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Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP001
Nanotoxicology
Experimental and Computational Perspectives
View Online
Issues in Toxicology
Series Editors:
Diana Anderson, University of Bradford, UK
Michael D. Waters, Michael Waters Consulting, USA

Timothy C. Marrs, Edentox Associates, UK
Editorial Advisor:
Alok Dhawan, CSIR-Indian Institute of Toxicology Research, India
Titles in the Series:

1: Hair in Toxicology: An Important Bio-Monitor
2: Male-mediated Developmental Toxicity
3: Cytochrome P450: Role in the Metabolism and Toxicity of Drugs and
other Xenobiotics
4: Bile Acids: Toxicology and Bioactivity
5: The Comet Assay in Toxicology
6: Silver in Healthcare
7: In Silico Toxicology: Principles and Applications
8: Environmental Cardiology
9: Biomarkers and Human Biomonitoring, Volume 1: Ongoing Programs
and Exposures
10: Biomarkers and Human Biomonitoring, Volume 2: Selected
Biomarkers of Current Interest
11: Hormone-Disruptive Chemical Contaminants in Food
.
12: Mammalian Toxicology of Insecticides

13: The Cellular Response to the Genotoxic Insult: The Question of
Threshold for Genotoxic Carcinogens
14: Toxicological Effects of Veterinary Medicinal Products in
Humans: Volume 1
15: Toxicological Effects of Veterinary Medicinal Products in
Humans: Volume 2
16: Aging and Vulnerability to Environmental Chemicals: Age-related
Disorders and their Origins in Environmental Exposures
17: Chemical Toxicity Prediction: Category Formation and Read-Across
18: The Carcinogenicity of Metals: Human Risk Through Occupational
and Environmental Exposure
19: Reducing, Refining and Replacing the Use of Animals in
Toxicity Testing
20: Advances in Dermatological Sciences
21: Metabolic Profiling: Disease and Xenobiotics
22: Manganese in Health and Disease
23: Toxicology, Survival and Health Hazards of Combustion Products
24: Masked Mycotoxins in Food: Formation, Occurrence and
Toxicological Relevance
View Online
25: Aerobiology: The Toxicology of Airborne Pathogens and Toxins

26: Chemical Warfare Toxicology, Volume 1: Fundamental Aspects
27: Chemical Warfare Toxicology, Volume 2: Management of Poisoning
28: Toxicogenomics in Predictive Carcinogenicity
29: Human Stem Cell Toxicology

30: The Comet Assay in Toxicology, 2nd edition
31: Computational Systems Pharmacology and Toxicology
32: Ecotoxicology and Genotoxicology: Non-traditional Terrestrial Models
33: Ecotoxicology and Genotoxicology: Non-traditional Aquatic Models
34: Oxidative Stress and Redox Signalling in Parkinson’s Disease
35: Nanotoxicology: Experimental and Computational Perspectives
.
How to obtain future titles on publication:

A standing order plan is available for this series. A standing order will bring
delivery of each new volume immediately on publication.
For further information please contact:

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Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-FP001 View Online
View Online
Nanotoxicology
Experimental and Computational
Perspectives
Edited by
Alok Dhawan
CSIR-Indian Institute of Toxicology Research, India
Email: alokdhawan@iitr.res.in
Diana Anderson
University of Bradford, UK
Email: d.anderson1@bradford.ac.uk
and
Rishi Shanker
CSIR-Indian Institute of Toxicology Research, India
Email: rishishanker@gmail.com
.
Issues in Toxicology No. 35
Print ISBN: 978-1-78262-158-4

PDF eISBN: 978-1-78262-392-2
EPUB eISBN: 978-1-78801-259-1
ISSN: 1757-7179
A catalogue record for this book is available from the British Library
r The Royal Society of Chemistry 2018
All rights reserved
Apart from fair dealing for the purposes of research for non-commercial purposes or for
private study, criticism or review, as permitted under the Copyright, Designs and Patents
Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not
be reproduced, stored or transmitted, in any form or by any means, without the prior
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The Royal Society of Chemistry is a charity, registered in England and Wales,

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For further information see our web site at www.rsc.org
Printed in the United Kingdom by CPI Group (UK) Ltd, Croydon, CR0 4YY, UK
Foreword
Over the past 20 years nanotechnology has been considered as the tech-
nology of the 21st century. A huge amount of development has occurred
during this period, not only of new applications and products already on the
market, but also of possibilities that may become available within the next
10 to 15 years. Besides innovation and market demands, products are in-
creasingly directly related to the use or implementation of nanomaterials.
The manufacturing of nanomaterials and the reinforcement of products by
the integration of nanomaterials leads directly to the interaction of nano-
materials with humans and the environment. With this increasing number
of scenarios for human exposure to the newly synthesized nanomaterials,
the evaluation of possible adverse effects of such materials is of utmost
importance. This is the reason for a flood of publications on the newly
created discipline of nanotoxicology, which was named as such in 2004 for
.
the first time. More than 25 000 publications have increased the knowledge
of the biological effects of nanomaterials in various species. However, we
have also realized that many such studies were undertaken in a misleading
sense as most of them have been mechanistic studies, but authors as well as
readers have often used them as toxicological studies. Moreover, as har-
monized test protocols and adapted OECD guidelines have not been avail-
able for nanomaterials in the past 15 years, many results are not reliable and
difficult to interpret and repeat. Therefore, we have to take up the challenge
of establishing such protocols and harmonize methods for a better tox-
icological research approach to nanomaterials in the near future.
But this is not the only challenge. Toxicology itself is being orientated into
a new direction. Animal testing is more and more under suspicion to deliver
false-positive or false-negative results, the existing in vitro methods, on the
other hand, also have many restrictions or shortcomings. Thus, the

Nanotoxicology: Experimental and Computational Perspectives
Edited by Alok Dhawan, Diana Anderson and Rishi Shanker
Published by the Royal Society of Chemistry, www.rsc.org
vii
View Online
viii Foreword
development of new testing strategies and new assay platforms is an urgent

necessity.
This book brings together a number of outstanding researchers in this
field to deliver an actual and comprehensive picture of the international
science of nanomaterials toxicology and to present an insight into future

opportunities. Here we find the actual knowledge about characterization of
newly synthesized nanomaterials as one of the major challenges for bio-
logists and toxicologists. Moreover, working with standardized operating
procedures (SOPs) is for many researchers an ‘‘old chestnut’’ and many
funding programmes are looking for the newest and most relevant project.
This neglects the fact that this is merely increasing the knowledge of new
mechanisms, but does not help legal regulation. Here we would need better
and more reliable data, which have probably been produced in round robins,
so we can rely on these during the regulatory steps. We should not forget the
necessity for rules in toxicological studies as otherwise we shall, in the long
term, fail in our attempts to regulate nanomaterials for the growing market.
This book is a helpful and excellent example of how we can proceed into a
future where toxicology is on the right track!
Harald Krug
Empa – Swiss Federal Laboratories for Material Science & Technology
Switzerland
.
Preface
The enabling nature of nanotechnology has infused engineered

nanomaterial-based products in the market worldwide. To date more than
1800 nano-based products in the domains of personal care, health and
fitness, electronics, textiles, sports, ceramics, energy, automotive, medicine,
agriculture and environmental remediation, are already in use. There are
more than 40 different types of engineered nanomaterials (ENMs) being
used in various products, these include ENMs of metals, metal oxides,
carbonaceous materials and composites. These ENMs are being inadver-
tently released into the aerial, terrestrial and the aquatic environment.
The need for test methods, validation of existing protocols, methodologies
and procedures for safety as well as, in vitro and in vivo toxicity assessment of
ENMs has been, and will continue to be the objective of studies in the area of
nanomaterial toxicology. The empowering nature of nanotechnology makes
.
the development and validation of toxicity assessment methodologies all

the more necessary if we are to engineer safe materials at the nanoscale,
exploit nanomaterials in drug delivery, determine the fate of ENMs in the
environment and safely dispose of nanoproducts.
This book, Nanotoxicology: Experimental and Computational Perspectives,
incorporates several comprehensive nanomaterial toxicity protocols, which
will serve as a highly useful and ready resource for research students and
scientists working in regulatory toxicology as well as biomedical, bio-
chemical and pharmaceutical sciences.
The authors have actively contributed to peer-reviewed scientific literature
in the area of nanomaterial toxicology. The book addresses the aforesaid
issues in thirteen chapters, each with a comprehensive discussion of the
methodology. The challenges faced in nanomaterial toxicity assessment
have been addressed in the very first chapter of the book to introduce the
reader to this new component of toxicological sciences.

ix
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x Preface
The initial chapters of the book describe the protocols for the chemical
synthesis of nanoparticles for a range of applications including usage in
biomedicine. The subsequent chapters describe the in vitro and in vivo
protocols for toxicity assessment of engineered nanomaterials, develop-
ments of alternate test models, emerging systems toxicology approaches,

organ-on-chip systems and needs in clinical toxicology assessment.
A component of the book also explores the need for safe nanoparticles for
biological and therapeutic use and computational approaches for modeling
of interactions of nanoparticles with biomolecules. The third section of the
book explores the paradigm of health hazard and risk assessment of these
novel materials in medicine and the environment. In this book the regu-
latory perspective, based on the risk associated with the application of
nanomaterials in nanomedicine, the status of existing assays and emerging
approaches to frame policy of health risk assesssment have been discussed.
The book also introduces the readership to international guidelines and
recommendations for safety and risk assessment.
This book epitomizes the long-term scientific association that the editors
have enjoyed with the authors and is a culmination of their collaborative
efforts.
Alok Dhawan
Diana Anderson
Rishi Shanker
.
Editor Biographies
Professor Alok Dhawan is currently Director of the CSIR-Indian Institute of

Toxicology Research (CSIR-IITR), Lucknow. He also served as Founding
Director of the Institute of Life Sciences, and the Dean for Planning and
Development at Ahmedabad University, Gujarat. Before joining as Director
of CSIR-IITR, he held a number of different scientific positions, such as –
Scientist, Senior Principal Scientist, Principal Scientist, etc. He obtained his
PhD in Biochemistry from the University of Lucknow, India in 1991. He was
awarded a DSc (Honorary) by the University of Bradford, UK in 2017, and was
a Visiting Scholar at Michigan State University, USA, and BOYSCAST Fellow
at the Universities of Surrey, Wales & Bradford, UK. Professor Dhawan
started the area of nanomaterial toxicology in India and published a guid-
ance document on the safe use of nanomaterials. His group elucidated
the mechanism of toxicity of metal oxide nanoparticles in human and
.
bacterial cells, and his work has been widely cited. He set up a state-of-the-
art nanomaterial toxicology facility at CSIR-IITR as well as at the Institute of
Life Sciences. Professor Dhawan has won several honours and awards in-
cluding the INSA Young Scientist Medal in 1994, CSIR Young Scientist Award
in 1999, the Shakuntala Amir Chand Prize of ICMR in 2002, and the Vigyan
Ratna from the Council of Science and Technology, UP in 2011. His work in
the area of nanomaterial toxicology has won him international accolades
as well, and he was awarded two Indo-UK projects under the prestigious
UK-IERI programme. He was also awarded two European Union Projects
under the FP7 and New INDIGO programmes. He founded the Indian
Nanoscience Society in 2007. In recognition of his work he has been elected
a Fellow of the Royal Society of Chemistry, UK; the National Academy of
Sciences, India; The Academy of Toxicological Sciences, USA; The Academy
of Environmental Biology; The Academy of Science for Animal Welfare;
The Society of Toxicology, India; The Indian Nanoscience Society; and of

xi
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xii Editor Biographies
The Gujarat Science Academy. He was also Vice President of the Environ-
mental Mutagen Society of India (2006–7), and Member of The National
Academy of Medical Sciences; The United Kingdom Environmental Mutagen
Society; and The Asian Association of Environmental Mutagen Societies,
Japan. He has to his credit over 125 publications in peer-reviewed inter-

national journals, 18 reviews/book chapters, four patents, two copyrights
and has edited two books. He is the Editor-in-Chief of the Journal of Trans-
lational Toxicology published by American Scientific Publishers and serves
on the Editorial Board of Mutagenesis, Nanotoxicology, Mutation Research
Reviews, and other journals of repute.
Professor Diana Anderson currently holds the Established Chair in

Biomedical Sciences at the University of Bradford, UK. She obtained her
first degree at the University of Wales and second degrees in the Faculty
of Medicine, University of Manchester. After tutoring at the University of
Sydney, Australia, she became a research worker in the Department of
Cancer Studies at the University of Leeds and at the Paterson Laboratories,
Christie Hospital, Manchester. In 1974, she was appointed Head of
Mutagenesis Studies at ICI’s Central Toxicology Laboratory. Professor
Anderson joined BIBRA International in 1981 as Head of Genetic and
Reproductive Toxicology and became Assistant Director and Group Forum
Co-ordinator in 1987. In 1992, she became Senior Associate and Co-ordinator
of External Affairs at BIBRA. She has attended various management courses.
She has served on the editorial board of 10 international journals, has over
450 publications, has edited/authored 9 books and guest-edited 9 special
.
issues of 4 international journals. She has been/is Series Editor of books in

Current Toxicology for John Wiley & Sons, and Issues in Toxicology for the Royal
Society of Chemistry. As an active Committee member, she has been Vice-
President of the Institute of Biology and was Chair of the Scientific Committee
of the International Association of Environmental Mutagen Societies. She is
Chief Examiner for the International Diploma in Toxicology under the aegis of
the Royal Society of Biology. As a successful supervisor for 28 PhD, 1 MSc and
2 MPhil students, she is currently supervising another 5 PhDs. She has been
external examiner for 27 PhDs and was External Examiner for the Department
of Genetics at the University of Wales, Swansea. She has been invited to speak
at many international meetings and chair many symposia. New research
laboratories in India and Korea have been established with her help under the
auspices of the British Council and UNIDO. Funded by various international
agencies, scientists from America, Australia, the Czech Republic, Italy, India,
Iran, Korea, Poland, Serbia, Spain and Turkey have received training under
her supervision. She has organized both national and international meetings
and was a member of various national (e.g., MRC Advisory Board and
Veterinary Products Committee) and international committees, including
the European Union Scientific Committee for Animal Nutrition (SCAN). For
2007–10 she won an award as Yorkshire Enterprise Fellow and was nominated
for an Albert Einstein Award In Science in 2016. She is a consultant for many
View Online
Editor Biographies xiii
international organizations, such as the WHO, NATO, TWAS, UNIDO and

the OECD.
Professor Rishi Shanker currently serves as Consultant to CSIR-Indian In-

stitute of Toxicology Research and Advisor to ABC Genomics (India) Private

Ltd at Biotechnology Park, Lucknow, India. He has served as Professor &
Associate Dean at the School of Arts & Sciences, Ahmedabad University,
Gujarat, India (2014–16). Prior to joining Ahmedabad University, he served
as Chief Scientist and Area Coordinator of the Environmental & Nanoma-
terial Toxicology Groups at CSIR-IITR, Lucknow (2001–13). He also served as
Principal Scientist at the CSIR-National Environmental Engineering Research
Institute, Nagpur and set up a state-of-the-art laboratory in the area of En-
vironmental Biotechnology (1991–2001). He obtained his Masters in Bio-
chemistry from University of Lucknow and a PhD in Environmental
Microbiology & Toxicology from CSIR-IITR & CSJM University (1985). Professor
Shanker’s post-doctoral research addressed methanogen microbiology, deep
subsurface microbiology and protein engineering at the University of London
and Pennsylvania State University, USA (1987–90).
Professor Shanker’s research contributions range from genetically
engineered bacteria for bioremediation and molecular probes for pathogen
detection to alternate models in toxicity assessment of chemicals and
engineered nanomaterials. He has successfully steered over 34 national and
international research projects including the Indo-US program: Common
Agenda for Environment, the Indo-Swiss Program in Biotechnology, the
Indo-German, EU FP7, EU New Indigo & Inno Indigo program and Unilever.
.
He has more than 80 publications and 20 reviews/book chapters to his credit

in aforesaid areas. He has mentored 11 PhD students and 20 research
projects of Masters’ students in life sciences.
His work on ‘pathogen detection and water quality’ received recognition in
form of Vigyan Ratna, conferred by the Government of Uttar Pradesh, India.
He was awarded the Visiting Research Fellowship of the Society for General
Microbiology, UK at the Anaerobic Microbiology Laboratory, Queen Mary
University of London. He has served as Visiting Scientist and Visiting
Professor at the University of Washington, Seattle, USA, the Pasteur Institute &
CEA, France, the and the Pohang University of Science & Technology, Republic
of Korea. He is a Founder Fellow of the Indian Nanoscience Society and a
Fellow of the Society of Toxicology, India.
.
Contents
Chapter 1 Nanotoxicology: Challenges for Biologists 1

Ashutosh Kumar, Sanjay Singh, Rishi Shanker and
Alok Dhawan
1.1 Introduction 1
1.2 The Hurdles in Toxicity Evaluation of NMs 2
1.3 ENM Interference with Toxicity Test Methods 4
1.3.1 Interference of NPs with Metabolic Activity
Detection Assays 4
1.3.2 Interference of NPs in Assays for Cell Death
Measurement 5
1.3.3 Interference of ENPs with Immunoassays 5
1.3.4 Interference of ENMs in Assays with
.
Enzymes 6
1.3.5 Interference with Measurement of
Free Radicals Generated due to ENM
Exposure 6
1.3.6 Interference in Cellular Uptake Assays 7
1.3.7 Interference with Cell Culture Media
Components 8
1.3.8 Interference due to Oxidation State Change
in Redox-active ENMs 9
1.3.9 Misinterpretation of TEM Images 9
1.3.10 Interference with the Comet Assay 10
1.3.11 Interference in Micronucleus Assays 11

xv
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xvi Contents
1.4 Conclusions 11
Acknowledgements 12
References 12
Chapter 2 Chemical Synthesis of Nanoparticles for Diverse

Applications 17
Dhrubajyoti Kalita and Jayesh R. Bellare
2.1 Introduction 17
2.2 Synthesis of Metallic/Bimetallic
Nanostructures 19
2.2.1 Solvothermal Synthesis 19
2.2.2 Reduction and Monolayer Capping in
Aqueous and Non-aqueous Media 21
2.2.3 Polymer-capped Metal Nanoparticles and
Bimetallic Nanoclusters 22
2.2.4 Synthesis in Microemulsion 24
2.3 Synthesis of Polymer Nanoparticles 25
2.3.1 Emulsification/Solvent Evaporation 25
2.3.2 Chemical Precipitation/Nanoprecipitation 27
2.4 Synthesis of Magnetic Nanoparticles 27
2.4.1 Co-precipitation 27
2.5 Conclusions 28
References 29
.
Chapter 3 Synthesis of Nanoparticles for Biomedical Applications 39

Sanjay Singh
3.1 Introduction 39
3.2 Synthesis of Gold Nanoparticles 40
3.2.1 Chemical Methods 40
3.2.2 Physical Methods 42
3.2.3 Biological Methods 42
3.2.4 Biological Applications of Gold
Nanoparticles 43
3.3 Synthesis of Magnetic Nanoparticles 46
3.3.1 Co-precipitation Method 47
3.3.2 Microemulsion Method 47
3.3.3 Sol–gel Method 49
3.3.4 Sonochemical Method 49
3.3.5 Flow Injection Method 49
3.3.6 Hydrothermal Method 50
View Online
Contents xvii
3.3.7Biological Applications of Magnetic

Nanoparticles 51
3.4 Synthesis of Carbon Nanotubes 52
3.4.1 Arc Discharge Method 53
3.4.2 Laser Ablation Method 54

3.4.3 Chemical Vapour Deposition Method 55
3.4.4 Biological Applications of CNTs 56
3.5 Synthesis of Quantum Dots 58
3.5.1 Biological Applications of Quantum Dots 60
3.6 Synthesis of Silica Nanoparticles 63
3.6.1 Stöber Method 64
3.6.2 Microemulsion Method 65
3.6.3 Biological Applications of Silica
Nanoparticles 66
3.7 Toxicity Considerations of Nanomaterials 68
3.8 Conclusions 69
Acknowledgements 70
References 70
Chapter 4 Protocols for In vitro and In vivo Toxicity Assessment

of Engineered Nanoparticles 94
Ashutosh Kumar, Violet Aileen Senapati and Alok Dhawan
4.1 Introduction 94
.
4.2 Cytotoxicity 96
4.2.1 MTT Assay 96
4.3 Live/Dead Assessment 99
4.3.1 Propidium Iodide Uptake Assay 99
4.3.2 Trypan Blue Exclusion Test 101
4.4 Genotoxicity 104
4.4.1 Single-cell Gel Electrophoresis Assay 104
4.4.2 The CBMN Assay 109
4.5 Immunotoxicity 113
4.5.1 Cytokine Release 113
4.5.2 Immunophenotyping 115
4.6 Oxidative Stress 119
4.6.1 ROS Generation 119
4.6.2 Glutathione Estimation 122
4.6.3 Lipid Peroxidation Determination 126
4.7 Conclusions 130
Acknowledgements 130
References 130
View Online
Nanotoxicology: Challenges for Biologists 5
to reduced cell metabolism) could be masked by the absorbance of these NMs

due to their optical density, thus providing a false impression of lack of
toxicity.20 Additionally, some ENMs can inhibit colour formation, thus ex-
hibiting falsely a cytotoxic effect. In the case of CNTs it has been seen that
Published on 03 November 2017 on http://pubs.rsc.org | doi:10.1039/9781782623922-00001
CNTs absorb formazan molecules and protect them from being metabolized
by cells.21 Under such circumstances, the decreased colour formation occurs
due to the direct effect of CNTs on the MTT dye rather than a decrease in the
number of living cells, thus leading to the false interpretation of a cytotoxic
effect. Aluminium NPs also demonstrate a strong interaction with MTT dye
resulting in significant misinterpretation of associated cytotoxicity.22
1.3.2 Interference of NPs in Assays for Cell Death

Measurement
Cell death measurement induced by the exposure of ENMs is usually
measured by lactate dehydrogenase (LDH) quantification in cell super-
natant. In principle, LDH reduces INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-
5-phenyl tetrazolium chloride) in the presence of NADH þ H1 (reduced
b-nicotinamide adenine dinucleotide) to give a pink water-soluble formazan,
which is quantified by light absorbance measurement in the visible region.23
The interference of ENM dispersions with the optical detection of INT might
have happened due to the intrinsic absorbance of ENMs in the visible region
(e.g., metallic NPs) and/or ENMs inducing reduction/oxidation under the
influence of cellular biochemical reactions.24 Engineered nanoparticles
(ENPs) may also react with INT leading to altered absorbance thus variability
.
in assay outcomes. Some ENMs are highly catalytically active, thus may
alter the intrinsic properties of assay reagents. Recently, experiments on
copper-containing compounds, such as CuCl2, CuSO4 and Cu powder,
showed interactions with LDH assay components.25 It was found that copper-
containing compounds incubated with LDH showed inhibition of LDH
calibrator detection depending on Cu salt dose. Recently, Kroll et al.26 reported
that inhibition of the LDH assay in the presence of fine-sized ZnO NMs was
dependent on the composition more than the size or surface. Han et al.,27
found Ag NPs (B35 nm) deactivate LDH due to interaction of synthesis
reagents with LDH whereas, TiO2 NPs (25 nm) were also found to interfere
with the LDH assay due to adsorption of LDH molecules on the surface.27
1.3.3 Interference of ENPs with Immunoassays

ENMs, due to their high surface area, are prone to adsorbing antibodies or
other immunoassay components on their exposed surfaces. CNTs have been
found to adsorb the antibodies on their surface, thus interfering with the
assay results leading to misinterpretation.21,28 Similarly, Kroll et al.26 also
reported TiO2 NPs as potential adsorbers of interleukins to their surfaces
leading to a reduced level of IL-8 into dispersion. This was found to be
concentration-dependent, where TiO2 concentrations below 10 mg cm2 did
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Co’o marquez, co’o grão senhor;
Porque como é perdedor
E mofino adredemente,
E faz um sangue excellente
A qualquer dos ganhadores,
Qualquer d’aquelles senhores
Por fidalgo egual o afóra.
Entendeis-me agora?
A UMA BRIGA
QUE TEVE CERTO VIGARIO COM UM OURIVES POR CAUSA DE
UMA MULATA
Naquelle grande motim,

Onde acudiu tanta gente,
A titulo de valente
Tambem veiu o Valentim:
Puxou pelo seu faim,
E tirando-lhe a barriga,
Você si quer que lh’o diga,
Disse ao ourives da prata,
Na obra d’esta mulata
Mette muita falsa liga.
Briga, briga.
É homem tão desalmado,

Que por lhe a prata faltar,
E estar sempre a trabalhar
Bate no vaso sagrado?
Não vê que está excommungado
Porque com tanta fadiga
A peça da egreja abriga
Numa casa excommungada,
Com censura reservada,
Pela qual Deus o castiga?
Briga, briga.
Porque com modos violentos
A um vigario tão capaz,
Sôbre os quatro que já traz,
.. lhe põe quatrocentos?
Deixe-se d’esses intentos,
E reponha a rapariga,
Pois a repô-la se obriga
Quando affirma que a possue;
E si ésta razão não conclue,
Vai ésta ponta á barriga.
Briga, briga.
Senhor ourives, você

Não é ourives de prata?
Pois que era essa mulata,
Que cobre ou tambaca é?
Restitua a moça, que
É peça da egreja antiga;
Restitua a rapariga,
Que se vingará o vigario
Talvez no confessionario,
Ou talvez na desobriga:
Briga, briga.
Á mulata ja lhe pêja

De trocar odre por odre,
Porque o leigo é membro podre,
E o padre é membro da egreja:
Sempre esta telha gotteja,
Sempre dá grão esta espiga:
E a obra da rapariga
Quer desfazer esta troca,
E deixando a vossa toca,
Quer fazer co’o padre liga:
Briga, briga.
Largue-lhe a mulata, e seja
Logo logo a bom partido,
Que como tem delinquido,
Si quer recolher á egreja:
Porque todo o mundo veja
Que quando a carne inimiga
Tenta a uma rapariga,
Quer no cabo, quer no rabo,
A egreja vence o diabo
Como outra qualquer estriga:
Briga, briga.
A PRISÃO DE DUAS MULATAS
POR UMA QUERELLA QUE D’ELLAS DEU O CELEBRE
CAPITÃO DOMINGOS CARDOZO, DE ALCUNHA O MANGARÁ,
PELO FURTO DE UM PAPAGAIO
CLAUSULAS
A quem não causa desmaio

Esta querella recente,
As mulatas na corrente
Em falta do papagaio?
Eu de verdade não caio
Nesta justiça em rigor:
Ora este tal prendedor
Quem seria, ou quem será?
Mangará.
Diz que em tudo tinha graça

A Jandaya, abrindo a boca,
Dizendo o seu toca toca,
Meu papagaio, quem passa?
Mangará, que vai á caça.
Porém na presente perda
Passará a beber ...,
Que não faltará quem vá.
Mangará.
As mulatas no seu mal
Vão disfarçando a paixão,
Pois lhe deu dura prisão
O papagaio real.
Diz que para Portugal
Lindamente dava o pé;
Mas uma articula que
O contrario provará.
Mangará.
Provará que elle gostára,

E que não satisfizera,
E muitas cousas dissera
Si o papagaio fallára:
Que o capitão intentára
Pagar-lhe em bens de raiz,
Pois sendo mangará quiz
Transfigurar-se em cará.
Mangará.
Pondo-se o pleito em julgado,

Dar testimunhas procura.
Com o primo Rapadura,
E um compadre seu Melado:
Mas ha de ficar borrado,
Como o tal primo ficou,
Quando a mulata o purgou
Naquelle triste araçá.
Mangará.
Na gaiola ou passarada,
Onde as duas pobres vejo,
A primeira entrou sem pejo,
Mas a segunda pejada:
Arrebentou de embuchada
Um presozinho pequeno,
Que creado com veneno
Damno jámais lhe fará.
Mangará.
Todo o povo, que isto vê,

Pergunta em seu desabono,
Não ao papagaio, ao dono,
Que casta de passaro é:
Eu por lhe fazer mercê
Dou definição cabal:
Um contrafeito asnaval,
Empenhado em Pirajá.
Mangará.
EPIGRAMMA
SOBRE VARIOS ASSUMPTOS
Sahiu a satyra má,

E empurraram-m’a os perversos,
Porque em quanto a fazer versos
Só eu tenho geito cá:
Noutras obras de talento
Só eu sou o asneirão;
Mas sendo satyra, então
Só eu tenho entendimento.
Acabou-se a Sé, e envolto

Na obra o Sete-caveiras
Enfermou de. .....,
E fez muito verso solto:
Tu, que o poeta motejas,
Sabe que andou acertado
Que pôr na obra o louvado
É costume das egrejas.
Correm-se muitos carneiros

Na festa das Onze mil,
E eu com notavel ardil
Não vou ver os cavalleiros:
Não os vou ver, não se espantem,
Que algum testimunho temo,
Sou velho, pelo que gemo,
Não quero que m’o levantem.
Querem-me aqui todos mal,
E eu quero mal a todos,
Elles e eu por nossos modos
Nos pagamos tal por tal:
E querendo eu mal a quantos
Me têm odio tão vehemente,
O meu odio é mais valente,
Pois sou só, e elles tantos.
Algum amigo que tenho,

Si é que tenho algum amigo,
Me aconselha que o que digo
O cale com todo o empenho:
Este me diz, diz-me este outro
Que me não fie d’aquelle;
Que farei, si me diz d’elle
Que me não fie aquelle outro?
O Prelado com bons modos

Visitou toda a cidade.
É cortezão na verdade,
Pois nos visitou a todos;
Visitou-se a pura escripta
O povo e seus comarcãos,
E os réus de mui cortezãos
Hão de pagar a visita.
A cidade me provoca
Com virtudes tão commuas,
Ha tantas cruzes nas ruas,
Quantas eu faço na boca:
Os diabos a seu centro
Foi cada um por seu cabo,
Nas ruas não ha um diabo,
Ha-os de portas a dentro.
As damas de toda côr,
Como tão pobre me veem,
As mais lástima me têm,
As menos me têm amor:
O que me têm admirado
É fecharem-me o poleiro
Logo acabado o dinheiro:
Deviam ter-m’o contado.
DESCREVE
O P. RACIONAL E VERDADEIRAMENTE QUEIXOSO OS
EXTRAVAGANTES MEIOS COM QUE OS EXTRANHOS
DOMINAM INDIGNAMENTE SOBRE OS NATURAES NA SUA
PATRIA
ROMANCE
Senhora Dona Bahia,

Nobre e opulenta cidade,
Madrasta dos naturaes,
E dos extrangeiros madre.
Dizei-me por vida vossa

Em que fundais o dictame
De exaltar os que aqui vêm,
E abater os que aqui nascem.
Si o fazeis pelo interesse

De que os extranhos vos gabem,
Isso os paisanos fariam
Com conhecidas vantagens.
E supposto que os louvores

Em bocca propria não valem,
Si tem força essa sentença,
Mór força terá a verdade.
O certo é, patria minha,

Que foste terra de alarves,
E inda os resabios vos duram
D’esse tempo e d’essa edade.
Haverá duzentos annos,
Nem tantos podem contar-se,
Que ereis uma pobre aldêa,
Hoje sois rica cidade.
Então vos pizavam Indios,

E vos habitavam Cafres,
Hoje chispais fidalguias,
E arrojais personagens.
A essas personagens vamos,

Sôbre ellas será o debate,
E Deus queira que o vencer-vos
Para envergonhar-vos baste.
Sae um pobrete de Christo

De Portugal ou de Algarve,
Cheio de drogas alheias
Para d’ahi tirar gages.
O tal foi sóta tendeiro

De um christão novo em tal parte,
Que por aquelles serviços
O despachou a embarcar-se.
Fez-lhe uma carregação

Entre amigos e compadres,
E ei-lo commissario feito
De linhas, lonas, beirames.
Entra pela barra dentro,

Dá fundo, e logo a entonar-se
Começa a bordo da nau
Co’um vestidinho flammante.
Salta em terra, toma casas,
Arma a botica dos trastes,
Em casa come balêa,
Na rua antoja manjares.
Vendendo gato por lebre,

Antes que quatro annos passem
Já tem tantos mil cruzados,
Conforme affirmam pasguates.
Começam a olhar para elle

Os paes, que já querem dar-lhe
Filha e dote, porque querem
Homem que coma e não gaste.
Que esse mal ha nos mazombos:

Têm tão pouca habilidade,
Que o seu dinheiro despendem
Para haver de sustentar-se.
Casa-se o meu matachim,

Põe duas negras e um pagem,
Uma rede com dous Minas,
Chapéu de sol, casas grandes.
Entra logo nos pelouros,

E sae do primeiro lance
Vereador da Bahia,
Que é notavel dignidade.
Já temos o canastreiro,
Que inda fede aos seus beirames,
Metamórphosis da Terra,
Transformado em homem grande:
E eis-aqui a personagem.
Vem outro do mesmo lote,
Tão pobre e tão miseravel,
Vende os retalhos, e tira
Commissão com coiro e carne.
Co’o principal se levanta,

E tudo emprega no Iguape,
Que um engenho e tres fazendas
O tem feito um homem grande.
E eis aqui a personagem.
De entre a chusma e a canalha

Da maritima bagagem,
Fica ás vezes um christão,
Que apenas benzer-se sabe.
Fica em terra resoluto

A entrar na ordem mercante,
Troca por covado e vara
Timão, balestilha e mares.
Arma-lhe a tenda um ricaço,

Que a terra chama magnate,
Com pacto de parceria,
Que em Direito é sociedade.
Com isto o marinheiraz

Do primeiro jacto ou lance
Bota fóra o .. breado,
As mãos assimilha em guantes.
Vende o cabedal alheio

E dá com elle em levante,
Vai e vem, e ao dar das contas
Diminue, e não reparte.
Prende aqui, prende acolá,
Nunca falta um bom compadre,
Que ou entretenha o credor,
Ou faça esperar o alcaide.
Passa um anno, e outro anno,

Esperando que elle pague,
Que uns lhe dão para que ajuncte,
E outros para que engane.
Nunca paga, e sempre come,

E quer o triste mascate,
Que em fazer a sua estrella
O tenham por homem grande.
O que elle fez foi furtar,

Que isso faz qualquer birbante,
Tudo o mais lhe fez a terra,
Sempre propicia aos infames:
Vem um clerigo idiota,

Desmaiado como um gualde,
Os vicios com seu bioco,
Com seu rebuço as maldades.
Mais sancto do que Mafoma

Na crença dos seus Arabes,
Lettrado como um matullo
E velhaco como um frade.
Hontem simples sacerdote,

Hoje uma gran’dignidade,
Hontem selvagem notorio,
Hoje encoberto ignorante.
A tal beato fingido
É força que o povo acclame,
E os do governo se obriguem,
Pois edifica a cidade.
Chovem uns e chovem outros

Co’os officios e os logares,
E o beato tudo apanha
Por sua muita humildade.
Cresce em dinheiro e em respeito,

Vai remettendo as fundagens,
Compra toda a sua terra,
Com que fica um homem grande:
Vêm outros lotes de requiem,

Que indo a tomar o caracter,
Todo o Reino inteiro cruzam
Sobre a chança viandante.
De uma provincia para outra

Como dromedarios partem,
Caminham como camellos,
E comem como selvagens.
Mariolas de missal,
Lacaios missa-cantantes,
Sacerdotes ao burlesco,
Ao serio ganhões de altares.
Chega um d’estes, e toma amo,

Que as capellas dos magnates
São rendas que Deus creou
Para estes Orate fratres.
Fazem-lhe certo ordinario,
Que é dinheiro na verdade
Que o Papa reserva sempre
Das cêas e dos jantares.
Não se gasta, antes se embolsa,

Porque o reverendo padre
É do sancto neque demus
Meritissimo confrade.
Com este cabedal juncto

Já se resolve a embarcar-se,
Vai para a sua terrinha
Com fumos de ser abbade:
Vêem isto os filhos da terra

E entre tanta iniquidade,
São taes que nem inda tomam
Licença para queixar-se.
Sempre vêem, e sempre callam,

Até que Deus lhes depare
Quem lhes faça de justiça
Esta satyra á cidade.
Tão queimada e destruida

Te vejas, torpe cidade,
Como Sodoma e Gomorra,
Duas cidades infames.
Que eu zombe dos teus visinhos,

Sejam pequenos ou grandes,
Gozos, que por natureza
Nunca mordem, sempre latem.
Porque espero entre os Paulistas
Na Divina Magestade,
Que a ti São Marçal te queime,
E a mim São Paulo me guarde.
RETRATO
DO GOVERNADOR ANTONIO DE SOUSA DE MENEZES
CHAMADO O BRAÇO DE PRATA
SYLVA
Oh! não te espantes não, dom Antonia,

Que se atreva a Bahia
Com expremida voz, com plectro esguio,
Cantar ao mundo teu rico feitio,
Porque é já velho em poetas elegantes
O cahir em torpezas similhantes.
Da pulga acho que Ovidio tem já escripto,

Luciano do mosquito,
Das rans Homero, e d’estes não desprézo,
Que escreveram materia de mais pezo
Do que eu, que canto cousa mais delgada,
Mais chata, mais subtil, mais esmagada.
Quando desembarcaste da fragata,

Meu Dom Braço de prata,
Cuidei que a esta cidade, tonta e fatua,
Mandava a Inquisição alguma estatua,
Vendo tão expremido salvajola,
Visão de palha sobre um mariola.
O rosto de azarcão afogueado,

E em partes mal untado,
Tão cheio o corpazil de godilhões,
Que o tive por um sacco de melões,
Vi-te o braço pendente da garganta,
E nunca prata vi com liga tanta!

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Nanotoxicology experimental and

computational perspectives 1st Edition

Computational Nanotoxicology Challenges and

Nanobiotechnology: Human Health and the Environment

Computational and experimental analysis of functional

Computational and Experimental Chemistry Developments

Computational and Manufacturing Strategies Experimental

Computational and Experimental Simulations in

Corrosion Preventive Materials and Corrosion Testing

Nanotoxicology : Toxicity Evaluation, Risk Assessment

Michael D. Waters, Michael Waters Consulting, USA

Titles in the Series:

12: Mammalian Toxicology of Insecticides

25: Aerobiology: The Toxicology of Airborne Pathogens and Toxins

29: Human Stem Cell Toxicology

How to obtain future titles on publication:

For further information please contact:

Issues in Toxicology No. 35

Print ISBN: 978-1-78262-158-4

r The Royal Society of Chemistry 2018

All rights reserved

The Royal Society of Chemistry is a charity, registered in England and Wales,

For further information see our web site at www.rsc.org

Issues in Toxicology No. 35

development of new testing strategies and new assay platforms is an urgent

science of nanomaterials toxicology and to present an insight into future

The enabling nature of nanotechnology has infused engineered

the development and validation of toxicity assessment methodologies all

Issues in Toxicology No. 35

ments of alternate test models, emerging systems toxicology approaches,

Professor Alok Dhawan is currently Director of the CSIR-Indian Institute of

Issues in Toxicology No. 35

xii Editor Biographies

Japan. He has to his credit over 125 publications in peer-reviewed inter-

Professor Diana Anderson currently holds the Established Chair in

issues of 4 international journals. She has been/is Series Editor of books in

Editor Biographies xiii

international organizations, such as the WHO, NATO, TWAS, UNIDO and

Professor Rishi Shanker currently serves as Consultant to CSIR-Indian In-

stitute of Toxicology Research and Advisor to ABC Genomics (India) Private

He has more than 80 publications and 20 reviews/book chapters to his credit

Chapter 1 Nanotoxicology: Challenges for Biologists 1

Issues in Toxicology No. 35

Chapter 2 Chemical Synthesis of Nanoparticles for Diverse

Chapter 3 Synthesis of Nanoparticles for Biomedical Applications 39

3.3.7Biological Applications of Magnetic

3.4.2 Laser Ablation Method 54

Chapter 4 Protocols for In vitro and In vivo Toxicity Assessment

Nanotoxicology: Challenges for Biologists 5

to reduced cell metabolism) could be masked by the absorbance of these NMs

1.3.2 Interference of NPs in Assays for Cell Death

1.3.3 Interference of ENPs with Immunoassays

Si hoje vos falla de perna

Naquelle grande motim,

É homem tão desalmado,

Senhor ourives, você

Á mulata ja lhe pêja

A quem não causa desmaio

Diz que em tudo tinha graça

Provará que elle gostára,

Pondo-se o pleito em julgado,

Todo o povo, que isto vê,

Sahiu a satyra má,