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.

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.

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.

Detection of DNA damage in human lymphocytes by alkaline single cell gel electrophoresis after exposure to benzene or benzene metabolites.

The alkaline single cell gel electrophoresis (Comet) assay was applied to study the occurrence of DNA damage in peripheral lymphocytes of human subjects with occupational exposure to low levels of benzene (twelve gasoline station attendants, with average benzene exposure of 0.3 mg/m3, 8 h TWA). The results obtained show a significant excess of DNA damage in lymphocytes of exposed workers, compared to matched unexposed controls (p = 0.028, Mann-Whitney U-test). Averaged tail moment values, based on 100 cells/individual, were 1.900 microns in the exposed and 0.936 micron in the unexposed group. In addition, exposed subjects showed a clearcut excess of heavily damaged cells, with tail moments > 90th percentile of the overall distribution (13.5 vs. 6.5%, p = 0.013, Mann-Whitney U-test). No correlation was found between the extent of DNA damage and the ages or smoking habits of the subjects. In order to assess the plausibility of the involvement of benzene in the results of the ex vivo study, further experiments were performed treating in vitro peripheral lymphocytes from unexposed donors with benzene metabolites hydroquinone, benzoquinone and benzenetriol. In these experiments, all benzene metabolites exerted a marked effect on resting lymphocytes, the lowest effective concentrations being below 1 microgram/ml. Conversely, far greater concentrations were required for the induction of significant DNA damage in parallel experiments with hydroquinone on mitogen stimulated lymphocytes. Addition of the DNA repair inhibitor cytosine arabinoside (Ara-C, 1-10 micrograms/ml) partially restored the sensitivity of stimulated cells to hydroquinone, an indication of the active processing of induced DNA lesions in growing cells. These results are discussed also in relation to the role of peripheral lymphocytes as target tissue in the biomonitoring of human exposure to genotoxic agents.

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.

Analysis of chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent damage resulting from low dose exposure to spindle poisons.

The chromosome malsegregation pattern produced by the spindle poisons vinblastine (VBL) and colchicine (COL) in human lymphocytes was investigated. For this purpose, the fluorescence in situ hybridization with centromeric DNA probes for chromosomes X and 1 was applied to cell cultures treated with cytochalasin B, a cytokinesis-blocking agent. With this method, chromosome segregation in daughter nuclei - retained in the same envelope - can be easily analysed, simultaneously determining the loss and non-disjunction of specific chromosomes. Preliminary experiments demonstrated that the aneugenic effects elicited by low dose exposure to spindle poisons were effectively detected with treatments from the S/G2 phase (43 h) to harvest of cell cultures (60 or 72 h), with no drug-free medium recovery. This exposure protocol was used in subsequent experiments, where COL and VBL were applied at concentrations which had no effect on the cell cycle ranging to producing marked mitotic block. To account for sex differences in chromosome X instability, lymphocyte cultures from both male and female donors were used to study X chromosome malsegregation. Chromosome 1 malsegregation, however, was analysed in female lymphocytes only. VBL induced reproducible, significant increases of micronuclei in cytokinesis-blocked cells at 5 ng/ml and over. In female lymphocytes, chromosome X loss was observed at 5 ng/ml whereas chromosome 1 loss was only observed at 10 ng/ml. In male lymphocytes, no significant chromosome loss was observed. On the other hand, non-disjunction of both chromosomes X and 1 was effectively induced in female lymphocytes even at 1.25 ng/ml, the lowest dose tested. In male lymphocytes, non-disjunction of chromosome X was observed at 5 ng/ml. Treatments with COL produced a significant increase of micronucleated cells only at the highest dose tested (20 ng/ml). No significant increase in the incidence of either chromosome X or chromosome 1 loss was observed. With cell cultures from donors of both genders, a significant increase in non-disjunction of chromosome X was observed at 5 ng/ml. At the same dose, chromosome 1 non-disjunction significantly increased. These results suggest that in cytokinesis-blocked human lymphocytes, non-disjunction is the prevalent error in chromosome segregation induced by low dose exposure to spindle poisons. Interestingly, non-disjunction was effectively induced even at doses which did not produce detectable detrimental effects on the cell cycle.

Sex chromosome loss and non-disjunction in women: analysis of chromosomal segregation in binucleated lymphocytes.

Chromosomal lagging and non-disjunction are the main mechanisms of chromosomal malsegregation at mitosis. To date, the relative importance of these two events in the genesis of spontaneous or induced aneuploidy has not been fully elucidated. A methodology based on in situ hybridization with centromeric probes in binucleated lymphocytes was previously developed to provide some insight into this matter. With this method, both chromosomal loss and non-disjunction can be simultaneously detected by following the distribution of specific chromosomes in the nuclei and micronuclei of binucleated cells. In this study, this approach was used for studying the role of chromosomal loss and non-disjunction in the age-related malsegregation of sex chromosomes in females. For this purpose, cultures of cytokinesis-blocked lymphocytes were established from 12 healthy women ranging in age from 25 to 56. The occurrence of malsegregation of X chromosomes in vitro was estimated in binucleated cells that contained four signals, which originates from the division of normal disomic cells. In this cell population, the frequencies of X chromosome loss and non-disjunction ranged from 0% to 1.69% (mean 0.75%), and from 0.20% to 1.33% (mean 0.57%), respectively. This indicates that both events contribute to malsegregation of X chromosomes in vitro. Moreover, a small but not negligible fraction of binucleated cells with two or six copies of the X chromosome was noticed in all donors. These cells, which are thought to arise from parental monosomic and trisomic types, may indicate the malsegregation of X chromosomes in vivo. The frequency of X chromosome aneuploidy both in vivo and in vitro significantly correlated with the age of donors. Analysis of chromosomal distribution in unbalanced cells demonstrated that both X homologues were frequently involved. The frequency of such multiple events (0.17%) was far greater than that expected by mere chance, indicating a tendency to multiple malsegregation events in the cell population investigated. Finally, parallel analysis of the segregation of chromosomes X and 1 in five of the donors confirmed the greater (about tenfold) susceptibility of X chromosomes to malsegregate compared with autosomes.

Genetic effects of petroleum fuels: cytogenetic monitoring of gasoline station attendants.

Workers in the petroleum distribution trades experience relatively high-level exposures to fuel vapours whose consequences have not been fully elucidated. In this study, the possible relationship between occupational exposure to petroleum fuels and cytogenetic damages in peripheral lymphocytes was investigated. Twenty-three male, non-smoking workers from the area of Rome were enrolled in the study, together with age-paired controls with no occupational exposure to fuels. Peripheral lymphocyte cultures were set up for the analysis of structural chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MN) in cytokinesis-blocked lymphocytes. Frequencies of CAs, SCEs and MN were compared between exposed and control groups, and evaluated in relation to blood lead level (as an indicator of engine exhausts exposure) for the whole group under study, and to yearly averaged exposure to benzene (8-h time weighted averages, as determined by repeated personal sampling) for fillingstation attendants only. Both CAs and SCEs were slightly increased in station attendants: 1.97 versus 1.46 aberrations per 100 cells, and 4.73 +/- 0.15 versus 4.48 +/- 0.11 SCEs/cell in exposed and control individuals, respectively. The difference between cumulative CA rates in the exposed and control populations was of borderline statistical significance (p = 0.066). However, when the exposed population was dichotomized for benzene exposure, a significant (p = 0.018) correlation of CAs with benzene exposure was found. The analysis of SCE data highlighted a significant increase of cells with more than 6 exchanges (HFCs), corresponding to the 75 degrees percentile of the overall distribution, in fillingstation attendants (relative risk (RR) = 1.3, 95% CI = 1.1-1.5) in comparison with controls. In the pooled population, the frequency of HFCs showed a statistically significant upward trend at increasing blood lead levels (chi 2 for trend = 27.8, p < 0.0001). A complex relationship between SCEs and benzene exposure was observed, with an increased frequency of HFCs in the medium exposure intensity class (RR = 1.5, 95% CI = 1.2-1.7), and no difference for exposure to higher benzene levels (RR = 1.0, 95% CI = 0.9-1.2), compared to reference subjects. Finally, the analysis of MN in both phytohemagglutinin- and pokeweed-stimulated cell cultures did not show significant excess of MN in binucleated lymphocytes of exposed workers with respect to the age-paired controls.

[Exposure to benzene and genotoxic effects among filling station attendants]. / Esposizione a benzene ed effetti genotossici tra gli addetti all'erogazione di carburanti.

Exposure to gasoline vapors is classified by the International Agency for Research on Cancer as possibly carcinogenic to humans, mainly on the basis of the established carcinogenicity of some component chemicals such as benzene. The mechanism of benzene toxicity, particularly its leukemogenic effects, is far from being fully understood. Different studies, aimed at evaluating the risk associated with exposure to benzene through fuels and coordinated by the Istituto Superiore di Sanità, are in progress in Italy. In an environmental monitoring survey on a sample of 111 service stations, conducted in Rome (Italy) in 1992, average yearly personal exposure to benzene, toluene and xylenes were estimated. Chemical determination of benzene and methylbenzene was carried out by GL-gas chromatography. From a sample of 27 service stations 34 fuel samples were collected, and their benzene content was measured by hr-gas chromatography. Subgroups of the filling station attendants undergoing the exposure assessment study, were included in biological monitoring surveys of early indicators of genotoxicity. In particular, 65 subjects were enrolled in a study aimed at evaluating the urinary concentrations of 8-hydroxydeoxyguanosine (8-OHdG), a biological marker of oxidative DNA damage, and 23 filling station attendants were selected for a survey of the frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral T lymphocytes. In the exposure assessment survey levels of 0.53, 0.71 e 0.32 mg/m3 in the average yearly personal exposure to benzene, toluene and xylenes, respectively, were estimated (individual means based on 6.5 repeated samples per employee). The daily quantities of super premium gasoline sold proved to be associated with the average yearly personal exposure to benzene, and current smokers showed a significantly lower exposure intensity compared with non-smokers. Among the latter, an increase of 0.11 ln mg/m3 in benzene exposure per unit increase (100 l) in gasoline sold (p < 0.001) was estimated by a multiple regression analysis with some personal characteristics of the subjects included in the model as potentially predictive variables (R2 = 0.17, p (F) < 0.05). Among smokers, however, only the age and the length of employment were able to predict the intensity of benzene exposure. On a sample of 27 filling station attendants, furthermore, the relationship between personal exposure to benzene and benzene fuel content was evaluated and an increase of 0.01 mg/m3 in the personal benzene exposure per unit increase (100 g) in the absolute quantity of benzene in the fuel sold was estimated (p < 0.0001, R2 = 0.50).(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous detection of X-chromosome loss and non-disjunction in cytokinesis-blocked human lymphocytes by in situ hybridization with a centromeric DNA probe; implications for the human lymphocyte in vitro micronucleus assay using cytochalasin B.

A methodology for the simultaneous detection of chromosome loss and gain in mammalian cells has been developed which is based upon the analysis of chromosome distribution in daughter nuclei of binucleated human lymphocytes. X-chromosome distribution was followed by in situ hybridization, using a commercial biotinylated DNA probe specific for the centromeric alphoid sequences of human X-chromosome. In order to optimize the experimental protocol for the use of cytokinesis-blocked lymphocytes in aneuploidy assays, the effect of harvest time and cytochalasin B (Cyt B) dosage upon chromosome distribution was investigated. To this end, lymphocyte cultures were treated 44 h after mitogen stimulation with different dosages of Cyt B and collected at 60, 66 and 72 h. High rates of binucleated cells with unbalanced chromosome distribution (two spots in one nucleus and none in the other in male cells; three spots in one nucleus and one in the other in female cells) and abnormal spot number (more than or less than two per male cell or four per female cell) were observed at 66 and 72 h in cultures treated with the lowest Cyt B dose (3 micrograms/ml). In contrast, low frequencies of unbalanced or abnormal binucleated cells were observed at 60 h with both 3 and 6 micrograms/ml Cyt B. These results indicate that binucleated lymphocytes with low background frequencies of malsegregation (required for the analysis of induced aneuploidy), can be obtained by harvesting lymphocyte cultures 60 h after stimulation (16 h after Cyt B block).(ABSTRACT TRUNCATED AT 250 WORDS)

An assessment of the in vivo clastogenicity of erythrosine.

In an investigation of the in vivo clastogenic potential of the food colouring erythrosine (ER), male B6C3F1 mice were treated by ip injection at doses of 50, 100 and 200 mg/kg, repeated 24 hr apart. Signs of toxicity were observed at the highest dose of ER administered. The three cytogenetic endpoints analysed were sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs), micronuclei in bone marrow polychromatic erythrocytes (PCEs), and micronuclei in peripheral blood reticulocytes (PBRs). SCE frequencies in PBLs were 4.13, 4.58, 4.33 and 4.60 SCE/cell at 0, 50, 100 and 200 mg ER/kg, respectively. At the same doses, the frequencies of micronucleated PCEs were 3.5, 3.2, 2.0 and 2.5/1000 PCEs. Micronuclei in PBRs ranged from 1.2 to 3.6 and from 1.4 to 3.0/1000 PBRs in control and treated mice, respectively. These results indicate that ER is inactive as a clastogen in mouse blood and marrow cells. This result supports the hypothesis of a non-genotoxic mechanism for ER carcinogenicity.

In vivo studies on chemically induced aneuploidy in mouse somatic and germinal cells.

Within the context of a coordinated program to study aneuploidy induction sponsored by the European Community, nine chemicals were tested in mouse bone marrow and spermatocytes after intraperitoneal injection. In somatic cells, cell progression delay, hyperploidy, polyploidy induction and induction of micronucleated polychromatic erythrocyte (MnPCE) were studied. In germ cells hyperploidy induction was evaluated. The chemicals selected were: colchicine (COL), econazole (EZ), hydroquinone (HQ), thiabendazole (TB), diazepam (DZ), chloral hydrate (CH), cadmium chloride (CD), pyrimethamine (PY) and thimerosal (TM). Using literature data on c-mitotic effects in bone marrow as a reference, the same doses were tested in somatic and germ cells in order to compare the effects induced. Bone marrow cells were sampled 18 or 24 h after treatment. Germ cells were sampled 6, 8 or 18 h after treatment. Effects of COL and HQ in bone marrow have been reported elsewhere. Somatic effects were induced by CH (hyperploidy and cell cycle lengthening), TB (MnPCEs and cell cycle lengthening) and by PY (MnPCEs). EZ, DZ, CD and TM did not induce any kind of somatic effects. An increase in the incidence of hyperploid spermatocytes was induced by COL, at three dose levels, and by one dose of HQ and TB. All the other chemicals did not induce germinal aneuploidy at any dose or time tested. The hyperploidy control frequency ranged between 0.4 and 1.0% in somatic cells and from 0.3 to 0.9% in germ cells. In both somatic and germ cells, the maximum yield of induced hyperploidy did not exceed 3.5%. The time period of target cell sensitivity is probably restricted and this, associated with the heterogeneity and the asynchrony of cellular maturation processes, may account for our data. Under these circumstances, the negative data should be interpreted with some caution, particularly in germ cells, where additional indicators of chemical-cell interaction and cell cycle effects were not provided by standardized approaches. The possibility of increasing the size of analyzed cell samples could be considered in the light of automatic scoring procedures.

Further in vitro and in vivo mutagenicity assays with thiram and ziram fungicides: bacterial reversion assays and mouse micronucleus test.

The fungicides thiram and ziram have been assayed in a battery of nine bacterial strains of different genetic specificity. The results obtained suggest the induction of excisable DNA lesion(s), and indicate similar mutability of strains with AT or GC base pairs at target sites. This mutagenic profile is clearly distinct from that of oxidative mutagens, and it does not support the proposed role of oxidative stress in the mechanism of dithiocarbamates mutagenicity in bacteria. Furthermore, the bone marrow micronucleus test has been carried out in B6C3F1 mice with intraperitoneal administration of high grade thiram (12.5-50 mg/kg) and ziram samples (2.5-10 mg/kg in males, and 5-20 mg/kg in females). Thiram produced a significant increase of micronucleated PCEs in male mice sampled 48 h after treatment with 25, 37.5, and 50 mg/kg. No significant increase was detected in treated females. Ziram, tested in a lower range of doses because of its higher toxicity, resulted negative in both sexes. Both the acute toxicity and the ratio polychromatic/normochromatic erythrocytes indicated some sex specificity in the toxic effects induced by these dithiocarbamates in the B6C3F1 mouse.