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.

Association of metabolic gene polymorphisms with alcohol consumption in controls.

The objectives were to study the association between metabolic genes involved in alcohol metabolism (CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1) and alcohol consumption in a large sample of healthy controls. Healthy subjects were selected from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). Subjects with information on both alcohol consumption and at least one of the studied polymorphisms were included in the analysis (n=2224). Information on the amount of alcohol consumption was available for a subset of subjects (n=844). None of the studied genes was significantly associated with drinking habits. A significant heterogeneity with age was observed when studying the association between CYP2E1 RsaI and alcohol drinking. CYP2E1 RsaI polymorphism was significantly associated with being a never drinker at older ages (odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2-4.8; at ages above 68 years), while the association was reversed at ages below 47 years (OR 0.5, 95% CI 0.2-1.4). For subjects with detailed information on alcohol intake, no association between alcohol quantity and polymorphisms in metabolic genes was observed; subjects carrying the NQO1 polymorphism tended to drink more than subjects carrying the wild-type alleles. Therefore, no significant association between CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1 polymorphisms and alcohol consumption was observed in healthy controls.

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.

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei.

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis-block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of "normal" values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin-B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5 per thousand and the interquartile range was between 3 and 12 per thousand. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14-24%). Statistical analyses were performed using random-effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods.

Metabolic polymorphisms and urinary biomarkers in subjects with low benzene exposure.

The effect of some common metabolic polymorphisms on the rate of trans,trans-muconic acid (TMA) and S-phenylmercapturic acid (SPMA) excretion was investigated in 169 policemen exposed to low benzene levels (<10 microg/m3) during the work shift. End-shift urinary concentrations of TMA and SPMA, normalized to unmetabolized blood benzene concentration, were used as indicators of individual metabolic capacity. CYP2E1, NQO1, GSTM1, and CSTT1 polymorphisms were analyzed in all subjects by polymerase chain reaction (PCR) restriction fragment length (RFL). The results obtained show significantly elevated levels of TMA and SPMA in urine of smokers compared to nonsmokers, whereas no correlation with environmental benzene was observed. TMA/blood benzene ratio was partially modulated by glutathione S-transferase (GST) genotypes, with significantly higher values in null individuals (GSTM1 and GSTT1 combined). However, a greater fraction of total variance of TMA/blood benzene in the study population was explained by other independent variables, that is, season of sampling, smoking habits, and gender. Variance in SPMA/blood benzene ratio was only associated with smoking and occupation, whereas no significant role was observed for the metabolic polymorphisms considered. These results suggest that in a population exposed to very low benzene concentrations, urinary TMA and SPMA levels are affected to a limited extent by metabolic polymorphisms, whereas other factors, such as gender, lifestyle, or other confounders, may account for a larger fraction of the interindividual variability of these biomarkers.

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.

Mismatch repair and differential sensitivity of mouse and human cells to methylating agents.

The long-patch mismatch repair pathway contributes to the cytotoxic effect of methylating agents and loss of this pathway confers tolerance to DNA methylation damage. Two methylation-tolerant mouse cell lines were identified and were shown to be defective in the MSH2 protein by in vitro mismatch repair assay. A normal copy of the human MSH2 gene, introduced by transfer of human chromosome 2, reversed the methylation tolerance. These mismatch repair defective mouse cells together with a fibroblast cell line derived from an MSH2-/- mouse, were all as resistant to N-methyl-N-nitrosourea as repair-defective human cells. Although long-patch mismatch repair-defective human cells were 50- to 100-fold more resistant to methylating agents than repair-proficient cells, loss of the same pathway from mouse cells conferred only a 3-fold increase. This discrepancy was accounted for by the intrinsic N-methyl-N-nitrosourea resistance of normal or transformed mouse cells compared with human cells. The >20-fold differential resistance between mouse and human cells could not be explained by the levels of either DNA methylation damage or the repair enzyme O6-methylguanine-DNA methyltransferase. The resistance of mouse cells to N-methyl-N-nitrosourea was selective and no cross-resistance to unrelated DNA damaging agents was observed. Pathways of apoptosis were apparently intact and functional after exposure to either N-methyl-N-nitrosourea or ultraviolet light. Extracts of mouse cells were found to perform 2-fold less long-patch mismatch repair. The reduced level of mismatch repair may contribute to their lack of sensitivity to DNA methylation damage.

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.

Reversal of methylation tolerance by transfer of human chromosome 2.

Human cell lines resistant to N-methyl-N-nitrosourea (MNU) were previously assigned to two complementation groups. Members of group I are defective in mismatch correction [S. Ceccotti, G Aquilina, P. Macpherson, M. Yamada, P. Karran, M. Bignami, Processing of O6-methylguanine by mismatch correction in human cell extracts. Current Biol. 6 (1996) 1528-1531]. To identify the mechanism responsible for the less pronounced phenotype of the second complementation group, we characterized the persistence of MNU-induced O6-methylguanine (O6-meGua) and mutation induction at the hypoxanthine guanine phosphoribosyl-transferase (HPRT) locus. Group II clones are unable to repair the premutagenic base O6-meGua and are as mutable by MNU as group I clones and the parental HeLaMR cells. MNU-induced SCE were undetectable in group I clones and drastically reduced in group II in comparison with the parental cells. These observations are consistent with a defective processing of DNA methylation damage by members of both groups. Group II clones exhibit a moderate spontaneous mutator phenotype at the HPRT gene but significant instability at mononucleotide repeat microsatellites. Introduction of a single human chromosome 2 (but not of chromosome 3 or 7) into group II cells partially reverts both MNU resistance and the increased spontaneous mutation rate. The properties of group II variants are consistent with methylation tolerance and a partially defective mismatch repair. We propose that members of group II are defective in the chromosome 2-based mismatch correction gene GTBP/hMSH6.

Analysis of chromosome segregation by means of fluorescence in situ hybridization: application to cytokinesis-blocked human lymphocytes.

The application of methods based on in situ hybridization to centromeric regions to cytokinesis-blocked cells provides a convenient way for the analysis of chromosome segregation in interphase cells. In this way, the reciprocal segregation patterns in daughter nuclei can be visualized and most of the problems related to the artefactual loss or gain of chromosomes which flaw other methods are avoided. In this work, the methodology has been applied to human lymphocytes to investigate the influence of donor age on spontaneous malsegregation rates, the occurrence of multiple malsegregation events, and the effect of the cytokinesis-blocking agent cytochalasin B (Cyt B) on spontaneous and induced chromosome malsegregation. The results obtained with 14 male donors, aged 22-57 years, demonstrated a significant (p < 0.001) increase in the frequency of micronuclei and X chromosome missegregation (both non-disjunction and chromosome loss) with the increasing age of the donors. Moreover, a similar association was observed with cultures hybridized with either chromosome 8 or 18 centromere probes, suggesting that the age-related loss of fidelity in chromosome segregation in vitro may be a general trait. The investigation of the distribution of multiple malsegregation events in cultured lymphocytes of eight male and nine female donors, with the simultaneous hybridization with pairs of centromeric probes (for chromosomes X and 8 or X and 18), demonstrated a large excess of multiple events with respect to that expected by random segregation. This fact may highlight the existence of cellular subpopulation(s) prone to malsegregate, or indicate that the malsegregation of one chromosome is able to affect the fidelity of segregation of the other chromosomes. Finally, the possible influence of Cyt B on chemically induced malsegregation has been investigated with the analysis of chromosomes X and 8 signals in nuclei of lymphocyte cultures treated with vinblastine (2.5-20 ng/ml) in the presence and absence of 6 micrograms/ml Cyt B. Vinblastine induced a small increase in hyperploidy of either chromosome X or 8 at 10 ng/ml in cultures treated with Cyt B. Without Cyt B, a significant increase of hyperploidy was only observed at the highest dose assayed (20 ng/ml). This vinblastine dosage had a severe inhibitory effect on cultures treated with Cyt B, where no binucleated cells were detected. At all doses, a relatively greater mitotic index was observed in cultures with Cyt B, suggesting a synergistic effect of this drug with vinblastine. Most notably, at the two highest vinblastine dosages (10 and 20 ng/ml), a large incidence of polyploid nuclei was observed in cytokinesis-blocked cultures, whereas none or far lower increases of polyploidy were found in the absence or Cyt. B. This results provides direct evidence of the potential of Cyt B to indirectly interfere with chromosome misdistribution induced by a spindle poison, to be considered before drawing firm conclusions from kinesis-blocked systems.

Induction of humoral immunity toward 2-acetylaminofluorene in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen.

In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti-2-acetylaminofluorene (anti-2-AAF) IgG were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatin conjugate, followed by a final immunogen injection 14 days later. At the end of treatment, the presence of specific anti-2-AAF antibodies in blood serum of all immunized animals was demonstrated. The immunization procedure did not affect liver metabolic activities, as evaluated using liver homogenates for the exogenous activation of 2-AAF to mutagen. After immunization, mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killed at the end of treatment. The determination of DNA adducts by ELISA in liver and spleen of treated animals revealed significantly (P < 0.01-0.001) lower 2-AAF adduct levels in both tissues of immunized mice with respect to non-immunized animals (both naive and pretreated with the adjuvant alone). This result suggests that the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.