genetic toxycology

MUTATION RESEARCH-GENETIC TOXYCOLOGY & ENVIRONMENTAL MUTAGENESIS
Vol.  557, issue 2, pp. 109-216. 10 Feb. 2004

Cytogenetic biomonitoring of a floriculturist population in Italy: micronucleus analysis by fluorescence in situ hybridization (FISH) with an all-chromosome centromeric probe
Claudia Bolognesi, Eleonora Landini, Emanuela Perrone and Paola Roggieri

Environmental Carcinogenesis Unit, National Cancer Research Institute, Largo R. Benzi 10, 16132, Genoa, Italy

Received 16 April 2003; revised 14 August 2003; accepted 24 September 2003. ; Available online 20 December 2003.

Abstract
Flower production in greenhouses associated with a heavy use of pesticides is very wide-spread in the western part of the Ligurian region (Italy). The formation of micronuclei in peripheral blood lymphocytes is a valuable cytogenetic biomarker in human populations occupationally exposed to genotoxic compounds.
In the present study we investigated the micronucleus frequency in peripheral blood lymphocytes of 52 floriculturists and 24 control subjects by use of the cytokinesis-block methodology associated with fluorescence in situ hybridization with a pan-centromeric probe that allowed to distinguish centromere-positive (C+) and centromere-negative (C-) micronuclei.
The comparison between floriculturists and controls did not reveal any statistically significant difference in micronucleus frequency, although an increase was observed with increasing pesticide use, number of genotoxic pesticides used and duration of exposure. An increase in C+ as well as in C- micronuclei and in the percentage of C+ micronuclei with respect to the total number of micronuclei was detected in floriculturists, suggesting a higher contribution of C+ micronuclei in the total number scored. The percentage C+ micronuclei was not related to the duration of exposure or to the number of genotoxic pesticides used, but a higher percentage (66.52% versus 63.78%) was observed in a subgroup of subjects using benzimidazolic compounds, compared with the floriculturist population exposed to a complex pesticide mixture not including benzimidazolics. These results suggest a potential human hazard associated with the exposure to this class of aneuploidy-inducing carcinogens.

Author Keywords: Cytogenetic biomonitoring; Pesticides; Chromosomal damage; Micronucleus test




Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization
A. Buschinia, A. Martinoa, B. Gustavinob, M. Monfrinottic, P. Polia, C. Rossia, M. Santorob, A. J. M. Dörrd and M. Rizzoni, b

a Dipartimento di Genetica Antropologia Evoluzione, Università degli Studi di Parma, Parma, Italy
b Dipartimento di Biologia, Università degli Studi di Roma "Tor Vergata", via della Ricerca Scientifica s.n.c., Rome, Italy
c Laboratorio di Ecologia Sperimentale e Acquacoltura, Dipartimento di Biologia, Università degli Studi di Roma "Tor Vergata", Rome, Italy
d Dipartimento di Biologia Animale ed Ecologia, Università di Perugina, Perugina, Italy

Received 8 April 2003; revised 11 July 2003; accepted 1 October 2003. ; Available online 20 December 2003.

Abstract
The detection of a possible genotoxic effect of surface water treated with disinfectants for potabilization is the aim of the present work. The Comet assay and the micronucleus test were applied in circulating erythrocytes of Cyprinus carpio. Young specimens (20-30 g) were exposed in experimental basins, built within the potabilization plant of Castiglione del Lago (Perugia, Italy). In this plant the water of the Trasimeno Lake is treated and disinfected for potabilization before it is distributed to the people in the net of drinkable water. A continuous flow of water at a constant rate was supplied to basins; the water was continuously treated at a constant concentration with one of the three tested disinfectants (sodium hypochlorite, peracetic acid and chloride dioxide), one control basin being supplied with untreated water. Three sampling campaigns were performed: October 2000, February 2001 and June 2001. Repeated blood samplings through intracardiac punctures allowed to follow the same fish populations after different exposure times: before introduction of the disinfectant, and 10 or 20 days afterwards. An additional blood sampling was performed 3 h after addition of the disinfectant in other, simultaneously exposed, fish populations. Genotoxic damage was shown in fish exposed to water disinfected with sodium hypochlorite and chloride dioxide. The Comet assay showed an immediate response, i.e. DNA damage that was induced directly in circulating erythrocytes, whereas micronuclei reached their highest frequencies at later sampling times, when a genotoxic damage in stem cells of the cephalic kidney is expressed in circulating erythrocytes. The quality of the untreated surface water seems to be the most important parameter for the long-term DNA damage in circulating erythrocytes.

Author Keywords: Water potabilization; Disinfectants; Micronuclei; Comet assay; Circulating erythrocytes; Cyprinus carpio




Assessment of DNA damage in glue sniffers by use of the alkaline comet assay
smet Çok, Semra arda, Ela Kadioglu and Eren Ozcagli

Department of Toxicology, Faculty of Pharmacy, Gazi University, Hipodrom, Ankara 06330, Turkey

Received 25 June 2003; revised 1 October 2003; accepted 6 October 2003. ; Available online 20 December 2003.

Abstract
Toluene is used widely, not only in industry, but also in households where toluene exposure and abuse can occur. To estimate the genotoxic risk of toluene exposure, DNA damage was determined in peripheral lymphocytes of 20 glue sniffers and 20 age-matched controls by use of the alkaline comet assay. Urinary hippuric acid and o-cresol excretion rates, which are used as a marker for toluene exposure, were also measured in sniffers and compared with historical control values. The increase in genetic damage in sniffers was statistically significant as compared to control subjects (P<0.0001). The mean values of the hippuric acid and o-cresol excretion rate for glue sniffers was 73- and 1582-fold higher, respectively, than in controls and confirms the putative exposure. Education of the general public and efforts to keep adolescents away from volatile solvent-based products, which may lead to a desire of sniffing in the future, would be advisable.

Author Keywords: Glue sniffing; Solvent abuse; Toluene; DNA damage; Single-cell gel electrophoresis; Comet assay

















Effects of paving asphalt fume exposure on genotoxic and mutagenic activities in the rat lung
H. W. Zhaoa, X. J. Yinb, D. Frazera, M. W. Bargera, P. D. Siegela, L. Millecchiaa, B. Z. Zhonga, S. Tomblyna, S. Stonea, J. K. H. Mab, V. Castranovaa and J. Y. C. Ma a

a Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA
b School of Pharmacy, West Virginia University, Morgantown, WV 26506, USA

Received 2 July 2003; revised 2 October 2003; accepted 8 October 2003. ; Available online 20 December 2003.


Abstract
Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung. In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied. Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay. To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored. Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo[a]pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029. This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3). The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner. In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479±33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S. typhimurium YG1024 or YG1029. At a higher total asphalt fume exposure level (1150±63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control. However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test. These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content.

Author Keywords: Paving asphalt fumes; DNA damage; Mutagenicity; CYP1A1; CYP2B1; Polycyclic aromatic hydrocarbons


The c-myc activation in cervical carcinomas and HPV 16 infections
Martín C. Abba, Rubén M. Laguens, Fernando N. Dulout and Carlos D. Golijow

Centro de Investigaciones en Genética Básica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Calle 60 y 118 s/n, B1900AVW, La Plata, Argentina

Received 28 May 2003; revised 8 October 2003; accepted 8 October 2003. ; Available online 20 December 2003.

Abstract
Despite the prominent role for Human Papillomavirus (HPV) infection in the development of genital cancer, other genetic or environmental co-factors have also been involved. Studies of c-myc activation in cervical carcinomas have reported that gene over-expression (mainly gene amplification) are common in cervical squamous cell carcinomas and may correlate with the biologic behavior of the neoplasm. Using PCR based technology, DNAs from 79 normal cervical samples and 225 abnormal cervical tissue scrapes were analyzed for HPV detection and typing and for c-myc gene amplification. Significant differences were found between the different cyto/histology groups (P<0.0001) and also with HPV high-risk infected samples (P<0.0002). In this sense, we showed that the average c-myc copy number increased according to the histological grade of the lesion (OR=6.3, CI=2.1–18.8). Also, the results showed that the infection with HPV 16 was tightly associated with c-myc amplification (OR=10.6, CI=3.1–36). These results could indicate that oncogene amplification take place in pre-invasive stages of cervical disease and could cooperate not only in tumor progression but also in cell transformation. Moreover, the results strongly associate the c-myc gene amplification to the infection with the oncogenic HPV 16, showing that the pattern of virus infection and oncogene activation could be specific for different viral genotypes.

Author Keywords: Cervical cancer; Human Papillomavirus; c-myc amplification













Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes
Katsuya Yamadaa, Takayoshi Suzukib, Atsushi Hakurac, Takaharu Mizutania and Ken-ichi Saeki a

a Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku, Nagoya 467-8603, Japan
b Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
c Drug Safety Research Laboratories, Eisai Co. Ltd., 1 Takehaya-machi, Kawashima-cho, Hashima-gun, Gifu 501-6195, Japan

Received 4 July 2003; revised 14 October 2003; accepted 14 October 2003. ; Available online 25 December 2003.

Abstract
We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.

Author Keywords: Aza-substitution; Polycyclic aromatic hydrocarbon; Human liver microsome; Ames test









Genotoxic effects of a complex mixture adsorbed onto ambient air particles on human cells in vitro; the effects of Vitamins E and C
Monika Lazarová and Darina Slameová

Cancer Research Institute of Slovak Academy of Sciences, Laboratory of Mutagenesis and Carcinogenesis, Vlárska 7, 83391, Bratislava, Slovak Republic

Received 23 May 2003; revised 29 September 2003; accepted 15 October 2003. ; Available online 20 December 2003.

Abstract
Genotoxicity of complex mixtures of organic compounds adsorbed onto ambient air particles (extractable organic matter, EOM) collected in Teplice (Czech Republic) as well as genotoxicity of the indirectly acting carcinogens benzo[a]pyrene (B[a]P) and 5,9-dimethyl-7H-dibenzo[c,g]carbazole (5,9-diMeDBC) was studied in human HepG2 and Caco-2 cells cultured in vitro. The level of DNA breaks was detected by conventional single-cell gel electrophoresis (alkaline comet assay). The level of DNA breaks+oxidative DNA lesions was assessed by modified single-cell gel electrophoresis. The indirectly acting chemical carcinogens studied were able to induce DNA breaks as well as oxidative DNA damage in both cell lines, but stronger DNA-damaging effects were observed in HepG2 cells, which contain a higher level of metabolic enzymes. Treatment of cells with the complex mixtures showed a dose-dependent increase of DNA breaks in HepG2 cells as well as in Caco-2 cells, with seasonal differences. Winter samples of EOM from Teplice (TP-W) were more effective in inducing DNA damage than summer samples (TP-S). Both mixtures caused significant oxidative DNA damage in HepG2 cells. The effect was less evident in cells treated with higher concentrations of TP-W, since the comet assay is limited by saturation at a higher level of DNA damage. Possible reduction of B[a]P-, 5,9-diMeDBC- or EOM-induced DNA damage by Vitamins E and C was evaluated in HepG2 cells only. Pre-treatment of these cells with either one of the vitamins considerably reduced the levels of both DNA breaks and oxidative DNA lesions induced by all compounds investigated.

Author Keywords: Complex mixture; Vitamin E; Vitamin C; Oxidative DNA damage; Comet assay









Anticlastogenicity of chlorophyllin in the different cell cycle phases in cultured mammalian cells
P. D. Negraesa, B. Q. Jordãoa, V. E. P. Vicentinib and M. S. Mantovani a

a Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Campus Universitário, Cx. Postal 6001, Londrina PR 86051-990, Brazil
b Departamento de Biologia Celular e Genética, Centro de Ciências Biológicas, Universidade Estadual de Maringá, Maringá, Paraná, Brazil

Received 11 June 2003; revised 28 August 2003; accepted 22 October 2003. ; Available online 23 December 2003.

Abstract
Chlorophyllin (Chln), a sodium–copper salt derivative of chlorophyll, like chlorophyll-a and -b found in green plants, has been studied for its protective action against the carcinogenic effects of various physical and chemical agents and in relation to the mutagenic and clastogenic activities of genotoxic agents. The aim of the present study was to evaluate chlorophyllin in different phases of the cell cycle for clastogenicity and anticlastogenicity, the latter in reversing DNA damage induced by ethyl methane sulfonate (EMS). The test for chromosomal aberrations was performed in cultured mammalian cells (CHO-K1). The three Chln concentrations tested (6.25, 12.5 and 25 ug/ml) were not clastogenic and damage induced by EMS (1240 ug/ml) was reduced in cells treated with Chln as well during S (25–48%) and G2/S (70–80%). The results demonstrate a greater protective effectiveness of Chln against EMS during G2/S.

Author Keywords: Chlorophyllin; Anticlastogenicity; Cell cycle phases; Chromosomal aberration; Cultured cells








Evaluation of DNA damage in a population of bats (Chiroptera) residing in an abandoned monazite mine
Kathleen A. Meehan, , a, Ernest J. Trutera, Jacobus P. Slabbertb and M. Iqbal Parkerc

a Faculty of Applied Sciences, Cape Technikon, P.O. Box 652, Cape Town 8000, South Africa
b National Accelerator Centre, Faure, South Africa
c Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Received 30 January 2003;  revised 17 October 2003;  accepted 22 October 2003. ; Available online 20 December 2003.

Abstract
Ionising radiation has the ability to induce DNA damage. While the effects of high doses of radiation of short duration have been well documented, the biological effects of long-term exposure to low doses are poorly understood. This study evaluated the clastogenic effects of low dose ionising radiation on a population of bats (Chiroptera) residing in an abandoned monazite mine. Bats were sampled from two chambers in the mine, where external radiation levels measured around 20 Sv/h (low dose) and 100 Sv/h (higher dose), respectively. A control group of bats was sampled from a cave with no detectable radiation above normal background levels. The micronucleus assay was used to evaluate residual radiation damage in binucleated lymphocytes and showed that the micronucleus frequency per 500 binucleated lymphocytes was increased in the lower radiation-exposed group (17.7) and the higher radiation-exposed group (27.1) compared to the control group (5.3). This study also showed that bats exposed to radiation presented with an increased number of micronuclei per one thousand reticulocytes (2.88 and 10.75 in the lower and high radiation-exposed groups respectively) when compared to the control group (1.7). The single-cell gel electrophoresis (comet) assay was used as a means of evaluating clastogenecity of exposure to radiation at the level of individual cells. Bats exposed to radiation demonstrated increased DNA damage as shown by the length of the comet tails and showed an increase in cumulative damage. The results of the micronucleus and the comet assays indicated not only a statistically significant difference between test and control groups (P<0.001), but also a dose-dependent increase in DNA damage (P<0.001). These assays may thus be useful in evaluating the potential clastogenecity of exposure to continuous low doses of ionising radiation.
Author Keywords: Ionising radiation; Chiroptera; Clastogenecity; Micronucleus; Comet; Biodosimetry
Abbreviations:Sv, microsievert; PHA, phytohaemagglutinin

An assay method for the prediction of tumor promoting potential of chemicals by the use of Bhas 42 cells
Kiyomi Ohmori, , a, Kiyoshi Sasakib, Shin Asadab, Noriho Tanakab and Makoto Umedab

a Chemistry Division, Kanagawa Prefectural Institute of Public Health, 1-3-1 Shimomachiya, Chigasaki, Kanagawa 253-0087, Japan
b Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan

Received 15 April 2003;  revised 27 October 2003;  accepted 27 October 2003. ; Available online 23 December 2003.

Abstract
It has become an important task to develop a simple in vitro method for the detection of non-genotoxic carcinogens, among which tumor promoters are included. Bhas 42 cells are v-Ha-ras-transfected BALB/c 3T3 cells and are regarded as initiated cells in the 2-stage transformation paradigm. We designed a method for detecting tumor promoters by the use of Bhas 42 cells at advanced passage generation. In this method, the cells are cultured in six-well plates for 17 days during which test chemicals are added in the medium for 11 days from days 3 to 14. The end-point of the assay is the induction of transformed foci. When the tumor promoter TPA was used, a significant number of transformed foci were induced concentration-dependently, whereas only a few foci were observed in control cultures. When various chemicals were examined by the method, a reasonable correlation was observed with the reported tumor-promoting ability in animal experiments. We propose that the Bhas 42 cell transformation method is practical and useful for the detection of tumor promoters.
Author Keywords: Tumor promoter; Transformation; Bhas 42 cell; BALB/c 3T3 cell; v-Ha-ras





Inhibition of B(a)P induced strand breaks in presence of curcumin
K. Polasa, , A. N. Naidu, I. Ravindranath and K. Krishnaswamy

National Institute of Nutrition, Indian Council of Medical Research, Hyderabad 500007, India

Received 29 July 2003;  revised 31 October 2003;  accepted 31 October 2003. ; Available online 23 December 2003.

Abstract
Incidence of cancer at different sites may be related to oxidative damage to host genome by genotoxicants. These oxidative actions may be modified by phytochemicals present in foods. The non-nutritive dietary constituents which possess antimutagenic property appear to be promising chemopreventive agents. This study reports the protective effect of curcumin on B(a)P induced DNA damage in human peripheral blood lymphocyte cells. The study group consisted of 10 male smokers, 10 non-smokers and 10 non-smoking females aged between 25 and 45. The DNA damage was assessed using comet assay. In all the groups curcumin showed a dose-dependent inhibitory effect. The effect appeared to be sex dependent. There was no correlation between DNA damage and GST-Mu levels and levels of micronutrients namely Vitamins A, E and beta carotene. The results of this study are in line with our earlier observations on turmeric/curcumin as a potential chemopreventer.
Author Keywords: DNA damage; Protective effect of curcumin; Strand breaks