Inhalation Toxicology, 19(Suppl.

1):189–198, 2007
Copyright c Informa Healthcare USA, Inc.
ISSN: 0895-8378 print / 1091-7691 online
DOI: 10.1080/08958370701496202

Genotoxicity of Poorly Soluble Particles

Roel P. F. Schins
Institut für umweltmedizinische Forschung (IUF) an der Heinrich-Heine-Universität Düsseldorf,
Düsseldorf, Germany

Ad M. Knaapen
Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute
Maastricht (NUTRIM), University of Maastricht, Maastricht, The Netherlands

Poorly soluble particles such as TiO2 , carbon black, and diesel exhaust particles have been
evaluated for their genotoxity using both in vitro and in vivo assays, since inhalation of these
compounds by rats at high concentrations has been found to lead to tumor formation. Two
principle modes of genotoxic action can be considered for particles, referred to as primary and
secondary genotoxicity. Primary genotoxicity is defined as genetic damage elicited by particles
in the absence of pulmonary inflammation, whereas secondary genotoxicity implies a pathway
of genetic damage resulting from the oxidative DNA attack by reactive oxygen/nitrogen species
(ROS/RNS), generated during particle-elicited inflammation. Conceptually, primary genotox-
icity might operate via various mechanisms, such as the actions of ROS (e.g., as generated
from reactive particle surfaces), or DNA–adduct formation by reactive metabolites of particle-
associated organic compounds (e.g., polycyclic aromatic hydrocarbons). Currently available
literature data, however, merely indicate that the tumorigenesis of poorly soluble particles in-
volves a mechanism of secondary genotoxicity. However, further research is urgently required,
since (1) causality between pulmonary inflammation and genotoxicity has not yet been estab-
lished, and (2) effects of inflammation on fundamental DNA damage responses that orchestrate
mutagenesis and carcinogenic outcome,that is, cell cycle arrest, DNA repair, proliferation, and
apoptosis, are currently poorly understood.

Cancer is believed to result from a sequence of events that
include genetic alterations involving activation of oncogenes,
inactivation of tumor suppressor genes, aberrant controls in cell
cycle checkpoint genes, and impaired DNA repair processes
(Hoeijmakers, 2001; Vogelstein & Kinzler, 2004; Feinberg
et al., 2006). DNA damage is considered as an important as-
pect of carcinogenesis, and therefore genotoxicity assays have
been introduced to allow for improved cancer risk assessment
strategies. Many tests are nowadays available to screen for gene
mutations, chromosomal mutations and aneugenic effects (ane-
uploidy), as well as to measure formation of DNA strand breaks,
DNA adducts, and the induction of DNA damage repair (Vainio
et al., 1992; McGregor et al., 1999).
Received 3 July 2006; accepted 23 November 2006.
This work is supported in part by grants from the German Research
Council (DFG-SFB503, IGK-738) [RS], and the Netherlands Organi-
sation for Scientific Research (NWO grant 916.46.092) [AK].
Address correspondence to Roel P. F. Schins, Institut für
umweltmedizinische Forschung (IUF) an der Heinrich-Heine-
Universität Düsseldorf, Auf’m Hennekamp 50, D-40225 Düsseldorf,
Germany. E-mail: roel.schins@uni-duesseldorf.de
Genotoxicity studies have also been performed in the field
of particle toxicology. This is notably the case for particles that
have been found to cause tumors in experimental animals, that is,
crystalline silica, titanium dioxide (TiO2 ), carbon black (CB),
and diesel exhaust particles (DEP). Key examples of in vitro
and in vivo genotoxicity studies, as performed with these particle
types, are listed in Table 1. Genotoxicity indicator tests that have
been specifically applied in particle research include assays to
determine DNA strand breakage (e.g., alkaline elution, comet as-
say), assays to detect DNA base damage or polycyclic aromatic
hydrocarbons (PAH)–DNA adducts (e.g., high-performance liq-
uid chromatography [HPLC], 32 P-postlabeling), chromosomal
damage tests representing possible clastogenic activity of par-
ticles (e.g., micronuclei), and measurements of gene mutations
(e.g., Ames test, HPRT assay, DNA sequencing) (Jaurand, 1996;
Schins, 2002). Apart from their importance for risk assessment
purposes (Greim et al., 2001), genotoxicity studies such as those
listed in Table 1 also provided benefit to our knowledge about
mechanisms that possibly drive carcinogenesis by particles (Hei,
2000; Knaapen et al., 2004). As such, for instance, genotox-
icity studies are highly relevant with regard to the observed

189
190 R. P. F. SCHINS AND A. M. KNAAPEN

TABLE 1
Particles that have been investigated for genotoxicity both in vitro and in vivo

Particle In vitro In vivo

Crystalline silica
Titanium dioxide
Carbon black
Diesel exhaust particles
or diesel exhaust∗
Note. (+) Positive studies; (−) negative studies; ∗ indicates studies with diesel exhaust.
(+) DNA strand breaks (Liu et al., 1996;
Zhong et al., 1997; Schins et al., 2002)
(+) Oxidative DNA damage (Schins et al.,
2002)
(+) Micronuclei (Nagalakshmi et al., 1995;
Liu et al., 1996)
(−) Chromosome abberrations
(Nagalakshmi et al., 1995)
(−) HPRT-mutations (Driscoll et al., 1997)
(+) Oxidative DNA damage (Gurr et al.,
2005)
(+) Micronuclei (Lu et al., 1998; Rahman et
al., 2002)
(+) Sister chromatid exchanges (Lu et al.,
1998)
(−) Chromosome abberrations
(Endo-Capron et al., 1993)
(−) HPRT-mutations (Driscoll et al., 1997)
(+) DNA strand breaks (Zhong et al., 1997;
Don Porto Carero et al., 2001)
(−) Bulky DNA-adducts (Borm et al., 2005)
(−) HPRT-mutations (Driscoll et al., 1997)
(+) DNA strand breaks (Don Porto Carero et
al., 2001; Dybdahl et al., 2004)
(+) Sister chromatid exchanges (Keane et
al., 1991)
(+) DNA strand breaks (Knaapen et al.,
2002a) (+) Oxidative DNA damage (Seiler
et al., 2001) (-) Oxidative DNA damage
(Albrecht et al., 2005) (+)
HPRT-mutations (Driscoll et al., 1997)
(−) Oxidative DNA damage (Rehn et al.,
2003)
(−) Bulky DNA adducts (Gallagher et al.,
1994)
(+) HPRT-mutations (Driscoll et al., 1997)
(+) Oxidative DNA damage (Gallagher et
al., 2003)
(−) Bulky DNA adducts (Gallagher et al.,
1994; Borm et al., 2005)
(+) HPRT-mutations (Driscoll et al., 1997)
(+) Oxidative DNA damage (Ichinose et al.,
1997; Risom et al., 2003, Dybdahl et al.,
2004)
(+) Oxidative DNA damage (Sato et al.,
2000) * (+) Bulky DNA adducts (Wong et
al., 1986)∗
(+) Bulky DNA adducts (Gallagher et al.,
1994)∗
(+) Mutation frequencies/spectra (transgenic
rat) (Sato et al., 2000)∗

epidemiological associations between ambient particulate mat-
ter (PM) exposure and lung cancer (e.g., Pope et al., 2002). Be-
cause of the highly complex and variable composition of PM, it
is by no means clear which components are causally linked to
the genotoxic and carcinogenic actions. Last but not least, the
requirement of validated genotoxicity assays will also remain
to enable appropriate hazard identification for newly developed
particles. This is especially the case for nanoparticles (i.e., di-
ameter <100 nm in at least one dimension), such as engineered
nanoparticles and nanotubes (Borm & Schins, 2006). Concerns
about issues of genotoxicity and carcinogenicity of nanoparticles
can be justified at least to some extent for the following reasons:
1. For well-investigated ultrafine particles (e.g., TiO2 , CB),
intrinsically greater toxicity is observed in rodent studies
when compared on equal mass dose with their non-ultrafine
counterparts (Donaldson et al., 2002; Oberdorster et al.,
2005).
2. Ultrafine particles including TiO2 and CB have been shown
to cause tumor formation in experimental animals after high
exposures (Borm et al., 2004).
3. Studies have revealed that some nanoparticles can penetrate
into cellular nuclei and hence may directly interfere with the
structure and function of the genomic DNA (Geiser et al.,
2005; Chen & von Mikecz, 2005).
 GENOTOXICITY OF POORLY SOLUBLE PARTICLES
INTERPRETATION OF GENOTOXICITY STUDIES FOR
RISK ASSESSMENT
As mentioned before, the predominant reason for the im-
portance of genotoxicity studies in particle research lies in the
fact that several particles have been shown to elicit tumors in
experimental animals. These include so-called poorly soluble
particles (PSP) of low toxicity such as CB and TiO2 , as well
as particles with intrinsically high toxicity, such as crystalline
silica particles (Greim et al., 2001; Borm et al., 2004). However,
for various reasons one should be cautious about the interpre-
tation and proper extrapolation of results obtained in genotox-
icity studies with particles. These are discussed in this review.
Above all, DNA damage represents only one constituent among
the overall highly complex processes that orchestrate carcino-
genesis, and many aspects of this disease are still inadequately
understood (Hoeijmakers, 2001; Vogelstein & Kinzler, 2004;
Feinberg et al., 2006). Moreover, particles have several unique
properties (Fubini et al., 2001), which may obscure the valid-
ity and interpretation of genotoxicity tests performed with these
materials. At least to some extent these properties also explain
for several apparent mechanistic and dosimetrical discrepancies
as observed among and between in vitro genotoxicity studies
and in vivo genotoxicity studies (see Table 1). These aspects are
discussed in detail in this article.
PARTICLE RISK ASSESSMENT AND THE CONCEPT OF
PRIMARY VERSUS SECONDARY GENOTOXICITY
In view of its relevance for risk assessment purposes, the con-
cept of the discrimination between primary and secondary path-
ways of genotoxicity has been introduced (Greim et al., 2001;
Knaapen et al., 2004) (see Figure 1). The aspect of secondary
(versus primary) genotoxicity originates from observations that
various poorly soluble particles are tumorigenic in rat lungs,
rather irrespective of their chemical composition, but obviously
after chronic high exposures that are associated with overload
and persistent inflammation (Greim et al., 2001; Borm et al.,
2004). Herein, secondary genotoxicity can be defined as ge-
netic damage resulting from reactive oxygen/nitrogen species
(ROS/RNS) (and possibly other mediators), which are known
to be generated during particle-elicited inflammation from acti-
vated phagocytes (macrophages, neutrophils). Of major impor-
tance for risk assessment, secondary genotoxicity is considered
to involve a threshold; its value is set to be determined by the
exposure concentration that will trigger inflammation and over-
whelm antioxidant and DNA damage repair capacities in the
lung (Greim et al., 2001). Notably, since the ability of particles
to elicit inflammation has been shown to depend on various prop-
erties, including particle solubility and surface reactivity, such
genotoxicity threshold is anticipated to vary with each type of
particle. As opposed to secondary genotoxicity, primary geno-
toxicity is defined as genetic damage elicited by particles in
the absence of inflammation. Hence, primary genotoxicity may
be principally indicated from in vitro genotoxicity/mutagenicity
191
FIG. 1. Pathways of primary and secondary genotoxicity as im-
plicated in particle carcinogenesis. In particle-induced carcino-
genesis, particles are considered to impact both on genotoxicity
and cell proliferation. Genotoxic events in proliferating cells
may, if subject to erroneous repair, result in irreversible genetic
changes (mutagenesis) and ultimately lead to cell transforma-
tion. To prevent mutagenesis, DNA damage induction is associ-
ated with processes of cell cycle arrest with increased allowance
for DNA repair, and/or the induction of apoptosis. With regard
to genotoxicity, two modes of actions of particles can be con-
sidered. Secondary genotoxicity is considered to be driven by
the oxidative attack of DNA by reactive oxygen/nitrogen species
(ROS/RNS) as generated during particle-elicited inflammation
from activated macrophages and/or neutrophils. Primary geno-
toxicity implies an induction of genetic damage by particles
in the absence of inflammation. Such primary damage may be
either direct (e.g., as may result from a physical interaction be-
tween a particles and the genomic DNA) or via various possible
indirect pathways (e.g., formation of ROS by metals and or-
ganic constituents, or, e.g., formation of bulky DNA adducts by
metabolites of available polycyclic aromatic hydrocarbons).
studies or in vivo studies performed with particle concentrations
that do not elicit significant pulmonary inflammation. As is dis-
cussed at a later stage, various physicochemical properties may
be involved in eliciting primary genotoxicity, and this may occur
via direct or indirect mechanisms (Table 2).
CHARACTERISTICS OF PARTICLE INDUCED
GENOTOXICITY
For particles, specifically also those that show tumorigenesis
in experimental animals, two predominant types of DNA damage
can be considered: (1) DNA damage by oxidative attack, and (2)
formation of bulky DNA adducts by organic constituents (e.g.,
polycyclic aromatic hydrocarbons, PAH). Obviously, the relative
importance of these respective insults depends on the physico-
chemical characteristics/chemical composition of the particle in
question, as well as on the relative impacts of primary versus
secondary pathways of genotoxicity.
192 R. P. F. SCHINS AND A. M. KNAAPEN

TABLE 2 First, oxidative attack of DNA may result from surface as-
Pathways of particle-mediated oxidant generation and their sociated free radicals or oxidative groups such as SiO. and
involvement in processes of primary and secondary SiO. 2 , which are present on crystalline silica particles (Fubini &
genotoxicity Hubbard, 2003). Oxidants may also be generated by particles in
aqueous suspension, for example, via Haber–Weiss reactions by
Process/mechanism Genotoxicity available metals (Hardy & Aust, 1995; Donaldson et al., 1997).
Intrinsic ROS generation from Primary (direct or Finally, organic compounds associated with particles, such as
particles. indirect) polycyclic aromatic hydrocarbons (PAH), can upon metabolic
• Surface associated free radicals or activation be converted into redox-cycling semiquinone radicals
oxidative groups (e.g., SiO. and SiO.2 (Squadrito et al., 2001; Li et al., 2003).
on crystalline silica) The second route of oxidant generation is represented by
• Oxidant generation by particles in the ability of particles to activate (epithelial) target cells for
aqueous suspension (e.g., (enhanced) intracellular ROS generation. Important mecha-
Haber–Weiss reactions by available nisms considered herein are the particle-elicited activation of
metals, semiquinone radical redox NAD(P)H-like enzyme systems (Deshpande et al., 2002) and
cycling of biotransformed PAH. particle-induced disturbance of the mitochondrial electron trans-
port chain function (Li et al., 2003).
ROS generation upon interaction of Primary (indirect) Third is generation of ROS (and RNS) during particle-elicited
particle with target cells. inflammation. Specifically, recruitment and activation of inflam-
• Damage to mitochondria/interaction matory cells is associated with excessive formation of ROS
with the electron transport chain and RNS in the inflamed lung (Kamp et al., 1994; Castranova
• Activation of NAD(P)H-like et al., 1998; Albrecht et al., 2005). Phagocyte-driven oxidant
enzyme systems. formation results from the activation of NADPH oxidase, nitric
• Disturbance of endogenous oxides synthase (NOS), and myeloperoxidase (MPO) in phago-
antioxidant defences cytes (e.g., macrophages, neutrophils).
In Table 2, the respective ways of oxidant generation are listed
Generation of ROS and RNS during Secondary in relation to their participation in primary and/or secondary
particle-elicited inflammation genotoxicity.
• Phagocytes: NADPH oxidase, nitric
oxide synthase, myeloperoxidase PARTICLES AND OXIDATIVE DNA DAMAGE
In general, ROS/RNS may cause damage to the DNA by
oxidation-, nitration-, depurination-, methylation- or deamina-
DNA DAMAGE RESULTING FROM OXIDATIVE ATTACK tion reactions. Of all ROS/RNS, the hydroxyl radical is the most
Likely the most dominating and relevant feature of many DNA-reactive one, causing a multiplicity of products from all
particles with regard to their genotoxic potential is their abil- DNA bases (Pryor, 1988). Probably the best known and most in-
ity to generate oxidants. These oxidants include reactive oxy- vestigated oxidative lesion caused by hydroxyl radical attack is
gen species (ROS) such as hydrogen peroxide (H2 O2 ), hydroxyl 8-hydroxydeoxyguanine (8-OHdG) (Marnett, 2000), which has
radicals (. OH), superoxide (O. − 1
2 ), and singlet oxygen ( O2 ), as been shown to be induced by various particles in vitro, including
well as reactive nitrogen species (RNS) including nitric oxide crystalline silica and TiO2 (see Table 1), asbestos fibers, coal fly
(NO) and peroxynitrite (ONOO− ) (Knaapen et al., 2004). The ashes, and PM (Chao et al., 1996; Xu et al., 1999; van Maanen
relevance for ROS and RNS in particle genotoxicity is supported et al., 1999; Prahalad et al., 2001; Shi et al., 2003). ROS and RNS
by the fact that these oxidants are clearly implicated in the ox- can also elicit exocyclic etheno-adducts, which can be formed
idative attack of DNA, which may lead to base-pair mutations, during lipid peroxidation with associated formation of muta-
deletions, or insertions, all being commonly observed in mutated genic electrophiles such malondialdehyde and 4-hydroynonenal
oncogenes and tumor suppressor genes (Wiseman & Halliwell, (Marnett, 2000). Such lipid peroxidation-mediated DNA dam-
1996). In relation to its impact on DNA damage, the generation age has been indicated from investigations with crystalline silica
of oxidants during particle exposure can be subdivided to occur and asbestos fibers (Shi et al., 1994; Howden & Faux, 1996). Var-
in three principal manners: ious particles including quartz, DEP, CB, and TiO2 (see Table 1)
have also been shown to elicit DNA single-strand breaks in vitro,
1. Intrinsic ROS generation from particles or their adsorbed which may occur by direct oxidative attack of the DNA backbone
constituents. (i.e., desoxyribose sugar), or during the process of oxidative
2. ROS generation upon interaction of particles with target cells. DNA damage repair (i.e., DNA base-excision repair, BER).
3. Generation of ROS (and RNS) during particle-elicited in- Overall support for a role of ROS/RNS in particle-elicited DNA
flammation. damage is also provided by the ability of free radical scavengers
 GENOTOXICITY OF POORLY SOLUBLE PARTICLES
and antioxidants to reduce or abrogate particle-induced oxida-
tive DNA damage (Shi et al., 1994; Xu et al., 1999; Dellinger
et al., 2001).
Beyond in vitro studies, also in vivo studies with particles
demonstrate oxidant-mediated DNA damage (see Table 1).
Indeed, many of these studies are in support of the role
of inflammation-mediated ROS/RNS, i.e., a mechanism of
secondary genotoxicity (see also Table 2). Elevated lung tissue
DNA levels or immunohistochemical staining intensity of
8-OHdG has been found in association with intensity or persis-
tence of pulmonary inflammation in rat lungs upon treatment
with crystalline silica, DEP or CB (Ichinose et al., 1997;
Seiler et al., 2001;Gallagher et al., 2003). In lacI transgenic
rats, enhanced 8-OHdG levels as observed in lungs following
diesel exhaust inhalation were found to correlate with in vivo
mutagenicity (Sato et al., 2000). Similar associations between
8-OHdG induction and mutagenicity in vivo were also shown in
the omentum major of rats following intraperitoneal instillation
of asbestos fibers (Unfried et al., 2002). In both these studies, the
spectrum of mutations was also analysed and showed increased
G to T transversions, which is in agreement with the pathway of
mutagenicity for 8-OHdG (Klungland et al., 1999). In vivo mu-
tagenesis has also been determined by the HPRT-mutagenicity
test, specifically in alveolar epithelial cells upon their isolation
from the lungs of rats that were instilled with crystalline silica,
CB or TiO2 . In this study, mutagenicity was only enhanced after
instillations of particle doses which also elicited a massive and
persistent lung inflammation (Driscoll et al., 1997). Interest-
ingly, further investigations also revealed that neither of these
particles could elicit enhanced HPRT-mutations in rat lung
epithelial cells in vitro (Driscoll et al., 1997). Further support
for inflammation-driven secondary particle-induced DNA
damage and mutagenesis also comes from in vitro experiments,
whereby activated neutrophils have been found to induce
DNA strand breakage and 8-OHdG in lung epithelial cells
(Knaapen et al., 1999, 2004), and lavage cells from particle-
treated rats were found to be mutagenic to lung epithelial
cells (Driscoll et al., 1997), both in an antioxidant-dependent
manner.
However, in contrast to the aforementioned studies, animal
data also exist wherein particle inflammation and associated for-
mation of ROS and RNS were not associated with enhanced
DNA damage or mutagenesis. For instance, in a comparative in-
halation study in rats with amorphous and crystalline silica, for
which the exposure levels were titrated to elicit similar extent of
inflammation, only the crystalline silica was found to increase
HPRT mutations in lung epithelial cells in vivo (Johnston et al.,
2000). More recently, we also showed absence of enhanced 8-
OHdG levels in rat lung tissue after intratracheal instillation with
crystalline silica, despite the presence of a marked inflamma-
tory response and considerable increases in pulmonary levels of
ROS and RNS (Albrecht et al., 2005). The lack of genotoxicity
as observed in both studies is considered to be due to respec-
tively concurrent processed of apoptosis (Johnston et al., 2000)
193
and compensating oxidative DNA damage repair induction (Al-
brecht et al., 2005).
In general, it needs to be emphasized that the causality of the
association between inflammation and genotoxicity in particle-
exposed animals still needs to be established. A specific ap-
proach to elucidate the role of phagocytic cell influx in (acute)
pulmonary genotoxicity upon particle exposure would be to
use animals that are depleted of neutrophils and/or alveolar
macrophages. For instance, Gavett and colleagues (1992) used
neutrophil-depleted rats to assess the role of neutrophils in gen-
eral pulmonary toxicity (unfortunately not genotoxicity) of crys-
talline silica. Interestingly however, the results suggested that
recruitment of neutrophils from the circulation into the alve-
olar region is not a necessary prerequisite for quartz-induced
acute lung injury (Gavett et al., 1992). In contrast, with regard
to genotoxic endpoints, Auten et al. (2002) showed that blocking
of neutrophil influx significantly reduced pulmonary oxidative
DNA damage in hyperoxia-exposed newborn rats. The applica-
tion of such animal models in particle research would not only
enhance our understanding of the involvement of pulmonary in-
flammation in particle-induced genotoxicity, but would possibly
also increase our insight into the capacity of particles to induce
primary (i.e., inflammation-independent) genotoxicity in vivo.
FORMATION OF BULKY DNA ADDUCTS AND
ASSOCIATED MUTAGENICITY
Particles including DEP and CB are known to contain var-
ious amounts of organic constituents, and the adsorption of
this organic matter to the particle core provides a fundamen-
tal mechanism whereby nonvolatile, lipophilic compounds can
be transported into the deep lungs. The mutagenicity of or-
ganic extracts of ambient PM has been clearly established for
several decades now. Significant contributors to this mutagenic
pathway include the polycyclic aromatic hydrocarbons (PAH).
Combustion-derived particles including DEP and CB, but also
PM samples from industrialized/urbanized regions, typically
contain various PAH, among which several are established muta-
gens and/or carcinogens. Apart from PAH, also nitro-PAH have
been implicated in particle carcinogenesis. With regard to DEP,
a major compound is 3-nitrobenzanthrone, which, because of its
extremely high mutagenicity and relative specificity to DEP, has
been referred to as “the devil in the diesel” (Arlt, 2005).
PAH are typically generated during combustion process (e.g.,
traffic exhaust) and are emitted in semivolatile as well as particle-
associated forms, whereby the particle-associated PAH are pre-
dominantly found in the ultrafine mode (Bostrom et al., 2002).
PAH can be biotransformed into electrophilic, highly DNA-
reactive intermediates (Hall & Grover, 1990). These interme-
diates include epoxides, which are known to form stable DNA
adducts, as well as radical cations, which form rather unstable,
rapidly depurinated adducts. Diol epoxides contain a particularly
high affinity for the exocyclic amino groups of deoxyguanine
(dG) and deoxyadenosine (dA), which may lead to mutations
following DNA replication. DNA lesions as induced by PAH as
194 R. P. F. SCHINS AND A. M. KNAAPEN
well as by aromatic amines are commonly referred to as bulky
adducts, and are characterized by their ability to cause major
distortion of the DNA double helix. If these helix distorting
DNA lesions escape from specific DNA repair processes (i.e.,
nucleotide excision repair), they may cause transcription and
replication blocks, ultimately leading to aberrant gene expres-
sion and mutagenesis (Luch, 2005; Gillet & Scharer, 2006).
The formation of bulky DNA adducts has been specifically
investigated for DEP and CB in vivo, in order to determine the
significance of PAH and related organic constituents in tumori-
genesis as observed in animal studies (see also Table 1). How-
ever, these investigations have yielded rather contrasting obser-
vations (reviewed in Borm et al., 2004). Several explanations
can be provided for these differences (see Figure 2). First of all,
the ability of organics such as PAH to lead to DNA adduct for-
mation will depend on desorption/leaching of these compounds
from the particles in a biological environment. Obviously, stud-
ies in which DNA adduct formation has been determined with
extracts of, e.g., PM or CB (e.g., Topinka et al., 2000; Borm et al.,
2005) entirely bypass this aspect of bioavailability. The ability of
adsorbed PAH and other constituents to become released from
the particle core depends on physicochemical properties of the
particles (e.g., surface area, porosity), as well as on the composi-
tion of the organic fraction and the concentrations of its individ-
ual constituents (Figure 2). Exemplary, DNA adducts could not
FIG. 2. Potential mechanisms implicated in DNA adduct for-
mation and its repair by particle-associated polycyclic aromatic
hydrocarbons. Polycyclic aromatic hydrocarbons (PAH) are well
known for their ability to form bulky DNA adducts in cells,
depending on the available biotransformation pathways. For
particle-associated PAH this necessitates leaching/desorption of
these compounds into the cellular environment, which is known
to depend on various particle aspects (e.g., surface area, porosity,
content of PAH and other chemical constituents). Meanwhile,
particles and their chemical constituents may also elicit various
other effects, such as via the generation of ROS. This may lead
to altered leaching and biotransformation of PAH, as well as in-
duction of cell processes including proliferation, apoptosis, and
possibly DNA adduct repair.
be detected in lung epithelial cells after in vitro treatment with
various CB types, in marked contrast to their extracts (Borm
et al., 2005). In fact, it was also shown in this study that a mix-
ture of freely added PAH rapidly adsorbs onto the high surface
of CB in this in vitro system. The level of DNA adducts will also
depend on the endogenous activity of specific biotransformation
pathways within the target cells, as well as their capacity to re-
pair these bulky lesions via the nucleotide excision repair (NER)
pathway (Gillet & Scharer, 2006). For instance, we showed that
exposure to DQ12 quartz increased the induction of cytochrome
P-450 1A1 (CYP1A1) in rat pulmonary epithelial target cells in
vitro and in vivo (Becker et al., 2006). Since CYP1A1 is crucially
involved in the metabolic activation of chemical carcinogens,
this indicates that the interaction between poorly soluble parti-
cles and target cells may directly affect DNA adduct formation.
Also, secondary inflammatory processes may have a crucial im-
pact on DNA damage by inhaled chemical carcinogens. For in-
stance, by releasing myeloperoxidase, activated neutrophils are
able to significantly enhance DNA adduct formation of a num-
ber of chemical carcinogens including PAH (review: Knaapen
et al., 2006). Hence, DNA adduct levels may vary with the level
of inflammation, as well as the target cell type, i.e., (1) as chosen
for the specific in vitro investigation, or (2) as hit by the rele-
vant particle upon its inhalation and subsequent particle-specific
deposition/translocation behavior. Finally, apart from delivering
PAH to target cells, particles may trigger various independent
cellular processes. For instance, ROS, as can be generated from
the particles and/or during particle-elicited inflammation, are
known to impact on biotransformation and metabolization of
PAH (Knaapen et al., 2006), cell proliferation, and induction of
apoptosis, and possibly also DNA adduct repair. Notably, such
effects will thus also impact on the ultimate mutagenic outcome
(see Figure 2). Taken together, each of these aspects can provide
some explanation for the apparent contrasts as observed in var-
ious in vivo studies with PAH-containing particles such as DEP
and CB.
Notably also, although bulky adduct formation has been
demonstrated in selected studies, several investigations suggest
that oxidative DNA damage rather than bulky adduct forma-
tion is implicated in the tumorigenesis of with particles such as
DEP and CB (Gallagher et al., 1994; Ichinose et al., 1997; Sato
et al., 2000; Gallagher et al., 2003; Borm et al., 2005). Such
observations are in support of a secondary mechanism of geno-
toxicity involving ROS/RNS generation by inflammatory cells,
although it cannot be entirely ruled out that these oxidative le-
sions (partly) result from oxidative DNA attack by redox-cycling
semiquinones (Park et al., 2006).
SUMMARY AND CONCLUSIONS
In this review, the hallmark mechanisms of genotoxicity by
poorly soluble particles of low toxicity as well as crystalline sil-
ica, representing a high-toxicity particle, have been discussed. A
considerable number of studies indicate that the action of ROS
and RNS during particle elicited inflammation may represent a
 GENOTOXICITY OF POORLY SOLUBLE PARTICLES
major operating mechanism for these particles, irrespective of
their intrinsic physicochemical properties and chemical compo-
sition. It should be emphasized, however, that many of the ob-
servations are of an associative nature, and causality still needs
to be determined in order to verify the importance of this mech-
anisms. Also, in vivo studies have usually been performed at
high particle concentrations and/or long term exposures, which
are associated with marked inflammatory and proliferative re-
sponses. As discussed in this paper, these effects may obscure
and/or modify genotoxicity readouts. It is also important to stress
that high treatment regimens are applied in most in vitro stud-
ies, typically several orders of magnitude above those that are
anticipated per unit lung surface following after inhalation. Al-
though such high concentrations are justifiable in investigations
that aim to unravel mechanisms of toxicity (Oberdorster & Yu,
1999), these data should be interpreted with caution for risk
assessment purposes.
The need for further investigations to unravel the concepts of
primary and secondary genotoxicity is also indicated from our
previous investigations on the DNA damaging properties of the
highly toxic respirable crystalline silica DQ12. Specifically, we
previously showed that DQ12 elicits DNA single-strand breaks
in lung epithelial cells from rats following a single instillation
(Knaapen et al., 2002a). In support of the role of inflammatory
cell-derived oxidants in these effects, this instillation treatment
was also found to result in a marked neutrophilic inflamma-
tion (Knaapen et al., 2002a). However, with in vitro studies in
a rat lung epithelial cell line using the identical genotoxicity
assay, DNA single-strand breaks could be significantly elicited
by DQ12 particles (Schins et al., 2002), as well as by phorbol
myristate acetate (PMA)-activated neutrophils (Knaapen et al.,
2002b). Even though a rather high particle dose was needed in
vitro, it cannot be entirely ruled out that (part) of the observed
DNA strand breakage in vivo resulted from the action of quartz.
Importantly, however, in contrast to our DNA strand breakage
observations, crystalline silica particles were able to elicit HPRT
mutations in rat lung epithelial cells after in vivo treatment, but
not in vitro (Driscoll et al., 1997). The discrepancies between
both series of investigations are on the one hand likely due to dif-
ferent treatment concentrations (5 mg/kg versus 10–100 mg/kg),
selected analyses time points (3 days versus 15 mo) and/or silica
samples (DQ12 versus Min-U-Sil). On the other hand, it is also
important to emphasize the differences in genotoxicity assays,
for example, a DNA single-strand breakage assay indicative of
genotoxicity, versus a mutagenicity assay that depends on a mu-
tagenic event and its fixation after cell division.
Similar to these considerations, also the role of organic con-
stituents such as PAH in the induction of DNA damage and muta-
genesis remains for further investigation. Here also, the extreme
exposure conditions as applied obscure the potential impact for
genotoxicity and mutagenicity of organic constituents. On the
one hand, the formation and persistence of bulky DNA adduct
may be altered due to the presence of marked inflammation. On
the other hand, at high exposures it remains to be elucidated
195
as to how far some of the observed oxidative DNA insults may
be due to semiquinones rather than the oxidants generated from
inflammatory cells. Notably, the more complex the chemical
composition of the particles, the more complex their potential
implication for genotoxicity. For instance, DEP (and to an even
greater extent PM from industrialized/urban regions) contain,
besides an organic fraction, metallic constituents also. Several
of these metals not only can elicit DNA damage, but also can in-
hibit DNA repair (Hei et al., 1998; Kessel et al., 2002; Hartwig,
2002; Tran et al., 2002; Kawanishi et al., 2002), and hence may
have impact on steady-state oxidative DNA damage as well as
bulky DNA adducts.
In conclusion, further studies are needed to unravel the mech-
anisms whereby particles can elicit genotoxicity under both re-
alistic in vivo conditions and doses for which the appropriate
endpoint of tumor formation is anticipated. To fully appreciate
the impact of genotoxic events for carcinogenesis by inhaled
particles, concomitant evaluation of DNA damage responses in-
cluding cell cycle arrest and DNA repair as well as the induction
of apoptosis and proliferation should preferably be incorporated
in such research. These investigations will lead to an improved
understanding of the mode of action of particles in relation to
their physicochemical properties and will accordingly allow for
improved risk assessment strategies
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