Cristina Andreoli

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.

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.

A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke

In the last few years tobacco companies have been developing several research strategies in order to reduce the risks associated with smoking. These strategies include, for example, the refining of alternative cigarette designs that reduce the amount of hazardous chemicals in the mainstream smoke by introducing modified filters, and/or reducing the amount of biologically significant ingredients in tobacco-burning cigarettes. In the last few decades numerous studies have been published to assess the biological activity of tobacco smoke using in vivo and in vitro test systems. In this scenario a general scientific consensus on how to measure and characterize the risk associated with cigarette smoke is still lacking. Short-term in vitro assays, which are widely accepted by regulatory agencies around the world, are useful tools to evaluate both the biological activity and the progress towards a reduction of tobacco smoke toxicity. These assays could be mainly applied to evaluate cytotoxicity and genotoxicity properties on whole cigarette smoke as well as condensates or fractions of whole smoke. Cytotoxicity induction can be measured as cellular viability and growth rates using different end-points. Otherwise, the target of genotoxicity studies is the DNA molecule. For genotoxicity evaluation, the end-points and cell systems should be chosen from those that are relevant and appropriate as clinical surrogate markers. In this respect, the occurrence of early biological effect markers, such as mutational or clastogenic events (point mutations, frameshifts, micronuclei, SCE, DNA adducts) in bacterial and mammalian cells should be studied in a tiered approach following the guidelines of regulatory agencies. The choice of criteria shall be matter of discussion.

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.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

Toxicology of halogenated aliphatic hydrocarbons: structural and molecular determinants for the disturbance of chromosome segregation and the induction of lipid peroxidation

The induction of mitotic chromosome malsegregation, mitotic arrest and lethality by a set of 55 halogenated hydrocarbons was investigated. To this aim, genetic assays in the mould Aspergillus nidulans, able to provide precise quantitative information on the end-points studied, were used throughout the work. The experimental data obtained were used to develop QSAR models for the induction of aneuploidy, which pointed to a major role of electrophilicity as molecular determinant for the aneugenic potential of the halogenated hydrocarbons investigated. Within the hypothesis of a link between the electrophilicity of haloalkanes and their propensity to undergo a reductive biotransformation, with production of free radical species, a subset of 27 compounds was also tested for the ability to induce lipid peroxidation in rat liver microsomes in vitro. The results obtained indicate a partial coincidence between the abilities to initiate lipid peroxidation and to disturb chromosome segregation at mitosis. The data base obtained was also used to investigate the relationship between chemical structure and peroxidative potential. The analysis indicated that electronic and structural parameters related to the ease of homolitic cleavage of the carbon-halogen bond play a pivotal role as determinants for the peroxidative character of haloalkanes.

The induction of mitotic chromosome malsegregation in Aspergillus nidulans. quantitative structure activity relationship (QSAR) analysis with chlorinated aliphatic hydrocarbons

The biological activity of 24 chlorinated aliphatic hydrocarbons has been studied in the mold Aspergillus nidulans. The ability to induce chromosome malsegregation, lethality and mitotic growth arrest has been experimentally determined for each chemical. These data, together with those of 11 related compounds previously investigated, generated a data base which was used for quantitative structure-activity relationship (QSAR) analysis. To this aim, both physico-chemical descriptors and electronic parameters of each compound have been calculated and included in the analysis. The QSAR analysis indicated that toxic effects induced by chlorinated aliphatics in A. nidulans are mainly dependent on steric factors, as indicated by the correlation with molar refractivity (MR). Conversely, the ease with which they accept electrons, parametrized by LUMO (energy of the lowest unoccupied molecular orbital), plays a prevailing role in determining the aneuploidizing properties. An involvement of free radicals, generated by the reductive metabolism of haloalkanes, is hypothesized as an explanation of the data.

Effects of cigarette smoking on circulating leukocytes and plasma cytokines in monozygotic twins

Abstract Background: Despite the well-documented role of cigarette smoke in the development of chronic obstructive pulmonary disease (COPD), lung cancer and cardiovascular disease, biomarkers for screening or monitoring disease progression and outcome remain elusive, particularly for COPD and lung cancer. Inflammatory cells and mediators are likely to be involved in the disease processes, but their importance is still poorly understood. The purpose of this study was to investigate early changes in immunological markers associated with smoking in healthy monozygotic twins without a detectable disease discordant for smoking, thereby minimising data variability due to genetic background. Methods: Twenty-two monozygotic twin pairs, aged 31.5±6.3 years, entered the study. One of each twin pair was a smoker and the other a non-smoker. None of the subjects reported any diseases or clinically defined respiratory symptoms or airflow limitation. Each subject donated blood samples for determination of total leukocytes and subpopulations, lymphocyte subpopulation plus pro-inflammatory mediators (interleukin-8, tumour necrosis factor-α, soluble tumour necrosis factor-α receptors and C-reactive protein). Results: We observed a significant increase in the number of circulating leukocytes and neutrophils in smokers compared to non-smokers. Smokers also had significantly higher numbers of B cells and CD4+ T cells, plus an increased CD4/CD8 ratio. The numbers of NK cells were statistically significant lower in smokers compared to non-smokers. Conclusions: While the prognostic significance of these changes is uncertain, results suggest that smoking is associated with immune changes, independent of genetic background and environmental conditions.

Prediction of rodent carcinogenicity of further 30 chemicals bioassayed by the U.S. National Toxicology Program.

Recently the U.S. National Toxicology Program (NTP) sponsored a comparative exercise in which different prediction approaches (both biologically and chemically based) were challenged for their predictive abilities of rodent carcinogenicity of a common set of chemicals. The exercise enjoyed remarkable scientific success and stimulated NTP to sponsor a second challenging round of tests, inviting participants to present predictions relative to the rodent carcinogenicity of a further 30 chemicals; these are currently being tested. In this article, we present our predictions based on structure-activity relationship considerations. In our procedure, first each chemical was assigned to an activity mechanism class and then, with semiquantitative considerations, was assigned a probability carcinogenicity score, taking into account simultaneously the hypothesized action mechanism and physical chemical parameters.