Francesca Marcon

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.

The detection and evaluation of aneugenic chemicals.

Although aneuploidy makes a significant contribution to both somatic and inherited disease the mechanisms by which environmental chemicals may induce numerical chromosome aberrations are only poorly defined. The European Union Project was aimed to further our understanding of those chemical interactions with the components of the mitotic and meiotic cell division cycle which may lead to aneuploidy and to characterise the parameters such as cellular metabolism which may influence the activity of aneugenic chemicals. C-mitosis can be induced by the highly lipophilic polychlorinated biphenyl and the completion of mitosis and cleavage can be modified by agents which deplete cellular levels of reduced glutathione. Modifications of the fidelity of chromosome segregation were produced by inhibiting the functioning of topoisomerase II during chromatid separation. In contrast, the modification of centromere integrity resulted in chromosome breakage as opposed to disturbance of segregation. Modifiers of tubulin assembly and centriolar functioning in somatic cells such as acrylamide, vinblastine and diazepam reproduced their activity in rodent bone marrow and male germ cells. The analysis of chromosome malsegregation in Aspergillus nidulans by a structurally related series of halogenated hydrocarbons was used to develop a QSAR model which had high predictive value for the results of fungal tests for previously untested related chemicals. Metabolic studies of potential aneugens in genetically engineered human lymphoblastoid cells demonstrated the detoxification of the aneugenic activity of chloral hydrate and the activation of 2,3-dichlorobutane, 1,1,2-trichloroethane and trichloroethylene by Phase I biotransforming enzymes. Cell transformation studies in Syrian hamster dermal cultures using a panel of 22 reference and or potential aneugens indicated that 15 of the 22 produced positive results following single exposures. Five of the aneugens which were negative following single exposures produced positive results where cultures were continuously exposed for up to 6 weeks to low concentrations following a single non-transforming exposure to the mutagen dimethyl sulphate. The transformation studies indicate that a significant proportion of chemical aneugens are potential complete carcinogens and/or co-carcinogens. To optimise the enumeration of chromosomes following exposure to potential chemical aneugens whole chromosome paints and centromere specific probes suitable for use in fluorescence in situ hybridisation (FISH) were developed for the rat, mouse and Chinese hamster and selected human probes evaluated for their suitability for routine use. Molecular chromosome probes were used to develop protocols for enumerating chromosomes in metaphase cells and centromeres and micronuclei in interphase cells. The analysis of segregation of specific centromeres in binucleate cells following cytochalasin B treatment was shown to be a potentially valuable system for characterising non-disjunction following chemical exposure. Whole chromosome paints and centromere specific probes were used to demonstrate the presence of dose-response thresholds following treatment with a reference panel of spindle inhibiting chemicals. These data indicate that the FISH technology is suitable for evaluating the relative hazards of low-dose exposures to aneugenic chemicals.

Assessment of individual sensitivity to ionizing radiation and DNA repair efficiency in a healthy population

Inter-individual variation in response to exposure to carcinogens has been ascribed to differences in carcinogen metabolism as well as to variability in DNA repair capacity (DRC). In order to investigate the role of inherited and acquired factors on individual variation in DNA repair capacity, a mutagen sensitivity assay was carried out on 31 healthy subjects. Fresh blood samples were irradiated with gamma-rays (2Gy) and the kinetics of DNA repair in leukocytes assessed by the comet assay 0, 15, and 30 min after irradiation. Whole blood cultures were set up to detect spontaneous and induced structural chromosomal aberrations in lymphocytes 48 h after irradiation. The results obtained were evaluated with respect to age, gender, smoking habits, occupational exposure to chemicals and metabolic genotype (NQO1, GSTM1 and GSTT1) of the study subjects. A higher frequency of radiation-induced aberrations was observed in GSTM1-positive individuals compared with GSTM1-null subjects (P=0.025), as well as in non-smokers compared with heavy smokers (P=0.05). Similar results were obtained by measuring residual DNA damage (RD) shortly after irradiation by means of the comet assay, with non-smokers showing a higher amount of RD compared with smokers (P=0.016). Moreover, a significant correlation (P=0.008) was observed between the amount of RD and the frequency of chromosome breaks after irradiation. The results of this pilot study suggest a modulator effect of smoking habits and GSTM1 genotype on the individual DNA repair capacity, possibly related to the higher expression of enzymes involved in the repair of oxidative DNA damage in heavy smokers and GSTM1-null subjects.

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.

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 orginates 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 chromosomex X and 1 in five of the donors confirmed the greater (about tenfold) susceptibility of X chromosomes to malsegregate compared with autosomes.

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.

Diet-related telomere shortening and chromosome stability.

Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.

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.

Toxic and genotoxic effects of oral administration of furan in mouse liver

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Novel Epigenetic Changes Unveiled by Monozygotic Twins Discordant for Smoking Habits

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.