DNA damage and oxidative stress induced by CeO2 nanoparticles in human dermal fibroblasts: Evidence of a clastogenic effect as a mechanism of genotoxicity.

The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 × 10(-5)-6 × 10(-3) g/l corresponding to a concentration range of 0.22-22 µM) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.

Ultrastructural interactions and genotoxicity assay of cerium dioxide nanoparticles on mouse oocytes.

Cerium dioxide nanoparticles (C(e)O2 ENPs) are on the priority list of nanomaterials requiring evaluation. We performed in vitro assays on mature mouse oocytes incubated with C(e)O2 ENPs to study (1) physicochemical biotransformation of ENPs in culture medium; (2) ultrastructural interactions with follicular cells and oocytes using Transmission Electron Microscopy (TEM); (3) genotoxicity of C(e)O2 ENPs on follicular cells and oocytes using a comet assay. DNA damage was quantified as Olive Tail Moment. We show that ENPs aggregated, but their crystal structure remained stable in culture medium. TEM showed endocytosis of C(e)O2 ENP aggregates in follicular cells. In oocytes, C(e)O2 ENP aggregates were only observed around the zona pellucida (ZP). The comet assay revealed significant DNA damage in follicular cells. In oocytes, the comet assay showed a dose-related increase in DNA damage and a significant increase only at the highest concentrations. DNA damage decreased significantly both in follicular cells and in oocytes when an anti-oxidant agent was added in the culture medium. We hypothesise that at low concentrations of C(e)O2 ENPs oocytes could be protected against indirect oxidative stress due to a double defence system composed of follicular cells and ZP.

Genotoxicity assessment of mouse oocytes by comet assay before vitrification and after warming with three vitrification protocols.

OBJECTIVE: To assess the genotoxicity of three oocyte vitrification protocols. DESIGN: Murine assay. SETTING: Biogenotoxicology research laboratory. ANIMAL(S): CD1 female mice. INTERVENTION(S): Three mouse oocyte groups were exposed to three commercialized human oocyte vitrification protocols. Protocols 1 and 2 contained dimethyl sulfoxide and ethylene glycol (EG), and protocol 3 contained EG and 1,2-propanediol (PrOH). DNA damage was first evaluated by comet assay after oocyte exposure to the three different equilibration and vitrification solutions. Comet assay was also performed after full vitrification and warming procedure and compared with a negative control group (oocytes stored in medium culture only) and a positive control group (oocytes exposed to hydrogen peroxide just before comet assay). MAIN OUTCOME MEASURE(S): DNA damage was quantified as Olive tail moment (OTM). Statistical analysis consisted of a Shapiro-Wilk test. Then, median protocol OTM was compared with the negative control group with the Mann-Whitney U test. The difference was considered to be statistically significant if the P value was <.05. RESULT(S): In both parts of our study, protocols 1 and 2 did not induce significant DNA damage, whereas protocol 3 induced statistically higher DNA damage compared with the negative control group. CONCLUSION(S): Vitrification protocols containing PrOH induced significant DNA damage on mouse oocytes, both before cooling and after warming. Therefore, for the moment, we prefer vitrification techniques without PrOH while we await more studies on PrOH toxicity and long-term evaluation.

EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: evidence for NADPH oxidase and mitochondrial stimulation.

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24 h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.

Influence of the length of imogolite-like nanotubes on their cytotoxicity and genotoxicity toward human dermal cells.

Physical-chemical parameters such as purity, structure, chemistry, length, and aspect ratio of nanoparticles (NPs) are linked to their toxicity. Here, synthetic imogolite-like nanotubes with a set chemical composition but various sizes and shapes were used as models to investigate the influence of these physical parameters on the cyto- and genotoxicity and cellular uptake of NPs. The NPs were characterized using X-ray diffraction (XRD), small angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Imogolite precursors (PR, ca. 5 nm curved platelets), as well as short tubes (ST, ca. 6 nm) and long tubes (LT, ca. 50 nm), remained stable in the cell culture medium. Internalization into human fibroblasts was observed only for the small particles PR and ST. None of the tested particles induced a significant cytotoxicity up to a concentration of 10(-1) mg·mL(-1). However, small sized NPs (PR and ST) were found to be genotoxic at very low concentration 10(-6) mg·mL(-1), while LT particles exhibited a weak genotoxicity. Our results indicate that small size NPs (PR, ST) were able to induce primary lesions of DNA at very low concentrations and that this DNA damage was exclusively induced by oxidative stress. The higher aspect ratio LT particles exhibited a weaker genotoxicity, where oxidative stress is a minor factor, and the likely involvement of other mechanisms. Moreover, a relationship among cell uptake, particle aspect ratio, and DNA damage of NPs was observed.

Is there a Trojan-horse effect during magnetic nanoparticles and metalloid cocontamination of human dermal fibroblasts?

This study investigates the issue of nanoparticles/pollutants cocontamination. By combining viability assays, physicochemical and structural analysis (to probe the As speciation and valence), we assessed how γFe(2)O(3) nanoparticles can affect the cytotoxicity, the intra- and extracellular speciation of As(III). Human dermal fibroblasts were contaminated with γFe(2)O(3) nanoparticles and As(III) considering two scenarios: (i) a simultaneous coinjection of the nanoparticles and As, and (ii) an injection of the nanoparticles after 24 h of As adsorption in water. In both scenarios, we did not notice significant changes on the nanoparticles surface charge (zeta potential ∼ -10 mV) nor hydrodynamic diameters (∼950 nm) after 24 h. We demonstrated that the coinjection of γFe(2)O(3) nanoparticles and As in the cellular media strongly affects the complexation of the intracellular As with thiol groups. This significantly increases at low doses the cytotoxicity of the As nonadsorbed at the surface of the nanoparticles. However, once As is adsorbed at the surface the desorption is very weak in the culture medium. This fraction of As strongly adsorbed at the surface is significantly less cytotoxic than As itself. On the basis of our data and the thermodynamics, we demonstrated that any disturbance of the biotransformation mechanisms by the nanoparticles (i.e., surface complexation of thiol groups with the iron atoms) is likely to be responsible for the increase of the As adverse effects at low doses.

Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation.

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.

Environmental nitration processes enhance the mutagenic potency of aromatic compounds.

This work is an attempt to establish if aromatic nitration processes are always associated with an increase of genotoxicity. We determined the mutagenic and genotoxic effects of Benzene (B), Nitrobenzene (NB), Phenol (P), 2-Nitrophenol (2-NP), 2,4-Dinitrophenol (2,4-DNP), Pyrene (Py), 1-Nitropyrene (1-NPy), 1,3-Dinitropyrene (1,3-DNPy), 1,6-Dinitropyrene (1,6-DNPy), and 1,8-Dinitropyrene (1,8-DNPy). The mutagenic activities were evaluated with umuC test in presence and in absence of metabolic activation with S9 mix. Then, we used both cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes on human lymphocytes in order to evaluate the genotoxic effects. Analysis of all results shows that nitro polycyclic aromatic hydrocarbons (PAHs) are definitely environmental genotoxic/mutagenic hazards and confirms that environmental aromatic nitration reactions lead to an increase in genotoxicity and mutagenicity properties. Particularly 1-NPy and 1,8-DNPy can be considered as human potential carcinogens. They seem to be significant markers of the genotoxicity, mutagenicity, and potential carcinogenicity of complex PAHs mixtures present in traffic emission and industrial environment. In prevention of environmental carcinogenic risk 1-NPy and 1,8-DNPy must therefore be systematically analyzed in environmental complex mixtures in association with combined umuC test, CBMN assay, and FISH on cultured human lymphocytes. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Assessment of 1,2-propanediol (PrOH) genotoxicity on mouse oocytes by comet assay.

OBJECTIVE: To assess the genotoxicity of 1,2-propanediol (PrOH) on mouse oocytes by comet assay. DESIGN: In vitro assay using murine model. SETTING: Biogenotoxicology research laboratory. ANIMAL(S): CD1 female mice. INTERVENTION(S): Three 40-oocyte groups were exposed to different PrOH concentrations (5%, 7.5%, and 15%). Each concentration was tested during both long and short exposures (1-2 hours and 1-5 minutes) in comparison with control groups. DNA damage was evaluated by a single-cell gel electrophoresis assay, also called "comet assay," and analyzed with Komet software. MAIN OUTCOME MEASURE(S): DNA damage was quantified as Olive tail moment (OTM). Interpretation was done on OTM with the use of χ(2). RESULT(S): High PrOH concentrations (7.5% and 15%) induced significant DNA damage on mouse oocytes. The OTM χ(2) values were 4.16 ± 0.40 and 6.80 ± 0.4 with 7.5% PrOH at 1 and 2 hours, respectively, 24.35 ± 1.60 with 15% at 1 hour, and for 2h at 15% the DNA damage was too drastic to calculate OTM χ(2). After 1 and 5 minutes, the OTM χ(2) values were, respectively, 5.19 ± 0.26 and 6.06 ± 0.42 with 7.5%, and 7.53 ± 0.33 and 16.81 ± 0.67 with 15%. CONCLUSION(S): High concentrations of PrOH (7.5% and 15%) induced significant DNA damage on mouse oocytes, whatever the exposure duration. These results should be interpreted with caution, because additional data are needed to evaluate PrOH genotoxicity and DNA oocyte reparation after exposure to high PrOH concentrations.

In smokers, swim-up and discontinuous gradient centrifugation recover spermatozoa with equally lower amounts of DNA damage than spermatozoa obtained from neat semen.

We compared the abilities of two commonly used semen preparation techniques to decrease the amount of benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in the spermatozoa of ten smokers. Semen processing by swim-up or discontinuous gradient centrifugation recovered spermatozoa showing an equally significantly lower amount of BPDE-DNA adducts than in unprepared spermatozoa from neat semen.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei.

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C-MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C-MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.

Tobacco consumption and benzo(a)pyrene-diol-epoxide-DNA adducts in spermatozoa: in smokers, swim-up procedure selects spermatozoa with decreased DNA damage.

OBJECTIVE: To analyze the distribution of benzo(a)pyrene-diol-epoxide (BPDE)-DNA adducts in spermatozoa selected and nonselected by a swim-up procedure with relation to smoking habits. DESIGN: Comparative study. SETTING: Public university and public university hospital. PATIENT(S): Seventy-nine men (37 smokers and 42 nonsmokers) who visited an infertility clinic for diagnostic. INTERVENTION(S): Tobacco and environmental exposure assessment, semen sample analysis, swim-up procedure, BPDE-DNA adduct immunolabeling. MAIN OUTCOME MEASURE(S): BPDE-DNA adduct quantification in selected (SEL-SPZ) and nonselected (NONSEL-SPZ) spermatozoa. Data were normalized by using a normalized fluorescence value (NFV). RESULT(S): The mean NFV (±SD) in SEL-SPZ was significantly higher in smokers than in nonsmokers (18.9±11.5 vs. 10.5±10.4, respectively). Within smokers, a paired analysis (SEL-SPZ and NONSEL-SPZ) showed that NFV was significantly lower in SEL-SPZ than in NONSEL-SPZ (20.0±11.3 vs. 31.5±16.0, respectively). Conversely, within nonsmokers, the mean NFV was higher in SEL-SPZ than in NONSEL-SPZ (10.3±10.6 vs 4.3±7.1, respectively). CONCLUSION(S): Tobacco consumption is associated with BPDE-DNA adducts in spermatozoa. In smokers, semen processing by swim-up recovers potentially fertilizing spermatozoa that show a significantly lower amount of BPDE-DNA adducts compared with NONSEL-SPZ. Further study is needed to improve the spermatozoa selection in smoking patients requiring assisted reproductive technologies.

Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells.

OBJECTIVE: To develop and validate an efficient comet assay on mouse oocytes without depellucidation. DESIGN: In vitro experiments using a murine model. SETTING: Biogenotoxicology research laboratory in Aix-Marseille II University, France. ANIMAL(S): CD1 prepubescent female mice. INTERVENTION(S): DNA lesions in oocytes were evaluated by the alkaline comet assay. After oocyte retrieval, we first studied the effect of zona pellucida (ZP) on comet morphology. For this study, we applied the comet assay to mature oocytes with and without ZP after exposure to simulated sunlight irradiation (SSI) compared with negative controls. Next, nondepellucidated mouse oocytes were exposed to three well-known genotoxic agents (SSI, methylmethanesulfonate [MMS], and hydrogen peroxide [H(2)O(2)]) and compared with negative controls. Images of oocytes were analyzed with Komet software. MAIN OUTCOME MEASURE(S): DNA damages were quantified and expressed as olive tail moment (OTM), defined as the product of the tail length and the fraction of total DNA in the tail. OTMχ(2) were calculated from OTM; they corresponded to the degrees of freedom (n) of each OTM distribution obtained from at least 50 oocytes. OTMχ(2) is an indicator of DNA lesions. The test was considered positive and statistically significant when OTMχ(2) increased in oocytes compared with the medium-only control cells. RESULT(S): There was no difference in comet aspect between oocyte groups with and without ZP. The three genotoxic agents significantly increased DNA damages as compared with the control groups. The OTMχ(2) values were (mean ± SD): 2.1 ± 0.07, 7.73 ± 0.35, 3.35 ± 0.15, and 12.4 ± 0.51 in control, SSI, MMS, and H(2)O(2) groups, respectively. CONCLUSION(S): Comet assay on non depellucidated mouse oocytes is a rapid and easy test. This assay would be useful to assess the genotoxicity on female germ cells of chemicals, drugs, or environmental pollutants and the efficiency of antioxidant molecules.

Secondary oxidation of cyclic 1,N2-propano and 1,N2-etheno-2'-deoxyguanosine DNA adducts. Consequences in oxidative stress biomarker development.

This work is an attempt to investigate the chemical stability of 1,N2-propano-2'-deoxyguanosine (pdG-HNE) and 1,N2-etheno-2'-deoxyguanosine (epsilondG) DNA adducts against hydrolysis and upon oxidation reactions. It includes both kinetic issues together with proposed degradation pathways. While both chemicals are stable in the 3.5-9 pH range, the results suggest that pdG-HNE adduct is less prone to in vitro oxidative transformation than epsilondG adduct. EpsilondG and pdG-HNE behave differently upon hydroxyl radical and one electron oxidation reactions. The exocyclic ring of epsilondG is mainly affected by oxidative processes leading to the regeneration of 2'-deoxyguanosine (dG) while the integrity of the exocyclic ring is preserved for pdG-HNE. Consequently, pdG-HNE might be a better biomarker than epsilondG for monitoring oxidative stress during environmental or occupational exposures to chemicals. Understanding the in vitro routes of etheno and propano DNA adduct degradation would probably help to guide the development of analytical methodologies for the reliable detection of these endogenous adducts.

Occupational exposures obtained by questionnaire in clinical practice and their association with semen quality.

In industrial countries, evidence suggests that semen quality has been steadily decreasing over the past 5 decades. We employed a short questionnaire to examine the association between self-reported physical or chemical occupational exposures and semen quality. The study included 402 men consulting for couple infertility (314 with oligospermia, asthenospermia, or teratospermia and 88 with normal semen; World Health Organization criteria). Exposure effects on global sperm quality and total sperm count, sperm motility, and sperm morphology were investigated. We found significant associations between semen impairment and occupational risk factors such as exposure to heavy metals (adjusted odds ratio [OR] = 5.4; 95% confidence interval [CI], 1.6-18.1), solvents (OR = 2.5; 95% CI, 1.4-4.4), fumes (OR = 1.9; 95% CI, 1.1-3.4), and polycyclic aromatic hydrocarbons (OR = 1.9; 95% CI, 1.1-3.5). Exposure to pesticides or cement was nearly significant (OR = 3.6; 95% CI, 0.8-15.8, and OR = 2.5; 95% CI, 0.95-6.5, respectively). Physical risk factors were associated with some sperm anomalies, such as mechanical vibrations with oligospermia and teratospermia as well as excess heat and extended sitting periods with impaired motility. Exposure to ionizing radiation and electromagnetic fields was not associated with semen impairment; these results, however, may be skewed, because very few subjects reported such exposure. Despite the small dataset, self-reported exposures were correlated with semen impairment. This approach may be recommended in routine clinical practice to seek relationships between occupational exposures to reprotoxic agents and impaired semen parameters. This knowledge would allow preventive measures in the workplace to be established and could be complemented by the use of biomarkers to better characterize exposure to chemical substances and their spermiotoxic effects.

[Exposure science: the need to articulate the data related to hazards, to the worker and to his activity]. / L'expologie ou la nécessité d'articuler les données relatives aux dangers, à l'homme et à son activité.

To optimise the prevention of occupational risks, it seems necessary to revise existing methodological approaches by organizing and better articulating the data concerning hazards, the worker and, his activity. In the context of occupational health, exposure science offers the opportunity to regroup and link knowledge and know-how in order to fully characterise hazardous occupational exposure. The authors illustrate the interest of this approach using the example of a field study on the characterisation of occupational exposures to polycyclic aromatic hydrocarbons.

Evaluation of the genotoxicity of river sediments from industrialized and unaffected areas using a battery of short-term bioassays.

The present investigation evaluated the capacity of the Salmonella mutagenicity test, the comet assay, and the micronucleus assay to detect and characterize the genotoxic profile of river sediments. Three stations were selected on an urban river (Bouches du Rhône, France) exposed to various sources of industrial and urban pollution (StA, StB, and StC) and one station on its tributary (StD). One station in a nonurban river was included (REF). The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were determined by HPLC, and the genotoxicity of the sediments was monitored by the Salmonella mutagenicity test (TA98 + S9, YG1041 +/- S9), the comet assay, and the micronucleus assay on CHO cells. Chemical analysis showed that the total PAH concentrations ranged from 23 microg kg(-1) dw (REF) to 1285 microg kg(-1) dw (StD). All the sediments were mutagenic in the Salmonella mutagenicity test. The mutagenicity was probably induced by the presence of nitroarenes (StA, StB, StC, and StD) and aromatic amines (REF) as deduced from the mutagenicity profiles of strains YG1041 +/- S9 and TA98 + S9. The comet assay revealed direct DNA lesions in REF, StA, and StB sediments and metabolization-dependent DNA damage in StC and StD. The micronucleus assay showed an absence of clastogenicity for StA +/- S9 and StC-S9, and a significant clastogenicity +/- S9 for the three other stations. The genotoxicity ranking determined by the comet assay + S9 matched the ranking of total and carcinogenic PAH concentrations, and this assay was found to be the most sensitive.

[Changes in chromosome number, genetic instability, and occupational exposures]. / Anomalies chromosomiques de nombre, instabilité génétique et expositions professionnelles.

Aneugenic compounds cause chromosome missegregation during cell division and induce aneuploidy in cells that do not die. Aneuploidy is a key step in the progression from a normal cell into a cancerous cell, and it could represent an early event in the carcinogenic process. Missegregation of chromosome during anaphase often originates from centrosome abnormality, which plays a key role in the formation of the mitotic spindle during cell division. Micronuclei (MN) are thought to be biomarkers of chromosome damage due to genetic instability or exposure to environmental mutagens or carcinogens (occupational exposure for example). The MN assay in combination with fluorescent in situ hybridization discriminates between MN containing acentric chromosome fragments (chromosome breakage) and MN containing whole chromosomes (chromosome loss), consecutively to clastogenic and aneugenic events, respectively. Centromere-positive micronuclei are due to alteration in mitotic apparatus proteins. Two pathways could be involved : chromosome migration impairment would lead to MN containing a single chromosome whereas centrosome amplification would induce MN containing several chromosomes. For biomonitoring purposes, numerous confounding factors (host factors, lifestyle, genetic polymorphism) influence the MN biomarker. Thus, the separated analysis of MN containing a single or several centromeres could be useful, as centrosome abnormalities seem to be linked with an increase in genetic instability.

In vitro synthesis of 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine by 2,4-dinitrophenol and 1,3-dinitropyrene in presence of a bacterial nitroreductase.

The formation of covalent nitro-PAH DNA adducts and nitro-PAH mediated oxidative lesions are two possible mechanisms for the initiation of nitro-PAH carcinogenesis. Sixty-minute incubation of 1,3-dinitropyrene (100 microM) or 1,4-dinitrophenol (100 microM) with a mixture of 150 microM NADH, 0.5 units of E. coli nitroreductase, 100 microM linoleic acid, 0.5 mM ferrous iron, and 100 microM 2'-deoxyadenosine (2'-dA) or 100 microM 2'-deoxyguanosine (2'-dG) were analyzed by liquid chromatography multistage mass spectrometry. Mixtures of 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) plus 4-oxo-2-nonenal (4-ONE) and 1,N(2)-etheno-2'-deoxyguanosine (epsilondG) plus 4-ONE could be detected from 2'-dA and 2'-dG, respectively. Addition of 2% propanol inhibited the formation of etheno adducts. Analyses of disappearance kinetics of dA and dG showed that dG was more rapidly eliminated than does dA (t[1/2] = 23.3 min and 98.3 min for dG and dA, respectively). Curves of formation kinetics revealed that the peak of epsilondG was at 55.6 min while that of epsilondA was at 186.9 min. These peaks represented 1.43% and 1.25% of the original dG and dA, respectively. In both cases, the peaks were followed by rapid degradations of etheno adducts. The results, obtained in this system, do not allow any extrapolation to realistic cellular responses; nevertheless, these data questioned the validity of the use of unsubstituted etheno adducts as reliable oxidative stress and nitro-PAH exposure biomarkers.

Evaluation of a battery of Salmonella typhimurium tester strains for biomonitoring of mutagenic polycyclic aromatic hydrocarbons, nitroarenes and aromatic amines.

Various combinations of Salmonella typhimurium tester strains and S9 mix for bioactivation (TA98+S9 mix, TA98S; YG1041+S9 mix, YG1041S) and strain YG1041 in the absence of S9 mix (YG1041) were used to evaluate the mutagenic activity of eight polycyclic aromatic hydrocarbons (PAHs), seven nitroarenes (NAs) and seven aromatic amines (AAs). Three cigarette smoke extracts and two extracts of smokers' urine (SUE) were also included. Urinary mutagenicity was then determined on 31 individuals, potentially exposed to PAHs, for 0 h, 7 h, 12 h and 24 h. Concentrations of urinary 1-hydroxypyrene (1OHP) and 3-hydroxybenzo[a]pyrene (3OHBaP), the levels of atmospheric pyrene (Py) and benzo[a]pyrene (BaP), and particulate concentrations in air (AP) were also measured. PAHs could be detected by TA98S and YG1041S, with TA98S being more sensitive than YG1041S. While NAs could be detected by all combinations, YG1041 and YG1041S were more sensitive than TA98S. Although both YG1041S and TA98S could detect AAs, YG1041S was more sensitive than TA98S. Cigarette smoke extract contained mutagenic AAs and NAs, but AAs were the only mutagenic compounds detected in the extracts of smokers' urine. The concentrations of 1OHP (7 h and 12 h) were significantly higher than those at 0 h, but no difference could be detected with 3OHBaP. Correlations were found between Py and 1OHP (7 h and 24 h) and between BaP and 3OHBaP concentrations (7 h, 12 h and 24 h). A significantly elevated urinary mutagenicity was detected with YG1041S at 7h in the group of smokers. A good correlation was determined between AP and the test results with TA98S (7 h) and with YG1041 (0 h and 7 h). Urinary 1OHP correlated with the test results with YG1041S (0 h, 7 h and 12 h) while 3OHBaP correlated with those obtained with YG1041S (7 h). Overall, 21/31 individuals were occupationally exposed to AAs, 15/31 individuals were exposed to NAs, and 2/31 were exposed to PAHs as indicated by the Salmonella mutagenicity assay. The urine mutagenicity test was not effective at monitoring occupational exposure to PAHs. However, the correlation with AP implied the presence of unknown mutagenic atmospheric substances that could modulate the urinary mutagenicity.