nanoparticles (marker
20 µm).
Such an event reminded us of the Burning Semen Disease suffered by

the wives of the veterans after their coming back from the war missions
in Iraq or Afghanistan during the 1st and 2nd Gulf War (see Cap. 5)
This findings had us redirect the investigations toward the patient’s
partner and we discovered that he had suffered from a testicle
cancer, operated on four years ago. So, we thought that analyzing his
pathological tissues could give us some important clues.
At the time of the disease, the partner worked in a tile factory as a
clerk, but his desk was located in a large room, close to a micronizing
tool used to ground the clay before it was mixed with water, and to a
special machine used to cut the edge of the tiles. The grinding and
cutting tools were made respectively of stainless steel and Tungsten
carbide.
The partner told us that during the period he was employed there, the
man who worked in direct contact with the latter machine had died of a
lung cancer.
The analyses on the four surgical samples we got revealed the
massive presence of micron-sized particles of silicates (tiles) and debris
of stainless steel and Tungsten.
The finding of this material inside a testicle had us put forward a
mechanism of dissemination and translocation: The partner had been
exposed to the materials making up the tiles and to the wear debris
coming from two different tool machines. Following a mechanism that
we cannot yet fully explain but that we have observed in many instances,
we think that those debris were entrapped in a testicle (we had no
samples from the contralateral one, so, nothing about it may be said)
after having been inhaled, and the metallic nanoscaled particles (stainless
steel and Tungsten) extravasated and contaminated the sperm which was
donated to our patient during sexual intercourse. The presence of non
biocompatible, not biodegradable particles in the girl’s vaginal mucosa
caused pain, inflammation and bleeding as occurs in the veterans’ wives.
The surgery that followed the bleeding episodes exposed a
compromised tissue, without any effective defense against sperm
contamination. As a matter of fact, the symptoms referred to the sclerosis
started a few months after the surgical operation.
The probable reasons why Tungsten, and not stainless steel, particles
were so widely disseminated were because of their size, much smaller
than that of the stainless steel particulate, or because of a physical-
chemical interaction withy the blood components, or both. Such
dissemination in the blood circulation had been the cause of the
vasculitis.
The particles extravasated and came into contact with the muscle
fibres, causing a fibrosis, which is the normal biological reaction to the
presence of non biocompatible foreign bodies.
Of course, we did not have the possibility to check but a few of the
girl’s tissues, but, as far as we saw, the particle dissemination seemed to
be ubiquitous. So, we suggested to have protected sex in order to avoid
further exposure, but the physical elimination of every single particle
from the organism looked impossible, at least to our experience and for
the time being.
In the case described, Tungsten acted as a marker and the search of its
specific source of pollution allowed us to identify the kind of exposure
and the gross mechanism of dissemination inside the organism. The
following images show the Tungsten particles found in the spermatic
vessel (Fig. 4.3), the stainless steel debris (Fig. 4.4) and the particles of
silicatic material from the ceramic tiles (Fig. 4.5, 4.6 and its spectrum).
140 Nanopathology
Fig. 4.3 Image of a Tungsten particle found inside the spermatic vessel (marker 50 µm).
Fig. 4.4 Image of a stainless steel particle (Iron-Chromium and Nickel) found in the
cancerous tissue of the testicle (marker 20 µm).
Fig. 4.5 Image of the part of the resected cancerous tissue of the testicle with the
spectrum of the normal biological tissue (marker 1.000 µm).
Fig. 4.6 shows the “dust” found in the resected tissue of the testicle
cancer. The samples contained many different particles, mainly silicatic
debris coming from the dust of the tiles (white particles indicated by
arrows). They are composed of Silicon-Aluminium-Calcium. Normal
biological tissue contains only Carbon-Oxygen-Sodium-Phosphorus-
Sulphur-Chlorine. We do not know if the cancer was induced by the
metallic particles or by the enormous pollution.
Fig. 4.6 Image of an agglomerate of silicatic nanoparticles trapped in a calcific tissue

(marker 50 µm).
142 Nanopathology
The 2nd Case
A professor of Mathematics started to betray some problems in

articulating the sound “r” during his lectures at the University. After
different neurological examinations, a diagnosis of Lateral Amyotrophic
Sclerosis was issued, a diagnosis he did not believe in and refused to
accept.
Thus, the professor started to search the Internet about that disease
and learnt that the one of the first, more common symptoms is a slight
claudication and this, among other symptoms he had read about, was not
something he suffered from.
In the enormous quantity of not always reliable information he found
in the Internet, he read that dental amalgam, because of its content of
Mercury, could be somehow related to the disease he had been
diagnosed.
So, the professor asked Dr Gatti her opinion as a biomaterial expert
and a teacher of Dental Materials at the University of Modena and
Reggio Emilia.
Actually, he had either amalgam restorations and dental prostheses in
his mouth and hardly in two teeth the amalgam did not look corroded. (It
is only in the course of corrosion that amalgam releases Mercury, Silver,
Tin and Copper ions which can be toxic, locally in the oral mucosa and
in a systemic way after ingestion).
But the observation of the mouth revealed an unusual situation: The
surface of the tongue was not symmetrical and the right half was not as
flat as the left one, but was corrugated and wavy. The surface of the
mucosa seemed to have a larger extension than the substrate, and that
created a sort of gibbosity, not adherent to the tongue base. In addition to
that, we noticed a discoloration in the internal cheek mucosa at the level
of the intersection of the teeth. We also observed that in the right part of
the mouth some porcelain prostheses had been implanted.
Having seen all that, we thought that if there is a wear phenomenon,
debris must be released, and those debris can be entrapped by the nearby
mucosa and induce a fibrosis. The tongue mucosa has a great aptitude to
absorb, so a passage of some fine particles through it cannot be
surprising and their entrapping occurs at the level of the underlying
tissue called corium. That formation is a thick network of fibrous

connective tissue strictly connected with numerous elastic fibres,
forming the septa between the muscular bundles of the tongue. It
contains the branches of many nerves and vessels that feed the papillae.
The tissue can react to a foreign body giving origin to a fibrosis.
A thickening and the following contraction of that layer could explain
the humped morphology of the mucosa surface and the loss of agility of
the tongue. It is also reasonable to think that all that exerts a compression
on the underlying muscle and nerves. But, in any case, a collection of
foreign bodies can give origin to a fibrosis, and a localized fibrosis could
be responsible for the problem in articulating the sound “r”, because a
fibrotic tissue is thick, less elastic than normal and does not allow fine
movement.
The first step we had to take was to demonstrate the presence of that
fibrosis with the inclusion of prosthetic debris in a biopsy of the internal
side of the cheek. As a matter of fact, the tissue presented a fibrosis and
some porcelain debris were present there.
The second step was to demonstrate a similar presence in a biopsy of
the right, ipsilateral side of the tongue. The hope was that if a fibrotic
reaction due to foreign bodies was present, probably the removal of this
tissue or a cut made on the tongue could interrupt the phenomenon and
its worsening. We thought that stopping the fibrosis’ progress could
probably prevent further damage to nerves and blood vessels. In fact, the
shrinkage of a fibrotic tissue can make the vessels occlude and stop of
the supply of nutritious elements and Oxygen. The afferent and deferent
nervous terminations can simultaneously stop the propagation of the
electrical signal, thus causing the opening of the electrical circuit.
While the histopathologist confirmed the presence of a fibrosis in the
bioptic sample, we saw some strange precipitates of Zinc there.
It is easy to tell the difference between exogenous particles and
endogenous precipitates: The EDS signal is high in the case of “stones”,
since the atomic structure is compact, while, in the case of precipitates,
the inorganic element (Zinc in this case) is bound to proteins, forming
insoluble metalloproteins. The density of these precipitates is very low
and also the signal they issue is low (proteins contain Carbon, Oxygen,
144 Nanopathology
Hydrogen and Nitrogen, all very light elements). Also the morphology is
characteristic.
In our case, Zinc ions must have been absorbed by the tongue mucosa
and in the sub-mucosa the interaction with proteins created insoluble
precipitates. The sub-mucosa is reach of nerves and nervous terminations
that can be compromised by the presence of toxic ions. Also the
formations of metalloproteic precipitates can interfere with the cellular
metabolism according to different mechanisms: protein depletion, protein
immobilization or a change in the local pH. A further possibility is the
entrance of ions within the cell where they can precipitate, forming
aggregates that disturb the intracellular metabolism. The sequestration of
proteins, namely the formation of insoluble precipitates, can interrupt
cascade reactions that cause “alternative metabolic solutions”.
It was not only Zinc what we detected, but a wide variety of particles
ranging 0.8 – 2 µm. Gold particles were there, and this was compatible
with the bridges the patient carried. 0.8 µm particles of Silicon,
Aluminium, Calcium, Magnesium, Potassium and Iron were also present,
and they could certainly be blamed for the fibrosis, the roughness of the
tongue surface and the discolouration of the mucosa.
Fig. 4.7 Image of nanoparticles of Phosphorus-Zinc-Magnesium-Aluminium-Silicon-

Calcium-Iron found in the biopsy of the mouth mucosa (marker 20 µm).
But what puzzled us most was the presence of Zinc (Fig. 4.7). That is
a metallic element commonly used as oxide because of its antibacterial
activity, but its presence is unexpected inside the oral mucosa and an
explanation for such a presence must be given.
As usual, we questioned the patient about his diet, the use of chewing
gum, drugs, etc., but no clue was to be found there.
The truth emerged unexpectedly when we asked the patient about his
visits to the dentist’s. The patient had been advised to use a mouthwash
and he had used it in an obsessive way, many times a day, for more than
3 years. The label of the product reported the presence of a Zinc salt as a
bactericide, but the solution contained also ethylic alcohol, a substance
that increases the permeability of the tongue epithelium, thus making the
entrance of the Zinc salt easier. That salt hydrolyzed, Zinc formed a
metalloprotein and that metalloprotein precipitated. Its presence was the
likely cause of the partial necrosis of the biological structures there
present.
Zinc is a well-known bactericide and is toxic to cells. Therefore, it
could interact with the myelin sheath of the nerves and damage it.
When we realized that, the patient had already quitted using the
mouthwash and the damage to his nervous system had grown
irreversible; so, all that was of no practical use to him and he died in six
months.
If our hypothesis is correct, this is a case where very simple
precautionary measures could have avoided the gravest of consequences.
The 3rd Case
The manager of a very important company consulted our laboratory for a

case of dermatitis afflicting his trunk, asking to check the biopsy of the
derma interested by the affection.
The observation of the very small sample showed the presence of
numerous particles of stainless steel (Fe-Cr-Ni).
Such a finding looked strange, because neither at home nor at the
patient’s working place stainless steel particulate should have been
present.
He lived an apparent healthy life in a “normally” polluted big city. He
had been a smoker in the past but in the last 10 years he had never lit a
cigarette.
146 Nanopathology
The patient told us that in the course of a yearly check up he had been
subjected to a chest X-ray examination that revealed light spots in his
lungs and, because of that, some biopsies of those spots had been
performed. The response was that the areas were calcified.
We took those bioptic samples, observed them with our usual
method, and found that stainless steel was present there in the form of
small micrometric and submicronic particles.
Having been found in the lungs, we supposed as a probable
hypothesis that those pollutants had been inhaled; some of them had
stayed in the lungs where they had triggered an inflammation with a
consequent calcification, and others had been disseminated throughout
the organism because of a size small enough to allow the passage
through the lung barrier and enter the blood stream.
Their presence in the skin (Fig. 4.8) could be explained with an
attempt of the organism to get rid of them, but they were halted by the
derma which is very compact and hard to cross, and there they caused the
irritation the patients complained of.
Such a hypothesis must be demonstrated, and to do that we had to
find out the origin of that particular pollution and the way our patient had
been exposed to it.
Questioning him, we got to know that he had suffered from nasal
congestion for more than twenty years and for that reason, to try and
breath better, he had been operated on his nasal septum one year ago. But
for the previous twenty years, he had put drops in his nose to decrease
the congestion. During all that period, he had used only two types of
herbal drugs, one coming from Italy and one from Switzerland, and we
checked both products from bottles the patient still conserved, even if he
did not use them any more after the successful surgery he had undergone.
What we saw was that only one of the two oily liquids contained
particles, and they were the same size and composition as those we had
detected in the pathological tissue.
That could be explained by guessing that the herbs used by that
particular laboratory were ground with a stainless steel tool that, because
of the obvious wearing of the machine, released particles to the product.
We wondered why the presence of metal particles did not induce a
more serious disease like, for example, a cancer?
A possible explanation is that the quantity of particles was relatively

small and the oil used as a vehicle protected them from corrosion, thus
preventing a release of Iron, Chromium and Nickel ions. Besides, that oil
was organic and the tissues had an interaction with something they
recognized and did not see the inorganic bodies below. So, all that
occurred was a comparatively mild inflammation with a consequent
calcification.
In that case, the exposure had been interrupted and nothing else could
be done as a preventive or therapeutic measure.
Fig. 4.8 Image of three particles of stainless steel embedded in a biopsy of derma (marker
20 µm).
Fig. 4.9 Image of a small stainless steel particle found in a lung biopsy (marker 20 µm).
148 Nanopathology
Fig. 4.10 Image of a stainless steel particle found inside the nose drops used by the
patient (marker 20 µm).
In Fig. 4.9, a single stainless-steel particles found in the patient’s lung

is visible. Fig. 4.10 and Fig. 4.11 show the contamination of Iron-based
particles detected after filtration in two different nasal drops used by the
patient.
Fig. 4.11 Image of the metallic contamination of the nose drops used by the patient, and
deposited on a paper filter (marker 200 µm).
The 4th Case
In this case, the patient was a 45-year-old lady suffering from a

peritoneal mesothelioma.
She had been operated on and some bioptic samples of her

peritoneum were sent to us to be analyzed.
There we found many debris singularly disseminated in the specimen:
metals like, for example, stainless steel were present, but one particular
chemical composition attracted our attention.
The EDS spectrum indicated that a number of submicronic particles
were a silicate containing, among other elements, Uranium and Thorium.
In that very period we were busy with specimens coming from Italian
and French veterans back from former Yugoslavia, and Uranium was
what we were looking for, but without any result: No trace of Uranium
was to be found in those pathological tissues.
Now we were confronted with the case of a lady who had never been
in a war theatre nor, to her knowledge, in the vicinity of a source of
radioactivity, but had spent her whole life in a quiet Italian town working
as a clerk in an office, yet she had Uranium and Thorium in her body.
So, we started a hunt to identify the origin of such an unusual and
unexpected particulate.
Being a silicate, we immediately suspected that the origin of the
pollution found could be an area about 20 kilometres south of the place
where the patient lived, where numerous ceramic factories are located.
As to Uranium, it is used to give a yellow colour to enamels and
Thorium is used as well. The study of the winds, though, seemed to rule
out such a hypothesis or, at least, to make it very unlikely.
Fig. 4.12 Image of a particular debris containing radioactive elements found in the
peritoneal mesothelioma (marker 20 µm).
150 Nanopathology
Fig. 4.12 shows a particle that contains Titanium-Zirconium-

Calcium-Iron-Aluminum-Yttrium-Niobium-Uranium-Thorium-Cerium-
Manganese-Copper. That can be originated by tiles industries that use a
silicatic or Zirconium-based earth for the tiles.
Fig. 4.13 Image of a particle found on a leaf surface. Its composition includes radioactive
elements (marker 20 µm).
Fig. 4.13 shows a particle composed of Silicon-Phosphorus-

Aluminium-Uranium-Thorium-Lanthanum-Cerium-Neodymium-Iron-
Potassium-Calcium. That composition is typical of a type of earth used
for ceramic tiles. Uranium and Thorium are likely to have been added as
components of some glazes.
Since those particles had been found in the peritoneum, i.e.
anatomically close to the digestive system, we assumed as a likely,
though not exclusive, hypothesis that ingestion could have been the way
they were introduced into the organism. According to that assumption,
we began to search the food the patient ate more frequently, despite the
fact that what was in the peritoneum could have entered long ago with a
food, if food was indeed the Trojan horse, that had been used in the past
and then forgotten. Nevertheless, we tried.
All the family composed by a husband and a girl, both healthy, was
involved in that sort of treasure hunting.
The patient told us that both her parents had died of liver cancer,
without any doubt something worth investigating, but we could not find
their biopsies and, of course, the histopathological records did not

contain anything that could help us.
Questioning the family about their preferences in the matter of food,
we got to know that the lady preferred vegetables to meat, unlike her
husband and daughter, and she was particularly fond of a kind of wild
chicory that her parents had been bringing to her at least once a week
from a village in the hills, about 30 kilometres away from town, where
she had moved at the age of 22 when she married. That chicory had been
a usual dish to her since her childhood and had never ceased to be.
Chicory was what we had to check and we went to the small valley
were it had always been picked.
It was winter time and no chicory could be found in that place nor
other edible vegetables, but, looking for possible clues of pollution, we
saw a plume of smoke in the distance and we drove there, four or five
kilometres away as the crow flies. There we saw a group of small
ceramic factories whose existence was unknown to us.
Then, we went back to the valley and picked some oak leaves still
hanging from a few trees that we brought to the laboratory and observed
under ESEM. On those leaves we detected particles containing silicatic
compounds with Uranium and Thorium.
According to what we discovered, the lady and her parents had been
eating something they believed to be very safe but which, in fact,
contained toxic (and slightly radioactive) particulate.
Now, a final piece was missing to the solution of the puzzle: why had
our patient contracted a peritoneal mesothelioma, while her parents had
died of liver cancer (granting the fact that the aetiology was the same)?
Twenty three years ago, our patient had spent her honeymoon in the
North African desert and, when a guest at some Bedouins’, she had
drunk tea which gave her a typhoid fever. Intestinal problems lasted a
long time and, probably, that inflammatory condition altered the
permeability of her intestinal mucosa which became more negotiable to
particles.
If we accept that hypothesis, had she had a healthy gut, she would
have contracted not a mesothelioma but a liver cancer, the way her
parents did.
152 Nanopathology
The 5th Case
A couple of somewhat unusual patients, two lady doctors working in the

neonatal department of a big hospital, came to our laboratory, asking us
if we could help solve their case.
Four doctors, including the chief of the department, and two nurses
suffered from different diseases: cancer, sarcoidosis, and two missing
diagnoses, as the operators developed symptoms that could not be
referred to any known disease.
One of the two ladies suffered from a lung sarcoidosis and inside the
granulomatous formations typical of that disease we found micro and
nanoparticles of stainless steel (Fig. 4.14) and silicates (Fig. 4.15).
Fig. 4.14 Image of a small stainless-steel particle (indicated by arrow) found in a lung
biopsy performed to confirm the diagnosis of sarcoidosis (marker 20 µm).
Fig. 4.15 Biopsy of a nodule containing Silicon-Aluminium particles (marker 10 µm).
Fig. 4.16 Bacterium (inside the circle) containing Aluminium particles collected by
gravity on a Carbon sensor (marker 10 µm).
The second case was different. The doctor-patient contracted a

bacterial infection in the ring finger of her left hand, an infection that
proved resistant to the specific antibiotic administered. Those bacteria
had been detected within the Intensive Care Unit of the department,
where the patient was usually busy (Fig. 4.16).
In a short time, the patient had started to suffer from fatigue, than
from acute episodes of sometimes monolateral and sometimes bilateral
pain at the lower limbs followed by the formation of nodules that were
surgically removed as many as 18 times without any advantage to the
patient, since the pathology continued its progress. Our investigations in
154 Nanopathology
those nodules discovered the presence of stainless steel and Aluminium

particulate. A section of those nodules was sent to the Harvard School of
Medicine in Boston and the diagnosis issued was of a vasculitis.
Our hypothesis was that phenomena of extravasation of those
particles, running in the circulatory system, had been occurring, and that
could explain the acute pain. Then, the presence of foreign bodies in the
muscular tissue could have reasonably induced a biological reaction that
drove to the formation of the nodules.
We had to look for the source of pollution that could have caused the
pathologies in doctors who had been living what for doctors is a normal
life.
So, we decided to sample the air in two adjacent rooms where the
doctors work more frequently and we did so by means of an air pump
that worked for one hour. The dust was collected on cellulose filters
where particles smaller than 2.5 µm were detected.
We were told that just outside the building where the department was
located, another building had been standing and that building had been
very slowly pulled down raising great clouds of dust. The still standing
broken stainless-steel reinforcement had been left on place for a long
time.
The pollution created was aspirated by the air conditioning system set
on the roof of the department. Every room was equipped with a filtration
system that can only stop particles larger than 2.5 micron, while smaller
particles can pass easily and their presence inside the rooms was
concentrated because of a recirculation system which created an
accumulation of fine pollutants.
It is only natural that everybody who works in such environmental
conditions is exposed to the effects of that sort of pollution.
The main difference between the two cases was the much more
regular presence of the second doctor in the Intensive Care Unit, where a
bacterial contamination had been found, and those bacteria were, in turn,
contaminated with Aluminium and had served as a carrier. Aluminium
particulate was inside the bacteria we found and that bulky presence
changed their morphology. It was probably for that change in shape that
the antibiotics administered did not have any effect. In the first case, the
inhalation of foreign bodies had induced a granulomatosic reaction,
while in the a second one the infective carrier of the pollution was
predominant.
To the same way of entrance, two different pathologies had ensued.
The cases proposed and the solution found should have us reflect.
The solution of the twin cases had been possible because we started
from looking for and finding the real exposure the patient had undergone.
Every patient has a personal history and epidemiological studies are not
always helpful or are not meaningful at all if the number of cases is
very restricted. So, a single-patient oriented, “customized” investigation
should be carried out in such circumstances. That is certainly far from
easy, expensive and time consuming, all things our society does not look
willing to accept, but we do not see any other way to deal with patients
like the ones we were confronted with.
Far from easy, expensive and time consuming but not useless. The
knowledge gained from such cases is of the utmost usefulness to prepare
prevention guide lines and, in a very pragmatic way, prevention means
saving money.
In the case in point, we got a further proof that any uncontrolled
release of nanoparticles in the working places must be avoided. Masks,
specific filtration means and effective systems to abate acute dust
pollution, especially when it is the nanometric size, must be studied and
readily adopted. When dust is released in the environment, everybody
must take proper care. How, it is the scientist’s task to say.
The 6th Case
The case of a dentist who had developed a lung sarcoidosis showed the
existence of a close correlation between his disease and the powder he
had used to bleach his patients’ teeth.
In 20 years he had used about 300 kilograms of that fine powder, a
part of which he had certainly inhaled and whose concentration in his
organism had probably exceeded his tolerable threshold concentration.
That dentist, male, 52 years old, never been a smoker, gradually
developed shortness of breath during exercise, cough and retrosternal
pain, which prompted for investigation of lung and heart function.
156 Nanopathology
Plain X-ray chest film showed signs of pulmonary fibrosis. A

subsequent CT scan confirmed that fibrosis and showed the presence of
several micro-nodules. All other diagnostic tests were normal.
Sarcoidosis was suspected and a bronchoscopy was performed,
during which a biopsy of the bronchial mucosa and bronchoalveolar
lavage were carried out.
Optical microscope observation of the biopsy revealed chronic
inflammation of the bronchial mucosa, with edema and eosinophilic
granulocytes.
Bronchoalveolar lavage showed an increase in total cell number and
CD4/CD8 lymphocytes ratio. A diagnosis of sarcoidosis was established
and a steroid therapy was started.
An investigation was performed in order to determine whether the
disease could be traced to an occupational environment agent.
The activity of dental surgeons is associated with several possible
hazardous exposures (such as X-ray, dental material components, blood-
borne pathogens and so on). Among these, aerosols of biological material
and inorganic substances are specifically responsible for airborne
exposure.
The instruments used during dental surgical procedures may produce
intense heat (electrocautery, laser) and such procedures usually generate
fumes containing biological material (even partially unburnt).
High-speed, air-driven dental handpieces, ultrasonic scalers, the
polishing of composite and ceramic restorations and the use of the
milling cutter on metallic prostheses disperse a large amount of aerosol
and spatter including fine particulate matter.
During the polishing and whitening of natural teeth, chemical
compounds (composed by particles of Sodium di-carbonate and
tricalcium phosphate) are sprayed on the tooth surface detaching part of
its enamel and dispersing particles in the environment, thus exposing
both patient and operator to risk of inhalation.
For example, a standard hygiene procedure involves the use of
prophylactic material and ultrasonic and/or manual instrumentation. The
prophylaxis phase can be substituted by an artificial bicarbonate aerosol
and that was what our subject had been doing. Artificial cleaning
aerosols are formed by a Mini-Clean device (Castellini, Bologna, Italy)
with air pressure set to 6 to 7 atmospheres and water flow to 1

atmosphere. Each individual run lasts 20-30 minutes for each patient.
It has been reported that 95% of the particles measure less than 5
micrometers and are mainly concentrated within 2 meters of the patient
where they can be easily inhaled by dental clinicians.
For this reason, the dentist always protected himself with a face mask
[3M ESPE 1942 F.B. (1800 NL) resistant molded face] whose level of
efficiency was 90-92%.
To test our hypothesis that sarcoidosis could be in this case related to
a possible environmental agent, a further examination of biopsy
specimens was performed. So, sections of the lung biopsy were sent to us
to be analyzed.
The label on the product used by our subject declared that the powder
contained particles of Sodium carbonate (Fig. 4.17) and tri-Calcium
phosphate (Fig. 4.18).
Fig. 4.17 Sodium-carbonate particle of and its EDS spectrum (marker 20 µm).
158 Nanopathology
Fig. 4.18 Tri-Calcium phosphate particles and their spectrum (marker 20 µm).
Fig. 4.19 Silica particles: a component non declared by the Manufacturer (marker
50 µm).
Fig. 4.19 shows other silicatic particles found in the polishing

material. Those particles were not declared in the product label.
Considering the patient’s long exposure to dust, a hypothesis was
advanced that the particulate matter could originate from the wear of
materials used during the abrasion and polishing operations.
It was hypothesized that the material used for dental cleaning
procedures could have a chemical composition similar to that of the
debris found. A sample was observed under ESEM and EDS analyzed.
One of the spectra obtained proved similar to that of the debris found in
the lung. (Fig. 4.19).
In the case we described, the diagnosis of sarcoidosis was issued
according to standard criteria (ATS 1999).
Additional information about the possible origin of the disease was
provided by ESEM with EDS of biopsy specimens (Fig. 4.20 and Fig.
4.21), which demonstrated that the material found inside the sarcoidotic
granulomas was identical to that found in the powder the dental surgeon
had used in large quantities for several years. More Calcium-phopshatic
particles were found in the bioptic specimens. Fig. 4.22 shows spherical
particles composed by Calcium-phosphate with Sodium and Magnesium
ranging from 0.5 to 2.5 µm. The prolonged inhalation of soluble tri-
Calcium-phosphate and Sodium-carbonate pollution provided a large
supply of Calcium, Phosphorus and Sodium ions. So, a logical
correlation can be established between the inhaled pollution and the
formation of those Calcium-phosphate entities.
Fig. 4.20 Particular of the lung biopsy (marker 200 µm).

160 Nanopathology
Fig. 4.21 Silica particle found in the lung biopsy (marker 20 µm).
Fig. 4.22 Spherules of Calcium-phosphate (marker 10 µm).
Such result cannot be considered a conclusive proof, but it does

however induce a strong suspicion that the disease could have been
initiated by the material used by the surgeon.
Chapter 5
War and Nanoparticles

___________
fter the 1st Gulf War many soldiers (mainly American, British and
$ French) became ill. After the Balkan War also Italian and in our
direct knowledge also Spanish and French soldiers, in the course
or soon after their peace-keeping missions, developed strange collections
of diseases, called later with the name of the site where that condition
seemed to have had its origin.
Then, after the 2nd Gulf War, other soldiers became ill.
Medicine can neither explain the collective, simultaneous presence of
all those symptoms nor cure them.
Also the epidemiological studies carried out on that problem are not
conclusive, also because they involve a relatively small number of cases,
in a limited time.
A new vision is now presented with some specific pieces of evidence
that clarify the impact of the new war technologies with human life.
It is a well-known fact that every kind of combustion generates
particles, and explosions can rightfully be classified as combustions.
Since the invention of gunpowder, probably in the middle of the
thirteenth century, explosions have been increasingly used in warfare and
have produced as a consequence clouds of dust, but it is only relatively
recently that blasting bombs have started to be widely used.
To our knowledge, the first connection between explosion and micro
and nanoparticles dates back to the late Seventies of the last century,
when the Air Force Laboratory, Armament Development and Test
Center, of Eglin Air Base, Florida, issued a report about the results of a
few experiments carried out on depleted-Uranium weapons which
included the Air Force GAU-8, the Army XM 774 and M735E1 and the
PHALANX gun systems (Technical Report, 1978).
161
162 Nanopathology
Depleted Uranium (DU) is a by-product of the enriching process of

natural Uranium meant to be used in nuclear reactors, when the fissile
and more radioactive isotopes are removed. Another, though less
common, source of DU is reprocessed spent reactor fuel.
DU is not so radioactive as natural Uranium and definitely less than
enriched Uranium (the specific activity [the activity in becquerels (Bq)
per unit mass i.e. one radioactive decay takes place per second] of DU is
about 15 Bq per mg compared with 24.5 Bq per mg for natural uranium),
but some not negligible radioactivity remains, and, in any case, DU is
toxic. Once natural Uranium has been deprived of much of its precious
235 isotope or enriched Uranium is not usable any more, DU must be
disposed of, and doing that is rather expensive. So, whoever faces that
problem is more than willing to rid himself of that metal in a quite
inexpensive way.
In the late 1940s, the US and the then existing USSR started their
respective nuclear programs and, in a short time, began to store huge
quantities of DU in stockpiles, in the hope that future technologies would
allow to further extract the 235 isotope, but the expectations of both
armies were disappointed.
In the 1970s, the USSR developed a particular armor plating for their
tanks that US ammunitions could not pierce, so the Americans began to
test materials dense and penetrating enough, and found that the best
compromise between efficiency and cost was DU, of which there was an
enormous availability.
Uranium, be it natural, enriched or depleted, is an excellent penetrator
and, in addition to that, is pyrophoric, i.e. gets oxidized and ignites
spontaneously upon impact with a solid target in the presence of Oxygen,
and the temperature that combustion reaches exceeds 3,000°C.
The study mentioned above and completed between October 1977
and October 1978 at the Ford Farm Firing Range, Aberdeen Proving
Ground, MD, placed a particular emphasis on size (just out of curiosity,
they describe the formation of ultrafine particles less than 0.1 µm in
diameter as an “unexpected phenomenon”), structure and stability of the
particles resulting from the impact against multiple-armor-plate targets of
penetrators consisting of 3.5 kg DU containing 0.75% Titanium by
weight.
War and Nanoparticles 163
The airborne particles were collected on a double-stick cellophane

tape which was cut in small portions, and those portions were placed on
Carbon-coated Aluminum stubs, coated with 100%-Gold and observed
under SEM.
The soil samples were sieved and treated in order to select and
concentrate particles in the respirable range and observed as described
above.
The elemental composition of the particles observed down to the size
of 0.5 µm in diameter was identified with an X-Ray energy spectrometer.
As could be expected, a large number of particles in a broad range of
sizes (< 50 µm - > 1 µm) resulted from the explosions, and most of them
were either spherical or ellipsoidal in shape, thus showing that the heat
generated was enough to melt Uranium and its oxides. On page 6, the
document reads: “Fragments produced are ignited spontaneously by a
combination of shock and friction heating at impact. Combustion of
fragments in air is exothermic and self-sustaining. Flash temperature
reached during impact of depleted Uranium penetrators with armor plate
have been shown to fall in the range of 5500 to 5600°F (…) Test results
show further a nearly constant (3037 to 3093°C) impact flash
temperature over the entire range of impact velocities from 4010 to 5560
feet per second. Such temperatures (…) are sufficient to initiate
combustion of the numerous particles produced.” And further on: “
Examination of numerous particles revealed several distinct
morphologies. The great majority of airborne particles exhibited a rugose
or convoluted structure which frequently appeared, at surface level, to
consist of large, interconnecting concave plates (…). Each plate, formed
independently from the solidification of molten material, was observed to
radiate from a common origin or focal point upon the surface. At or near
the junction of adjacent plates considerable overlapping and fusion
occurred, resulting in distinct but irregularly delineated boundary edges.
Higher magnification revealed numerous imperfections in the overall
crystalline structure along the surface, thereby serving to further
subdivide each major plate. (…) some rough particles exhibited a more
uniform surface morphology, devoid of major plate divisions. The
convoluted surface, although similar to that previously described,
consisted of deeper, more numerous folds and extensive dimpling.
164 Nanopathology
Fissures and pore-like invaginations were frequently observed and

progressively developed into deep fractures, presumably as a
consequence of thermal expansion or through collisions with other
objects. These fractures, which traversed irregular courses along the
convoluted folds, were the eventual cause of extensive particle breakup
(…). Internal morphology was frequently revealed by examination of
fractured particles, particularly those in which large portions had become
detached. Both solid and hollow particles were observed, the latter
clearly demonstrated by the presence of hemispherical fragments (…).
Although wall thickness varied greatly, the inner surface morphology of
hollow particles consistently resembled that of the outer surface. (…)
Prior to weathering, the surfaces of most airborne particles were covered
to a varying extent by immense numbers of nearly spherical, ultrafine
particles less than 0.1 µm in diameter (…) Identity of these particulates
as pure or alloyed Uranium was confirmed by X-ray analysis.
“An example of the uniform dispersal of ultrafine particulates on the
surface of a depleted Uranium/iron particle is demonstrated (…) Any
further accumulation, however, generally resulted in extensive
coagulation and hence the formation of billowing aggregates (…).
“Although generally found in association with larger particles, the
ultrafine particulates were also detected in the free state directly upon the
surface of the collective tape. At low magnification these particulates
appeared as large concentric masses often reaching several hundred
micrometers in diameter (…). At greatly increased magnification (…),
these masses were revealed as consisting of vast numbers of small
aggregates and long angular chains. The ultrafine spherical particles
comprising these aggregates measured approximately 0.01 to 0.1 µm in
diameter. (…)
“Morphology of particles removed from soil samples was quite unlike
that of their airborne counterparts. Far greater numbers of irregularly
shaped fragments were present, presumably the result of interaction and
fusion with sand and other materials within the soil (…). Spherical
particles, although quite numerous, generally lacked the convoluted
surface morphology so apparent in airborne samples. Their surfaces were
consistently smoother and frequently speckled with knobby blebs (…).
Some of these blebs were clearly continuous with the surrounding

surface whereas others appeared nearly detached.
“The relative fragility of these Uranium particles was clearly evident
following brief exposure at the laboratory to ultrasound (…). Although
sonification lasted no longer than 15 seconds, particles showed extensive
fracturing and in many instances compete disintegration.
“(…)
“The elemental composition of individual particles was qualitatively
determined by energy dispersive X-ray spectroscopy. Depleted Uranium
particles frequently contained iron, aluminum, silicon, magnesium,
potassium, titanium, and tungsten as a result of contamination during
impaction and settling.”
Unfortunately, a document of such importance was never published
to the scientific community and is still very hard, if not impossible at all,
to come by, in spite of what is declared in the preface: “This report has
been reviewed by the Information Office (OI) and is releasable to the
National Technical Information Service (NTIS). At NTIS, it will be
available to the general public, including foreign nations.”
It was only in the 1990s, starting from the first Gulf War, that DU
weapons were largely and regularly employed in warfare, and it was
soon apparent that both soldiers and civilians involved in that conflict
showed unexpected health problems whose nature was unclear. The
collection of symptoms they suffered from was called “Gulf Syndrome”
since they did not fit in any already classified pathology and its origin
was attributed to various causes, from the radioactivity and toxicity of
Uranium to the improper use of vaccines and other drugs, but that was
done without any convincing scientific evidence. Much of it was due to
an emotional wave caused by the fact that some people, especially
journalists, had discovered that the Allied Forces had used bombs
containing DU, and Uranium, with all that that name recalls, looked an
excellent scapegoat and also the spectre of the diseases induced by the
radioactivity of the A-bomb resurfaced.
Uranium is dangerous if it is inhaled, ingested or its debris are
incorporated in biological tissue. It is chemically toxic and its
radioactivity can induce lethal effects in biological tissues. The effects of
the radiations emitted by Uranium can be deterministic or stochastic.
166 Nanopathology
That means that in the former case, if a threshold dose is reached, the
probability to contract a medullar aplasia or cutaneous lesions or sterility
is equal to 1; in the latter case, a malignant disease originates in a
fortuitous way without a critical dose having been administered.
Not much later, another undeclared war broke out, that time in what
remained of Yugoslavia, and DU weapons were used there as well. Like
in Iraq, also there soldiers and civilians showed symptoms of disease,
mainly, but far from exclusively, of oncologic nature, and those
symptoms were classified under the common expression of “Balkan
Syndrome”. A few mistakes were made in the epidemiologic studies that
were implemented in order to try and quantify the actual extent of what
someone described as a disaster and others as something of no
importance when non existing at all, and that delayed and then slackened
the scientific investigations. In any case, it was a matter of fact that the
collection of symptoms was not consistent with any already known
pathology and that alone was enough to stimulate our curiosity.
Stress may be blamed for disorders like fatigue, shortness of breath,
headache, sleep disturbance, forgetfulness and impaired concentration,
but cancer, diseases of the genitourinary system (GC Gray et al., 1996),
diseases of the blood and the haematopoietic organs, and some forms of
mental disorders can probably be ascribed to other causes. But also now,
long after the end of the war, final reports are delivered from the
Departments of the Veterans Affairs and new suspicions of associations
with known diseases, (but of unknown origin) are rising; among others,
for instance, with the Lou Gehrig’s disease (web ref. 1, 2001), and
unexpected deaths (web ref. 2, 2002)
Studies on cohorts of soldiers who had served in different wars had
already shown the existence of previously unknown collections of
symptoms related to the particular conflict they had taken part in.
Already in the XIX century, after the US civil war, the so-called Da
Costa Syndrome was described (JM Da Costa, 1871). Then, after the
first World War, the so-called effort Syndrome was observed, while
psyconeurosis was reported as a consequence of the participation in the
second World War. Anxiety, neurosis, panic disorders, mitral-valve
prolapse and chronic fatigue Syndrome were seen in soldiers employed
in the Korean conflict, and a post-Vietnam Syndrome was described in
veterans from the long war fought in Vietnam, but in that conflict an
important contribution was most probably given by chemically toxic
agent as Agent Orange, i.e. dioxin.
The Syndrome suffered by the soldiers who were engaged in the first
Gulf War included fatigue, headache, muscle and joint pain, diarrhoea,
frequent fevers, skin rashes, shortness of breath, chest pain, sleep
disturbance, irritability and depression, but also, in some cases, increased
birth defects among the veterans’ children. In the UK that condition was
called SSIDC, by that acronym meaning Symptoms and Signs of Ill-
Defined Conditions.
The symptoms suffered by the veterans from the Balkans were
somewhat different from those of the ones who took part in the Gulf
War, but, in that latter case, some veterans had died in a very short time.
Then, between the end of the 1990s and the beginning of the new
century, the claims that radioactivity was to be blamed grew louder and
louder. To verify the truthfulness of that claim a specific Committee
from the U.N. Environment Protection Agency checked the radioactivity
in ex-Yugoslavia on January 2001. After 340 samplings in 11 sites, only
a few of them resulted to have a high level of radioactivity apparently
linked to DU ammunitions (web ref. 3).
Though not overwhelming, evidence that DU ammunitions was not
only used (something NATO had then already admitted) but a certain
radioactivity was still present was there. Circular red lines are still
present in the ground in many areas, delimiting an area around a DU
bullet non exploded entrapped in the soil. Nevertheless, a few Italian
soldiers who were not deployed in those zones reported some of the
symptoms listed above, so the probability that radioactivity could be the
cause of the diseases grew much weaker.
Another group of people identified the cause of the Syndrome in the
toxicity of multiple vaccinations that part of the soldiers were subjected
to in a short period of time, but also for this claim there was no scientific
evidence strong enough, though that factor may not be ruled out.
The multifarious and often unexplained symptoms shown by a
comparatively high number of veterans of the Desert Storm Conflict
induced many researchers to call it a Syndrome, commonly referred to as
the Gulf War’s. As a matter of fact, the collection of symptoms was not
168 Nanopathology
consistent with any already known pathology. Also the delay in showing
up and the duration of the symptoms was something hard to explain still
today.
The studies on soldiers who had served in different wars previously
mentioned had already shown symptoms related to that particular
conflict:
1 - after the US civil war: DaCosta Syndrome
2 - I World War: so-called Effort Syndrome
3 - II World War: Psyconeurosis
4 - Korean conflict: anxiety, neurosis, panic disorders, mitral valve
prolapse and chronic fatigue Syndrome
5 - Vietnam war: post-Vietnam Syndrome (In the course of that war,
an important contribution was given by chemically toxic agent as Agent
Orange [dioxin])
6 - Gulf War Syndrome: fatigue, headache, muscle and joint pain,
diarrhoea, frequent fevers, skin rashes, shortness of breath, chest pain,
sleep disturbance, irritability and depression, but also (in some cases)
increased birth defects among the veterans’ children. In the UK that
condition was called SSIDC, i.e. Symptoms and Signs of Ill-Defined
Conditions.
There is a similarity of symptoms as fatigue, headache, shortness of
breath, forgetfulness, sleep disturbance, impaired concentration, but they
can found also in adult population under psychological stress. It is
singular the symptom of diarrhoea.
The variety of the pathologies and their intensity, that in some cases
led to death, seem to have different aetiologies. That is the conclusion of
many researchers even if many veterans have expressed concern that
their unexplained illnesses may result from their experience in the war.
Also after the end of the war in what was Yugoslavia, the soldiers
engaged there denounced a collection of symptoms that has been called
the Balkans Syndrome.
The symptoms were different from those of the Gulf War, but, in this
case, some veterans died in a very short period. After the first casualties,
somebody tried to explain why young and healthy (before the war)
soldiers died and to find a possible correlation among pathologies like
the various forms of cancer and their presence in the war theatre.
Some people, especially journalists, discovered that the allied forces

used bombs containing Depleted Uranium, and Uranium looked an
excellent scapegoat.
Uranium is a high density, metallic element that, in its natural
form, consists of three radioisotopes: 238U, 235U, 234U.(2). It decays
spontaneously emitting alpha, beta and gamma radiations. This
peculiarity was sufficient to suspect that some soldiers were highly
exposed to its radioactivity due to the enormous quantity of DU
bombs used in the conflict and developed cancers and others diseases.
The correlation radiation = cancer was already been demonstrated
in people who lived in Nagasaki and Hiroshima during the second
World war.
Uranium is dangerous principally (Grandolfo et al., 2003) if it is
inhaled, ingested or debris are incorporated in biological tissue. It is
chemically toxic and its radioactivity can induce lethal effect in the
biological tissues.
The rumours of weapons containing radioactive materials dropped
in the Balkans were verified by a specific Committee from the
United Nations for the Environmental Protection Agency on January
2001. After 340 samplings in 11 sites, only three sites resulted to have a
high level of radioactivity apparently linked to the DU ammunition,
for instance Hadzici, a small village, 60 km far from Sarajevo.
(web ref. 3).
So there is evidence that DU ammunition were used and a certain
radioactivity is still present, but some ill Italian soldiers were not
deployed in those zones, so the radioactivity cannot be the cause of the
diseases.
170 Nanopathology
Another cause was identified in the toxicity of multiple vaccinations

in a short period of time, but also for this cause there is no scientific
evidence.
The Italian Commission (web ref. 4) considered 44 cases reported in
Tab. 5.1 and the first conclusion was that there was no relationship with
their permanence in the war theatre. One year later the Commission
elaborated again the data and reached a different conclusion. They found
that there was a slightly higher incidence of Hodgkin’s lymphoma and
Acute Lymphatic Leukaemia among the military people deployed in ex-
Yugoslavia or in close contact with DU ammunition in fire grounds.
Specific observations were carried out in soldiers exposed to high-
temperature combustion processes involving the blast of Depleted
Uranium weapons. Our original aim was to find Uranium nanoparticles
in the bioptic samples we received, but what we found instead of
Uranium was metal dust coming from target and bomb volatilized by the
heat generated by Uranium and re-condensed under nanoparticle form.
Inhaled and ingested nanoparticles can negotiate either the alveolar and
the digestive system walls, migrate to the blood and be carried virtually
to any organ. Sperm samples belonging to dead soldiers who were active
in war territories were referred to our laboratory and we developed a
novel technique to be able to test them. In all cases, nanoparticles were
present, while nothing was found in similar samples coming from
healthy subjects we used as reference. [See chapters 3 and 5]
Having found dust dispersed in the sperm, we checked if malformed
human and animal feti contained that particular form of pollution.
Tab. 5.1 Cases examined by the Mandelli Commission in Italy.
Pathology N. of Cases
Hodgkin’s lymphoma 12
non-Hodgkin’s lymphoma 8
Acute lymphatic leukaemia 2
Thyroid Carcinoma 3
Colon or rectum cancer 4
Larynx and Pharynx cancer 1+1
Lung and Bronchus cancer 1+1
Kidney cancer 1
Stomach cancer 1
In her capacity of coordinator of the European project on

nanopathology and consultant of the 2nd Italian Governmental
Commission, Dr Gatti had the opportunity to check a small cohort of 52
soldiers (for a total of 59 samples, because from autopsy we had more
samples for 1 subject) with our technique. The aim was to look for
Depleted Uranium inside the pathological tissues of the sick veterans.
Some of the Italian soldiers had already died before getting a clear
diagnosis, while others had developed symptoms typical of well-known
diseases (for instance, Parkinson’s or Alzheimer’s). All declared to suffer
from a common symptom: chronic fatigue, a symptom usually
complained for also by the New York fire fighters who took part in the
rescue operations of 9/11. So we analyzed autoptic specimens, biopsies,
surgical samples or “fresh” specimens as blood or sperm. (See Tab. 5.2)
In eleven of them, colon cancer had been diagnosed and the rest
suffered from non-Hodgkin’s lymphoma. In all the small specimens
inorganic particulate could be detected and its chemistry verified. The
samples were very small, and there is the possibility that further debris
included Uranium could be entrapped in other organs and tissues, but
what we found is meaningful of a certain contamination and it responds
to a logic behavior of the bombing: it creates a new “special”
environmental pollution.
172 Nanopathology
Tab. 5.2 Type of samples analyzed.
Sample No. of Cases

Bone marrow 12
Liver 6
Lymph node 8
Thyroid 3
Bowel 3
Larynx and Pharynx 2
Lung 5
Kidney 3
Spleen 3
Stomach 2
Bladder 1
Sperm 6
Gonads 2
Brain 1
Skin 2
Total 59
In none of the cases we checked could we find trace of DU debris,

either micro- or nano-sized. In many of them, instead, we detected the
presence of nanoparticles, often showing very strange elemental
chemical compositions, different from case to case. The explanation to
that phenomenon is relatively easy: As described above, when a bomb
goes off, the explosion induces a great quantity of heat and involves a
large number and variety of objects and materials. Those objects and
materials volatilize and most of the molecules they are made of break
into chemical elements that recombine, often under the form of an alloy,
as soon as the atmosphere cools down. Since all that matter is cast far
away from the spot where the explosion had taken place, it finds an
environment cold enough to allow it to recombine very quickly, in a
matter of seconds. Because of the variety of the materials that take part in
the explosion and the fact that the soldiers we checked had been
deployed in a comparatively vast territory, the alloys we found were
rather different from each other.
That finding agrees with the deployment of the soldiers. They did not
go in the same bombed places and at the same time.
Like in many other, not war-related, cases we could study,

nanoparticles were scattered in many different tissues, including red cells
(an excellent carrier), brain and gonads.
Fig. 5.1 Image of red cells spread on a plastic sheet, of a contaminated spouse, observed
under ESEM, with a white particle of Antimony –Cobalt attached (marker 5 µm).
The first of the cases we checked was particularly interesting and was
related to the 1st Gulf War (web ref. 5).
A Canadian officer with a history of amateur marathon runner and,
also because of that, enjoying an excellent physical condition, was
repatriated after a six-month stint on a wheel-chair, despite the fact that
he had never been injured, but for three days he had been highly exposed
to bombing pollution.
The symptoms since February 1991 were loss of motor control,
chronic fatigue, respiratory difficulties, chest pain, insomnia, short-term
memory loss, testicle pain, aching bones, diarrhea, and depression.
After his return he survived for 8 years. During that time he
developed symptoms specific of different pathologies, included
Parkinson’s and Alzheimer’s diseases.
174 Nanopathology
Among the unusual aspects of this Syndrome there was the change of
his eye colour, originally brown, but after 2 years turned to greyish, then
to intense blue. Then, just before his death, they turned to grey again. His
widow did not find any doctor who could give an explanation to this
strange phenomenon.
Besides his difficulty in walking, he had problems with his sexual
life, as, after sexual intercourse, his wife felt a smart burning sensation in
her vagina, which started to bleed and, in a short time, developed sores
which did not respond to any therapy. That particular condition, later on
reported by other partners of veterans when we interviewed them, was
called “Burning Semen Disease” (see also Chapter 3).
Blaming a particular chemical composition for that disease does not
seem possible, but the cases we had the chance to study so far are still
few and deeper investigations on a sufficient number of cases are
necessary to be able to say something conclusive.
The analyses were carried out on 4 different types of samples: the
lung (Fig. 5.2), the spleen (Fig. 5.3), the liver (Fig. 5.4) and the kidney
(Fig. 5.5).
In the liver we found Cobalt and Antimony nanoparticles. The
presence of Cobalt debris attracted our attention and pushed out our
fantasy, beyond the border of the present knowledge.
Cobalt is blue (Fig. 5.4). The patient’s eye became blue and all his
body contained Cobalt nanoparticles. All his body contained metallic,
toxic nanoparticles, finely disseminated. To explain the colour eye
mystery we can remind the symptoms of a genetic disease called
Wilson’s Syndrome. In this case the lack of a protein does not allow to
metabolize Copper that precipitates, mainly in the liver. One of the
symptoms considered for the diagnosis is a yellow circle around the iris.
Copper is brown-yellow in colour. Can this comparison be sustainable?
For now there is no possibility of epidemiological studies, since that was
a unique case. The particles we found in the liver were present in the
blood circulation and reached also the eye microcirculation. We found
also nanoparticles of Mercury-Selenium in the kidney. A Canadian
doctor performed analyses on his urine and he found traces of Uranium,
but in the samples we analyzed the amount of toxic particles was
sufficient to explain the symptoms.
This case was deeply analyzed since we had sections from autoptic
samples of different organs, and we could understand that the
dissemination is total, and some of the particles accordingly to their
chemistry can be trapped selectively in some organs. The disease and the
symptoms are related to this wide contamination.
Fig. 5.2 ESEM image of a solitary spherical nanoparticle of Bismuth-Chlorine-Sodium

found in the lung (marker 20 µm).
Fig. 5.3 ESEM image of a post mortem section with spherical nanoparticles of
Antimony-Chlorine found in the spleen (marker 20 µm).
176 Nanopathology
Fig. 5.4 ESEM image of a post-mortem sample of the liver. Nanoparticles of Cobalt were
found (marker 10 µm).
Fig. 5.5 ESEM image of a post-mortem sample of the kidneys. Nanoparticles of toxic
Mercury-Selenium were found (marker 20 µm).
Especially in the kidney the particles were found as singlets

(Fig. 5.5). This fine dissemination is peculiar. Until now it is impossible
to reproduce that phenomenon in an in-vitro simulation test for the
capacity of clustering of engineered nanoparticles.
We analyzed also soldiers not directly involved in the war, but who
served in peace-keeping missions in bombed sites.
The Italian soldiers’ samples we analyzed were provided by the
patients themselves or by their families. We present a selection of some
results. It is possible that if we had the opportunity to analyze other
tissues we could have found more or less debris with further
compositions, since a selective capture from an organ of specific

chemistry is possible. The soldiers we analyzed served in the Balkans in
different periods and in different areas, so the difference in compositions
of the pollution is a logic consequence. Many slept and/or worked in
destroyed buildings, other visited polluted areas or were busy destroying
weapons. It is a common procedure, after the destruction of enemy
ammunition collected inside a hole dug in the ground, to verify directly
on the explosion site, on the smoking ashes, if everything had been
actually destroyed. That is a dangerous procedure: the risk exists of
inhaling the pollution generated by the explosion. That habit was kept
also during the 2nd Gulf War. That means that soldiers slept close to
primitive open-air incinerators, no more than holes in the ground where
they dispose of daily wastes of food, but also many other kinds of
unwanted materials, including captured weapons.
The first 3 cases (Fig. 5.6, Fig. 5.7, and Fig. 5.8) show images of
pollution inside biopsies of lungs and bone marrow of Italian soldiers.
Fig. 5.6 Image of micro and nanoparticles of compounds containing Iron-Silicon-

Phosphorus-Aluminum-Sulphur-Chlorine-Sodium-Potassium (marker 20 µm).
178 Nanopathology
Fig. 5.7 Image of nanoparticles of stainless steel inside the bone marrow (marker 50 µm).
Fig. 5.8 Image of spherical particles of Iron-Manganese-Chromium-Silicon found in a

bone marrow biopsy (marker 5 µm).
Figs. 5.7 and 5.8 show different particles found in a bone marrow
sample. They are metallic debris both irregularly and, in most cases,
round-shaped; their origin was from combustive processes at a
temperature higher than 900°C.
Fig. 5.9 Image of a cluster of nanoparticles of Iron found inside the liver (marker 10 µm).
Fig. 5.10 Image of particles of a compound of Zirconium-Sulphur-Chlorine-Calcium

found in the liver (marker 10 µm).
Figs. 5.9 and 5.10 are related to the same soldier who died after
having contracted a lymphoma. His organs were contaminated mainly by
Iron-based particles, found mainly embedded in the bone marrow. Also
Zirconium-based particles where found in the spleen biopsies after the
organ was removed.
180 Nanopathology
Fig. 5.11 Image of a debris of Antimony found in the lung (marker 20 µm).
Fig. 5.12 Image of a particle of Tungsten found in the lung (marker 20 µm).
Figs. 5.11 and 5.12 are related to the case of a soldier who had a lung
neoformation removed. Solitary particles of Antimony and Tungsten
were found inside. Those are recognized as chemically toxic materials.
Fig. 5.13 Image of a cluster of Cobalt particles inside a bladder cancerous tissue (marker
10 µm).
Fig. 5.14 Image of round-shaped particles inside a bladder cancer (marker 2 µm).
Figs. 5.13 and 5.14 show particles found in the same sample of
bladder cancer. They belong to a mine clearer who had served for 25
years in the Army and had been present at very numerous explosions for
the elimination of ammunition. For that reason, he was exposed to the
pollution created by that kind of combustion. Inside his pathological
tissue we found nanoscaled particles of Cobalt, Tungsten, stainless steel,
etc.
182 Nanopathology
Fig. 5.15 Image of a solitary particle of stainless steel surrounded by red cells (marker
50 µm).
The picture above (Fig. 5.15) is relative to a rhino-laryngeal

neoformation in a soldier affected by non-Hodgkin’s lymphoma.
Fig. 5.16 Image of a wide dissemination of debris of stainless steel inside the bone
marrow in an (marker 50 µm).
The sample of Fig. 5.16 is related to an acute mieloid leukaemia a

veteran from the Balkan War suffered from.
Fig. 5.17 Iron-Zinc micro and nanodebris inside a calcific tissue found in the liver
(marker 20 µm).
The soldier whose liver biopsy was observed in Fig. 5.17 suffered
from pancytopenia. The tissue contained a wide variety of particles with
different compositions. Calcium was detected together with Iron, Zinc,
Magnesium, Silicon, Phosphorus and Sulphur.
Fig. 5.18 Image of an Antimony-Cobalt debris found inside the sperm of a soldier who
served in a fire ground (marker 10 µm).
Hodgkin’s lymphoma was the disease diagnosed to the soldier,

now died, whose sperm we examined. It contained 800-micron sized
Antimony-Cobalt particles (Fig. 5.18). The patient was never deployed in
the Balkans but served in a firing ground in Sardinia.
184 Nanopathology
Fig. 5.19 Image of Iron micro and nanoparticles disseminated in a brain tissue affected by
glioblastoma (marker 20 µm).
Fig. 5.19 shows Iron-Silicon-based nanoparticles finely disseminated

in a soldier’s brain affected by glioblastoma. The presence of Silicon
witnesses that the particles are of exogenous origin.
Fig. 5.20 Image of a solitary debris of Lead-Chlorine-Antimony found in a

ganglioneuroma (marker 20 µm).
Fig. 5.20 and Fig. 5.21 present two solitary particles disseminated in a
ganglioneuroma (Antimony-Lead-Chlorine) and in an adrenal gland
(Mercury-Silver). The elements composing those particles are recognized
as toxic.
Fig. 5.21 Image of a Mercury-Silver-Calcium-Phosphorus singlet particle found in an

adrenal gland. (marker 20 µm).
Fig. 5.22 Image of two Strontium-Sulphur-Calcium-Sodium debris found in an adrenal

gland (marker 20 µm).
186 Nanopathology
Fig. 5.23 Image of three Copper-Iodine-Sulphur particles found in an adrenal gland.

(marker 20 µm).
Figs. 5.21, 5.22 and 5.23 show three different types of particles
found in a removed adrenal gland. They contain chemically very oddly
composed toxic materials. The pathology that affected the patient who
served in a peace-keeping mission in the Balkans was adrenal adenoma.
Fig. 5.24 Image of a 5-micron sized particle of a silicate containing also radioactive
materials like Uranium-Thorium-Yttrium-Cerium-Neodimium (marker 10 µm).
Fig. 5.25 Image of micron- and nano-sized (white arrow) debris of a silicatic
composition. The debris on the left is composed of nanoparticles found in a bone marrow
sample (marker 2 µm).
Figs. 5.24 and 5.25 show mostly nanometric debris found in the same
bone marrow biopsy that contains mainly ceramic materials, some of
them radioactive (Fig. 5.24). The patient had served in Bosnia and
developed a thyroid carcinoma with lymph-nodal metastases, and acute
leukemia. In that biopsy, we found ceramic debris containing, among
other materials, radioactive elements like Uranium, Thorium and
Yttrium. We do not know when and where the soldier was exposed, but
these materials may come from the pollution of buildings, perhaps
containing tile with radioactive glazes. After the buildings were
destroyed, a part of them was aerosolized.
Among the samples we received, there were some coming from a few
French soldiers who had taken part in the peace-keeping missions in the
Balkans.
One of those soldiers had served for six months as a clerk in an office
based in Kosovo and had developed a Hodgkin’s lymphoma because of
which he eventually died. In the biopsies we got, we found nanoparticles
composed of alloys containing Silver and Gold, among other elements
like Iron, Chromium, etc. (Fig. 5.26).
No such alloy is to be found in any metallurgy manual or, at least to
our knowledge, in any commercially available product. As a matter of
fact, Gold is very often used for its anti-corrosive properties and is never
188 Nanopathology
mixed with stainless steel, which is what the rest of the alloy looked like.
So, as happens in many cases, we guessed that that strange composition
could be the result of a combustion and the consequent interaction of the
elements occasionally present. Explosions are obviously very common in
war theatres, and explosion is a form of combustion. But the subject we
were dealing with had never been involved in battlefield actions or was
close to actual war theatres.
Fig. 5.26 Image of the cluster of nano-debris composed of Silver-Gold-Chlorine-Iodine-

Chromium,-Iron- Gold,-Magnesium-Silicon-Sulphur (marker 5 µm).
In the course of our investigations, though, we were informed of a

kind of exposure our subject might have undergone.
He had slept and eaten for two months in a truck, and that truck had
been previously used in Iraq, from where it had been moved to Kosovo
without having been cleaned.
Of course, we did not have any chance to get samples of the dust
contained inside the truck, but it is only natural that such a condition can
rouse suspicion about the possibility that that dust may have polluted the
people who had stayed there for a relatively long time. Long enough, in
any case, to get contaminated. The sleeping bags could have been
particularly interesting to check, as dust trapped there would have kept
the soldiers closely exposed to particles for many hours a day.
The small size of the particles found and their composition made their
origin clear: combustion at high temperature. But we have no answer to
the question about their origin, Balkans or Iraq.
One of the questions we wondered about was: Can only Depleted

Uranium and Tungsten bombs create nanoparticles, or is the temperature
developed by collections of more conventional weapons enough to make
them?
Thus, we carried out a simple experiment with the Italian
Governmental Commission Dr Gatti worked with.
Since the Italian soldiers stationed in Baghdad confiscated all kinds
of small armaments and bombs, put them in large holes and had them
blow, we had gravimetric passive sensors set at distances of 100, 200 and
300 meters from the hole and exposed their surface just a few seconds
before the explosion. Then let them exposed to the environment for 30
minutes and analyzed them to check for the presence of nanopollution.
In all samples we found a great quantity of nanosized dust and, in
addition to that, we detected there particles with the same, unusual
composition we had already found in the bioptic specimens of soldiers
we had had the possibility to observe. That was a simple demonstration
that very fine pollution can be generated also by “low-technology”
weapons. The next images show the pollution created during the firing of
a pool of ammunitions.
Fig. 5.27 and Fig. 5.28 show particles with their chemical
composition found 100 m away from the explosion, while Fig. 5.29 and
Fig. 5.30 represent the dust collected 200 m away.
190 Nanopathology
Fig. 5.27 Image of dust created 100 m away from the firing of a pool of weapons in
Baghdad. The big 20 micron-sized particle is a cluster of nanoparticles (marker 5 µm).
Fig. 5.28 Image of a 700 nm-sized round-shaped particle of Iron-Aluminium-Silicon

found at 100 m from the explosion (marker 10 µm).
Fig. 5.29 Low-magnification image of the dust collected at 200 m from the explosion
(marker 200 µm).
Fig. 5.30 Image of a spherical Zirconium-Sulphur-Sodium particle found 200 m away

from the site of explosion (marker 20 µm).
192 Nanopathology
Fig. 5.31 Image of another group of particles found 200 m away from the explosion site.
(marker 20 µm).
Fig. 5.31 shows debris collected on the passive sensor 200 m away
from the explosion site.
Micro and nanoparticles were found in all the three points of measure
(100, 200, 300 m). Some showed strange chemical compositions like that
of Fig. 5.31 and Fig. 5.32. The first spectrum shows the presence of Lead
together with Chromium, Iron, Silicon, Aluminium, Calcium. That
composition is the result of an occasional melting. This alloy is not be
found in any metallurgy handbook and is the occasional result of
elements present when high-temperature combustions occur. The particle
of Fig. 5.32 shows a round-shaped morphology formed by other smaller
“balls” composed of Iron, Silicon, Sulphur, Aluminium, Sodium and
Calcium.
Fig. 5.32 Image of a composite round-shaped particle composed of Iron, Silicon, Sulphur,
Aluminium, Sodium and Calcium from the explosion occurred 200 m away (marker
10 µm).
We had the opportunity to analyze the oil used in the engine of trucks
that had worked for 2 years in Iraq. That oil contained dust of different
compositions (Fig. 5.33 and Fig. 5.34). Some of the particles detected
presented the same morphologies and chemical compositions as we
found in some pathological tissues. Zirconium, one of the rather unusual
elements detected in the oil, was also present in the specimens of two
soldiers.
Fig. 5.33 Image of a round-shaped, hollow particle of Iron found inside the engine oil
used in the truck in Baghdad (marker 5 µm).
194 Nanopathology
Fig. 5.34 Image of a Zirconium bar found inside the engine oil used in the trucks in
Baghdad (marker 10 µm).
So, we observed something that probably did not need any particular
demonstration, but just common sense, i.e. that explosions cause a new
pollution contaminating the environment. For how long? Are those
particles somehow degradable? In our opinion, seen their composition,
we can hardly believe that they, or, at least, the vast majority of them,
can “disappear”, even after a long time. And that persistence applies both
to the environment and to the organism.
Once they have been released in the atmosphere, they float in the air
and can cover long distances, carried by the wind. When they eventually
fall to the ground - and rain and snow can be the occasional carriers of
particles - a gust of wind is enough to raise them again and have the
circle restart.
As to the organism, we have demonstrated that particles, no matter
how they are introduced, can escape most physiological barriers and
settle virtually everywhere. Their presence in the lymph nodes, where
some physiological waste collects, looks almost natural, but their being
in the bone marrow, in the brain and in the sperm is somewhat less
expected.
Now, having observed all that evidence, one may ask whether their
presence in the pathological tissues is actually related to the pathology?
Is their chemistry a crucial, or even just an important, factor? And do all
subjects polluted in the same way develop identical symptoms and

disease?
We try and answer those questions checking places where explosions
occur on a regular basis, i.e. in firing grounds.
Italy has a few of them, but one in particular attracted our attention
because of the rumors and the actual complaints we heard from the
people living in its vicinity.
The firing ground of Salto di Quirra is located in the middle of the
east coast of Sardinia, close to a village called Quirra, along a beach of
“uncontaminated” beauty. There are no industries worth mentioning in a
radius of many tens of kilometers and traffic is scarce. The village has no
more than 150 inhabitants.
The firing ground specializes in testing missile aiming systems (web
ref. 6) and is used not only by different Italian services but also by
private companies producing weapons and foreign armies.
A few soldiers serving there had died in the same short period of time
of Hodgkin’s disease. Among those soldiers we could analyze the case of
a 22-year old boy whose Hodgkin’s lymphoma had not been diagnosed
in time and he had died.
In a supra-clavicular neoformation we found particles of Antimony
and Iron-Copper, while in his sperm we found round-shaped particles,
about 1-micron-large particles of Antimony-Cobalt and Iron-Chromium-
Sulphur. Their spherical shape and chemical composition witnessed a
high-temperature combustive origin.
Some of the inhabitants of the village had developed forms of cancer
and the activities of the firing ground had been blamed by them as the
responsible of the diseases, and in another village of the inland called
Escalaplano, twelve malformed babies were born, but that was limited to
the period between 1988 and 1990.
By request of the authority responsible for public health of that
district, we analyzed pathological samples (see Tab.5.3) from ten of the
inhabitants of the village of Quirra, located closer to the firing ground
than Escalaplano.
196 Nanopathology
Tab. 5.3 List of the pathologies and the examined samples.
Pathology Examined sample

Melanoma Skin
Non-Hodking’s lymphoma Lymph node
Chronic Myeloid leukemia Aspiration biopsy
Breast cancer Breast
Adenocarcinoma Uterus
Thyroid carcinoma Lymph node
Lobular carcinoma Breast
In order to verify if this pollution was related to the activities of the

firing ground, a specific investigation was carried out inside the area with
the permission of the military authority and under their supervision.
At the time of the investigations, no activities were planned, so, we
collected on adhesive disks the dust already present in that environment,
touching the areas behind the missile launching pads on the ground and
on the special metallic devices that adsorb the fire.
We collected samples also from a pool under a platform where
special, big engines for rockets were tested (Fig. 5.35). The water of the
pool served to absorb the heat of the exhausts.
Fig. 5.35 Particle found attached to the wall of the pool. It is composed of Lead-
Phosphorus-Iron-Copper-Silicon-Aluminium-Sodium-Calcium (marker 10 µm).
The samples collected there contained dust with different

composition, but one attracted our attention since it contained Lead. A
specific research verified that the engine used a solid red propellant that
contains Lead. The sample was not analyzed in our laboratory, but by
RIS (Crime Scene Investigative Institute) of Parma (Italy) which gave us
the result shown in Fig. 5.36.
:
Fig. 5.36 Image of a particle of solid fuel with its spectrum. It is composed of Carbon-
Oxygen-Lead and Aluminium (marker 20 µm).
Lead was important for our study, since some patients showed its
presence in their pathological tissues.
198 Nanopathology
5.1 Civilians living around a firing ground
In some cases the patients living around the firing ground and who
developed a disease presented metallic particles, some of them Lead-
based. That presence is puzzling for three reasons: Firstly, the village
where those people live is very small, with little traffic car and without
any industry. Secondly, this contaminant is nanosized and its production
needs a very high temperature. Finally, Lead is bound to elements like
Titanium, Chromium, Iron, Silicon, Aluminium and such an alloy does
not exist in any metallurgical handbook and can only be the result of
an occasional meeting of all those elements, which is typical of a
combustion where some presences are accidental.
For these reasons, we think that there is a certain correlation
among some activities of the firing ground (rocket engines or weapon
elimination), the chemistry of the pollution generated and some cases of
the patients examined. The activities of launching missiles are controlled
and the population is alerted when a launch is planned, but the bench
tests of the rocket engines might be considered a non risky activity, so,
no precaution is taken.
There are also other specific activities we had no opportunity to
investigate, that probably were responsible for the creation of the
Antimony–Cobalt pollution we found in the samples of the soldiers.
Fig. 5.1.1 Image of a cluster of nanoparticles found in a lymph node affected from non-
Hodgkin’s lymphoma (marker 10 µm).
In the technical report of 1978, Dr. Joe A. Framer declares that

“Depleted Uranium as the most desirable candidate is based primarily
upon on its 1-high density 2- pyrophoricity 3-metallurgcalproperty, 4-
availability and 5- relatively low cost.” But after the test, he says that
“additional research is necessary to better define the physical and
chemical nature of the fragmentary depleted Uranium generated as a
consequence of its military use as weaponry. Foremost attention should
be focused on its potential for dissemination within the environment and
entry into biological systems, particularly that of man”
In addition to that, “Such specific knowledge is required for
determination of potential safety hazards associated with the respiration
and deposition of Depleted Uranium aerosol within the lungs. Particles in
the 0.1 – to 0.5 micron size range are of great concern because of high
efficiency for deposition in the lungs. This range has been defined
appropriately as the respirable size range.”
Back in 1978, nobody talked about nanoparticles and
nanopathologies, but the concept is clear.
The concern for the environment and humans caused by the effects of
explosions was already expressed almost three decades ago, but nothing
was made to control or to avoid the problem. Now we extend the
concept: All the particulate matter generated by high-temperature
combustive processes is greatly aggressive to environment and human
and animal health.
5.2 A few reflections
We are all aware that the new technologies we invented or what we call,
without much insight, progress have always a more or less important
negative side.
In this case we see that bombing creates a new environmental
pollution, sometimes nanoscaled, but always with a chemical
composition that can be very dangerous for human and animal life.
The new wars create very small bullets, impossible to see and for now
undetectable by soldiers since they have no sensors for them. But
nanotechnologies can help find the right solution to develop equipment
200 Nanopathology
sensitive enough. Through them we can develop sensors for

nanoparticles, filters to capture them, masks to avoid their inhalation,
personal protections for contamination. But for now no technology is
available or even thinkable for an environmental remediation. When the
environment (ground, vegetables, water) is contaminated, it is practically
impossible to clean it.
The effects of such contamination will be paid not only by the
populations living there, where that contamination had actually occurred,
but by the neighbouring countries as well, perhaps not directly involved
in the war but, unfortunately, pollution does not stop at frontier stations.
Wind carries particles far from their origin, but so does water through
torrents and rivers that flow into the sea. Fish eat particles along with
food and are contaminated, thus becoming a contamination carrier to
animals they are food for.
Our planet is too small to stand all that. So, before starting another
war, governments ought to be aware of the hardly soluble problems they
are triggering, last but not least among them that of the money that will
be spent in what is going to be the unsuccessful attempt to reclaim a so
polluted territory.
5.3 Bibliography
Da Costa, J. M. (1871) On irritable heart: a Clinical study of a form of functional cardiac

disorders and its consequences Am. J. Med. Sc., 61, 17-52
Grandolfo, M., Mele, A., Ferrigno, L., Nuccetelli, C., Risica, S and Tosti, M. E. (2003).
Uranio impoverito e linfomi di Hodgkin nei soldati italiani in Bosnia e Kossovo:
una possibile associazione?, Not Ist Super Sanità; 16 (7/8)
Gray, G. C., Coate, B. D., Anderson, C. M., Kang, H. K., Berg, S. W., Wignall, F. S.,
Knoke, J. D. and Barrett-Connor E. (1996). The postwar hospitalization experience
of U.S. veterans of the Persian Gulf War, N Engl J Med. 1996 Nov
14;335(20):1505-1513.
Technical report of the Air Force Armament Laboratory – Armament development and
test Center, Eglin Air Force Base, Florida, USA, From October 1977 to October
1978, Project n° 06CD0101
Web Ref. 1: www.gulfwarvets.com/gehrig.htm,
Web Ref. 2: www.gulfwarvets.com/study.htm, The Washington Post Company 4 March
2002, by Suzanne Gamboa
Web Ref. 3: http://balkans.unep.ch

Web Ref. 4: www.uranioimpoverito.it/mandelli.htm
Web Ref. 5 (http://www.umrc.ney/riordon.aspx)
Web Ref. 6: www.perdasdefogu.it/MILITARE.htm
Chapter 6
Nanoparticles in the Environment and

Working Places
___________
6.1 Introduction
3
ollution taken in a broad sense depends both on the source and
the environment. Restricting the subject to particles generated at
high temperature, their composition depends mostly on what is
being burnt and, in part, on the physical conditions at which that
combustion takes place. The environment where particulate matter is
issued affects in a determinant way its concentration and scattering. It is
only natural that, in general, a windy place will suffer less from
concentrated dust pollution than a place where the calm of the wind is a
predominant condition and, even more so, an indoor environment will be
very critical in comparison with the open air, because of stagnation. Also
atmospheric pressure and inversion phenomena influence diffusion in the
environment of that dust.
The traceability method we use takes all those factors into
consideration. A few examples are described in this chapter where
traceability has been of the utmost importance to understand some
phenomena otherwise difficult to make out. In some of those examples, a
chemical element has been taken as the marker and the guiding thread
that led us to the solution of some cases.
There are instances where the technical attempt of solving a problem
linked to pollution leads involuntarily to the creation of different,
unexpected problems that, in some circumstances, can be more serious
than the original ones. Filters used in Diesel cars (FAP, see Chap. 6.5) to
capture the particulate matter produced by the combustion of gasoil are
203
204 Nanopathology
just one possible example and are briefly taken into consideration as the
producers of a novel form of pollution. Another example is that of
incinerators, meant to solve the problem of getting rid of wastes but
causing the formation of pollutants far more dangerous than what was to
be eliminated.
Distribution of particulate pollutant is of the utmost importance and
may be expressed in terms of mass, volume, surface area or number.
Already back in 1998, toxicological studies on rat models showed that
nanoparticles are much more toxic than coarser ones made of the same
components (K. Donaldson et al., 1998) and even earlier, in 1995,
ultrafine particles were demonstrated to be able to penetrate in the
pulmonary interstitium, thus causing cardiovascular diseases (A. Seaton
et al., 1995), something that larger particles are unable to do.
As already explained in Chapter 1.1, nanoparticles are dominant in
the particle number count and are significant in contributing to surface
area, but mean little in terms of mass.
Tab. 6.1 shows this concept.
Tab. 6.1 List of the ratio size-number and surface of nanoparticles.
Diameter (in µm) Relative number Relative surface

10 1 1
1 103 102
0.1 106 104
0.01 109 106
Nevertheless, it is a well-known fact that size is a critical parameter

when the point of view is that of human health, as the smaller the
particles, the easier they can penetrate the organism and the deeper they
can reach. It is another fact that the larger the surface area of those
particles, the more chemically reactive they become. And, finally, count
is also determinant, since it represents the number of impacts the
organism may undergo. So, evaluating particulate pollution in terms of
weight alone has very little significance, at least when health is the issue.
Condensation of non volatile materials from supersaturated hot
combustion gases is a process responsible for the formation of airborne
nanoparticles, the size of about 1-2 nm. Those particles can grow very
Nanoparticles in the Environment and Working Places 205
rapidly in size by condensation of low-volatility materials. It must be

remembered that inorganic, microsized particles formed at high
temperature are often fragile and can break into much smaller, often
nanosized, particles. In that case, the number of actual particles is
increased and so is their surface area, while their mass stays constant.
The following examples are presenting places and situation were
nanopollution is already unintentionally released in the environment.
This knowledge can be very useful since, being aware of the problem, we
can study precautionary measures, preventive procedures and counter-
measures.
6.2 Chimneysweeps: a historical case
“Ramazini [sic, correct spelling is Ramazzini] has written a book De

Morbis Artificium. The colic of Poictou is a well known distemper; and
every body is acquainted with the disorders to which painters, plumbers,
glaziers, and the workers in white lead, are liable: but there is a disease
as peculiar to a certain set of people, which has not, at least to my
knowledge, been publicly noticed; I mean the chimney-sweepers’ cancer.
It is a disease which always makes its first attack on, and its first
appearance in, the inferior part of the scrotum; where it produces a
superficial, painful, ragged, ill-looking sore, with hard and rising edges:
the trade call it the soot-wart. I never saw it under the age of puberty,
which is, I suppose, one reason why it is generally taken, both by patient
and surgeon, for venereal; and being treated with mercurials, is thereby
soon and much exasperated. In no great length of time, it pervades the
skin, dartos, and membranes of the scrotum, nod seizes the testicle,
which it enlarges, hardens, and renders truly and thoroughly
distempered; from whence it makes its way up the spermatic process into
the abdomen, most frequently indurating and spoiling the inguinal
glands: when arrived within the abdomen, it affects some of the viscera,
and then very soon becomes painfully destructive.”
That is how Dr Percivall Pott (1819) [The Chirurgical Works of
Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital with his
last corrections. To which are added, a short account of the life of the
206 Nanopathology
Author, a Method of curing the Hydrocele by injection, and occasional

notes and observations by Sir James Earle, F. R. S. Surgeon
Extraordinary to the King, &C. First American, from the last London
edition. In two volumes. Vol. I. Philadelphia: Published by James
Webster, No. 24, South Eight Street. William Brown, Printer. 1819] in
one of his numerous texts on this subject, starts to describe a sort of
cancer that had affected chimneysweeps for centuries and for which no
therapy had been found.
The chimneys built in North Europe especially in the XVIII century
had a very small diameter and followed long and tortuous courses. So,
people with a very slight build and a nimble body, and scantily dressed to
reduce their bulk, were needed to clean them and, in fact, children were
usually employed to do that job. And, already in their early adolescence,
a non negligible number of those young workers contracted scrotum
cancer.
What Dr Pott did was to perform a surgical ablation of the tissues
involved, but, unfortunately, to no avail, thus inflicting further sufferings
and pain to his patients.
Already in 1775 Pott had realized that soot was responsible to the
disease, in this way admitting that some “poison” could induce a cancer.
The practical problem was solved in a very pragmatic way by the
Danish government that obliged all chimneysweeps to take daily baths,
thus avoiding a too long exposure of the skin (children worked all but
naked) to the soot that smeared their body. Great Britain followed later
with a law that forbade the employment of children under eight years of
age and imposed weekly baths (G. Paganetto. 2001)
The same type of disease was observed at the first half of the 20th
century in mule spinners in cotton textile factories, where they were
exposed to shale oil, which was used as a lubricant for cotton spindles.
Between 1920 and 1943, 824 out of the 1,303 skin carcinomas identified
in textile-industry workers affected the scrotum (S.A. Henry 1943).
In both cases, for the onset of the pathology polycyclic aromatic
hydrocarbons (PAHs), particularly some 3-, 4- and 5-ring PAHs such as
benz(a)pyrene and dibenz(a,h)anthracene were blamed.
Two things are particularly noteworthy: An environmental agent was
recognized as cancerogenic, and effective laws were passed on the sole
basis of medical observations without waiting for a complete theoretical

explanation of the physical, chemical and biological mechanisms
involved, the three of them then completely missing.
6.3 Welding metals
Welding is used to join different metal parts by having them coalesce,

and that is done by melting them. Many different energy sources can be
used to carry out that process, and, among them are gas flame, electric
arc, laser light, electron beam, friction, and ultrasound. All those
methods generate particles that scatter in the environment. Their shape is
generally spherical and their size depends mostly on the temperature at
which the procedure has occurred (Fig. 6.3.1). Most of those particles are
composed by a thin, crystalline skin, are hollow inside and are very
fragile.
Fig. 6.3.1 Image of a hollow particle obtained by laser welding (marker 20 µm).
208 Nanopathology
Even a sight collision can break them up and the fragments thus
originated become particles in their turn. Those particles are obviously
irregular in shape and smaller then the objects they come from, growing
that way more numerous and more aggressive. Many of them are likely
to be nanometric in size, so a single microparticle may give birth to a
high number of nanoparticles (Fig. 6.3.2 and Fig. 6.3.3).
It is only natural that welding is more critical from the point of view
of the health impact it may have, when it happens in a closed space, and
among the effects the dust produced can cause is its being trapped in the
welder’s clothes. Besides rather uncommon circumstances, those clothes
are eventually handled, often to be washed, by the welder’s wife or, in
any case, by somebody who is not the welder himself and it is not rare
the case when that person, never directly involved in the workman’s job,
falls ill with the same occupational diseases that typically affect welders.
Fig. 6.3.2 Image of an agglomeration of spherical particle of Iron-Copper-Zinc-Silicon-

Sulphur-Calcium found on the ground around a welding site (marker 5 µm).
Fig. 6.3.3 Image of spherical Zinc debris collected by air filtration during a flame
welding (marker 10 µm).
Some of the particle have a submicronic size and, if inhaled, can pass
through the lung barrier and enter the blood circulation, causing a
chemico-physical pollution. Also the microsized particles that have been
blocked in the alveoli because of their large size and the weakness of the
aggregation bounds can degrade along the time. Or, better, the material
that induced their aggregation degrades, releasing all the nanosized
debris, that, being so small, have a high probability to pass through the
lung barrier. That means that pollution can have a delayed effect,
practically impossible to predict.
In the case of these workers, it is mandatory that they use an
effectively protective mask when at work; that they wear overalls that are
never brought back home but are washed at the working place; that they
do not eat in the working place; that they change their cloth when they
eat.
Many working places are potential sources of pollution. So, a
dedicated risk analysis is necessary, along with the proper education of
every single worker. In most cases, the protective measures necessary
are simple enough and even inexpensive.
210 Nanopathology
6.4 Toner
Nowadays printer toner is widely used in offices and even in household

environments. No particular attention is usually reserved to it, but when
there are many printers gathered together in the same room, black dust is
often visible on the furniture and on the machines themselves. We had
the chance of checking a few toners made by different producers and
saw that the Carbon granules contain metal micro and nanoparticulate
whose composition depends on the effect the manufacturer wants
to obtain. Fig. 6.4.1, Fig. 6.4.2 and Fig. 6.4.3 show Carbon-based
spherules containing respectively Iron, Titanium-Tin-Antimony-Silicon
and Titanium-Strontium-Sulphur nanoparticles.
Fig. 6.4.1 Image of dust of toner (Olivetti) composed of tiny Iron particles contained in
larger Carbon-Oxygen particles (marker 5 µm).
Toner can be dangerous particularly when the cartridge is being

changed, and especially so if that operation is made in room without any
proper air aspiration. Repeated exposure, like the one undergone by
technicians assigned to service of photocopiers, may be particularly risky
because of accumulation of this kind of dust.
Fig. 6.4.2 Small Carbon-Oxygen particles contaminated by Titanium-Tin-Antimony-

Silicon (marker 5 µm).
Fig. 6.4.3 Coloured toner containing Titanium-Strontium-Sulphur nanoparticles (marker

10 µm).
212 Nanopathology
6.5 The particulate active filter or FAP
Because of a more favourable taxation as to gasoline engines come into

force decades ago, Diesel cars have grown very popular in Europe. One
of their drawbacks, though, is the great quantity of particles they give
out, a major problem in European cities where pollution has already gone
beyond any sustainable limit, and PM10 may not exceed by law a certain
mass per cubic metre (now, in 2007, the limit is 40 µg/m3), a mass limit
that will become smaller and smaller along with the approval of new
rules.
In the year 2000, a filter invented the year before, meant to capture
that dust, started to equip some French cars. Owing to its capability of
having particles coalesce, Cerium oxide contained in a small tank is let in
the filter, so that the dust becomes coarse enough to be detained. In order
to avoid obstruction and keep the filter clean, every 300-400 kilometres
the system breaks up what has been captured and emits gases and
ultrafine, mostly Carbon-based, particles from the tailpipe. Those
particles contain inorganic nanodust and are much finer than the
relatively big ones produced originally by the combustion in the engine.
That “cleaning” operation occurs when the car is running at high enough
speed for a comparatively long time, so, presumably, not in urban areas,
but nevertheless, from the nanopathological point of view, the inorganic
dust let free is far more dangerous than the one that comes out of cars
that are not equipped with that device and, because of its size, it can stay
suspended in the atmosphere a long time and travel very long distances.
Another problem is Cerium, an element belonging to lanthanides
whose toxicity has not been exhaustively investigated but which is not
negligible, which is excreted from the organism rather slowly when in
ionic form (but, as explained all over this book, particles behave in a
different way) and which we start finding under particulate form in urban
surroundings. Fig. 6.5.1 shows a Cerium particle detected in the
environmental pollution of Mantua, an Italian 48,000-inhabitants town.
In the case of the Cerium particles of Fig. 6.5.2, they contain also smaller
Platinum particulate that may be used as a marker indicating a particular
source of pollution.
Fig. 6.5.1 Image of a Cerium particle found on a trafficked street in Mantua (Italy)
(marker 2 µm).
Fig. 6.5.2 Platinum nanoparticles contained in a larger Cerium particle (marker 500 nm).
Such pollution was unknown until a few years ago. In fact, we

performed a similar analysis on a trafficked street of two North-Italy
streets, but the dust we found, in spite of its complexity, did not contain
any Cerium.
214 Nanopathology
One of the elements we found was Silver in particle form, maybe

coming from the coating of low-cost catalytic mufflers (Fig. 6.5.3).
During the periodic ignition and killing procedures, the muffler is
subjected to heating and cooling cycles that cause the detachment of the
Silver coating and the expulsion of the debris in the environment.
Environmental pollution can depend on current technology and,
therefore, can change along with progress. An example of that can be
that of Sapporo (Japan) where snow tyres with steel spikes where largely
used. The wear debris of those spikes was related to an increase of lung
cancer incidence and, out of prudence, those particular tyres were
forbidden. Epidemiologic studies will tell if that measure was indeed
effective.
Fig. 6.5.3 Micro and nanoparticles of a Silver compound released by cars in a trafficked
street (marker 20 µm).
From Lavoisier’s principle’s point of view (the total mass of an

isolated system is unchanged by interaction of its parts), it is obvious that
no inorganic pollutants is disposed of, particles are made smaller and, as
a consequence, more penetrating and, in addition to that, a new element,
in this case Cerium, is introduced into the mass of pollutants.
What is surprising is that this aspect of the problem has never been
taken into consideration and cars equipped with those devices are
allowed to circulate when car traffic is forbidden by municipal
authorities because pollution limits have been exceeded.
If more sensible, more adherent to science, regulations were enforced,

devices like that would have never been proposed by industry.
6.6 The environment around a foundry
In January 2006 we had the chance to check the territory around a huge
steelworks at the outskirts of Udine, a medium-sized town in North Italy
(95,000 inhabitants).
As it often happens in similar circumstances, the people leaving in a
radius of a few kilometres from the plant had complained for years about
the poor quality of the air, the ubiquitous presence of dust that made it
impossible to hang out the washing and forced them to keep their
windows constantly closed, and the fact that fruit trees and greens
withered and died. An unusually high rate of cancer was also unofficially
reported, but, not unlikely what happens in most similar cases, no
epidemiologic evidence (or, to be sure, serious research) existed to
support that widespread persuasion.
Fig. 6.6.1 Image of micro and nanoparticles of an Iron-based compound found in the
vicinity of a foundry (marker 5 µm).
Fig. 6.6.1 and Fig. 6.6.2 show some examples of round-shaped

pollutants found in the environment.
216 Nanopathology
Fig. 6.6.2 Image of an Iron-Manganese particle (marker 10 µm).
The environment around the factory was full of the material worked
inside and dispersed in the environment through the chimneys. Dross
was also heaped outside, that contributed to disperse particles in the
environment (Fig. 6.6.3).
Fig. 6.6.3 Images of the two morphologies of the Iron-based particles released in the
environment. The first is hollow but filled with smaller spherical debris, the second one is
an aggregation of very small debris on an Iron sphere (marker 5 µm).
So, the municipality of Udine together with those of three

neighbouring small towns interested by the same problem asked us to
carry out an investigation limited to ten specimens sampled inside and in
the vicinity of the plant.
Unfortunately, the person in charge of the factory did not allow us to
enter the premises where metals were actually worked nor to take
samples from the dross that were heaped outside the buildings. So, we
had to be content with what we could gather in the air, on some
vegetables and a few standing objects within about 800 meters from the
area of the plant.
What we found was a huge quantity of micro- and nanoparticles
ranging from 0.1 to 20 µm, in many cases spherical in shape and with an
elemental chemical composition mainly based on Iron, but containing
also other elements.
Failing a way prescribed by law to characterize particles and in
presence of an undeniable pollution, car traffic is generally the source
blamed.
In this particular case of Udine, the spherical shape prevalent in the
particles, the fact that those spheres were hollow, the nanometric size of
most of them and the unusually high content of Iron, in that case
considered as a marker, made it evident that the origin could not be
traffic exhausts. Non spherical, nanometric particulate matter was also
present, and also in that case, due to its size and composition where Iron
was again predominant, blaming cars did not look reasonable. Besides, in
the case in point, traffic was not particularly heavy and no other industry,
big enough to justify such a degree of pollution, stood in the territory. It
must also be considered how fragile hollow particles are and how the
breaking fragments inevitably released in the environment are nothing
else but irregularly shaped nanoparticles.
That the particles detected were formed at high temperature is further
proved by their composition. Just as an example, in one sample we saw
the presence of 12 elements (C, Fe, O, Zn, Si, Ca, Mn, S, Al, Mg, Cl,
Cr), while in another one, the elements were as many as 14 (Fe, O, Ca,
Si, C, Al, Mn, Mg, Cr, P, K, S, Ti, Zn). In both cases- but similar
conditions are present in the majority of the particles examined - those
elements are alloyed and the alloy is formed in a fortuitous way,
218 Nanopathology
according to the elements present in the crucible that vaporize and

subsequently combine as soon as they go up and find a cooler
environment, a fact, this, shared by all similar cases like, for example,
those that pass in the course of explosions.
A comparatively small number of particles were also detected, whose
origin was most probably different from the former. Calcium, Potassium
and Magnesium are typically found in the soil and it is only natural that
some particles thus composed are airborne and are detected among the
others. And it is as likely that traffic, however scarce, is responsible for a
fraction of the pollution present in that territory, though with a particulate
which is not the same as the one generated by the steelworks.
All samples were taken in places where dust had the time to
accumulate, with the exception of a sample, which was obtained sucking
air for 45 minutes in a place 200 meters away from the factory. Also in
that case a great quantity of Iron-based micro and nanoparticles were
filtered, most of which were spherical and clustered like those harvested
elsewhere.
Fig. 6.6.4 shows a fully contaminated acarus found on a leaf picked
about one kilometer away from a foundry. Also its mouth (Fig. 6.6.5), a
sort of open door to its organism, is surrounded by the metallic debris.
Unfortunately, only the first part of the investigation could take place.
What should have followed to come to a meaningful and much more
useful conclusion, i.e. a research on biopsies of people suffering from
diseases likely to be due to particulate pollution, could not be carried out
because of pressures that convinced the municipalities not to pursue the
matter further.
Fig. 6.6.4 Image of an acarus found on a leaf. It is widely contaminated by the Iron-based
particles (marker 500 µm).
Fig. 6.6.5 Image of the mouth of the acarus at higher magnification with the chemical
composition of the homogenous pollution (marker 100 µm).
220 Nanopathology
6.7 The environment around a power plant
The Italian river Po flows into the Adriatic Sea forming a delta, and that
delta was transformed into a regional park. Between 1970 and 1984, an
electric power station was built on a small island called Polesine
Camerini situated in those wetlands, and the combustible it used was
heavy oil at a yearly rate of 3 million tons.
The not many people living in that territory started soon to complain
about occasional oil fallouts and, in a relatively short time, about some
health problems that they ascribed to the pollution generated by the
power station.
Their suspicion was impossible to prove, as no epidemiological study
had ever been carried out and the population was too small to justify
such an undertaking. In addition to that, epidemiology is often tainted by
mistakes in what should be the population taken as a reference, as in
many cases that population is living in conditions that are not the ones
where contamination is absent.
In 2005, together with other experts in fields different from ours, we
started to investigate the problem on behalf of the cognizant criminal court.
Our idea was to cover the whole course the possible particulate
pollutants, if any were present, had followed.
We had, then, the possibility to check a certain number of samples
that were suspected to contain micro and nanoparticles with the obvious
aim of discovering if the power plant could be blamed for the pollution
of the park it was accused of. The samples had been collected years
before, when the plant was fully operational, since at the time of the trial
it was closed.
The first specimen we took into consideration was a cotton singlet
which had been washed and then hung to dry in the open. After a short
time, yellowish oily drops had fallen on that garment, leaving easily
visible spots.
Different varieties of oil had been used in the station, according to
their content of Sulphur, and we analyzed all of them according to our
method. Then, we checked the different ashes deriving from combustion.
Lichens are considered excellent biological indicators and we check
numerous samples of those vegetables. We also received dried salad
leaves picked in the territory, some of them grown outside and some
inside greenhouses. Those grown indoor did not carry particulate
pollutants, unlike those grown outside.
Another kind of specimens we checked were the particles captured by
the filters of the devices used by the local agency for the protection of the
environment.
The following images (Fig. 6.7.1) show the content of the oil
polluted by Lead, Iron-Sulphur, Barium-Sulphur-Strontium and Silicon-
Aluminium-Sulphur-Iron and some Vanadium-Sulphur compounds. Part
of them remains in the ashes, but part is released through the chimneys to
the environment, with a chemical composition that a combination of
these elements.
Fig. 6.7.1 Image of a drop of oily fuel. The oil contains Carbon-based material and also
different particulate matter, for each of which the relative spectrum is shown (marker 100 µm).
222 Nanopathology
After having been burnt, the elements contained in the oil may
recombine in particulate form. In addition to the spectra shown, small
quantities of elements such as Vanadium, Calcium and Bismuth were
also present and, as a consequence, were found in the ash (Fig. 6.7.2 and
Fig. 6.7.3).
Fig. 6.7.2 Image of the ash with its chemical composition. It contains mainly Barium
sulphates, Vanadium-Calcium-Iron-Nickel-Strontium-Magnesium-Silicon (marker 400 µm).
Fig. 6.7.3 Image of a particular of the ash. The particle is round-shaped and is composed
of Iron-Sulphur-Vanadium-Magnesium-Aluminium-Silicon (marker 20 µm).
The flying ash is particularly light and is easily carried by the wind.
For that reason, it can be found in the environment, including vegetables
like, for example, lichens (Fig. 6.7.4).
Fig. 6.7.4 Particulate pollution in dry lichens (marker 10 µm).
In the dry lichens we analyzed, we found a great number of particles.

Most of them were spherical, as is typical of a combustive origin. The
spectra of Fig. 6.7.4 shown two different types of particles: the former
contains mainly Sulphur and Iron with a ratio similar to that found in the
oil, along with other elements in a much smaller quantity. The latter
contains a mixture of many elements like, for instance, Silicon, Lead,
Titanium, Iron, Zinc, etc.
224 Nanopathology
Fig. 6.7.5 Round-shaped hollow debris made of Iron, Aluminium, etc. found in a lichen
(marker 5 µm).
Fig. 6.7.6 Sulphur-Iron-based nanoparticle cluster found in a lichen (marker 2 µm).
Fig. 6.7.5, Fig. 6.7.6 and Fig. 6.7.7 show single and composite round-
shaped particles found over the lichen surface. Though they have
different chemical compositions, they contain a combination of the same
elements present in the oil.
Fig. 6.7.7 Round-shape particle containing, among other elements, Vanadium (marker
10 µm).
The lichens examined contained particulate whose composition

recalls that of the elements found in the oil. The same elements have
been combined in a different way as a consequence of combustion. It is
important to remember that the power plant was placed in a regional
park, close to the sea and rather far from any other pollution source.
Besides lichens, we analyzed also some lettuce on which we found
Sulphur and Iron in the same ratio as in the oil and in the particles
detected on the lichens. It is only natural that the lettuce grown in
greenhouses, which we examined as well, was free from particulate
contamination (Fig. 6.7.8).
226 Nanopathology
Fig. 6.7.8 Spherical particles on a lettuce leaf, containing Sulphur-Iron and Sulphur-
Barium (marker 20 µm).
On the basis on the evidence described, along with that of other

experts, the criminal court condemned to damages the property of the
plant and sentenced its president and its manager to imprisonment
(suspended sentences).
On behalf of the criminal court, we checked about thirty cases of
cancers suspected to be related to the power-plant’s pollution. Not all the
specimens we received were particularly useful for our kind of
investigation, since they were not taken from the interface between
cancerous and healthy tissue. In the case of pathological lymph nodes,
particles are inside their structure and, for that reason, we did not meet
any problem. In ten of those samples, we found pollution compatible
with the combustion of the oil previously checked.
In Fig. 6.7.9 particles are shown with the same Iron-Sulphur ratio
detected in oil, lichens and lettuce.
Fig. 6.7.9 Debris detected in a supra-clavicular lymph-node in a patient living in the

power-plant’s area, suffering from Hodgkin’s lymphoma (marker 20 µm).
The same kind of Iron-Sulphur particles were found in dust fallen on

car bodies. That dust induced corrosion of the metal parts. The obvious
question is: If those particles are aggressive enough to corrode a car, can
they be accepted by the organism?
6.8 The case of a ship
A ship is a sort of maze made up of an unbelievable number of rooms.

Some of them are enormous, some are very small. Some received an
abundant supply of air directly from the atmosphere, some, and those are
the majority, are ventilated only through air which is forced there
through a complex network of pipes.
One of our investigations concerned a ship under construction and we
had thus the possibility to observe men at work, the particulate pollution
they produced and how those particles behaved in such an environment.
Samples were taken close to welders busy in a huge shed placed in
the dock yard. As could be expected, the vast majority of the particles
were made of Iron and were spherical. A very similar type of particles
we found in the air inside the ship, where workmen were doing the same
job as their colleagues in the shed, and those particles had been captured
by the bulk insulators and the varnish used to paint the walls of the
228 Nanopathology
rooms. Obviously, those particles may be slowly released in the scarcely

ventilated room and be a continuous source of pollution.
6.9 Incinerators
We would have much liked to study the pollution generated by

incinerators, but that proved all but impossible. The reason is not a
technical one, but is due to the huge economical interests involved in the
building and running of those systems used to dispose of urban and
industrial wastes, interests that involve powerful lobbies of builders,
businessmen and politicians that oppose any form of serious control.
Those lobbies describe incineration as a way to have waste disappear, but
such a claim is in jarring contrast with the law of conservation of matter,
that states that the mass of a closed system of substances will remain
constant, regardless of the processes acting inside the system, provided
no nuclear reactions take place. That means that matter may change
form, but cannot be created or destroyed, a notion that was already
familiar to Greek philosophers of the fifth century B.C. and was
experimentally demonstrated by Antoine Lavoisier in 1786.
It is hard to think that burning tons and tons of matter every day,
whatever that matter is made of, produces just steam, Carbon monoxide,
Nitrogen oxides and little more. Besides organic pollutants as dioxins,
furans, polychlorinated biphenyls (PCB), polycyclic organic matter
(POM) and a very long list of further pollutants, many of which probably
unknown, non existent in the original load but the result of chemical
transformations, mainly oxidations, and depending on what is being
burnt, incinerators process virtually all sorts of materials and many
components of those materials are of inorganic origin. Since chemical
elements cannot be transformed, at least in the working conditions of an
incinerator, into other elements and experience proves that combustion,
when occurring in the presence of inorganic materials, generates
inorganic particles, it is evident that those particles must be taken into
consideration when dealing with the fumes coming from waste
incineration. Those particles can either form directly where combustion
occurs or farther than there, in the latter circumstance beyond the
filtration system any incinerator is equipped with. In the former case, it

must be kept in mind that the majority (not in mass but in number) of the
particles generated are most probably nanometric in size and escape any
possibility of capture by the filters used, hardly mattering what filters are
made of. In the latter case, what passes through the filter has not yet
condensed into particulate and, for that reason, the filter is hardly
effective. In both cases, even in the event the filter has trapped all the
nanodust generated upstream, something that waits to be proved in an
indisputable way, what the fate of that material is remains dubious and
the subject is always dealt with in an ambiguous and far from exhaustive
way. As a matter of fact, that dust is somehow, sooner or later,
introduced into the environment. As an example, activated charcoal may
be cited. Because of the high surface area of its granules, activated
charcoal is sometimes used as a filter, but as soon as it grows no more
usable because it has already captured all it could, it is thrown into the
incinerator the way any waste is, thus introducing the dust it had trapped
into the cycle again.
In a way, incineration as a means to dispose of waste is a sort of naïve
conjuring trick. It is a fact that most of what is burnt is not visible any
more, but Lavoisier teaches us that nothing has actually disappeared, but
must have been transformed into something else. And that something
else is heavier than the rubbish we did not want to see. The reason for
that is that burning means oxidizing, and, in the case in point, oxidizing
means involving atmospheric Oxygen in the reaction, i.e. adding mass.
But more mass is being added, as, depending on the different techniques
used, chemicals like, for example, bicarbonate, lime and ammonia, are
employed for various reasons. Water is also generously used to cool parts
of the plant and that water is poured in streams at a higher than natural
temperature, thus influencing negatively the habitat of plants and
animals. It is only natural that also those substances contribute to
increase the mass that results from the combustion the original waste
undergoes. So, if we consider all the addenda, what comes out of the
process weights more or less twice as much as what we wanted to get
rid of.
Setting aside organic pollutants, in most cases much more aggressive
than the original rubbish, and setting also aside what are called secondary
230 Nanopathology
particles, i.e. those that form in the atmosphere from gaseous pollutants,
particularly Sulphur dioxide, Nitrogen oxides, ammonia, and volatile
organic compounds and that can be the carriers of other particulate,
incineration produces a wide variety of particles, presumably often in the
form of alloys whose composition is actually impossible to prognosticate
because of the almost infinite assortment of materials entered into
combustion. So, guessing what their toxicity will be is impossible. All
we can say is that thinking that dust is not formed by incineration is a
preposterous notion and one of the many dangers is that particles, even if
microsized, are very likely to break up into a higher quantity of
nanoparticulate.
We had the chance of analyzing some of the ash produced by an
incinerator and scattered on the ground for a radius of many hundred
metres and we also collected dust from vegetables grown in the vicinity
of one of such plants, but we were never allowed to carry out more
systematic investigations.
Biomass like dead trees, tree branches, yard clippings, left-over crops,
wood chips, bark and sawdust from lumber mills is used in some
circumstances as a fuel to produce energy. Apart from the generation of
pollutants like, for instance, dioxins because of the presence of Chlorine
in the wood, such process is responsible for the release in the atmosphere
of inorganic particulate, since inorganic molecules and metallic elements
are contained in vegetables.
In spite of the fact that vegetable biomass used for that purpose
should not contain chemical additives, in fact, in the vast majority of
cases, those vegetables contain pesticides and evident traces of chemical
fertilizers. In other, almost as numerous cases, the timber used is the
industrial production reject of furniture, frames or other wooden objects,
containing all the chemicals used to enhance the characteristics of those
materials, chemicals that end up, in chemically and physically changed
forms, often as more toxic substances, in the fumes given off by through
the chimneys.
In many circumstances, because actual biomass coming from
comparatively short distances is not enough to fuel this kind of plants,
they are gradually transformed into waste incinerators.
Also in this case, we have never been allowed to investigate.
6.10 Tobacco smoke
About 15 billion cigarettes are smoked every day in the world, but,
though the most popular, cigarettes are not the only system to inhale
burnt tobacco, and pipes, cigars and less common systems are currently
used.
Smoking tobacco is a concentrate of particulate atmospheric pollution
because its leaves are relatively large and dust falls on their surface. In
order to be suited for consumption, those leaves must be desiccated and
that procedure makes them loose an enormous fraction of their weight.
So, dust, being not volatile, concentrates on the final product.
Smokers inhale smoke on a voluntary basis, and that smoke,
produced at a temperature of about 800°C, contains more then 4,000
known pollutants, among which are Carbon monoxide, Nitrogen oxides,
Hydrogen cyanide, ammonia, and other toxic irritants such as acrolein
and formaldehyde. Inorganic particulate is more difficult to define
chemically as it depends on fortuitous, variable circumstances like the
place where the plant has grown and dried and what was actually present
in the atmosphere during those periods of time.
Everybody, particularly a non-smoker, is aware of the fact that
tobacco smoke does not interfere exclusively with smokers but reaches
also, though in a more diluted way, the respiratory system of whom is
unwilling to breath burnt tobacco. That way, cigarettes, cigars and pipes
become the producers of environmental pollution which is particularly
perceptible and aggressive in confined rooms, and that pollution contains
suspended particles.
The following pictures show the particles of what we found in some
of the cigarette tobacco we observed and their relative elemental
spectrum.
232 Nanopathology
Fig. 6.10.1 Biological morphology of a tobacco leaf with its spectrum (marker
1,000 µm).
The elemental composition shown in Fig. 6.10.1 is the one typical of

tobacco leaves. The elements shown (Potassium, Carbon, Oxygen,
Calcium, Chlorine, Magnesium, Phosphorus and Sulphur) in the
spectrum are homogeneous throughout the whole leaf and are to be
subtracted from the following spectra.
A few pictures of particulate pollution found in cigarettes from
Sarajevo, Baghdad and the US are shown below. Some of the particles
are nanosized, while others are microsized but equally interesting as they
contain typical war pollutants, some of which are radioactive. Those
cigarettes had been produced in 2003, but we do not know when that
tobacco had been grown.
Fig. 6.10.2, Fig. 6.10.3 and Fig. 6.10.4 show the pollution on the
tobacco leaf strips in cigarettes (Drina King Size and Aura Light) from
Sarajevo after the war, in 2000. Fig. 6.10.5 represents the war pollution
in a cigarette from Baghdad, (Sumer Star King Size) in 2004.
Fig. 6.10.2 Different debris found in a cigarette from Sarajevo (marker 100 µm).
The heavy metals present in the particles are notoriously toxic and
their penetration may be enhanced by the hot surface of burning tobacco.
Fig. 6.10.3 Drina King Size Cigarette from Sarajevo (after the Balkan war). The particles
found contain Uranium and Thorium (marker 20 µm).
234 Nanopathology
Fig. 6.10.4 Aura Light Cigarette from Sarajevo with very unusual particulate:
Lanthanum, Praseodymium, Neodymium, Cerium and Samarium are present in its
composition (marker 20 µm).
Fig. 6.10.5 Sumer Star King Size Cigarette from Baghdad with particles containing
Uranium and Thorium (marker 50 µm).
Fig. 6.10.6 Cigarette from the US (Marlboro) polluted by particles containing rare earths
like Lanthanum-Cerium-Neodymium (marker 10 µm).
Fig. 6.10.7 Cigarette from the US (Marlboro) polluted by particles containing rare earths
and Lead (marker 10 µm).
The Fig. 6.10.6, Fig. 6.10.7 and Fig. 6.10.8 show the environmental
pollution on an American tobacco leaf. During smoking this pollution is
burnt and inhaled.
236 Nanopathology
Fig. 6.10.8 Cigarette from the US (Marlboro) In this case, the particles polluting the
leaves contained Gold, among other inorganic pollutants.
Nowadays world is vastly polluted by anthropic activities and sooner

or later we will realize that we have no other choice left but face the
problem instead of clumsily try to hide it the way most politicians are
doing. A political action, common to all countries, is inescapable and the
sooner the better. But, beyond politics, the problem is also a
technological one, as, in many circumstances, no available technology
can clean where we have made a mess and something must be contrived.
Our work is meant to contribute to solve this problem, showing the
presence of a very particular, infinitesimally small but highly dangerous
pollution inside Man and animals in the deepest parts of their body, and
that presence is mainly caused by Man himself. As soon as this
contamination exceeds a certain threshold now impossible to tell, and do
it in a continuous and stable way, our species is doomed to extinction.
Let us call all that catastrophism, if we like it better, but let us get up and
do something.
6.11 Bibliography
Donaldson, K. and McNee, W. (1998). The mechanism of lung injury caused by PM10 In
Issues of environmental science and technology, Ed. R. E. Hester and R. M.
Harrison, no. 10, pp- 21-32. The Royal Society of Chemistry
Henry, S. A. (1943). Cancer of the Scrotum in Relation of Occupation”, 16, Oxford
University Press,
Paganetto, G. (2001). Percorsi storici di cancerogenesi chimica. Basell Poliolefine Italia.
Ferrara
Seaton, A., McNee, W., Donaldson, K. and Godden, D. (1995). Particulate air pollution
and acute health effects. Lancet 345, 176-178
The Chirurgical Works of Percivall Pott, F. R. S. Surgeon to ST. Bartholomew’s Hospital
with his last corrections. To which are added, a short account of the life of the
Author, a Method of curing the Hydrocele by injection, and occasional notes and
observations by Sir James Earle, F. R. S. Surgeon Extraordinary to the King, &C.
First American, from the last London edition. In two volumes. Vol. I. Philadelphia:
Published by James Webster, No. 24, South Eight Street. William Brown, Printer.
1819. (http://www.mindfully.org/Health/Chirurgical-Works-Percivall-Potts.htm.)
Chapter 7
Nanoparticles in Food, Cosmetics and

Other Products
___________
1
anotechnological items are already on the market and we interact
with them everyday even if we are not informed and we are
not aware. Label on the product rarely contains the definition
“nanotechnological” or the notice “contains nanoparticles” or something
of the kind, because industries are afraid of a negative perception people
may have and the a-priori rejection of a nanoproduct.
Singular is the case occurred in Germany involving “Magic Nano”, a
protective glass and bathroom sealant in the form of a spray, which
claimed to have antibacterial activity (web ref. 1).
Within a few days, almost 100 people were hospitalized for
pulmonary problems, some of them edema. The producer Kleinmann
GmbH, a multinational industry, assured that it did not contain
nanoparticles or other nano-things, as confirmed also by Rene Zimmer of
the Federal Institute for Risk Assessment (BfR) in Berlin (web ref. 2),
(web ref. 3), (web ref. 4), (web ref. 5) and that the toxicity was due to the
propellant. No matter what the origin of the problem was, the product
had to be withdrawn in a hurry. We do not know what the truth is, but
propellants are contained in many sprays and there is no information
about other similar cases. In any case, people rightly or wrongly,
perceived that “nano” is suspicious. And that event made also the
scientists ill at ease.
L’Oreal, the French cosmetics company, invented liposomes,
nanostructures capable of crossing the derma and penetrate deeply. They
are investing a lot in these technologies and the hope is that they are
putting money also in safety, because beauty is certainly something
239
240 Nanopathology
pleasant that may be rightfully pursued, but safety is far more important.
So, biocompatibility of the nanostructures they add (web ref. 6) and their
long-term reactions must be ascertained beyond any possibility of doubt.
Matter at nanoscale level can hide surprises.
For instance, Aluminium is stable in bulk shape, but explosive
at nano-levels. Carbon nanostructures are not soluble and not
biodegradable. So, taking advantage of that characteristics, they are used
for the construction of golf balls, tennis rackets and skis. From our point
of view, these items can be considered safe, since the nanomaterials used
are trapped in the solid structure and cannot be released.
“Nanotea” by a Chinese company will increase tenfold the amount of
Selenium adsorbed with green tea through nanocapsule engineered to
bypass the acid environment of stomach and dissolve in the bowel.
Canova Activa oil contains nanocapsule-delivered chemicals in
rapeseed cooking oil that will stop cholesterol entering the bloody
stream.
Slim Shake chocolate is a powdered drink that uses nanotechnology
to cluster cocoa cells.
Among their all but infinite applications, nanotechnologies can be
applied also to create sensors for food. In fact, there is a project using
that technology to detect bacteria inside meat, doping 60-nm sized silica
nanoparticles with molecules of fluorescent Ru(bpy) dye. The scientists
of the University of Florida attached antibodies for antigens that are
present on the surface of the bacteria to nanoparticles. That can work for
Escherichia Coli bacteria present in mincemeat and the test takes only 20
min.
Nanotechnological devices have started to be used in washing-
machines, air conditioners and freezers made by two industries: Samsung
and Daewoo. (web ref. 7), (web ref. 8), (web ref. 9)
Samsung inserted a device that releases “Silver Nano” ions (Ag+)
that bind themselves with the fabric at a molecular level. “This draws out
impurities and bacteria from the clothes, leaving them completely
sterilized and totally fresh”. “Silver Nano employs the safe and sanitizing
power of silver to eradicate airborne bacteria and germs”.
Nanoparticles in Food, Cosmetics and Other Products 241
Obviously, something like nano-sized silver ions (Ag+) is a nonsense.

Does the device release nanoparticles or does it release ions? The
difference is enormous.
When the advertisement of this product line appeared for the first
time, it publicized Silver nanoparticles and nanotechnology. The toxic
activity of Silver nanoparticles is well-known (see Chapter 9) and is
being taken advantage of by Monsanto that use it in pesticides to control
the Colorado potato beetle. FDA say they do not have the authority to
regulate pesticides and that is an EPA’s job. EPA say that FDA has
responsibility for requiring a label, because potato is a food (web ref.
10), (web ref. 11).
What is occurring is that nanotechnologies, creating new products,
create at the same time new situations that are not regulated. So, when
Samsung changed their advertisement from just Silver nano into Silver
nano ions, it is possible that they did so as soon as they knew of the
possibility that the material used in the device is a pesticide.
Daewoo uses Silver particles mixed with a plastic resin. “Using Nano
Poly Technology in “Pulsator” & “Tub U” in Washing Machines many
hurtful bacteria in clothes shall be sterilized perfectly”. That is what they
claim (web ref. 9).
All that is very exciting, but we cannot exclude that there may be a
risk that we inhale Silver nanoparticles directly from our clothes and/or
dispersed in our house from air conditioners. And no studies have been
undertaken about a potential skin contamination. But further hypotheses
can be put forward. The waste water from the washing machine is
drained to the water treatment plant through the sewer system, but at
present there are no filters that can capture nanoparticles. So, there is a
real possibility that they reach the sea, through the rivers, and are
ingested by fish. The end of the story is that we can eat a material
released by our own washing machine.
That waste water can also pollute the environment, contaminating
through irrigation the grass eaten by animals as well as the vegetables
that are our food.
So, we should not be too surprised by what we found in a hamburger
bought in a shop: a cluster of Silver nanoparticles (Fig. 7.1).
242 Nanopathology
Fig. 7.1 Image of a cluster of Silver nanoparticles contained in the meat of a hamburger
(marker 20 µm).
We do not the actual origin of that Silver, but our hypothesis is not
unreasonable.
After some time from this finding we had the possibility to analyze
dried hay in a small Italian farm where a case of mad cow had been
reported. The hay contained the environmental pollution we usually find
in that area, but we detected also numerous Silver particles, though in a
composition containing also Magnesium, Aluminium and Silicon (Fig.
7.2). We supposed that they could come from a pesticide, but we had no
possibility to check.
Fig. 7.2 Image of small Silver particles on the surface of a hay blade (marker 50 µm).
Silver or Silver oxide nanoparticles are already present in socks and

T-shirts, as the aversion for body smell induces industries to make yarn
with bactericidal properties.
Fig. 7.3 shows a thread coated with an already scratched Silver layer.
What is the appearance of this layer going to be after many washings? If
the metal coating is no more there, we will have good reasons to think
that probably some fish, clams and other beings have been contaminated.
Fig. 7.3 Image of a thread of a T-shirt with Silver-oxide nanoparticles (top marker 20 µm;
bottom marker 2 µm).
There are famous examples of “translocation” of environmental

pollution.
The people of Minamata City in Japan suffered from the toxicity of
Mercury eaten along with contaminated fish (web ref. 12). That Mercury
was ingested in particulate form and not as ion, but the corrosion the
particles underwent in the body had Mercury ions released and their
toxicity induced the pathology.
244 Nanopathology
Another example not yet entirely investigated is related to the Balkan

war. A refinery in Pančevo was bombed for three days and a 800-m
column of soot was created that moved also toward the American
headquarters (web ref. 13).
Airplanes took off and bombed the cloud with Silver iodide in the
attempt to make it precipitate with the rain. Unfortunately, that operation
caused the river Danube to be contaminated by this tiny, not
biodegradable pollution as well as the Black Sea, where the Danube
flows. Having fish polluted by Silver-iodide particles is a logic
probability, but nobody seems to show any particular interest in that and
no proper prevention measures are taken, except a control for Uranium
and a few other heavy metals on game and mushrooms imported to Italy
from former Yugoslavia.
Heavy-metal pollution is something we detected in 140 samples of
bread or biscuits coming from 10 different countries in three continents.
40% of them were contaminated by environmental or industrial
particulate pollution.
Fig. 7.4 Image of Tungsten-Cobalt micro and nanoparticles detected in non-industrial

bread (marker 1000 µm, image above and 20 µm, image below).
Fig. 7.4 shows a metallic contamination in a bread sample. Because

of the particle shape, we may say that that is probably due to the wear of
some crushing or milling tools, probably from the anti-wear metal used
as coating of those machines.
In “nanotechnological” food, non biodegradable nanoparticles can be
added intentionally, for commercial, technological or aesthetic reasons.
One clear example is represented by a chocolate bar called Mars
where Titanium dioxide nanoparticles are added to avoid the separation
of cocoa and cocoa butter due to aging and changes of temperature.
Inside our body, there is no way to degrade titania nanoparticles. They
are insoluble, non biodegradable and can remain as foreign bodies inside
the digestive system or migrate elsewhere. There is no evidence that they
are eliminated with defecation and, to our knowledge, no studies have
been undertaken on the subject.
246 Nanopathology
Fig. 7.5 Image of Iron-Chromium nanoparticles in a chocolate (Mars) (marker 10 µm).
Fig. 7.5 shows nanoparticles of a type of stainless steel that were

probably unintentionally released in the chocolate during a specific
industrial manufacturing process.
Fig. 7.6 shows titania nanoparticles contained in a chewing gum.
It contains also silicates and silica micro particles added to remove
food debris from the teeth.
Fig. 7.6 Image of a cluster of titania nanoparticles in a chewing gum (marker 2 µm).
But nanoparticles can be present also in toothpastes and even as

coatings of toothbrushes (head and bristles). Nano-UP, a Japanese
company, has a product line composed of toothbrushes (Fig. 7.7) and
toothpastes where Gold and Silver as coating and nanoparticles
respectively are used. Their toothpaste contains Silver and few coarse
Gold debris.
Fig. 7.7 Image of a toothbrush bristle with Silver nanoparticles (marker 100 µm).
It is only obvious that the presence of unwanted, toxic, not

biodegradable contaminants (pesticides, dioxin, etc.) in food decreases
its nutritive value and has a negative impact on human health. Among
the numerous contaminants that can be present, heavy metals make up a
well-known group.
Recently, with the diffusion of nanotechnology, the problem of
toxicity related to nanoparticle size (and not just to its chemical
composition) has started to be investigated (Donaldson, K. et al., 2004),
(Kipen, H. M. et al., 2005). Indeed, we can say that some in-vivo studies
suggest that nanoparticles are not inherently benign (Hillyer, J. F. et al.,
2001), and that they affect the biological behaviour at the cellular, sub-
cellular and protein levels (Donaldson, K. et al., 2001), (Zhao, Z. et al.,
2006)
The possible presence of metals as salt has been investigated in food
(Charbonneau, J. E. 2001), (Szefer, P. et al., 2003).
248 Nanopathology
So far, inorganic pollutants not as soluble salts (i.e. ions) but under
particulate form has not been investigated in depth. The previous
chapters proved that exogenous, inorganic, micro- and nanoparticles can
be found in biological and, more specifically, human pathological
tissues, in tissues affected by pathologies of unknown origin like Crohn’s
disease (a granulomatous disease of the colon) or various forms of cancer
(e.g. colon, stomach cancer).
Food could be an important carrier for “dust” since, in many cases,
row materials grow in polluted environments, more and more
contaminated by industrial wastes.
Nanotechnological food can be very useful, but also very dangerous
for human and animal life.
The information gathered will be used to set up prevention program.
Then we should study food “from the fork to the farm” and detect what
the sources polluting it are. The study should also include animal feed.
As our method allows to work on archived biological samples, “mad
cow” disease could and should be part of the study, in order to verify if
cattle were actually fed with exhausted lubricants mixed with animal
flour as is sometimes reported. A focused study could be carried out on
the poultry food in order to verify if there is a nano-contamination. If so,
the infected animals must be verified for this contamination and the
nanoparticle-virus interaction should be evaluated.
It is desirable to analyze as basic research ice carrots in the Antarctica
to check if environmental nanopollution was already present in the past
and to what extent. Hiroshima and Nagasaki destruction and Chernobyl
accident with its Cesium dust could be particularly interesting.
Then, again as basic research, it would be desirable to know how
bacteria and parasites behave and develop after having interacted with
nanodust.
It would be of great interest to study how viruses adhere to
nanoparticles and how (and if) they change, for example in
pathogenicity, after having interacted.
Nanodust seems to be teratogenic and this is an issue that certainly
deserves to be deeply investigated.
We should also develop traps and filters to capture nanodust and

sensors to detect and measure it. Without any doubt, this will prove very
useful to nanotechnology industries to protect workers.
Then, it would be very useful to develop a network of infrastructures
to monitor nanodust in the environment, capable of alerting people in
case of danger (see, for example, what was not available at Chernobyl).
Setting up courses and schools to educate scientists and technicians in
this new, particular field of environmental science is of the greatest
importance and an urgent necessity to nanotechnology companies
producing or working with engineered nanoparticles.
Special attention must be paid to security, since nanoparticles can be
inexpensive and silent bullets to the cell nucleus and could be used as
such.
Further research directions can be the study of the so-called industrial
sterility, malformed feti in specific industrial areas, rare diseases and new
pathologies investigated, but not yet understood. Epidemiological studies
with homogenous cohorts of patients should be carried out, where,
starting from the analysis of the embedded particulate matter, exposure is
verified and the source of pollution is identified.
The results of our research have been readily applied by the Italian
senatorial commission dealing with the problem of pathologies
contracted by peacekeeping troops in former Yugoslavia. A non
negligible number of them were in the vicinity of the explosion of high-
temperature rounds (e.g. depleted Uranium or Tungsten weaponry) or
were involved in the destruction of weapons which was carried out by
heaping them up in large holes dug in the ground and having them burn.
Some of those soldiers were also close to burning oil wells. In all those
cases, they inhaled particulates produced be the combustion of large
quantities of matter and may also have ingested them. Part of those
soldiers developed diseases that can be classified as nanopathologies.
7.1 Bibliography
Charbonneau, J. E. (2001), Investigation of corrosion and container integrity in metal

food containers using scanning electron microscopy-X-ray microanalysis,
Scanning, May-Jun;23(3):198-203.
250 Nanopathology
Charbonneau, J. E. (2001), Investigation of foreign substances in food, Scanning, Jan-

Feb;23(1):51-7.Donaldson, K., Stone, V., Tran, C. L., Kreyling, W., and Borm, P.
J. (2004b), Nanotoxicology. Occup.Environ.Med. 61, 727-728.
Donaldson, K., Stone, V., Clouter, A., Renwick, L., and MacNee, W. (2001a). Ultrafine
particles. Occup.Environ.Med. 58, 211-6, 199
Hillyer, J. F. and Albrecht, R. M. (2001), Gastrointestinal persorption and tissue
distribution of differently sized colloidal gold nanoparticles, Journal of
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Kipen, H. M. and Laskin, D. L. (2005), Smaller is not always better: nanotechnology
yields nanotoxicology. Am J Physiol Lung Cell Mol Physiol, Nov;289(5):L696-7.
Szefer, P., Domagała-Wieloszewska, M., Warzocha, J., Garbacik-Wesołowska, A. and
Ciesielski, T. (2003), Distribution and relationships of mercury, lead, cadmium,
copper and zinc in perch (Perca fluviatilis) from the Pomeranian Bay and Szczecin
Lagoon, southern Baltic, Food Chemistry 81, 73-83.
Zhao, Z., Hyun, J. S., Satsu, H., Kakuta, S. and Shimizu, M. (2006), Oral exposure to
cadmium chloride triggers an acute inflammatory response in the intestines of
mice, initiated by the over-expression of tissue macrophage inflammatory protein-2
mRNA, Toxicol Lett. Jul 1;164(2):144-54. Epub 2006 Jan 18.
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www.washingtonpost.com/wp-dyn/content/article/2006/04/05/AR2006040502149.html
Web Ref. 2:
http://www.bfr.bund.de/cms5w/sixcms/detail.php/7750
Web Ref. 3:
http://www.quackwatch.org/01QuackeryRelatedTopics/PhonyAds/silverad.html
Web Ref. 4:
http://www.fda.gov/bbs/topics/ANSWERS/ANS00971.html
Web Ref. 5:
www.smalltimes.com/Articles/Article_Display.cfm?ARTICLE_ID=270664&p=109
Web Ref. 6:
www.wired.com/medtech/health/news/2005/09/68683)
Web Ref. 7:
www.samsung.com/in/products/washingmachine/thesamsungwashingmachinea
dvantage/index.htm)
Web Ref. 8:
www.samsung.com/ph/presscenter/samsunginthephilippines/productnews_20040608_00
00058341.asp)
Web Ref. 9:
www.daewoo-electronics.de/eu/products/living_washing_glos.asp
Web Ref. 10:
(www.biotech-info.net/seeds.html,
Web Ref. 11:
(www.lawbc.com/fda.html).
Web Ref. 12:
(www1.umn.edu/ships/ethics/minamata.htm)
Web Ref. 13:
(www.ieer.org/reports/bombing/index.html)
Chapter 8
New York 9/11

___________
n 9 September 2002, Dr Gatti sent the following message to
2 the Herald Tribune, as an answer to a specific request by the

editor to remind the anniversary of the Towers’ collapse with a
100-word message.
“I was in London, at Harrod’s, when I watched the Twin Towers

collapse.
The immediate slaughter was something everybody could understand,
but there was something more sadly sophisticated I thought of. All people
who survived the disaster inhaled and ingested particles of dust which
got stored in the tissues of their respiratory and digestive tracts. There
they will induce more or less slow, but often deadly, reactions, what a
European research started relatively recently calls a ‘nanopathology’.
(An innovative technique makes it possible to detect and diagnose them.)
As a scientist involved in the research, I know that New York is bound to
suffer more mourning that will pass unrecognized.”
Probably somebody remembered that letter, since a little less than one
year later we received a phone call from New York, and the person who
called was a lady who had been heavily involved in the event of 9/11.
She was one of those people who had their working place inside one of
the Twin Towers and survived the attack; but she inhaled a huge quantity
of dust and, because of that and the respiratory problems that followed,
she spent two months inside an Oxygen tent. She called to know
something about the theory of Nanopathology mentioned in the letter and
about the possible existence of remedies. So, we started to exchange
messages.
251
252 Nanopathology
After two years, she developed an angioma in her brain, she was
operated on and eventually recovered after a long convalescence, during
which she followed what looked a strange detoxification program (Web.
Ref. 1)
The Council of Down Manhattan had accepted the proposal made by
the Foundation for the Advancement of Science and Education located in
Los Angeles to apply a particular procedure aimed at detoxifying a
certain number of workers employed by the Council and exposed to
dust: firemen, sanity workers etc. That detoxification consisted of a 3-
week treatment based on physical exercise, sauna and a sort of vitamin
cocktail. Dr Gatti was invited to take part in meetings where medical
doctors and patients presented their data and discussed their experiences
and, in case, their progress. At the beginning, she was sceptical, but,
before rejecting any theories or hypotheses, knowing them well enough
is necessary, in order to be able to find out their possible weak points, if
any. So, every judgement was left suspended.
From what is a traditional scientific point of view, such a treatment
looked indeed much of a nonsense and a very poor remedy. Moreover,
we are accustomed to believing that the higher the cost, the more
effective the result is, and, in that case, the cost of the treatment proposed
was in fact very low.
The particulate pollution generated by the collapse was extremely
fine, in our opinion especially the one formed in the part of the buildings
crossed by the aircrafts that had “disappeared” there. In that particular
point, the temperature had been very high, high enough to have the
metallic structure and the airplanes vaporize and it was particularly so in
the vicinity of the blast, and that blast involved a great variety of
different things, from glass to plastics, from steel to computers.
Everything was inside disappeared from sight. But, in fact, as every
student knows, nothing had disappeared: everything had been sublimed
and transformed into something else: an aerosol whose composition
depended on the elements present in the reaction matter. That new
pollution thus generated had a huge variety of chemical compositions
that did not exist before.
The Environmental Protection Agency (EPA) workers looked for
asbestos in the pipelines of the air conditioning systems three years after
New York 9/11 253
the collapse to demonstrate a possible correlation between the diseases

become manifest and the presence of such pollution. But blaming just
asbestos is a limited way of facing the problem which is much bigger and
with more culprits than just that.
As a consequence of the collapse of the buildings, another form of
pollution was created and that was the one originated from the
pulverization of concrete, whose particles were larger in size than the
ones generated by the parts of the buildings where the planes had
entered.
The constant, persistent cough of the firemen is a demonstration of
the inhalation and entrapment of those micro-sized particles that, because
of their larger size, had not penetrated deep enough to reach the alveoli
but had remained in touch of the bronchial walls where the ciliary
activity tried, not so successfully, to get rid of them.
One of the subjects belonging to the group had started his work
September 11, 2001, but grew seriously ill next May. After such a
comparatively long time, that person developed chronic fatigue, that did
not allow him to stand up from his bed without exhausting all his
energies. A number of other, apparently not homogeneous, symptoms
like blurred vision, nightmares, depression, etc. affected him and, for all
those reasons he was unable to work. For him, like for all the other
patients, no current drug was effective against those symptoms and some
of the subjects even suffered from the side effects those drugs produce.
So, this person entered the group and underwent the “detoxification“
treatment after which he recovered and we lost his track.
During the meetings held at the place where doctors and patients
gathered and carried out their program, and thanks to long discussions
with the patients, Dr Gatti got to know interesting details about the onset
of those people’s diseases, exposure, latency period, symptoms, their
getting better and their personal feelings. In particular, a sentence of a
survivor attracted her attention: while he talked about his impressions
during the detoxification treatment, he told of his surprise when he
observed the dramatic change in colour of his own sweat which had
turned to a rich brown. None of us had ever heard of such a phenomenon.
Sweat’s main functions are regulation of body temperature and
elimination of some waste products and it was probably those products
254 Nanopathology
that had caused that reaction. As we work on inorganic debris, we

thought it natural to check if that liquid contained them and, in case,
what could their size and composition be. Had we found what we wanted
to look for in those people’s sweat, the next question was: are those
things, expelled from the organism, responsible for the partial recovery
experienced by the majority of those subjects participating in the
programme?
If the particulate matter inevitably inhaled, dispersed in the blood
circulation and in the internal organs, can induce a body reaction with
those unusual collections of symptoms, its complete, or even only partial,
elimination can eliminate or even just decrease the biological reaction.
So, we proposed to analyze the sweat of those patients and some of
them collected it during their saunas following a protocol simple enough
not to be a bother, but strict enough not to yield polluted samples. We
asked the patients to clean their skin scrubbing it with alcohol, not to use
soap, creams or other potential contaminants. They were provided with a
sterile vial where they collected the sweat drops during the sauna, mainly
from the breast and the arms, and not from the armpits.
The analyses on the sweat we got were very interesting. They
revealed that Nature is not unkind and a possibility exists to get rid of so
unusual a pollutant from the pores of the skin. When the body
temperature rises and sweat is produced, the pores open and a certain
amount of liquid is thus eliminated. In that liquid we actually found the
debris we were looking for.
The following images show the particles we found in the fluids
examined.
We present below a series of unusual chemical compositions found in
the particles detected in the sweat, that could be related to the collapse.
New York 9/11 255
Fig. 8.1 Particles of n. 1 rescue-worker’s sweat containing a particle composed of

Copper-Zinc-Gold-Chlorine-Potassium (marker 20 µm).
Fig. 8.2 Particle from n. 2 worker’s sweat containing Zinc-Lead-Iron-Chlorine-Silicon-

Calcium (marker 5 µm).
Fig. 8.1 and Fig. 8.2 show small particles found in two different
workers that present two different peculiarities. The first chemical
compound (Copper-Gold-Zinc) contains Gold and Copper, typical of a
commercial golden alloy, but in commercial alloys Zinc is never present.
Such a composition is not used in any known material. The second one
contains again Zinc but now that element is bound to Phosphorus-Lead-
Iron-Chlorine-Calcium-Silicon. The oddity is represented by the hard-to-
explain presence of Phosphorus. This can be the result of an occasional
combustion: a residual of the melting of pipelines of water-gas, window
structures, etc.
256 Nanopathology
Fig. 8.3 Particle from n. 3 rescue-worker’s sweat containing Copper-Silver-Gold-Zinc

(marker 20 µm).
Fig. 8.4 Particle from n. 4 worker’s sweat containing Iron (marker 10 µm).
Fig. 8.3 shows an 8-micron sized particle of Copper-Gold-Silver-

Zinc. It is strange to find golden compounds in the sweat of two different
workers who shared only one comparable exposure. The presence of
Gold and Silver as parts of computers, furnishings, jewels kept inside
safes as well as Copper in electrical wires is understandable. Zinc is
another element whose presence can be explained as it may derive from a
coating (zincing), but is never used to form golden alloy. That means
that this type of particles can be originated by an accidental, local,
uncontrolled combustion.
Fig. 8.4 presents a particle containing a mixture of elements
coming from two different origins. Chlorine-Sodium is the elemental
composition of a salt commonly and predominantly present in the sweat,
New York 9/11 257
so of biological origin. Iron-Sulphur, instead, with the specific

morphology observed (a sharp-cornered debris) is of exogenous origin.
The particle appears immersed in the sweat and surrounded by other
nanosized debris (Fig. 8.5).
Fig. 8.5 Image of a Sodium chloride crystal trapping other nanometric particles
containing Iron-Barium-Sulphur-Magnesium-Aluminium-Silicon-Calcium-Potassium
(marker 20 µm).
Thanks to a lucky coincidence, we could get the dust deposited on an

object on display in a shop that was located inside one of the Towers that
its owner found completely covered with dust just after the collapse, and
on a fireman’s helmet. Unfortunately, we did not have the possibility to
analyze the flying ashes that hovered over what is today called Ground
Zero and flew toward the other boroughs of New York. Those are likely
to preserve the memory of the two airplanes and the parts of the
skyscrapers where they stroke.
The following images show the dust collected on the firemen’s
helmet used for 3 months and put inside a bag together with his overalls.
Neither helmet nor overalls were cleaned. After 3 years from the
collapse, when we formulated the hypothesis that the dust on the helmet
represented the exposure the subject had suffered and caused the disease
he suffered from, he asked us to check and try to explain why his weight
had increased and why his breath had grown short. The helmet was very
dusty, and the main composition was concrete (Fig. 8.8). In that dust we
found some particles with compositions similar to those we had found in
258 Nanopathology
his sweat samples. Much of our attention was focused on golden alloys,
not common in the normal pollution, and on debris composed of
numerous elements, since this is one of the clues left by a random
combustion.
Fig. 8.6 Image of micro and nanosized debris containing Gold-Silver-Copper-Calcium-

Silicon (marker 50 µm).
Fig. 8.7 Image of particles of the dust collected on the helmet. They contain Sulphur-
Copper-Silicon-Calcium-Chlorine (marker 20 µm).
New York 9/11 259
Fig. 8.6 and Fig. 8.7 show particles detected in the dust found on the
rescue worker’s helmet. They have different morphology and size. The
bigger and sharp-edged ones are concrete, while the smaller have
different origins. Some of them present a matrix where very small
particles are embedded. The first contains noble metals that were also
present in the sweat. That should not look strange, but what is just
obvious to find in such circumstance.
When the disaster occurred, the atmospheric temperature was still hot
and many air conditioners were working. (In New York, air conditioners
are kept on even if there is no actual need of them.) For that reason, the
dust produced, much of which was very thin and behaved more or less
like a gas, entered the pipes. That transferred and probably even
concentrated that kind of pollution indoors. A problem that is somehow
similar to that is due to the rubble and remains of the collapse moved
elsewhere and disposed of in sorts of landfills. Those too were the
obvious origin of dust. Since some of those particles are still nested
somewhere, are actually impossible to get rid of and can have only been
diluted in the atmosphere, it is only natural that they can still have an
impact on the people who live in the territory.
Fig. 8.8 Debris of the concrete found at Ground Zero with the spectrum (marker 100 µm).
260 Nanopathology
Fig. 8.9 Image of round-shaped Iron-Sodium-Silicon-Sulphur-Chlorine-Calcium debris.

(marker 20 µm).
Fig. 8.10 Image of composite debris found on the overalls of the fireman. It includes
nanosized, round-shaped Lead-Iron-Sodium-Aluminium-Silicon-Phosphorus-Chlorine-
Calcium particles (marker 5 µm).
Fig. 8.9 and Fig. 8.10 show particles of compounds of Iron and Lead:
similar debris were found also inside the sweat specimens. There is a
correlation between the chemical compositions of the particles found in
the sweat and those found in the pollution.
New York 9/11 261
Fig. 8.11 Image of a round, submicronic Chlorine-Cerium-Sodium-Magnesium-

Aluminium-Silicon-Sulphur-Calcium-Iron particle (marker 20 µm).
Fig. 8.11 shows a 500-nanosized spherical particle with a strange

composition: Chlorine-Cerium-Sodium-Magnesium-Aluminium-Silicon-
Sulphur-Calcium-Iron, found on a firefighter’s fatigue dress.
Fig. 8.12 Image of New York debris. The whiter particle is composed of Gold-Copper-
Nickel-Zinc-Sodium-Aluminium-Silicon-Chlorine-Potassium-Calcium-Iron (marker 10 µm).
262 Nanopathology
Fig. 8.13 Image of a round-shaped particle containing Calcium-Lanthanum-Cerium-

Neodymium-Sodium-Magnesium-Aluminium-Silicon-Sulphur-Potassium-Iron. (marker
10 µm).
Fig. 8.12 and Fig. 8.13 show particles detected on the object found
intact below the ruins, inside a shop of sanitary fixtures by the owner. He
collected that as a memento of his destroyed activity and life. Also the
chemistry of this dust containing Gold-Copper, but also Zinc, Iron,
Nickel was related to that found in the sweat samples. Once again, it is a
random combination of elements that are not related to the melting of a
single material or object, but to the presence of many other objects in
the same crucible. In many of these spectra there is the presence of
Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, i.e. is the basic
composition of a ceramic glass material. It has been observed that during
the melting in some parts of the Towers, there was the formation of glass
debris entrapping other materials. The composition of the image of
Fig. 8.13, a spherical debris, is rather peculiar. Its spectrum contains
Sodium-Magnesium-Aluminium-Silicon-Calcium-Iron, but that spectrum
could be due to the part that surrounds it. Certainly, it contains earth
elements like Cerium-Lanthanum-Neodymium, and Sulphur. In our
experience, though not exclusively, the earth elements’ triplet signal may
be related to an environmental contamination of tobacco smoke. A more
accurate investigation about the history of the object, of the shop and of
the owner (was he a smoker?) could explain much about those presences.
In any case, nothing can be completely destroyed.
New York 9/11 263
Most of the dust we analyzed was related to concrete. In this chapter

we presented the more meaningful or unusual debris to try and
demonstrate if a passage from inside the organism to outside is possible.
We considered those strange compositions as traceability markers and
the conclusion we came to was that a passage looks possible. Can this
finding help to devise an efficient detoxification method? There is a
possibility, but a limited one, since, probably, the passage is allowed
only to the dust trapped in the fat. For the one remained in the blood or in
other tissues there seems to be no way to be exteriorized. Let us hope
that scientists and technologists are smart enough to find a solution.
8.1 Bibliography
Web. Ref. 1 http://www.nyc.gov/html/doh/html/wtc/index.html

Chapter 9
The Future and Prevention Criteria

___________
9.1 The state of the art
an has always lived with nanoparticles: proteins and colloids,
0 when observed according to their size, may be so classified, but

proteins and colloids are organic compounds and, at least in most
cases, can be tolerated by man or even used for his metabolism.
Inorganic micro and nanoparticles are constantly generated by nature:
belching volcanoes throw out huge quantities of minerals in the form of
tiny specks of dust, fires produce particulate, sand is blown by the wind
in the atmosphere and part of the products of rock erosion caused by the
weather ends up in the air.
So, man has been confronted all along with that kind of pollution, but
very little can be said of its interactions with his organism, since, with
rare exceptions, that has always been considered as an inescapable
condition, almost a sort of background noise, wherein man is bound to
live. Therefore, little or no attention at all has been paid by medicine to
that phenomenon that in most cases was not seen as a problem.
One of the characteristics that distinguishes man from all other
animals is his unique way of using the planet where he lives. Man is the
only animal who can lit a fire, and this now apparently very simple and
harmless action has been the first source of non natural pollution, as all
combustions produce particulate matter whose composition is different
from the one of the materials it derives from. For millenniums, though,
fire was very sparingly used, and in the vast majority of applications just
to warm oneself or cook food, and the pollution generated represented
a comparatively trifling problem in a planet inhabited by a scanty
population. Energy has not been in great demand for the largest part of
265
266 Nanopathology
human presence on the Earth: Muscles, be they offered by animals or by

man, have been the primary form of energy for a very long time, only
much later boosted by natural sources like wind and flowing water, and it
was only in Europe, in the second half of the eighteenth century, with the
so-called First Industrial Revolution, that combustion started to be used
on a relatively large scale, and that was done to heat water meant to
produce steam exploited to drive machinery. A few decades later, with
the Second Industrial Revolution, coal became quantitatively the most
important source of energy, thus supplanting wood and peat as a fuel and
transforming water-driven mills into plants whose output was strictly
dependent on man’s decisions. Thus, man started to impinge noticeably
upon nature by producing through combustion particulate matter that,
when inorganic, could not be degraded.
Nowadays the sources of anthropic micro and nanoparticulate are all
but endless. Heat has become very easy to come by and very cheap, and
higher and higher temperatures can be reached with no technical
difficulties. This allows us to produce energy and materials we
commonly and often absently use in our every-day life without
considering that one of the by-products is inevitably dust, and the higher
the temperature, the tinier the particles. And what we produce nowadays
is particles much smaller than and very different from those produced by
nature.
One of the most obvious and visible responsible for this kind of
pollution is certainly car traffic. And not only with its exhaust fumes, but
also with the wear of the tyres and the brakes. Heating systems, when
using fossil fuels, are also polluters, and so are all plants that have
recourse to combustion: incinerators, cement works (often used, more or
less legally, as incinerators as well), power plants, foundries and many
factories in general. But great quantities of heat can also be generated by
the explosion of weapons, especially when they are high-technology ones
like those based on depleted uranium or tungsten, and much heat and
dust was generated in New York when the World Trade Center
collapsed.
Besides unintentionally produced, noxious nanodust, there is dust
of the same size we manufacture and whose properties, for which we
keep finding new applications, we exploit more and more frequently.
The Future and Prevention Criteria 267
Engineered nanoparticles are already used in a number of commercially

available products, while scientists and technicians are busy
experimenting new possibilities for their use.
Huge amounts of money and human resources are being invested in
nanotechnologies and great expectations, both technical and economical,
are placed in the indeed extraordinary properties of nanosized matter.
If, on one hand, one can share this enthusiasm, some prudence is
necessary if we don’t want to run the risk of being sorely disappointed.
There are a few applications, mainly in food and in medicine, that require
the introduction of nanoparticles in the organism, and that is done
without having carried out a sufficient experimentation and without
having allowed enough time to elapse to observe and evaluate the
consequences of such use. In too many cases it is taken for granted that
our organism can get rid of those particles, but no scientific evidence
does really exist to confirm such an assumption. As far as we have been
the possibility to see, we could not detect any biological mechanism of
elimination for inorganic particles once they have been trapped in a
tissue, and evidence is being collected that shows that those foreign
bodies are far from harmless. In our opinion, this should be regarded as
an invitation not to act in a hurry in the hope of outrunning one’s
competitors.
9.2 What is next?
Nanotechnologies, creating new materials and structures not far from an

atomic level, at least from the dimensional point of view, will represent a
societal revolution. But the other side of the coin inherent in every
progress may be not so rosy and it is impellent and mandatory to find it
out before the side effects are the cause of phenomena that may be hard
to tackle or even be or become irreversible.
The synthesis of new matter and the discovery of its incredible
properties excite not just the researchers involved but also businessmen
and a market always searching for new possibilities of gain.
The previous chapters showed pieces of evidence that are hard, when
not impossible at all, to deny.
268 Nanopathology
Everybody remembers how GMOs (Genetically Modified

Organisms) flopped partly because of a generally bad perception but,
above all, because research organizations and media were not wise
enough and lied on the possible effects those organisms could induce on
human bodies. So, now, great care is taken on the news released by
scientists, and this, in some cases awkward, prudence may cause a bad
perception of nanotechnologies. Booklets are prepared for the instruction
of scientists in order to alert them of the danger and teach them how to
present the subject to the public opinion. Specific European projects
(web ref. 1) of communication in the specific field are being financed
and a sort of censorship has been set up by the European Community in
order to prevent the release of news that may be unfavourably interpreted
by the public.
But if, from one side, there is a need to control information in order to
protect the investments and avoid that huge amounts of money go
wasted, from another, certainly more important, point of view, we need
to set up all the possible preventive measures we are capable of in order
to avoid risks for the workers, the end-users and the environment.
It is a matter of fact that a number of nanotechnological products are
distributed on the market without informing the consumer of that
particular origin and something like that cannot be accepted. The fear of
a flop surrounding these new technologies due to a possible bad,
emotional, reaction of the public, induces the producers perhaps not to
lie, but to issue only partial information. When, within 3-4 years, no
adverse side effects are reported, it is very likely that they will not only
reveal but even advertise the nanotechnological origin of their product.
But not to run risks in the event of something having gone wrong and
their products having caused some sort of damage to their users,
producers have no interest in furthering the research on nanotoxicity so
to be able to maintain that there is no evidence to blame their goods. One
of the dangers inherent in that kind of attitude is that the full
understanding of what is a scientific problem involving health be delayed
and a possible wide dissemination of the diseases originated by a misuse
of nanotechnology, deriving from the globalization of the market and the
dissemination of the products worldwide, go unrecognized even by the
cleverest epidemiologist. In such a scenario, what will be registered will
be nothing more than a “normal” increase of different types of cancer,

leukaemia, lymphoma, etc. of “hard-to-explain” origin.
Another problem is the attitude of some scientists, hopefully a
minority, but in some instances an important one, at least from the
acquired prestige point of view, to yield to the allurements of money or
power. The way it happened some decades ago with tobacco, those
scientists agree to write papers that are then published even by serious
journals, and in those papers the essence of science gets twisted and, in
the most favourable of hypotheses, truth is concealed and facts are
passed over in silence. People who obtained money for their research or
people who invest their money to develop nanotechnological products,
will hardly look for side effects. So, all that is used as an alibi based on a
mistaken feeling of authority to escape from responsibilities and, in our
case, the risk is that the use of nanoproducts and its relevant exposure do
not enter among the parameters examined and are not considered in the
statistical evaluations.
What is written above must not be seen as a rejection of
nanotechnology but just a sort of warning intended to make it stronger,
more effective and much less open to attack.
A discipline that comes often into the picture is epidemiology.
Research in that field requires huge numbers of cases and a long time,
and, for that reason, a research whose conclusions are presented today
was actually started ten or fifteen years ago when the conditions, due to
the rapid evolution of pollution, were not comparable with the present
ones. If, as an example, we take into consideration a population exposed
to the pollution originating from an incinerator built according to the
most modern technology, we must know that the particulate matter it
eventually produces as a final result of its processes is probably much
finer than that produced by the older plants. So, the modern particulate’s
behaviour in the environment is much more similar to a gas than the old
one’s and limiting the epidemiologic evaluation to areas were gross
particles are likely to fall to the ground becomes meaningless and
deceptive. And equally meaningless and deceptive is considering just
respiratory, oncologic and cardiovascular diseases, neglecting all those
belonging to nanopathologies, including, for example and among others,
270 Nanopathology
Parkinson’s and Alzheimer’s diseases, diabetes, fetal malformations, etc.

On top of that, many studies are published only if they are “reassuring”
and some of them draw conclusions that should be rejected by any
serious epidemiologist or, in any case, looked at with suspicion. It is not
exceptional to find articles maintaining that increasing pollution has no
effect on the morbidity of diseases notoriously due to pollution, and even
taking for granted that that had been written in good faith, something like
that, in contrast with science and common sense, should be considered as
a false negative, waiting for more and stronger evidence.
We must keep in mind that nanoparticles behave in a very peculiar
way, unsuspected until not long ago, and probably much is still to be
learnt and psychologically accepted. Nanoparticles are sets of atoms, but,
because of their size, they have properties that are not ruled by the
atomic-physics laws. And, in more than a way, their adverse effects are
different from those of the chemicals containing the same elements but in
a form that is not that of nanoparticulate.
At certain threshold concentrations, nanoparticles have been found to
be toxic (Brayner et al.,2006), and capable of inducing oxidative stress
(RDS) to cells (Nell, A. et al., 2006), but under that critical concentration
they can present new, maybe long-term, toxic aspects never investigated
or suspected before. (Hansen, T. et al., 2006)
This reveals the particular physico-chemical aspect of inorganic
nanoparticles. In fact, in addition to the particle chemistry, their
inorganic nature and particulate structure makes them respond in a
special way in a biological environment. They have greater densities
(and greater inertia, in a world small enough to be dominated by
Brownian motion), high electron densities (the response to light is very
different and this is the basis of the remote photoablation), they have
strong redox potentials, they have a marked thrombogenic activity, etc.
Some of them have another peculiarity: they are not soluble and not
biodegradable, so, when they penetrate the organism, since it is likely
that we have no elimination mechanisms made available by nature, they
stay there forever.
An example of such a behaviour can be found in silicosis. Inhaled
silica microparticles are attacked and incorporated by macrophages that
try to digest them but, because of their absolute non biodegradability,

they do not succeed. So, when those macrophages die, they are
“digested” by the organism the way all macrophages are. Not so their
content which is indestructible and is released again in the biological
environment to be, in case, attacked by another macrophage. This
continuous, repetitive behaviour causes a state of disease. Single
nanoparticles represent a very small stimulus to cell, too small to be
recognized by the membrane sensors and activate the normal cell
defences (the activation of the macrophagic reaction), but they disturb
the cellular metabolism.
So, the presence of inorganic nanoparticles in biological media, even
without being directly toxic to the cell, may cause environmental stress.
Cells are very sensitive to signals from the environment. For
example, cellular reproduction is regulated by complex exterior signals
and synchronized with the neighbouring cells. In the presence of a stress
(i.e. a non expected modification of the environment), the cell stops
temporarily its reproduction cycle until it re-adjusts to the new situation.
The responsible for that are the Stress-Activated Proteins (SAP), kinases
which transmit the alarm signal to the cell. If this process does not
function properly, erroneous cellular duplication may be induced, which
might result into tumour cells.
In addition to that, nanoparticles can be a reservoir of perturbing or
toxic cations which are slowly released and induce noxious effects in
tissues. Furthermore, nanoparticles may modify ternary structure of
proteins, changing their functionality. In addition to that, they can be not
recognized as self, and give an immunological disease.
Prion-related pathologies as Creutzfeldt-Jacob’s and amyloidosis as
Alzheimer’s are classified as conformational diseases where misfolded
proteins induce nearby proteins to change their conformation. Moreover,
the biodistribution of the molecule attached to a nanoparticle can be very
different from the monomeric form. For example, compared to free
chemotherapeutic molecules, delivery to cancerous tissues may be
favourably biased by several mechanisms, including particle size (Tang
Z. et al., 2001) and by attaching targeting ligands to the surface of the
particles.
272 Nanopathology
All this is enough to understand that conventional tests to assess the

cyto- and eco-toxicity are not designed nor suited to study nanoparticles–
cell interaction and that the study of acute toxicity may not be as relevant
as other slower processes, where inorganic contamination progressively
induces stress and damages tissues.
There are no homogenous and certain results about the in-vitro and
in-vivo toxicity of nanoparticles, so floods of words are being written to
debate the problem. Failing data, writing has the effect to remove the fear
of the unknown.
Many articles, reports, strategic documents, risk assessments, contain
recommendations. An article by Dr. Maynard of the Woodrow Wilson
International Center for scholars in Washington (Maynard, A. D. et al.,
2006) is a series of good intentions mixed with fundamental, theoretical
rules of risk assessment and management. The most used words are “we
must develop”, and, in particular, what we must develop is instruments,
tests, validations of tests, and models to predict toxicity. That sounds
rather premature since, without knowing the laws ruling the nanoworld,
it is impossible to predict anything meaningful. The incredible properties
we verified in some nanoparticles witness that nanoworld presents rules
that can be either wonderful or terrifying.
All the reports of different associations and organizations underline
the “must” task (Research Needs on Nanoparticles, 2005), (web ref. 2),
(web ref. 3)
A far from negligible quantity of money is dedicated to research,
much of it by the European Community and the US. But now there is a
risk: nanotechnologists who had new institutes built, money and honours
as pioneers may not want to kill “the goose that lays the golden egg”, so
it may be not so likely that they declare the existence of a possible risk.
The death of Richard Smalley, co-discoverer of the so-called “bucky-
balls” and Nobel Prize laureate, occurred October 31, 2005, could be a
sign of danger not to be ignored, as he died of lung cancer and
leukaemia, simultaneously.
There is a possibility that such an unusual combination of diseases be
the expression of the exposure to the nanoparticles he himself had
created and that possibility should be considered among the risks related
to handling nanoparticles.
A hypothesis that can be put forward is that part of the inhaled

particulate matter forming clusters may have remained trapped in the
lungs where, because of its non biodegradability and non
biocompatibility, it may have induced a cancer. The nanoparticles that
did not form clusters or with a size small enough, for example, below
100nm, may have passed rapidly, before having the time to cluster,
through the lung barrier and gone into the bloody stream. There, they
may have created bounds with some proteins or cells specific of this
environment, so they were not filtered by organs but went to the bone
marrow.
We do not know for certain if the cell reaction is due to the particle’s
chemistry or its physical structure or only because nanoparticles
are foreign bodies interfering with the normal cell metabolism, but a
reaction is possible at the deeper levels.
What is sure is that this approach leads to a “customized medicine” to
cure diseased people rather than diseases as a sort of abstract entities,
since each individual has necessarily undergone his own exposure to
pollutants. This implies more time and more money dedicated to patients
and this, in its turn, means that only affluent people will be able to afford
it. If we want to avoid such a situation, the only possible solution is
primary prevention, that set of measures aimed at removing the causes of
a disease, rather than diagnose it precociously or cure it when it has
already grown manifest. At present, no pharmaceuticals show any real
activity against the effects the interaction between non biodegradable and
non biocompatible nanoparticles and organism has, and the most they
can do is a simple palliation, and that in case they are correctly
prescribed, something that means a correct diagnosis has been issued.
From our point of view, we have already a basic knowledge
to understand what the possible risks are and how and where they
can be expressed. Identifying risks does not mean at all rejecting
nanotechnologies on principle and the business hoped for as some
scientists and entrepreneurs claim and fear, but means the intention to
drive their development safely in order to exploit their great potential in
the best and most lasting way. Nanotechnology-based industries must
have taken precautions and probably check their staff from the health
point of view, but the question is: How? And how much do they really
274 Nanopathology
want to know? If, for example, one of their employees develops a cancer,
how willing are they to investigate on a possible correlation between
working place exposure and disease? Cancer morbidity is growing more
and more common in industrialized areas, and children are not spared by
that trend, too often hastily dismissed as an inescapable fatality caused
by wrong parental genes. One of the likely causes, almost certainly not
the sole but nevertheless not negligible, is particulate pollution and there
is no reason to believe that engineered particles differ under this special
aspect from unintentionally produced ones. But there are also diseases
other than cancer that must be kept under control, and among them
troubles like chronic fatigue, insomnia, loss of memory and irritability
that are often attributed to stress and treated accordingly but that may
have nanoparticles as cause.
For all these reasons, referable to the prudence of common sense, we
suggest to apply the precautionary principle before an intentional,
uncontrolled, systematic, massive release of engineered nanoparticles in
the environment occurs.
Something we will be bound to face soon is the nanoscaled pollution
unintentionally released in some specific working places and, later, in the
environment.
If it can be easy or, at least, feasible to check the release of
engineered nanoparticles in the place where they are produced or
worked, it is extremely complicated and expensive, if not sometimes
impossible, to control them when they are already disseminated in the
environment.
The need to be competitive in the market has already induced
industries to develop a fair number of nanotechnological products. It is
the case of Samsung and Daewoo that constructed washing machines
with the NanoSilver technology. Inside the machines there is a tablet
releasing Silver nanoparticles (Silver oxide is well-known for its
bactericide activity). So, their presence in the fabric guarantees no odours
since the bacteria responsible for the problem are killed. Those
manufacturers claim also a lesser use of detergents. The use of molecule
of Silver oxide as a bactericide is regulated in “Private area and public
health area disinfectants and other biocidal products” under the Directive
98/8/EC (web ref. 4) but in the case of Silver oxide in nanoparticulate
form, its properties are due mainly to their being nanoscaled, so, the
reference to the disinfectants regulation is improper and should not be
considered valid. It may be curious to observe that after the news was
spread that the US EPA (Registration Review Schedule: Antimicrobial
Pesticides of October, 4, 2006) had declared that Silver Oxide (AgO) is
a pesticide, the Italian advertisements for those products changed and
they claim a release only of Silver ions and not nanoparticles. A direct
measure of this presence in the washing water could solve the mystery.
With a similar technology, Samsung make also air conditioners and
refrigerators (web ref. 5), (web ref. 6), (web ref. 7). From our point of
view, the clothes, having come out of washing full of “free”
nanoparticles, are very close to people who wear them and, as a
consequence of such a proximity, those people can inhale them. But
those particles can also be disseminated in the house and outside and the
waste water, not filtered efficiently at nanolevel, can disperse the
nanoparticles in the rivers and in the sea, providing unwanted “food” to
clams, fish, etc.
It is somehow absurd that the scientists that discovered the nano
phenomena be still busy assessing their potential toxicity, that the
funding agencies be just starting broadly funding the assessments of risks
and funding research, while industry is already prepared to release
massively and in an uncontrolled way biocide nanoparticles. Such
nanoparticles, despite being used as a substitute of bleach to disinfect
water for a long time, should be banned from therapeutic use because
their mechanism of action is still unknown, because, in contrast with
common antibiotic agents, nanoparticles may persist indefinitely active
since they are not degraded during their bacteria killing function and one
has to bear in mind that attacking the microbe world means to attack the
life substrate itself, and, for example, it has been found that biocide
Al2O3 nanoparticles reduce root growth in different crops, due to the
perturbation of the soil composition (Yang, L. et al., 2005).
Of course, releasing nanoparticles through air conditioners inside a
close ambient is a nonsense. We fear to inhale dust from incinerators,
from cement plants, from diesel engines, etc. and allow industries to
install a source of indoor pollution with nanotechnological particles in
the house. This pollution can be inhaled by children or pregnant women,
276 Nanopathology
which is something that must be taken into very serious consideration

and not dealt with so superficially as we are doing now.
The case of refrigerators releasing bactericide nanoparticles is
somewhat different. Only when its door is open the Silver pollution is
released in the domestic environment, but that pollution is likely to
contaminate food. Thus, the same device can potentially give two
different pathologies, the former starting from inhalation, the latter from
ingestion.
Special attention must be paid to what happened with asbestos. It was
used in brakes, buildings and innumerable further applications, from
where it was expected not to be released, but actually it was. The next
observation was that it accumulates in the lung, but as it did not produce
acute toxicity, it was happily overlooked until many years later with a
burst of lung and brain (where they may have entered through the
olfactory nerves) diseases related to cancer.
The results obtained through our research should put scientists,
technicians and politicians on the alert and, maybe, induce them to revise
their way of understanding the impact of environmental pollution upon
our health. We should start by considering a simple natural law, stating
that nothing can be created and nothing destroyed, but everything can
just be transformed. It is a matter of fact that all forms of combustion
change matter and produce micro- and nanoparticulate whose size
depends mainly on the temperature reached in the crucible, and that most
of that particulate, which, in many instances, is neither biodegradable nor
biocompatible, gets dispersed in the environment. So, the air, the soil,
water and vegetables are polluted and both humans and animals are the
victims of that condition through the inhalation of air and the ingestion of
food. The situation may grow worse to humans who eat animals whose
flesh is polluted, and nowadays that pollution may be imported from
places that are very far from where those humans live. In our century, it
is particularly hard to set boundaries to everything and pollution is no
exception.
What the majority of the systems aimed at getting rid of wastes do is
reduce them to a very small size, sometimes incomparably smaller than
the original, and disperse them. As already pointed out, the problem is
that the smaller the size of that dust, the more penetrating and aggressive
to the organism it is, and making it smaller and smaller does not seem
particularly wise.
One of the most promising results of our research is the possibility it
offers to trace back the source of pollution and the kind of exposure the
subject underwent, by comparing what we detect in his pathological
specimens with the environment where that subject lives or work, or the
food he eats. So, it will not be too difficult to understand whether the
strategy of having wastes “disappear” by making them small enough to
become hard to detect or we had better resort to different solutions.
To summarize, there are some pathologies strictly related to the
environmental pollution; so, in order to understand them it is necessary
to know and characterize that.
The following Tab. 9.1 summarizes the parameters we need to know
to explain these pathologies.
Tab. 9.1 Scheme of the factors that can trigger a disease.

278 Nanopathology
As a result of our research, but much is still ignored, being a foreign

body seems to be the most important among the factors inducing
pathologies. No matter what particles are made of, they are not
recognized as compatible with the environment they have entered and
that lack of compatibility is mainly due to their being a physical entity.
This is something that could be somewhat hard to classical toxicologists,
accustomed to reasoning rather in terms of ions or molecules and their
chemical noxiousness.
Size is also of the utmost importance, since, as often mentioned, the
smaller particles are, the worse their effect on organisms is. Clustering in
a tissue is a common phenomenon and, under certain aspects, for
instance in inducing the formation of granulation tissue, clustered
particles may behave like larger entities, but the sum of their surface
areas is in any case much larger than the one of bigger particles and the
result is an enhanced reactivity.
As just mentioned, surface area also influences pathogenicity and, in
particular, its ratio with volume, as the higher it is, the higher reactivity
particles have.
Another factor worth considering is particle shape. According to our
experience, still in need of being confirmed by more observations, a
needle-like shape like that of asbestos fibres is more capable of
penetrating tissues than a bulkier one.
Concentration is also a critical factor: unless particle penetrate cell
nuclei, in which case this consideration may lose much of its meaning, it
seems that a comparatively low concentration of particulate matter can
pass unnoticed and be tolerated by the host tissue that, on the contrary,
reacts when a threshold concentration is exceeded. That such
concentration exists is a fact hard to doubt of, but how to measure it is
now beyond our possibilities. We strongly suspect that that may depend
on a number of factors including size, shape, surface-area/volume,
chemical composition, tissue involved and its health condition, along
with the subject’s health and habits. If and how other chemicals or
pollutants in general interfere with that hypothetical threshold we do not
know, though it is reasonable to think so, and even to think that in some
cases a sort of synergy may occur.
A phenomenon we have often noted is how intake velocity can

influence the onset of a disease. In some cases, that can be rather easily
explained: Let us take a non-smoker and a smoker and have them inhale
quickly the same amount of dust. A non-smoker is likely to be able to get
rid of it because of the better efficiency of his respiratory system and, in
particular, of his muco-ciliary cells. In other instances, a quick intake
may happen when a certain organ is especially prone to capture those
particular particles and the consequence is they are not more evenly
distributed throughout the organism.
As to radioactivity, we have no direct experience, since we never
found any trace of it in the cases of soldiers we had a chance to observe
and in the case of peritoneal mesothelioma described in Chapter 4 we did
not measure it. So, radioactivity has been listed as a likely factor but, as a
matter of fact, we have no direct experience to support its influence.
Though probably not so important as being a foreign body, chemistry
is certainly a weighty factor of pathogenicity. It is only obvious that a
particle made of Arsenic is more toxic than an Iron particle sharing the
same shape and size. Most of the particles we observed, though, are
alloys and their behaviour is notoriously not the same as the sum of each
of their elemental components. If a particle gets corroded in a tissue, the
substances resulting from that chemical phenomenon are something
different from the material composing the original entity and, in that
case, the resulting toxicity can be the one well-known by toxicologists. It
is reasonable to think that chemistry influences the possibility of a
nanoparticle to be bound, for example, by a protein and thus acquire a
different dynamics.
It should not be surprising that every individual reacts to nanodust in
a way that can be different from his neighbour’s. The same individual
variability occurs with exposure to bacteria, viruses, parasites and most
other aggressions.
280 Nanopathology
9.3 The future
As described in Chapter 5, we know from war experience that as soon

as the hot, volatilized materials generated by explosions cool down,
nanosized particles are created and scattered in the environment. The
inhalation or ingestion of those, mainly metallic, particles by humans and
animals can bring about pathological effects. But for the formation of
nanoparticles as pollutants, there is not only warfare to blame. Car
engines, industry, incineration and, in general, all high-temperature
procedures are just a few examples of particulate pollutants producers.
So, it is easy to guess that in more than one case, the environment is
already contaminated.
As far as we know now, it is not possible to reclaim the environment
from nanoparticles and even in the future, with better technologies than
those available today, that may be extremely difficult. In many cases, the
proposed environmental remediation obtained with techniques making
use of nanoparticles does not solve the problem but simply shifts it from
a variety of pollution to another, this time induced by the nanoparticles
employed, nanoparticles that in most cases are not biodegradable and
stay in the environment, perhaps not the same as the one that had been
“cleaned”, but still somewhere on this planet, forever. But, being aware
of their possible adverse effects on the human health, even now it is not
so hard to confine them in a laboratory and study the safest procedures to
handle them.
At present, we have sufficient knowledge to suggest some simple
criteria to try and avoid toxicological problems in a “not alerted”
population, criteria that should be adopted as a precautionary measure.
The following ten “golden rules” can be adopted immediately and at
trifling costs:
1 - Nanoparticles contained in any product should be declared in the
product’s label, so that consumers are free to make a conscious selection.
2 - A notice should be clearly visible in any rooms or sites where
nanoparticles are released (e.g. some new air conditioners).
3 - Do not disperse intentionally, non biodegradable nanoparticles in
the environment, specially indoors, and take all necessary measures not
to disperse them unintentionally.
4 - Do not add non biodegradable nanoparticles in food.

5 - Do not introduce in any way (inhalation, ingestion, injection,
instillation, etc.) non biodegradable, non biocompatible nanoparticles in
living organisms.
6 - Do not put non biodegradable, non biocompatible nanoparticles in
contact with external or internal parts of living organisms.
7 - Do not have sexual intercourse with persons who were exposed to
micro- and nano-pollution. In case, the use of condoms is mandatory.
8 - Do not procreate if the male partner was exposed to pollution.
Assisted fertilization can help in such a situation, if only decreasing risk.
9 - Do not eat vegetables grown in a polluted area. Water can be
polluted as well.
10 - Do not smoke tobacco leaves grown in a polluted environment,
especially the one created by war. Weapon explosion can create a new,
mainly inorganic, form of pollution.
The criteria above are simple enough and should be applied
immediately for the sake of human and animal health, but more will
certainly be added as our knowledge advances. Those criteria give also
clear indications to the laboratories and companies busy in the
fabrication of new nanoproducts.
That does not mean that nanotecnologies must be sacked, it means
that certain products are safe, other that release nanoparticles not. We
need nanotechnologies to face the nanosized world and contrast that for
our safety and of our society.
9.4 A few reflections
“Memento, homo, quia pulvis es et in pulverem reverteris.” (Remember,

man, that you are dust and unto dust you shall return.) That is what
priests say when they put a pinch of ash on the head of the members of
their flock on Ash Wednesday. Is that sentence adapted from the Genesis
a prophecy?
Like a parasite, man has already destroyed a part of his habitat and
any being doing that is doomed to extinction. If he has done so in an
irreversible way is hard to say.
282 Nanopathology
Every day we can create forms of pollution that did not existed before
and whose impact on this planet where vegetables, animals and men
belong on an equal basis, has never been experimented. Can living
beings adapt themselves to the attack of those unexpected pollutants? In
many cases the question has no answer. What we can say is that Nature
rests on a delicate equilibrium and her times are long, much longer than
today’s Homo sapiens sapiens.
Nanopollution is a stimulus living beings had never been confronted
with before; at least, not to the present extent. We should not be mistaken
and think it does not have an effect or has a minor one just because those
particles are so small. What they do in comparison with the larger dust
generated by Nature or by less technological human activities, is change
their target. As an example, let us take 10-micron particles and 0.1-ones.
When inhaled, the former are likely to penetrate no farther than the
alveoli, and that just in case they can get that deep; the latter, instead,
reach the alveoli very easily, stay there no longer than few seconds and
enter quite easily the blood circulation to invade virtually all the
organism and be sequestered inside it by some tissue impossible to guess.
What we did in the last few years is create new forms of pollution as
side effects of novel, very sophisticated technologies. At the beginning,
we did not pay attention to it. We did not want to. And now that
pollution has already shown some of its possible interferences upon
human and animal organisms, but we are not entitled to say that we have
already seen everything. Much is likely to be still unknown and we shall
have to wait many years before being able to say something conclusive.
Let us take as an example asbestos. We can inhale it again and again and
in most cases nothing visible happens for a long time. But we know that
mesothelioma may take up to forty years after exposure before growing
manifest and we have not the least evidence allowing us to say that
something similar is not happening with nanodust. Nature does not come
to terms with what we do: she just lives according to her rules and is
blind and deaf to any protest. Once again, the way we have been doing
for millennia using our brain to develop more and more deadly weapons,
challenging Nature instead of living in harmony with her as all living
beings do, we are showing all our naïve arrogance and lack of wisdom.
As it always happens, those who will suffer the severest

consequences of nanopollution belong to the weaker part of society:
children and old people. Children can be affected even before they are
born as may be seen reading what is reported in Chapter 3 on malformed
feti, while old people are obviously more prone to be affected because of
their generally poorer health condition and less efficient defence systems.
But, if we look at the problem from a social standpoint, we must admit
that less affluent people will suffer more or, better, will be the first to
suffer. In many cases, large factories and incineration plants are built
in the poorest quarters of cities and in already environmentally
compromised areas where the rich do not live in a sort of more or less
conscious attempt to escape disaster, a disaster, however, that in the long
run cannot be dodged. As already described, nanoparticles, the most
aggressive particulate, behave like gases and their distribution is
homogeneous over large areas, knowing no boundaries. In addition to
that, as far as we know, physiological known barriers are hardly
effective.
Anthropic pollution will probably cause an increase in sterility and
miscarriages may become more and more common. This is not
psychological terrorism: more than one medical association has already
alerted governments and international agencies about that danger, while
symposia, congresses, meetings on nanotoxicity are being held all over
the world, articles are being written, TV and radio programs are being
broadcast, but the truth is that no meaningful political decision has ever
been taken while we keep approaching a no-return point at increasing
speed. Politicians are not only short-sighted but are often in bad faith, as
what they keep trying to do is hiding the problem. And they do that by
hiring scientists available to produce literature fit for that aim, and by
calling terrorists all the others.
So, an honest, effective political action on a global scale is
mandatory, as we cannot afford to live the way we are doing, with a
small - small from the population size point of view - country like the
United States consuming a disproportionate amount of energy resources
and being responsible for not much less than half the production of
greenhouse gases. All that, while emergent countries like China and
India dream to adopt as behavioural models our most reprehensible
284 Nanopathology
habits in terms of respect for planet we live on. We are spoilt children
and are the model which billions of people aim at.
Nature does not care for money, political colours, philosophical ideas,
sociology or any other superstructure we created. Nature does not even
care too much for Man. Man is just another animal and if the new
equilibrium is not compatible with Man’s life, let Man be extinct.
Dinosaurs reigned on this planet for a very long time, far longer than we
Men have done. Yet, something happened that had the scale tip just a
little and those enormous animals simply disappeared. We are not sure,
but perhaps a meteorite hit the Earth creating a huge, long-lasting cloud
of very fine dust, and that dust became an impassable screen to some
solar radiations, causing the extinction of organisms that were at the base
of the food chain. And the same dust has certainly been inhaled by
animals and ingested along with vegetables so that only the more
resistant to that kind of unusual aggression survived.
That pollution may cause disasters is something even some historians
maintain. One of the factors they add to those which led to the fall of the
Roman empire is heavy-metal pollution in their aqueduct pipelines and
in their wine. And if we look at the tragedies of Hiroshima and Nagasaki
in the light of what we know now, we must accept that the two very
high-temperature explosions which destroyed the cities and had all sorts
of materials sublime must have created millions of tons of nanodust
whose existence is impossible to deny and is proved by the black rain
that fell about half an hour after the bombing. And if nanodust was
present, it must have behaved the way all nanodust does. So, it is
reasonable to think that part of the pathologies that affected the
inhabitants of those cities and the malformations so common in their
offspring is due to particulate matter entered in their tissues.
One of our present themes of research is aimed at finding out if that
hypothesis is correct and will be one of the subjects of our next book.
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Appendix
Our analyses are mainly made with an Environmental Scanning Electron

Microscope (ESEM-Quanta, FEI-Company, The Netherlands)
The reason why that instrument is called “Environmental” is because,
besides being able to work in high and medium vacuum, it can analyze
samples at room, i.e. environmental, conditions. Thanks to that feature, it
can easily accept biological specimens. (Report, 1996)
In addition to that, there are 3 major characteristics that make ESEM
excellent for our use:
Pure secondary-electron detection;
Compatibility with water;
High chamber pressure.
1- Secondary electrons come from the atoms of the sample that derive
from interactions with the primary electrons of the beam. Those electrons
are responsible for the best sample resolution at low energies. ESEM can
detect pure secondary electrons in a gaseous environment. In
conventional scanning electron microscopes (SEM), the electronic gun
must work at high-vacuum conditions because of the high voltage it
needs. Because of that, the equipment accepts only dried and clean
samples, since humidity and dirt are incompatible with high vacuum. In
addition to that, the samples must be electron-conductive and so they
must be coated inside a vacuum chamber through a sputtering process
with Carbon or with a Gold/Palladium alloy. In order to remove the
electrostatic charge created by the electron beam, their morphology must
be flat and simple enough, so as to allow the coating to be homogeneous.
2- The imaging process is directly affected by the ionization

characteristics of the gas inside the chamber. Different gases can be
employed. There is no difficulty in having water vapour ionise, and that
yields excellent imaging performance, so biological samples can be kept
fully hydrated virtually indefinitely.
287
288 Nanopathology
3- The lowest pressure necessary to keep water in a liquid state is

about 4.6 Torr. In a conventional low-vacuum scanning electron
microscope, the highest chamber pressure does not exceed 2 Torr. High
chamber pressure permits to study samples with high outgassing rate as
well as to perform dynamic experiments like re-crystallization, or to
observe living cells in the medium.
Our ESEM is equipped with a Tungsten filament, an ionisation

secondary-electron sensor, a back-scattered sensor, SW controlled
Peltier cooled specimen stage and an Energy Dispersive System (EDS by
EDAX, USA).
The electron beam issued by the ESEM hits the sample surface and
produces X-ray fluorescence from the atoms in its path. The energy of
each X-ray photon is typical of the element which generated it. The EDS
microanalysis system collects the X-rays, sorts and plots them by energy,
and allows to identify the elements that produced the peaks in this energy
distribution. The system can identify the elemental composition of the
materials imaged for all elements with an atomic number greater than
Boron. Most elements can be detected at concentrations of order 0.1%.
In general, when analyzed by the EDS, biological samples from
healthy tissues show just the peaks of Carbon and Oxygen, but some
specialized tissues reveal also a content of other elements like Calcium
and Phosphorus in the bone, Iron in the liver and Phosphorus in the
brain.
When their ion size is below the sensitivity threshold of the system,
some elements undoubtedly present in a tissue are not detectable with
this method. Iron bound to haemoglobin in the red cells, for example,
cannot be seen, while when it precipitates in the liver as it does in
patients affected by siderosis, detecting it becomes quite easy.
If the tissue to be observed has been fixed chemically, dehydrated and
then embedded in paraffin, it can show small peaks of elements like
Sulphur or Chlorine not belonging to the specimen, and that depends on
the chemicals present in the liquids used for the treatments. In this case,
the spectrum of the biological tissue is necessary as a reference and must
be subtracted from that of the foreign body contained in it.
Appendix 289
Sample Preparation
The biological sample must be properly prepared in order to detect

inorganic particulate it contains.
Biological samples can be divided into two groups:
fresh tissues (not chemically fixed), bulk or sections; and
chemically fixed and paraffined tissues.
a – Fresh tissues are: 1- the bulk samples from surgery, including
bioptic and autoptic specimens, 2 – the blood , 3 – the sperm, and 4 –
the living cells from in-vitro simulation.
As briefly described above, they can be observed in wet conditions,
keeping the sample at a temperature of 5°C degree and at a relative air
humidity of 90-95%. Since these samples did not undergo any additional
chemical treatment, no pollution is possible.
According to the kind of sample, different preparation methods have
been developed.
The samples obtained from surgery are usually bulk and, to compare
them with the histo-pathological sections on which diagnoses are
performed, they are frozen at –20°C and then cut into 20-micron slices.
Then, they are deposited on an acetate sheet and observed under
ESEM.
Acetate was selected since, under Energy Dispersive Spectroscopy, it
emits only minor signals for Carbon and Oxygen, ever-present elements
in any biological specimen. When the section is very thin, the contribute
from the acetate substrate is just added to that from the biological
specimen and does not affect the measure carried out on the inorganic
debris.
Since every fresh sample represents a potential a risk of infections
(HIV, hepatitis) for the operators, we decided to subject them to a half-
hour’s immersion in formalin before handling it.
Bulk samples may be subjected to a critical-point dehydrating process
through the exchange of water with Nitrogen.
The blood and the sperm are smeared on the acetate sheet and then
covered with another sheet of the same material. A few seconds later,
they are delicately turned and separated in order to obtain a cell
290 Nanopathology
monolayer. The sheet is glued with a Carbon disc on a stub and observed
immediately.
The living cells, not treated in any way, are put on a support and
observed under ESEM directly in the medium. As, because of the
medium surface, observing the cell morphology is impossible, humidity
is slightly and smoothly decreased, thus allowing a mild dehydration
without prejudice to the morphology.
Sometimes, fixation is performed (a few drops of 2,5%
glutaraldehyde) so the morphology is preserved and, what is most
important, the interaction between cells and nanoparticles is preserved as
well.
b – The following method allows the observation of old samples
preserved in archives. Those samples had already been fixed, dehydrated
and embedded in paraffin and, from those blocks, we cut 20-micron-
thick sections. The slices are then suspended in warm water and
deposited on the acetate sheet. To remove the paraffin, the samples are
covered with a few drops of xylol and 98%-alcohol and, a few seconds
after, the excess of liquid is slid along the sheet edge and absorbed in
blotting paper. The samples can be observed in different modalities: in
high and low vacuum; in secondary and in backscattered electron mode.
Bibliography
Report from Environmental Scanning Electron Microscopy: An Introduction to ESEM by

Philips Electron Optics, Eindhoven, The Netherlands 1996, Robert Johnson
Associates California.
Index
1st Gulf War, 161 antibodies, 21

2nd Gulf War, 161 Antimony, 125
9/11, 251 anti-wear, 245
arteries, 45
A-bomb, 165 arthrosis, 5
acarus, 218 Asacol, 106
acrolein, 231 asbestosis, 22
Acute Lymphatic Leukaemia, 170 ash, 230
acute renal failure, 97 atelectasis, 49
adenocarcinoma, 65
adenoma, 99 Baghdad, 189, 232
adrenal adenoma, 186 Balkans War, x
adrenal-gland, 99 Barium, 3
Adriatic Sea, 220 beauty and sun-screen creams, 24
Agent Orange, 167 bicarbonate, 229
air conditioners, 259 biocompatibility, 16
Air Force Laboratory, Armament biocompatible, 16
Development and Test Center, biomass, 230
of Eglin Air Base, Florida, 161 biscuits, 244
Allied Forces, 165 Bismuth, 222
alloy, 9, 95 Black Sea, 244
Aluminium, 2 Blebs, 62
alveoli, 43 blood, 1
alveolitis, 55 blood-brain barrier, 18
Alzheimer’s disease, 13 blood-placenta barrier, 23
amalgam, 13 blurred vision, 253
ameloblastoma, 102 bombing, 54
amiotrophic lateral sclerosis, 13 bone, 47
ammonia, 229 bone marrow, 177
amyloidosis, 271 Bonfield, William, vii
angiogenic, 26 Boraschi, D, 24
animal feed, 248 Boron, 288
Antarctica, 248 Bosnia, 187
291
292 Nanopathology
bowels, 4 Chromium, 1
brain, 18 chronic fatigue, 40, 253
brakes, 266 cigarette, 22, 231
bread, 244 cirrhosis, 87
bronchial mucus, 136 clams, 275
bronchoalveolar lavage, 156 clothes, 208
Brownian motion, 270 coal, 266
bucky-balls, 272 Cobalt, 2, 24
Burning Mouth Disease, 102 colloids, 265
Burning Semen Disease, 121 colon, 5
combustion, 7
Cadmium, 21 composites, 13
calcification, 100 concrete, 259
Calcium, 2 coronary vessel, 78
cancerogenic, 28 cough, 155, 253
cancerogenicity, 40 Council of Down Manhattan, 252
Canova Activa, 240 COx, 46
Capitani, Federico, vii Creutzfeldt-Jacob, 271
car bodies, 227 criminal court, 220
Carbon, 1 Crohn’s disease, 5, 40
Carbon monoxide, 42 cryptogenic diseases, 39
cardiogenic mortality, 79 customized medicine, 273
cardiovascular, 23 cystic fibrosis, 5
cardiovascular diseases, 77 cytokine, 15
caval filtration, 5
cell, 16 Da Costa Syndrome, 166
cellular duplication, 271 Daewoo, 240
Cerium, 55 Danube, 244
Cesium, 248 De Morbis Artificium, 205
Chernobyl, 248 defecation, 245
chewing gum, 24 dendrimers, 17
children malformation, 116 dental filling resins, 13
chimney-sweepers’ cancer, 205 dental prosthesis, 5
China, 283 dental restorations, 13
Chlorine, 1, 76 Departments of the Veterans
chocolate, 245 Affairs, 166
cholangiocarcinoma, 93 depleted Uranium, x, 54
cholestasis, 4 depression, 167, 253
Index 293
derma, 145 explosions, 161

dermatitis, 145 exposure, 39, 253
Desert Storm Conflict, 167 eye, 174
detoxification, 252
diabetes, 91
faeces, 14
digestive system, 5
fat, 263
dioxin, 8, 42, 167
fatigue, 83
DNA, 12
FDA, 241
Drexler, Kim Eric, 6
Federal Institute for Risk
drug, 85
Assessment (BfR), 239
FEI-Company, 287
EDAX, 288
edema, 239 fertilizers, 230
Effort Syndrome, 168 feti, 41
enamel, 156 fever, 85
endometrium, 137 fibrosis, 3
endothelial cells, 23 fibrotic capsule, 29
endothelium, 45 filters used in Diesel cars (FAP),
Energy Dispersive Spectroscope 203
(EDS), 1 firemen, 252
energy, 266 firing ground, 121
engineered particles, 274 First Industrial Revolution, 266
environment, 22 fish, 200
Environmental Scanning Electron Fluorine, 104
Microscopy, 86 food, 88, 248
enzymes, 18 Ford Farm Firing Range, Aberdeen
EPA, 241 Proving Ground, MD, 162
epidemiologic studies, 166 foreign-body, 3, 20
epidemiological studies, 249 forgetfulness, 168
epidemiology, 269 formaldehyde, 231
Escalaplano, 195 fossil fuels, 266
Escherichia Coli, 240 Foundation for the Advancement of
ESEM, 28 Science and Education, 252
Europäische Akademie, 11 foundries, 266
Europe, 266 Framer, Joe A, 199
European Commission, vii Free Royal Hospital of London, 4
European Community, 5, 43 fullerene, 18
excipient, 102 furan, 8, 42, 228
294 Nanopathology
Gadolinium, 20 HIV, 289

Gambarelli, Andrea, vii Hodgkin’s lymphoma, 115
ganglioneuroma, 184 hydrocarbons, 42
gastrointestinal system, 4 Hydrogen, 2
Gatti, 171 hydroxyapatite, 14, 15
Germany, 239
giant cells, 58 immune system, 50
Gitelman’s syndrome, 54 immunological disease, 20
glass, 8 incinerators, 8
glioblastoma, 184 India, 283
globalization, 268 industrial sterility, 121
GMOs (Genetically Modified infarction, 45
Organisms), 268 inflammation, 4
Gold, 5 inflammatory pathologies, 39
gonads, 173 Information Office (OI), 165
granulation tissue, 278 inhalable, 19
granuloma, 31 inhalation, 22
granulomatosic reaction, 28 insomnia, 173
granulomatosis, 3 internalization, 23
granulomatous tissue, 29 interstitial pneumonia, 55
Ground Zero, 257 intestinal mucosa, 4, 151
Gulf War, 54 Iodine, 99
Gulf War Syndrome, 43, 95 Iraq, 54
Iron, 1
Hadzici, 169 irritability, 167
haemolysis, 4 Istituto Tumori (Cancer Institute)
haemolytic anaemia, 2 of Milan, 5
hamburger, 241 Italian Commission, 170
Harrod’s, 251 Italian senatorial commission, 249
Harvard School of Medicine in
Boston, 154 Kleinmann GmbH, 239
headache, 167 knee, 5
heart, 19 Korean conflict, 166
heavy oil, 220 Kosovo, 188
hepatic echinococcosis, 83
hepatitis, 289 landfills, 259
hepato-splenomegaly, 2 Lanthanum, 55
Herald Tribune, 251 Lateral Amyotrophic Sclerosis, 142
Index 295
Lavoisier, 214 metalloproteic precipitates, 144

law of conservation of matter, 228 metalloprotein, 145
Lead, 21, 91 metastasis, 19
LeGeros, Rachel, 101 mieloid leukaemia, 182
leiomyomatosis, 64 Minamata City, 243
lettuce, 225 miopathy, 135
lichens, 223 Molybdenum, 1, 111
lime, 229 monocytes, 30
liposomes, 239 Monsanto, 241
London, 251 mouth, 44
Los Angeles, 252 mouthwash, 145
Lou Gehrig’s disease, 166 muco-ciliary cells, 279
lung, 5 mule spinners, 206
lung barrier, 209 multi-organ failure, 100
lymph-nodal metastases, 187 multi-organ-diseases, 43
lymph-nodes, 19 multiple sclerosis, 13
lymphocytes, 30 muscles, 266
lymphomas, 40
Nagasaki and Hiroshima, 169
macrophage, 15 Nano Poly Technology, 241
mad cow disease, 248 nanocomposites, 13
Magic Nano, 239 nanopollution, 135, 205
Magnesium, 1, 2 nanoproduct, 239
malformed lambs, 121 nanospheres, 19
Malta, 121 Nanotea, 240
Manganese, 150 nanotechnologies, ix
Mantua, 212 nanotube, 18
Mars, 245 nasal congestion, 146
masks, 155 National Technical Information
mast cells, 30 Service (NTIS), 165
Maynard, 272 NATO, 167
medical devices, 16 neck, 20
Medical Geology, 43 Nemery, Ben, 69
medullar aplasia, 166, 169 Neodymium, 55
Mercury, 13 nephrocalcinosis, 43
mesenchymal tumors, 31 Neu-Laxova syndrome, 121
mesothelioma, 46 neurological, 19
mesoworld, 6 New York, 171, 251
296 Nanopathology
Nickel, 1 polychlorinated biphenyls, 228

nightmares, 253 polycyclic aromatic hydrocarbons,
Niobium, 66, 150 8, 206
Nitu, Lavinia, vii polycyclic organic matter, 228
non-Hodgkin’s lymphoma, 43 post-Vietnam Syndrome, 166
NOX, 46 Potassium, 2
nucleating agents, 71 power station, 220
Praseodymium, 234
oil wells, 249 precautionary principle, 274
olfactory nerves, 276 pre-neoplasia, 32
osteoblast, 15 prevention, 87
oxidative stress, 27, 270 primary electrons, 287
Oxygen, 3 pro-inflammatory, 27
prostate, 19
paints, 103 protein, 16, 265
Palladium, 287 pseudotumour, 49
Pančevo, 244 psyconeurosis, 166
pancreas, 91 pulmonary, 19
pancytopenia, 183 pulmonary intestitium, 204
Parkinson’s disease, 13 pulmonary thromboembolism, 70
peace-keeping missions, 176 Pulsator, 241
Percivall Pott, 205 PVC, 24
peritoneum, 150 pyrophoric, 162
pesticides, 230
phagocytosis, 80 quantum dots, 21
Phosphorus, 1 quartz, 22
photoablation, 270
Phynox, 2 Racemose ossification, 48
physiological barriers, 121 Ramazzini, 205
platelets, 114 rats, 28
Platinum, 102, 212 red cells, 65
PM10, 22 redox potentials, 270
PM2.5, 43 renal failure, 2
pneumoconioses, 22 respiratory, 23
pneumothorax, 5 respiratory system, 5
Po, 220 retrosternal pain, 155
Polesine Camerini, 220 Revell, Peter, 4
pollutants, 8 rhabdomyosarcoma, 32
Index 297
rheumatoid arthritis, 81 soldier, 99

RIS (Crime Scene Investigative SOx, 46
Institute) of Parma (Italy), 197 sperm, 42
risk factors, 22 spleen, 84
Ru(bpy), 240 squamocellular carcinoma, 52
stainless steel, 72
Salto di Quirra, 195 steatosis, 89
Salvatori, Roberta, vii steelworks, 215
Samarium, 234 stomach, 44
Samsung, 240 Stress-Activated Proteins (SAP),
Sapporo, 214 271
Sarajevo, 169 stroke, 19
sarcoidosis, 5 Strontium, 95
Sardinia, 121 Sulphur, 2
sclerodermia, 135 sunscreen creams, 103
sclerosis, 139 surface/size-ratio, 22
Second Industrial Revolution, 266 sweat, 254
second World War, 166 Symptoms and Signs of Ill-Defined
secondary electrons, 287 Conditions, 167, 168
secondary particles, 229
Selenium, 240 talc, 113
September 11, 253 Taniguchi, Norio, 11
Shake, Slim, 240 Tantalum, 66
shale oil, 206 tea, 240
ship, 227 teeth, 13
shortness of breath, 155 Teflon, 104
siderosis, 87 teratogenic, 248
Sighinolfi, Gianluca, vii testicle, 121, 205
Silicon, 1 Thorium, 109, 150
silicosis, 22 threshold, 10
Silver, 14 thrombectomy, 70, 78
Silver Nano, 240 thrombi, 5
skin, 205 thrombus, 23
skin carcinomas, 206 thyroid, 187
skin pesudolymphoma, 43 tiredness, 2
sleep disturbance, 167 Titanium, 2
Smalley, Richard, 272 tobacco, 55, 231
Sodium, 2 Tokyo Science University, 11
298 Nanopathology
toner, 210 Vanadium, 221

tongue, 142 vein thrombosis, 40
toothbrushes, 247 vena cava filter, 1
toothpaste, 14, 247 veterans, 116
Tossini, Daniela, vii Vietnam, 167
toxicity, 212 Virchow’s Triad, 77
traceability, 203 volcanoes, 265
Tub U, 241
tuberculous pleuritis, 46 wall paints, 24
Tungsten, 66 Washing Machines, 241
Twin Towers, 251 wastes, 204
typhoid fever, 151 water, 266
tyres, 266 wear, 14
Wegener’s granulomatosis, 43
U.N. Environment Agency, 169 welding, 207
U.N. Environment Protection white cells, 114
Agency, 167 wind, 266
Udine, 215 Woodrow Wilson International
ultrafine, 23 Center, 272
umbilical circulation, 41 World Trade Center, 266
University of New York, 101
University of Siena (Italy), 5 X-ray, 288
Uranium, 54, 150
urine, 2 Yttrium, 109, 150
US, 272 Yugoslavia, x, 116
USSR, 162
uterus, 118, 137 Zimmer, Rene, 239
Zinc, 21
vaccines, 165 Zirconia, 13
vagina, 137 Zirconium, 13
vaginal mucosa, 118

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N A N O PAT H O L O G Y

THE HEALTH IMPACT OF

British Library Cataloguing-in-Publication Data

Rhaimie - Nanopathology.pmd 1 10/3/2007, 5:24 PM

Nanotechnologies can represent a real innovation for human society

Recent European research projects (Nanosafe1, Nanosafe2,

“The ability to think differently today than yesterday is

1. How the Whole Thing Began or the Logic Path Towards a

2. In-vitro and in-vivo Biological Behaviour of Micro and Nanoparticles 11

3. Clinical Cases: Lung, Blood, Liver, Kidney, Digestive System,

4. Six “Detective Stories” 135

4.5 The 5th case ...................................................................................... 152

5. War and Nanoparticles 161

6. Nanoparticles in the Environment and Working Places 203

7. Nanoparticles in Food, Cosmetics and Other Products 239

8. New York 9/11 251

9. The Future and Prevention Criteria 265

How the Whole Thing Began or the

So, a percutaneous echo-assisted biopsy was performed in the liver

surprisingly enough but undoubtedly environmental dust, while in one

fragments which are obviously smaller and, as a consequence,

In-vitro and in-vivo Biological Behaviour of

To give an idea of the smallness of these things, we can compare

Fig. 2.1 Scale of dimensions.

Applications in the medical field are already possible and a brief

2.2 Nanoparticles and medical devices

Applications of nanotechnologies are being experimented in a number of

At present, dental restorations employ amalgams, composites, and Gold

The improvement of these properties is only partial, since the wear

Besides dental implantology, another field of application of

possibly, self-lubricating. A possible solution can be the development

2.2.3 Nanostructured surfaces

Implantable medical devices are bound to employ biocompatible

catheters in order to avoid blood clotting was not particularly successful,

2.2.4 Drug delivery

Other emerging and especially promising applications are those in the

Nevertheless, it has been demonstrated that different drugs bound to

2.3 The results of the nanopathology project

In 2001, we coined a new word: “nanopathology”, meaning by that the

The activation of IL-8, E-selectin and ICAM-1 is regulated by the

that leads to the formation of in-vitro capillaries within a three-dimen-

The Cobalt nanoparticles showed an angiogenic behaviour, that can

2.3.1 In-vivo experiments

The 5 different materials mentioned above (Cobalt, Nickel, silica, titania,

The other materials (PVC, silica, and titania) developed only a

Tab. 2.3.1 Synopsis of the histological findings.

Material 6-month period 12-month period

Note: B= bulk, NP= Nanoparticles; * Animals were euthanized after 8 months. # No

TiO2 – In detail: a thin, fibrous, moderately capillarized pocket

bulk showed discrete chronic inflammatory infiltrates. No particles could

Fig. 3.1 serves to understand the paragraphs that follow, as it

Medical Geology is a new discipline of investigation that studies the

We selected the particles shown according to peculiarity of

Tab. 3.2.1 List of the investigated samples of the lung.

Much literature exists describing an increase of lethal episodes in

L. A. et al., 2007), (Schwarze, P. E. et al., 2006), (Newby, J. A. et al.,

Pleural mesothelioma has been described in literature for a long time

disease were never exposed to that pollutant. It is a well-known

Fig. 3.2.3 Asbestos fibre in a pleural mesothelioma. (marker 10 µm).

Fig. 3.2.5 shows a case of round atelectasis (Stark, 1982) (Menzies

Like all sarcoidoses, lung sarcoidosis is characterized by the presence

Fig. 3.2.7 concerns a case affected from tubercolo-silicosis. At optical

Fig. 3.2.7 Low-magnification image of tubercolo-silicosis with wide Calcium phosphate

Fig. 3.2.8 High-magnification image of tubercolo-silicosis. At such magnification, Silicon-