Sabrina Rossi

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.

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.

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.

Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice

Abstract The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl3. Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl3 oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl3 bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.

DNA damage response in monozygotic twins discordant for smoking habits

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.