Influence of DNA repair polymorphisms on biomarkers of genotoxic damage in peripheral lymphocytes of healthy subjects.

DNA repair polymorphisms may represent susceptibility factors affecting DNA integrity, and possibly cancer risk, in human population. In order to elucidate the influence of a few widely studied DNA repair polymorphisms on individual levels of DNA damage and their possible interaction with lifestyle and environmental exposures, 171 subjects from a well-characterized human population enrolled in a previous study on genetic effects of air pollution were genotyped for the XRCC1 Arg280His and Arg399Glu, XRCC3 Thr241Met and ERCC2 Lys751Gln polymorphisms. The association between DNA repair genotype, alone or in combination with metabolic genotype, on the levels of SCE, micronuclei and tail moment values in peripheral lymphocytes was evaluated. A significant influence of the ERCC2 genotype on SCE frequency was observed. Subjects with ERCC2 751 Gln/Gln genotype had significantly higher risk of high (above the median) SCE/cell with respect to Lys/Lys referents (OR 4.55, 95% CI 1.48-13.99). A non-significantly elevated OR was also observed in Gln/Lys heterozygotes, suggesting a gene dosage effect. When subjects were categorized by smoking habits and professional exposure, the variant ERCC2 751 Gln/Gln genotype was associated with elevated SCE rates in non-smokers and in exposed subjects, but not in smokers. The results of this study support the hypothesis that some DNA repair polymorphisms exert a modifying effect on individual levels of DNA damage in healthy subjects, possibly also modulating cancer risk.

Genotoxicity of the disinfection by-products resulting from peracetic acid- or hypochlorite-disinfected sewage wastewater.

Wastewater disinfection is routinely carried out to prevent the spread of human pathogens present in wastewater effluents. To this aim, chemical and physical treatments are applied to the effluents before their emission in water bodies. In this study, the influence of two widely used disinfectants, peracetic acid (PAA) and sodium hypochlorite (NaClO), on the formation of mutagenic by-products was investigated. Wastewater samples were collected before and after disinfection, in winter and in summer, at a pilot plant installed in a municipal wastewater-treatment plant. Samples were adsorbed using silica C18 cartridges and the concentrates were tested for mutagenicity in the Salmonella typhimurium reversion test with strains TA98 and TA100. Non-concentrated water samples were tested with two plant genotoxicity assays (the Allium cepa root anaphase aberration test and the Tradescantia/micronucleus test). Mutagenicity assays in bacteria and in Tradescantia showed borderline mutagenicity in some of the wastewater samples, independent of the disinfection procedure applied. Negative results were obtained in the A. cepa anaphase aberration test. These results indicate that, in the conditions applied, wastewater disinfection with PAA and NaClO does not lead to the formation of significant amounts of genotoxic by-products.

[Genotoxic and ecotoxic effects of urban waste water disinfected with sodium hypochlorite or peracetic acid]. / Effetti genotossici ed ecotossicologici di acque reflue urbane sottoposte a disinfezione con ipoclorito di sodio o acido peracetico.

Genotoxic and ecotoxicologic effects of urban wastewater disinfected with sodium hypochlorite or peracetic acid were analyzed. The formation of genotoxic species was studied by determining clastogenic and mutagenic activity of aqueous samples and their extracts with in vivo and in vitro tests, respectively. In particular, we have applied citogenetic tests to Allium cepa roots and Tradescantia inflorescence (Allium cepa test and Tradescantia/micronuclei test) and reversion test to Salmonella typhimurium according to the microsuspension procedure (Kado test). The latter is the method of choice for the analysis of complex matrices due to its high sensitivity and specificity. The mutagenic activity of disinfected effluents was similar to the corresponding untreated wastewater both sampled in four different periods. Therefore, the disinfection process did not seem to contribute to aquatic mutagenicity in the examined range of biocide concentration. The potential toxicity of disinfected wastewater for aquatic organisms was evaluated using Daphnia magna. The acute toxicity of peracetic acid in sewage was 0.4 mg/L (24 h E(L)C50). By comparing this value with peracetic acid concentrations detected in effluents from a pilot plant it is expected that treated wastewater would show acute toxic effects on aquatic organisms. Dissociation compounds (hydrogen peroxide and acetic acid) and possible by-products of peracetic acid did not seem to contribute significantly to the toxicity of sewage treated with peracetic acid.

Folate status, metabolic genotype, and biomarkers of genotoxicity in healthy subjects.

Gene-environment interactions play an important role in folate metabolism, with a potential impact on human health. Deficiencies in the uptake of key micronutrients and variant genotypes can affect the folic acid cycle, modulating methyl group transfer in key processes and leading to increased cancer risk and Down syndrome incidence. So far, the significance of folate status and metabolic genotypes on baseline levels of DNA damage in normal individuals has not been fully elucidated. In this study, the possible modulation of SCE, micronuclei and tail moment values in peripheral lymphocytes by plasma levels of folic acid, homocysteine and vitamin B12, and by the methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G polymorphisms was investigated in 191 healthy subjects. The results obtained show a highly significant (P = 0.001) positive association between plasma levels of vitamin B12 and frequencies of both SCE and high frequency cells (HFC, above 90 degrees percentile) in smokers. No significant effect was observed in non-smokers. Moreover, after correction for age, gender and GSTM1 genotype, a significant association (P = 0.026) between the MTRR 66GG variant genotype and higher micronucleus rates was observed. Tail moment values were not affected by any of the independent variables considered. Overall, the results obtained suggest that both folate status and relevant metabolic genotype can influence background levels of DNA damage in normal subjects. The significant association observed in smokers between plasma vitamin B12 and SCE frequencies may highlight the effect of methylation status on DNA damage and repair, although the role of other, unidentified dietary factors cannot be ruled out. At the same time, micronucleus data indicate that the MTRR 66GG variant may represent another individual trait of relative genomic instability, thus supporting epidemiological data on increased risk of Down syndrome conception in MTRR 66GG subjects.

Analysis of micronuclei in peripheral blood lymphocytes of traffic wardens: effects of exposure, metabolic genotypes, and inhibition of excision repair in vitro by ARA-C.

The cytokinesis-block micronucleus (MN) assay in peripheral lymphocytes was used to assess the genetic effects of the occupational exposure to traffic fumes in policemen from the Municipality of Rome. The study population consisted of 192 subjects engaged in traffic control (exposed, 134 subjects), or in office work (controls, 58 subjects). Groups were balanced for age, gender, and smoking habits. The average benzene exposure during the workshift was 9.5 and 3.8 microg/m(3) in exposed individuals and controls, respectively. All subjects were genotyped for CYP1A1, CYP2E1, GSTM1, GSTT1, and DT-diaphorase polymorphisms. The incidence of micronuclei and micronucleated cells was recorded in 1,000 binucleated cells harvested 66 hr after mitogen stimulation. Regression analysis of data showed that MN frequency was mainly modulated by the age (P = 0.001) and gender (P = 0.001) of the study subjects (relatively higher in the elderly and females), whereas it was unaffected by the occupational exposure to traffic fumes and smoking habits. A weak (P = 0.02) association between lower MN frequency and the GSTM1 null genotype was also observed. In order to improve the sensitivity of the method to excision-repairable lesions, a modified protocol, with exposure of cells to the repair inhibitor cytosine arabinoside (Ara-C) during the first 16 hr of growth, was applied to 78 subjects (46 exposed and 32 controls). The results confirmed the higher MN frequency in females (P < 0.05), but failed to demonstrate any significant effect of chemical exposure (occupational or related to smoking habits). When the frequency of MN induced by Ara-C (i.e., spontaneous values subtracted) was considered, a significant inverse correlation with age was observed (P = 0.005), possibly related to the age-dependent decrease in repair proficiency.

Biomonitoring of exposure to urban air pollutants: analysis of sister chromatid exchanges and DNA lesions in peripheral lymphocytes of traffic policemen.

In order to elucidate the health effects of occupational exposure to traffic fumes, a few biomarkers of early genetic effect were investigated in Rome traffic policemen. One hundred and ninety healthy subjects engaged in traffic control (133 subjects) or in office work (57 subjects) participated the study. For all subjects, detailed information on smoking habits and other potential confounders were recorded by questionnaires. Average exposure of the study groups to benzene and other aromatic hydrocarbons was evaluated in a parallel exposure survey. All workers were genotyped for the following metabolic polymorphisms: CYP1A1 (m1, m2, and m4 variants), CYP2E1 (PstI and RsaI), NQO1 (Hinf1), GSTM1 and GSTT1 (null variants). In this paper, the results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported. No statistically significant difference in the frequency of SCE or high frequency cells (HFC) was observed between traffic wardens and office workers (controls), despite the significantly higher exposure to benzene of the former (average group exposure 9.5 versus 3.8microg/m(3), 7h TWA). Conversely, both SCE per cell and HFC were highly significantly (P<0.001) increased in smokers compared to nonsmokers, showing a significant correlation (P<0.001) with the number of cigarettes per day. Multiple regression analyses of data, with metabolic polymorphisms, smoking habits, alcohol consumption, age, gender, and family history of cancer as independent variables, showed that smoking habits, and possibly the CYP2E1 variant genotypes, were the main factors explaining the variance of both SCE and HFC. Within smokers, an association of borderline significance between the CYP1A1 variant genotypes and increased SCE (P=0.050) and HFC (P=0.090) was found. This effect was mainly observed in light smokers (<15 cigarettes per day). The analysis of DNA damage by comet assay did not highlight any statistically significant difference between the exposed and control workers. Moreover, no significant model explaining tail moment variance was obtained by multiple regression analysis using the independent variables shown above. On the whole, these results indicate that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells. This evidence does not rule out any possibility of adverse effects, but strongly suggests that in urban residents life-style related factors, such as tobacco smoking, give the prevailing contribution to individual genotoxic burden.

Detection of 1cen--1q12 lesions in different phases of the cell cycle: dual colour FISH analysis of peripheral lymphocytes from subjects with occupational exposure to petroleum fuels.

Dual colour FISH was used to assess the genotoxic effects of exposure to petroleum fuels and low benzene levels in peripheral lymphocytes of 12 gasoline station attendants. Labelled DNA probes were used for hybridization of the 1cen and 1q12 contiguous regions of chromosome 1, allowing simultaneous detection of hyperploidy and breakages in both interphase and metaphase cells. The analysis of interphase cells (either unstimulated or mitogen stimulated) showed a prevalence of cells with signal separation in exposed workers compared to matched controls. This difference was highly significant (P < 0.001) in stimulated lymphocytes (9.9 +/- 3.3 and 6.5 +/- 1.5 per thousand in exposed and controls, respectively). Far lower incidences of breaks, with no relation to chemical exposure, were detected in metaphase cells (0.3 +/- 0.8 versus 0.7 +/- 1.0 per thousand, respectively). The analysis of post-mitotic, cytokinesis-blocked cells again showed a relatively high incidence of nuclei with displacement of fluorescent signals (7.2 +/- 2.4 and 5.6 +/- 1.7 per thousand, respectively), suggesting that chromatin decondensation, rather than alteration of DNA strand integrity, led to signal separation in interphase nuclei. Even though the mechanism leading to the separation of alpha and classical satellites in interphase nuclei has not been elucidated, the significant association between cytogenetic findings and intensity of benzene exposure (as shown by the analysis of internal exposure biomarkers) suggests that signal displacement in 1cen-1q12 may be a marker of chemical exposure.

Detection of antibodies to the benzo(a)pyrene diol epoxide-DNA adducts in sera from individuals exposed to low doses of polycyclic aromatic hydrocarbons.

The exposure to DNA reactive carcinogens is known to elicit a specific humoral immunological response, with the production of antibodies toward the carcinogen adducts. Consequently, the presence of circulating anti-carcinogen antibodies has been proposed as a marker of carcinogen exposure, and as a potential modulating factor in chemical carcinogenesis. In this work, the presence of serum antibodies to 7beta,8alpha-dihydroxy-9alpha10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-DNA (BPDE-DNA) adducts was determined in two groups of workers occupationally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs), i.e. policemen (194 subjects) and workers in the aluminum industry (105 subjects). Specific anti BPDE-DNA antibodies were detected in 5.7% (11/194) of policemen and 13.3% (14/105) of aluminium industry workers. Among policemen, a small, not significant (p=0.09), prevalence of positives was observed in traffic wardens compared to office workers. A borderline significant (p=0.052) prevalence of positives was also observed in heavy smokers compared to light smokers among aluminium industry workers. These results basically support previous findings on the association between chronic exposure to polycyclic aromatic hydrocarbons and formation of anti-BPDE-DNA antibodies, even though such association appears to be weak, possibly biased by individual factors which are still largely unidentified.

Environmental and biological monitoring of traffic wardens from the city of Rome.

A molecular epidemiological study on Roman policemen is ongoing. The results of a first assessment of the occupational exposure to aromatic compounds of 66 subjects engaged in traffic control and of 33 office workers are presented in this paper. Passive personal samplers and urinary biomarkers were used to assess exposure to benzene and polycyclic hydrocarbons during work shifts. The results obtained indicate that benzene exposure in outdoor workers is about twice as high as in office workers (geometric mean 7.5 and 3.4 micrograms/m3, respectively). The distribution of individual exposure values was asymmetrical and skewed toward higher values, especially among traffic wardens. Environmental benzene levels recorded by municipal monitoring stations during work shifts (geometric mean 11.2 micrograms/m3) were in the first instance comparable to or greater than individual exposure values. However, several outlier values were observed among personal data that greatly exceeded average environmental benzene concentrations. Among the exposure biomarkers investigated, only blood benzene correlated to some extent with previous exposure to benzene, while a seasonal variation in the excretion of 1-hydroxypyrene and trans-muconic acid was observed in both study groups. In conclusion, these results suggest that outdoor work gives a greater contribution than indoor activities to benzene exposure of Roman citizens. Moreover, relatively high-level exposures can be experienced by outdoor workers, even in the absence of large-scale pollution episodes.

Exposure to benzene in urban workers: environmental and biological monitoring of traffic police in Rome.

OBJECTIVES: To evaluate the contribution of traffic fumes to exposure to benzene in urban workers, an investigation on personal exposure to benzene in traffic police from the city of Rome was carried out. METHODS: The study was performed from December 1998 to June 1999. Diffusive Radiello personal samplers were used to measure external exposures to benzene and alkyl benzenes during the workshift in 139 policemen who controlled medium to high traffic areas and in 63 office police. Moreover, as biomarkers of internal exposure to benzene, blood benzene, and urinary trans, trans-muconic and S-phenyl mercapturic acids were measured at the beginning and at the end of the workshift in 124 traffic police and 58 office police. RESULTS: Time weighted average (TWA) exposure to benzene was consistently higher among traffic police than among indoor workers (geometric mean 6.8 and 3.5 microg/m(3), respectively). Among the traffic police, the distribution of individual exposures was highly asymmetric, skewed toward higher values. Mean ambient benzene concentrations measured by municipal air monitoring stations during workshifts of traffic police were generally higher (geometric mean 12.6 microg/m(3)) and did not correlat with personal exposure values. In particular, no association was found between highest personal exposure scores and environmental benzene concentrations. Among the exposure biomarkers investigated, only blood benzene correlated slightly with on-shift exposure to benzene, but significant increases in both urinary trans, trans-muconic and S-phenylmercapturic acids were found in active smokers compared with non-smokers, irrespective of their job. CONCLUSION: The exposure to traffic fumes during working activities in medium to high traffic areas in Rome may give a relatively greater contribution to personal exposure to benzene than indoor sources present in confined environments. Smoking significantly contributed to internal exposure to benzene in both indoor and outdoor workers.

Threshold-mediated mechanisms in mutagenesis: implications in the classification and regulation of chemical mutagens.

Chemical mutagens are currently regulated and labelled on the basis of their hazardous properties defined in hazard classification schemes. The strength and type of experimental evidence is used as the only criterion for classification in categories which express different levels of concern for the possibility of adverse effects - notably transmissible genetic alterations - in humans. Differently from the classification of carcinogens, no consideration is given to potency, nor to the mechanism of action. The rationale of such hazard based classification is that the hazardous property of a chemical is an intrinsic feature, which is expressed independently of dosing. Changing of dose level results in a mere change in the probability to observe an adverse effect, but not in its potential occurrence. The lack of theoretical threshold underlying this approach can be envisaged, in principle, for stochastic processes such as DNA damage, which can be triggered by single molecular interactions. On the other hand, indirect mechanisms of genotoxicity, involving multiple interactions with non-DNA targets, are expected to show a threshold. At variance to DNA reactive agents, chemicals acting with threshold-mediated mechanism do change also qualitatively their toxic properties depending on the dose level. Possible problems arising in the application of hazard based schemes for the evaluation of chemicals with threshold-mediated mechanism of action are discussed, using the spindle poisons benzimidazole fungicides as an example.

The effect of humoral immunity against adducted benzo[a]pyrene on DNA damage elicited by acute carcinogen exposure in Swiss mice.

Immunoglobulins G (lgG) specific for benzo[a]pyrene-DNA adducts were elicited in Swiss mice by repeated subcutaneous injections of a high molecular weight benzo[a]pyrene-DNA conjugate-adjuvant mix. The immunization procedure resulted in the production of specific antibodies against adducted benzo[a]pyrene B[a]P in all treated animals. One week after completion of the immunization procedure, groups of ten immunized and ten non immunized female mice were treated by single intraperitoneal injection with two different doses of B[a]P. The mice were sacrificed 48 hours after treatment, and both liver and bone marrow cells were isolated for subsequent determinations of DNA binding and micronucleus induction, respectively. Covalent benzo[a]pyrene adducts in liver DNA were detected by competitive ELISA and the incidence of micronucleated polychromatic erythrocytes was evaluated by scoring one thousand cells per animal. The determination of DNA adducts in liver revealed significantly (p < 0.05) lower levels of B[a]P adducts in immunized mice compared to non-immunized animals at both doses, whereas no significant difference was observed between controls. Administration of benzo[a]pyrene produced moderate, dose-related increases in the incidence of micronucleated polychromatic erythrocytes in all treated groups, with no significant difference between immunized and non-immunized mice. The decrease of covalent DNA adducts in the liver of immunized mice suggests that the specific humoral immunity elicited by repeated carcinogen exposure may act as a relevant modulating factor in chemical carcinogenesis.

Chromosome damage and aneuploidy detected by interphase multicolour FISH in benzene-exposed shale oil workers.

A multicolour tandem-labelling fluorescence in situ hybridization (FISH) procedure was used to detect chromosome alterations in peripheral blood cells of a group of Estonian petrochemistry workers. Twelve workers employed in benzene production and five cokery workers, together with eight unexposed rural controls, were enrolled in the study. The methodology employed, based on the in situ hybridization of adjacent centromeric and pericentromeric regions, allowed the simultaneous detection of both chromosome breakage, involving damage-prone pericentromeric regions, and hyperploidy in interphase cells. Blood smears from all subjects were hybridized with chromosome 1 specific probes, in order to detect genotoxic damage in circulating lymphocytes and granulocytes. Moreover, lymphocyte cultures were established, harvested 48 h following mitogen stimulation and hybridized with the tandem chromosomes 1 and 9 probes. No significant difference in the incidence of breakage was detected in the nucleated cells of blood smears of exposed vs. control subjects. In contrast, modest but significantly increased frequencies of breakage affecting both chromosomes 1 and 9 were observed in the cultured lymphocytes of the benzene-exposed workers compared to the unexposed controls, suggesting an expression of premutagenic lesions during the S-phase in vitro. Across the entire study group, the frequencies of breakage affecting chromosomes 1 and 9 in the stimulated lymphocytes were highly intercorrelated (p < 0.001). No significant difference was found in the incidence of hyperploidy among the study groups, although a tendency to higher values was observed in benzene-exposed workers. Although the relatively small size of the study groups does not allow firm conclusions on the role of occupational exposure, the observed patterns are suggestive of effects in the benzene-exposed workers. This work also shows that tandem labelling FISH can be usefully applied in human biomonitoring, allowing the simultaneous detection of both hyperploidy and chromosome breakage at interphase in different cell types.

Analysis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects.

Chromosome malsegregation in peripheral blood lymphocytes of 24 healthy male subjects was analysed by means of fluorescence in situ hybridization with centromeric probes of chromosomes 7, 11, 18 and X. On the basis of the distribution of centromeric signals in cytokinesis-blocked cells, both loss (leading to centromere-positive micronuclei) and non-disjunction (resulting in an unbalanced distribution of signals in the main nuclei) of the hybridized chromosomes in vitro were identified. In addition, the incidence of binucleated cells with two hyperploid nuclei, possibly arising from mitotic division of trisomic types, was determined. In this way, the incidence of chromosome malsegregation in vivo and in vitro could be compared in the same cell samples. The results obtained show that ageing is positively correlated with the incidence of malsegregation of chromosome X in peripheral lymphocytes of male subjects and confirm the higher susceptibility of chromosome X to malsegregation in comparison with autosomes. A positive correlation between in vitro and in vivo malsegregation rates was observed for both chromosome X and for autosomes. Finally, relatively high frequencies of multiple malsegregation events, greater than expected for independent events, were recorded for both chromosome X and for autosomes, indicating that the abnormal segregation of chromosomes may be connected to a general dysfunction of the mitotic apparatus. The correlation observed between in vitro and in vivo malsegregation frequencies and the association of both parameters with ageing suggest that analysis of chromosome malsegregation in binucleated cells is a useful tool in the study of genomic instability in human populations.

Processing of DNA damage induced by hydrogen peroxide and methyl methanesulfonate in human lymphocytes: analysis by alkaline single cell gel electrophoresis and cytogenetic methods.

The persistence of induced DNA damage in human lymphocytes after mitogen stimulation and its relationship to subsequent cytogenetic alterations were investigated. The analysis of single-strand breaks and alkali-labile sites by single cell gel electrophoresis (SCGE) showed the almost complete repair of damage induced in resting lymphocytes by methyl methanesulfonate (MMS, 140-210 microM) and hydrogen peroxide (H(2)O(2), 25-100 microM) during the first 16 h of culture. On the other hand, DNA damage was shown to persist to a large extent when cells were cultured in the presence of the repair inhibitor cytosine beta-D-arabinofuranoside (Ara-C) (1 microg/ml). Although highly effective in the induction of DNA lesions detectable by SCGE, both agents failed to significantly increase the rate of micronucleus formation in cytokinesis-blocked cells harvested 66 h after treatment. However, when Ara-C was present during the first 16 h of culture, micronuclei were significantly increased at all doses. Conversely, sister chromatid exchange (SCE) rates were increased by chemical treatments to a higher extent in cultures without Ara-C. Delayed treatments, 16 h after mitogen stimulation, led to a significant induction of micronuclei in the case of MMS but not with H2O(2). These results suggest that only a minor fraction of DNA damage induced in resting lymphocytes is available for fixation through misreplication, because of its effective repair prior to S phase. However, the processing of damage through recombination pathways can lead to increased SCE rates in treated cells. These features of the processing of DNA damage in human lymphocytes should be taken into account when structural cytogenetic alterations in cultured lymphocytes are used in monitoring human exposure to genotoxic agents.

Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test.

Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70-80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.

DNA damage by hydroquinone in human white blood cells: analysis by alkaline single-cell gel electrophoresis.

The genotoxicity of hydroquinone (HQ) in human white blood cells was investigated by means of alkaline single-cell gel electrophoresis (SCGE). The exposure of purified lymphocytes to HQ (0.5-50 microg/ml) produced significant and dose-related increases in DNA migration; conversely, no induction of DNA damage was observed in leukocytes after in vitro treatment of whole blood samples (100-500 microg/ml). Similar differences in DNA damage between whole blood samples and purified lymphocytes were observed after treatments with hydrogen peroxide (H2O2, 50 microM). The DNA damaging activity of HQ was significantly (p<0.001, U-test) inhibited by exogenous catalase (250 U/ml), indicating the generation of peroxides in the mechanism of genotoxicity of HQ. Parallel experiments using the standard SCGE protocol, and an acellular method entailing the lysis of cells before HQ treatment, provided fairly similar results, indicating that HQ oxidation does not require endogenous metabolism. Experiments to compare the effectiveness of HQ in the induction of single-strand breaks and alkali-labile sites in resting cells and micronuclei in cytokinesis-blocked cells indicate that despite the extensive DNA damage detected by SCGE immediately after treatment, a significant excess of micronuclei is not observed after stimulation and in vitro cultivation. These data explain the apparent discrepancy between the high DNA damaging potential of HQ in human lymphocytes, as revealed by SCGE, and the relatively low activity reported in most cytogenetic assays with HQ on the same cell type.

Genetic effects of petroleum fuels: II. Analysis of chromosome loss and hyperploidy in peripheral lymphocytes of gasoline station attendants.

Molecular cytogenetic methods were applied to investigate the effect of the occupational exposure to low concentrations of benzene and petroleum fuels on genomic stability. Twelve male gasoline station attendants (average benzene exposure of 0.32 mg/m3 as 8h TWA) and 12 age- and smoking-matched unexposed controls were selected for the study. The incidence of hyperploidy and polyploidy in peripheral lymphocytes was evaluated through in situ hybridization of interphase cells, harvested 24 hr after stimulation, with centromeric probes of chromosomes 7, 11, 18, and X. For half of the subjects, metaphases harvested 24 hr later were analyzed. The incidence of chromosome loss in vitro was determined in cytokinesis-blocked cells, harvested at 66 hr, through the hybridization of micronuclei with a pancentromeric probe. Ten thousand chromosomes (more than 200 metaphases equivalent) and 2,000 binucleated cells/person were scored for hyperploidy and micronucleus analysis, respectively. The results obtained did not show any exposure-related excess of hyperploidy or micronucleus formation. Conversely, the age of the subjects was significantly correlated with several markers of genomic instability, such as the incidence of chromosome X and chromosome 18 hyperploidy, total hyperploidy and polyploidy, and close to statistical significance with chromosome loss. Smoking habits did not appear to contribute significantly to the effects measured. The parallel analysis of hyperploidy and polyploidy in interphase nuclei in 24-hr cultures and in metaphase cells harvested 24 hr later showed basically similar incidences of aneuploid cells, indicating that no significant selection against hyperploid and polyploid types occurred during the first cell cycle in vitro.

Analysis of micronuclei and DNA single-strand breaks in mouse splenocytes and peripheral lymphocytes after oral administration of tetramethylthiuram disulfide (thiram).

The fungicide thiram (tetramethylthiuram disulfide, TMTD) was administered by repeated oral intubations to groups of male B6C3F1 mice at 100, 300 and 900 mg/kg body weight for 4 consecutive days, or at 300 mg/kg for 8 and 12 days. 24 hr after the last treatment animals were killed, and splenocyte cultures were set up for the analysis of micronuclei by the cytokinesis-block method. DNA single strand breaks (ssb) and alkali labile sites were also analysed by the single cell gel electrophoresis (Comet) assay in splenocytes and lymphocytes of animals receiving the 8- and 12-day treatments. Parallel experiments with human peripheral lymphocytes were carried out to assess the ability of thiram to induce micronuclei and DNA ssb and alkaline labile sites under in vitro conditions. No significant increase of micronucleated splenocytes was observed in treated animals, despite some evidence of treatment-related cellular toxicity. A borderline excess of DNA damage was suggested by the Comet assay on circulating lymphocytes, whereas negative results were obtained with splenocytes. In vitro, positive results with both genetic end points were obtained in assays with human lymphocytes in the dose ranges 0.5-24 microg/ml and 0.1-8 microg/ml for micronucleus and Comet assays, respectively. These results suggest that thiram, despite its established genotoxicity in vitro, is devoid of appreciable clastogenic and/or aneugenic activity in vivo after oral administration to mice at the maximum tolerated dose.