Assessment of micronuclei in lymphocytes from workers exposed to vapours and aerosols of bitumen

We investigated the micronucleus frequencies in peripheral blood lymphocytes of 225 mastic asphalt workers (age 17-62 years) and 69 non-bitumen-exposed road construction workers (age 18-64 years) in Germany before and after the working shift. Median shift exposure to vapours and aerosols of bitumen of exposed workers was 3.0 mg/m3. Micronuclei (MN) were determined with a standard method using cytochalasin B. Median MN frequency was 6.0 (interquartile range (IQR) 4.0-8.5) MN/1,000 binucleated lymphocytes (MN/1,000 BNC) in exposed workers and 6.0 (IQR 4.0-8.3) MN/1,000 BNC in non-exposed workers before shift. After shift, we observed 6.5 (IQR 4.4-9.3) MN/1,000 BNC in exposed workers and 6.5 (IQR 4.0-9.0) MN/1,000 BNC in non-exposed workers. Regression models were applied with the log-transformed MN frequency as the dependent variable in order to estimate the effects of exposure to vapours and aerosols of bitumen and of potential confounders. Age was the strongest predictor of MN formation in both exposed workers and referents. Our data suggest that MN formation was not associated with concentration of vapours and aerosols of bitumen during shift at the individual level. Although similar MN frequencies were observed in both groups, the modelling of factors potentially influencing MN frequency revealed a weak group difference in the post-shift model. We conclude that this small difference cannot be judged to be a relevant mutagenic effect of exposure to vapours and aerosols of bitumen, also with regard to the lack of adjustment for multiple testing and the lack of a group effect in the original data.     

Urinary metabolites of polycyclic aromatic hydrocarbons in workers exposed to vapours and aerosols of bitumen

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons (PAH) were investigated as potential biomarkers of bitumen exposure in a cross-shift study in 317 exposed and 117 non-exposed workers. Personal measurements of the airborne concentration of vapours and aerosols of bitumen during a working shift were weakly associated with post-shift concentrations of 1-hydroxypyrene (1-OHP) and 1-, 2+9-, 3- and 4-hydroxyphenanthrenes (further referred to their sum as OHPHE), but not 1- and 2-hydroxynaphthalene (OHNA). Smoking showed a strong influence on the metabolite concentrations, in particular on OHNA. Pre-shift concentrations of 1-OHP and OHPHE did not differ between the study groups (P = 0.16 and P = 0.89, respectively). During shift, PAH metabolite concentrations increased in exposed workers and non-exposed smokers. Statistical modelling of post-shift concentrations revealed a small increase in 1-OHP by a factor of 1.02 per 1 mg/m(3) bitumen (P = 0.02) and 1.04 for OHPHE (P < 0.001). A group difference was observed that was diminished in non-smokers. Exposed non-smokers had a median post-shift 1-OHP concentration of 0.42 μg/l, and non-smoking referents 0.13 μg/l. Although post-shift concentrations of 1-OHP and OHPHE were slightly higher than those in the general population, they were much lower than in coke-oven workers. The small content of PAHs in vapours and aerosols of bitumen, the increasing use of additives to asphalt mixtures, the strong impact of smoking and their weak association with airborne bitumen limit the use of PAH metabolites as specific biomarkers of bitumen exposure.     

Air sampling and determination of vapours and aerosols of bitumen and polycyclic aromatic hydrocarbons in the Human Bitumen Study

The chemical complexity of emissions from bitumen applications is a challenge in the assessment of exposure. Personal sampling of vapours and aerosols of bitumen was organized in 320 bitumen-exposed workers and 69 non-exposed construction workers during 2001-2008. Area sampling was conducted at 44 construction sites. Area and personal sampling of vapours and aerosols of bitumen showed similar concentrations between 5 and 10 mg/m(3), while area sampling yielded higher concentrations above the former occupational exposure limit (OEL) of 10 mg/m(3). The median concentration of personal bitumen exposure was 3.46 mg/m(3) (inter-quartile range 1.80-5.90 mg/m(3)). Only few workers were exposed above the former OEL. The specificity of the method measuring C-H stretch vibration is limited. This accounts for a median background level of 0.20 mg/m3 in non-exposed workers which is likely due to ubiquitous aliphatic hydrocarbons. Further, area measurements of polycyclic aromatic hydrocarbons (PAHs) were taken at 25 construction sites. U.S. EPA PAHs were determined with GC/MS, with the result of a median concentration of 2.47 μg/m(3) at 15 mastic asphalt worksites associated with vapours and aerosols of bitumen, with a Spearman correlation coefficient of 0.45 (95% CI -0.13 to 0.78). PAH exposure at mastic-asphalt works was higher than at reference worksites (median 0.21 μg/m(3)), but about one order of magnitude lower compared to coke-oven works. For a comparison of concentrations of vapours and aerosols of bitumen and PAHs in asphalt works, differences in sampling and analytical methods must to be taken into account.     

The Human Bitumen Study: executive summary

Bitumen has attracted attention from the scientific community and regulating agencies. The debate on health effects of exposure to vapours and aerosols of bitumen during the hot application of bitumen ranges from respiratory and neurological effects to carcinogenicity. In 2000, the German Hazardous Substances Committee (AGS), in collaboration with the German Bitumen Forum, initiated the examination of a group of mastic asphalt workers and a same number of construction workers without exposure bitumen using a cross-shift design. The study was then extended to the Human Bitumen Study, and the recruitment was finished in 2008 after examination of 500 workers on 80 construction sites. Three hundred and twenty workers exposed to vapours and aerosols of bitumen at high processing temperatures and 118 workers at outdoor construction sites were included. In the Human Bitumen Study external exposure to vapours and aerosols of bitumen, internal exposure to PAH by analysing urinary 1-hydroxypyrene, the sum of hydroxyphenanthrenes and the sum of 1- and 2-hydroxynaphthalenes, irritative effects in the upper and lower airways and genotoxic effects in blood cells were investigated. The study turned out to be one of the largest investigations of workers exposed to vapours and aerosols of bitumen under current exposure conditions. The present paper summarizes its background and main topics.     

Occupational exposure to polycyclic aromatic hydrocarbons and DNA damage by industry: a nationwide study in Germany

Exposure to polycyclic aromatic hydrocarbons (PAH) and DNA damage were analyzed in coke oven (n = 37), refractory (n = 96), graphite electrode (n = 26), and converter workers (n = 12), whereas construction workers (n = 48) served as referents. PAH exposure was assessed by personal air sampling during shift and biological monitoring in urine post shift (1-hydroxypyrene, 1-OHP and 1-, 2 + 9-, 3-, 4-hydroxyphenanthrenes, ΣOHPHE). DNA damage was measured by 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and DNA strand breaks in blood post shift. Median 1-OHP and ΣOHPHE were highest in converter workers (13.5 and 37.2 μg/g crea). The industrial setting contributed to the metabolite concentrations rather than the air-borne concentration alone. Other routes of uptake, probably dermal, influenced associations between air-borne concentrations and levels of PAH metabolites in urine making biomonitoring results preferred parameters to assess exposure to PAH. DNA damage in terms of 8-oxo-dGuo and DNA strand breaks was higher in exposed workers compared to referents ranking highest for graphite-electrode production. The type of industry contributed to genotoxic DNA damage and DNA damage was not unequivocally associated to PAH on the individual level most likely due to potential contributions of co-exposures.     

Bitumen workers handling mastic versus rolled asphalt in a tunnel: assessment of exposure and biomarkers of irritation and genotoxicity

Emission levels of vapours and aerosols of bitumen are different when processing rolled asphalt compared to mastic asphalt, with working temperatures up to 180 and 250°C, respectively. During the Human Bitumen Study, we examined six workers handling rolled asphalt and mastic asphalt in two consecutive weeks at the same construction site in a tunnel. In addition to the determination of exposure to bitumen and polycyclic aromatic hydrocarbons (PAH) during shift, we examined urinary PAH metabolites, irritative and genotoxic effects before and after shift. Median personal shift concentration of vapours and aerosols of bitumen was 1.8 (range 0.9-2.4) mg/m(3) during the application of rolled asphalt and 7.9 (range 4.9-11.9) mg/m(3) when mastic asphalt was applied. Area measurement of vapours and aerosols of bitumen revealed higher concentrations than the personal measurements for mastic asphalt (mastic asphalt: 34.9 mg/m(3); rolled asphalt: 1.8 mg/m(3)). Processing mastic asphalt was associated also with higher PAH concentrations. Urinary 1-hydroxypyrene and the sum of 1-, 2+ 9-, 3- and 4-hydroxyphenanthrene increased slightly during shift without clear difference between mastic and rolled asphalt application. However, the post-shift urinary PAH-metabolite concentrations did not reflect the different PAH exposure during mastic and rolled asphalt application. Individual workers could be identified by their spirometry results indicating that these data reflect more chronic than acute effects. In most cases, an increase of 8-oxodGuo adducts was observed during shift that was independent of the asphalt application. 8-oxodGuo and (+)-anti-BPDE-DNA adducts were higher than in exposed workers of the Human Bitumen Study independent of the asphalt application. The DNA-strand breaks were considerably higher pre-shift and decreased during shift. In this study, mastic asphalt application led to significantly higher exposure to vapours and aerosols of bitumen, as well as to airborne PAH, compared to rolled asphalt application. Nevertheless, no differences in the excretion of urinary PAH metabolites, lung function impairment and genotoxic markers were detected. However, higher levels of genotoxicity markers on both examination days compared with the results of the Human Bitumen Study may indicate a possible influence of the specific tunnel setting.     

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.     

Influence of hOGG1, XRCC1 and XRCC3 genotypes on biomarkers of genotoxicity in workers exposed to cobalt or hard metal dusts

Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. We examined whether variations in genes involved in base-excision (hOGG1, XRCC1) and double strand break (XRCC3) DNA repair contribute to inter-individual differences in genotoxic effects induced in the lymphocytes of 21 cobalt (Co) exposed, 26 hard metal (WC-Co) exposed and 26 matched control male workers. Genotyping was performed by PCR-RFLP. DNA single strand breaks and alkali-labile sites were measured by the alkaline Comet assay. Chromosomal rearrangements resulting from chromosome loss or acentric fragments were assessed as micronucleated mononucleates (MNMC) and binucleates (MNCB) with the cytokinesis-block micronucleus test. Urinary 8-hydroxydeoxyguanosine (8-OHdG) levels were used as an indicator of systemic oxidative DNA damage. A significantly higher frequency of MNMC was observed in WC-Co exposed workers with variant hOGG1(326) genotype. Multivariate analysis performed with genotypes, age, exposure status, type of plant, smoking and their interaction terms as independent variables indicated that MNMC and Comet tail DNA (TD) were influenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MNMC frequencies. Further studies will demonstrate whether genotyping for hOGG1 and XRCC3 polymorphisms is useful for a better individual monitoring of workers.     

Demonstration of Benzo(a)pyrene-Induced DNA Damage in Mice by Alkaline Single Cell Gel Electrophoresis: Evidence for Strand Breaks in Liver but Not in Lymphocytes and Bone Marrow

Abstract: Alkaline single cell gel electrophoresis (also known as the‘comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3×100 mg benzo(a)pyrene/kg b.wt.). No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3×150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.     

Levels and determinants of exposure to vapours and aerosols of bitumen

Bitumen (referred to as asphalt in the United States) is a widely used construction material, and emissions from hot bitumen applications have been a long-standing health concern. One objective of the Human Bitumen Study was to identify potential determinants of the exposure to bitumen. The study population analysed comprised 259 male mastic asphalt workers recruited between 2003 and 2008. Personal air sampling in the workers' breathing zone was carried out during the shift to measure exposure to vapours and aerosols of bitumen. The majority of workers were engaged in building construction, where exposure levels were lower than in tunnels but higher than at road construction sites. At building construction sites, exposure levels were influenced by the room size, the processing temperature of the mastic asphalt and the job task. The results show that protective measures should include a reduction in the processing temperature.     

DNA strand breaks in the lymphocytes of workers exposed to diisocyanates: indications of individual differences in susceptibility after low-dose and short-term exposure

Diisocyanates are chemically reactive and induce asthma, but data on genotoxic effects of diisocyanates in humans are limited. The investigation presented here used short term diisocyanate chamber exposure to study DNA strand breaks in lymphocytes of 10 healthy individuals and of 42 workers, with airway symptoms, who had previously been exposed to diisocyanates. The alkaline version of the Comet assay was used to analyse DNA strand breaks in lymphocytes. In addition, blood samples of 10 further control individuals without any exposure to diisocyanates were studied. Substances studied were 4,4-methylenediphenyldiisocyanate (MDI, n=25), 2,4-toluenediisocynate and 2,6-toluenediisocyanate (TDI, n=5), and 1,6-hexamethylenediisocyanate (HDI, n=12), at concentrations between 5 and 30 ppb for 2 h. Lymphocytes isolated from the subjects before exposure and 30 min and 19 h after were used to evaluate DNA damage. No significant changes in DNA strand-break frequencies were measured, as Olive tail moment (OTM), either between groups or before and after diisocyanate exposure. OTM was similar in subjects with an asthmatic reaction (MDI, n=5; TDI, n=1; HDI, n=1) and in subjects without such a reaction. However, a small and susceptible group (about 10% of the individuals studied) could be identified with higher frequencies of DNA strand breaks in lymphocytes after chamber exposure. The occurrence of DNA damage in this group may be based on indirect mechanisms such as oxidative stress or apoptosis.     

Modulation of oxidative DNA damage by repair enzymes XRCC1 and hOGG1

The influence of DNA repair gene polymorphisms (XRCC1: Arg194Trp, Arg280His, Arg399Gln; APE1: Asp148Glu; hOGG1: Ser326Cys) on oxidative DNA damage is controversial and was investigated in 214 German workers with occupational exposure to vapors and aerosols of bitumen,compared to 87 German construction workers without exposure, who were part of the Human Bitumen Study. Genotypes were determined by real-time polymerase chain reaction (PCR), and actual smoking habits by a questionnaire and cotinine analysis. Oxidative DNA damage in white blood cells (WBC) collected pre- and postshift was measured as 8-oxodGuo adducts/10(6) dGuo by a hjigh-performance liquid chromatography electron capture detection (HPLC-ECD) method, followed by calculation of the difference between post- and preshift values (Δ8-oxodGuo/10(6) dGuo). The 214 bitumen exposed workers showed higher median Δ8-oxodGuo values than the 87 references. In the whole study group (n=301) there was a trend for increasing adduct values for XRCC1 Arg(GG)399Gln(AA) during a shift, especially in nonsmokers (n=108. Referents (n=87) displayed a similar trend for hOGG1 Ser(CC)326Cys(GG). In contrast, XRCC1 Arg(GG)280His(AA) showed a decrease of median Δ8-oxodGuo/10(6) dGuo values in workers with exposure to vapors and aerosols of bitumen (n=214), especially in smokers (n=145). XRCC1 Arg194Trp and APE1 Asp148Glu displayed no marked association with Δ8-oxodGuo levels. Data indicate that the combination of different variants in DNA damage repair enzymes may modulate the production of 8-oxoguanine adducts in WBC produced by xenobiotics during a shift.     

Pre- and postshift levels of inflammatory biomarkers and DNA damage in non-bitumen-exposed construction workers-subpopulation of the German Human Bitumen Study

Circadian variations in immune defense and tissue repair may interfere with shift effects of occupational exposure when investigating biomarkers in cross-shift studies. This investigation compared biomarkers of inflammation and DNA damage in 59 nonsmoking and 59 smoking male construction workers pre- (6-10 a.m.) versus postshift (4-7 p.m.). Cellular compositions were analyzed in blood, induced sputum (IS), and nasal lavage fluid (NALF) and soluble inflammatory biomarkers were analyzed in IS and NALF. DNA damage was measured as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) adducts and DNA strand breaks (alkaline Comet assay) in white blood cells (WBC). Apoptosis was quantified as percent apoptotic cells by annexin V and 7-amino-actinomycin staining in blood lymphocytes using flow cytometry. In nonsmokers higher preshift than postshift levels of interleukin-8 (IL-8) in IS and more DNA strand breaks were detected. However, more DNA adducts were found postshift. Among smokers, the cellular composition of IS and NALF differed between pre- and postshift samples, in particular more neutrophils pre- than postshift. In contrast, more cells in early apoptosis were observed post shift in both smokers and nonsmokers. These results indicate a potential influence of circadian rhythms on several biomarkers used in epidemiological studies. Data suggest interference with shift-work effects of occupational exposure in cross-shift studies and also the need to consider smoking as a modifying variable.     

Genotoxicant induced DNA damage and repair in early and late developmental stages of the grass shrimp Paleomonetes pugio embryo as measured by the comet assay

In this study, data are presented which link frequency of DNA strand breaks and repair capability to developmental stage. Stages 4 and 7 embryos of the grass shrimp (Palaemonetes pugio) were exposed to various concentrations of benzo[alpha]pyrene (BalphaP), Cr(VI) and hydrogen peroxide. Following exposure, responses were measured as changes in hatching rates and DNA strand breaks (using the comet assay). The comet assay was modified by treatment of isolated nuclei with endonucleases which cleave DNA at oxidative lesions in DNA prior to electrophoresis. DNA repair was followed by transfer of toxicant exposed embryos to clean water and periodic determination of strand breaks. DNA strand breaks were higher in stage 7 embryos than in stage 4 embryos after exposure to the same concentration of different genotoxicants. However, when samples were treated with endonucleases to measure oxidative lesions, the total amount of DNA damage between stages 4 and 7 were similar. After toxicant exposure and transfer to clean water, DNA strand breaks in stage 7 embryos returned to background levels more rapidly than in stage 4 embryos. Similarly, samples treated with endonucleases during DNA repair studies showed that oxidative lesions were repaired more rapidly in stage 7 than in stage 4. These findings suggest that because of rapid DNA repair in late embryo stages that early embryo stages are more likely to have developmental effects after genotoxicant exposure.     

Pulmonary exposure to carbon black by inhalation or instillation in pregnant mice: effects on liver DNA strand breaks in dams and offspring

Effects of maternal pulmonary exposure to carbon black (Printex 90) on gestation, lactation and DNA strand breaks were evaluated. Time-mated C57BL/6BomTac mice were exposed by inhalation to 42 mg/m(3) Printex 90 for 1 h/day on gestation days (GD) 8-18, or by four intratracheal instillations on GD 7, 10, 15 and 18, with total doses of 11, 54 and 268 μg/animal. Dams were monitored until weaning and some offspring until adolescence. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) 3-5 days after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Persistent lung inflammation was observed in exposed mothers. Inhalation exposure induced more DNA strand breaks in the liver of mothers and their offspring, whereas intratracheal instillation did not. Neither inhalation nor instillation affected gestation and lactation. Maternal inhalation exposure to Printex 90-induced liver DNA damage in the mothers and the in utero exposed offspring.     

DNA damage in workers exposed to lead using comet assay

Lead (Pb) is a ubiquitous and toxic metal. Secondary Pb recovery unit workers are prone to possible occupational Pb exposure. Hence, this investigation was conducted to assess the genotoxic effect of Pb exposure in these workers. In the study, 45 workers were monitored for DNA damage in blood leucocytes. Simultaneously 36 subjects were used as control group in this study. All the subjects were estimated for Pb content in whole blood by ICP-MS. The alkali single cell gel electrophoresis assay (comet assay) was adopted for detecting the DNA damage. The air inside the premises of the unit had Pb concentrations of 4.2 microg/m(3). The level of DNA damage was determined as the percentage of cells with comets. The mean Pb content was found to be significantly higher in the study group (248.3 microg/l) when compared with the controls (27.49 microg/l). Significantly more cells with DNA damage (44.58%) were observed in the study group than in the control persons (21.14%). Smoking had a significant effect on DNA damage in the control group whereas an insignificant effect was noticed in the exposed workers. Study as well as the control group failed to show a significant effect on DNA damage with age (P>0.05). Pb content and years of exposure significantly correlated with DNA damage in the study group (r=0.602, r=0.690; P<0.01). The increased levels of DNA damage observed in the exposed workers, justifies the use of the comet assay for the evaluation of genotoxic effects in humans exposed to Pb.     

Genotoxicity and ecotoxicity assays using the freshwater crustacean Daphnia magna and the larva of the aquatic midge Chironomus riparius to screen the ecological risks of nanoparticle exposure

Genotoxic and ecotoxic assessments of widely used nanoparticles, cerium dioxide (CeO(2)), silicon dioxide (SiO(2)) and titanium dioxide (TiO(2)), were conducted on two aquatic sentinel species, the freshwater crustacean Daphnia magna and the larva of the aquatic midge Chironomus riparius. CeO(2) may have genotoxic effects on D. magna and C. riparius, given that the DNA strand breaks increased in both species when exposed to this nanoparticle; whereas, neither exposure to SiO(2) nor TiO(2) had a genotoxic effect on either species. A statistically significant correlation was observed between DNA damage and mortality in the CeO(2)-exposed C. riparius, which suggests that CeO(2)-induced DNA damage might provoke higher-level consequences. SiO(2) did not seem to affect the DNA integrity; whereas, the mortality of both the SiO(2)-exposed D. magna and C. riparius increased. The TiO(2) nanoparticle did not lead to significant alterations in geno- or ecotoxic parameters of both species. Overall, these results suggest that CeO(2) nanoparticles may be genotoxic toward aquatic organisms, which may contribute to the knowledge relating to the aquatic toxicity of the most widely used nanomaterials on aquatic ecosystems, for which little data are available.     

Comparative evaluation of DNA damage by genotoxicants in primary rat cells applying the comet assay

Various compounds known to cause DNA damage (hydrogen peroxide, visible light-excited methylene blue, N-nitrosomorpholine and benzo[a]pyrene) were tested with different primary rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) to determine the range of induced DNA damage applying the comet assay. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all cell types studied. The most prominent effect was observed in lymphocytes, whereas only a slight increase of DNA breaks was observed in hepatocytes. Visible light-excited methylene blue caused significant oxidative DNA damage, which did not significantly differ between the cell types used with the exception of hepatocytes, for which a lower level of DNA damage was observed. N-Nitrosomorpholine and benzo[a]pyrene induced a moderate but significant increase of DNA strand breaks in pneumocytes and hepatocytes while in lymphocytes no effect was observed. Our results clearly demonstrate that due to their differential function which is also expressed by the level of drug metabolizing and/or antioxidant enzymes, freshly isolated rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) respond differently to the exposure to genotoxic agents as detected by comet assay.     

Genotoxic pressure of vineyard pesticides in fish: field and mesocosm surveys

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2mugL(-1) for diuron and 0.5-1mugL(-1) for axoxystrobin). A single 6h pulse of pesticide (14mugL(-1) of diuron and 7mugL(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.     

Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine

S-(trans-1,2-dichlorovinyl)-L-cysteine (DCVC) is a recognized nephrotoxin. To investigate the genotoxic effects of DCVC on the kidney, DNA strand breaks were measured as an indicator of DCVC induced damage. To ascertain if bioactivation of DCVC occurred in the kidney, 3 experimental systems were used: in vivo; isolated perfused kidneys; and isolated proximal tubules of albino male rabbits. A dose-dependent increase in strand breaks in the kidney tubular DNA occurred after in vivo dosing with 5-100 mg/kg DCVC and after in vitro exposure to 10(-5)-10(-2) M DCVC. These results demonstrate the genotoxic effect of this compound on renal tissue.