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Risk assessment of workers exposed to fumes of bitumen: genotoxic effects and urinary metabolites of naphthalene, phenanthrene, and pyrene
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Abstracts / Toxicology Letters 172S (2007) S1–S240
Kanpur, an industrial metropolis of India. These Crcompounds have polluted the aquifers in and around this city. Of these compounds, Cr-III and Cr-VI are potential carcinogens and are known to be involved in lung carcinogenesis. The protooncogene c-kit, a transmembrane receptor kinase, is implicated in lung carcinogenesis with frequent changes in Exons 9, 11 and 17. In the above background, we have planned to analyze the most vulnerable exon 11 of c-kit gene in chromium exposed human subjects, suffering from lung problems. Blood samples from 37 Cr-exposed persons suffering from lung problems were collected from a hospital in Kanpur, India, on their consent. Atomic Absorption Spectrophotometry was used for the estimation of Cr in blood samples, using standard protocol. DNA was extracted by using standard Phenol chloroform Method. PCR SSCP was done for the Exon 11 region of c-kit gene. Amplified fragment was sized for 256 bp with commercial ladder. The results reveal statistically significantly of higher concentration of Cr in human subjects suffering from lung ailments than normal ones. PCR, SSCP showed no shift in banding pattern in all human subjects, indicating that exon 11 is possibly not involved in lung ailments in these patients; and some other site(s) is (are) affected in Cr intoxication. Exon 11 of c-kit gene is supposed to be not involved in the pulmonary problems. doi:10.1016/j.toxlet.2007.05.439
General frequency of aberrant cells, average number of aberrations per aberrant cell, number of multiaberrant cells, and intercellular distribution of aberrations in aberrant cells were determined. The results demonstrate that in dosage studied cytogenetic damage under gammairradiation was much higher when compared with those under thiotepa. At the same time thiotepa demonstrated higher cytotoxicity (by mitotic index). Results obtained under seedlings growing in 241 Am solutions (calculated internal doses of alpha-irradiation were 0.37–37 cGy) demonstrated any visual cytogenetic effects. It is supposed to be due to unequal distribution of 241 Am in cells and its concentration on cell wall. Thus our results revealed particularities of cytogenetic effects of genotoxic factors studied in Allium-assay. doi:10.1016/j.toxlet.2007.05.440 G22 Risk assessment of workers exposed to fumes of bitumen: genotoxic effects and urinary metabolites of naphthalene, phenanthrene, and pyrene Boleslaw Marczynski 1 , Monika Raulf-Heimsoth 1 , Heiko Udo K¨afferlein 1 , Katrin F¨orster 2 , Anne Spickenheuer 1 , Beate Pesch 1 , Dietmar Breuer 3 , J¨urgen Angerer 2 , Peter Welge 1 , Rainer Bramer 1 , Jens-Uwe Hahn 3 , Thomas Mensing 1 , Thomas Br¨uning 1 1 Berufsgenossenschaftliches
G21 Comparative analysis of genotoxicity induced by irradiation and thiotepa in Allium assay Nataliya Kutsokon, Namik Rashydov Institute of Cell Biology and Genetic Engineering, Kyiv, Ukraine It is supposed the different mutagenic factors should induce the different ratios of chromosome aberrations types. This is of particularly importance in the cases of retrospective analysis of damaging agents, for example for assessment of environmental pollutions. We studied several cytogenetic characteristics to evaluate particularities of genotoxic effects of gamma-, alphairradiation and triethylenethiophosphoramide (thiotepa). Allium cepa L. air-dry seeds were irradiated by 60 Co gamma-rays and grown in distilled water during 3 days. Cytogenetic analysis of root tip cells was also performed after 72 h of seedlings growing in 241 AmCl3 and thiotepa solutions. The mitotic index and chromosome aberrations in each anaphase cell were analysed with in details.
Forschungsinstitut f¨ur Arbeitsmedizin (BGFA), Ruhr-Universit¨at Bochum, Bochum, Germany; 2 Institut und Poliklinik f¨ur Arbeits-, Soziales- und Umweltmedizin, Universit¨at Erlangen-N¨urnberg, Erlangen, Germany; 3 Berufsgenossenschaftliches Institut f¨ ur Arbeitsschutz (BGIA), St. Augustin, Germany We conducted a cross-shift study to determine the associations between biomarkers of external and internal exposure and genotoxic effects in 202 bitumen-exposed workers and 55 construction workers without exposure to bitumen. Fumes of bitumen during the shift were determined by personal air sampling. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine. 8-Oxo-7,8-dihydro2 -deoxyguanosine (8-oxodGuo) adducts and DNA strand breaks (Comet assay) were analysed in white blood cells. Personal air monitoring of exposed persons revealed a median shift concentration of fumes of bitumen of 3.7 mg/m3 . Concentrations of fumes of bitumen showed a moderate association with 1- and 2-naphthol (NOL, rs = 0.18, P < 0.01,), 1-,2 + 9-,3- and
Abstracts / Toxicology Letters 172S (2007) S1–S240
4-hydroxyphenanthrene (rs = 0.36, P < 0.001) and 1hydroxypyrene (1-OHP, rs = 0.25, P < 0.001) after shift. Significantly increased 8-oxodGuo adduct levels were observed after shift in exposed and non-exposed persons (P < 0.0001). However, decreased DNA strand break frequencies were observed after shift in both groups (P < 0.05). Significantly higher levels of DNA damage were found in exposed workers compared with reference subjects. DNA strand break frequencies after shift were only moderately associated with NOL (rs = 0.20, P = 0.01) and 1-OHP (rs = 0.19, P = 0.01) in urine after shift at the group level but not on the individual level. In conclusion, mastic asphalt workers show higher levels of oxidative DNA damage and DNA strand breaks frequencies. However, the observed effects are not dependent on the dose of fumes of bitumen. Additionally, associations between DNA strand break frequency and PAH metabolites of naphthalene and pyrene in urine after shift were observed. doi:10.1016/j.toxlet.2007.05.441 G23 See W9, abstract 51. doi:10.1016/j.toxlet.2007.05.083 G24 Mechanistic study on fenofibrate-induced hepatocarcinogenesis in a 2-stage hepatocarcinogenesis model of rats: Involvement of oxidative stress Jihei Nishimura 1 , Jihei Nishimura 2 , Yasuaki Dewa 1 , Yasuaki Dewa 2 , Toshiya Okamura 2 , Toshiya Okamura 4 , Jin Mailan 1 , Jin Mailan 3 , Masaomi Kawai 1 , Masaomi Kawai 2 , Yukie Saegusa 1 , Yukie Saegusa 2 , Takashi Umemura 4 , Kunitoshi Mitsumori 1 1 Laboratory
of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo, Japan; 2 Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan; 3 Biochemistry and Biotechnology, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, Tokyo, Japan; 4 Division of Pathology, National Institute of Health Sciences, Tokyo, Japan To clarify whether oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by fenofibrate (FF), PPAR alpha agonist, male F344/N rats were fed diet containing 6000, 3000 or 0 ppm of FF for 13 weeks after DEN initiation. Two-
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third partial hepatectomy was applied one week after treatment of FF. The number of hepatocellular foci was increased in FF-treated groups, but the number and area of GST-P positive foci were decreased in these groups. There was a significant increase in hepatocytes positive for anti-Ki-67 antibody in FF-treated groups. In gene expression analysis, significant up-regulations of Aco and Cyp4a1 related to lipid metabolism, Gpx2, Cat, Cyp2b15 and Ugt1a6 related to metabolic oxidative stress, Apex1, Mgmt, Xrcc5 and Gadd45a related to DNA repair, and CyclinD1 related to cell cycle were observed in FF-treated groups, while the expression levels of Cyp1a2, Gsta2, Gstm2 and Gstm3 related to phase I or II metabolism, Mlh1 and Top1 related to DNA repair, and p21, p27, chek2 and Gadd45b related to cell cycle/apoptosis were significantly decreased in these rats. In addition, microsomal ROS formation in the liver, 8-OHdG in the liver DNA and lipofuscin deposition in the liver were significantly increased in FF-treated groups. These results suggest the possibility that oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by FF. doi:10.1016/j.toxlet.2007.05.442 G25 Emblica officinalis prevents oxidative stress and genotoxicity against arsenic induced toxicity in Swiss albino mice Ambika Sharma, Mukesh Kumar Sharma, Madhu Kumar Department of Zoology, University of Rajasthan, Jaipur 302004, Rajasthan, India Health hazards caused by heavy metals have become a great concern to the population. All human populations are exposed to arsenic and its compounds through occupational or environmental processes. DNA damage and genetic instability induced by the inorganic arsenicals present in water are thought to be prerequisites for the initiation of carcinogenesis. The present study was undertaken to evaluate the protective efficacy of Emblica officinalis fruit extract in arsenic induced toxicity. Arsenic treated animals showed clastogenicity in form of chromatids and chromosomal breaks, centric rings, dicentrics and acentric fragments and exchanges. Highly significant (P < 0.001) enhancement in micronuclei (MN) frequency and highly significant depletion in various antioxidant/detoxification enzymes was also observed following arsenic intoxication. Pre and post treatment of Emblica fruit extract is effec-
Abstracts / Toxicology Letters 172S (2007) S1–S240
Kanpur, an industrial metropolis of India. These Crcompounds have polluted the aquifers in and around this city. Of these compounds, Cr-III and Cr-VI are potential carcinogens and are known to be involved in lung carcinogenesis. The protooncogene c-kit, a transmembrane receptor kinase, is implicated in lung carcinogenesis with frequent changes in Exons 9, 11 and 17. In the above background, we have planned to analyze the most vulnerable exon 11 of c-kit gene in chromium exposed human subjects, suffering from lung problems. Blood samples from 37 Cr-exposed persons suffering from lung problems were collected from a hospital in Kanpur, India, on their consent. Atomic Absorption Spectrophotometry was used for the estimation of Cr in blood samples, using standard protocol. DNA was extracted by using standard Phenol chloroform Method. PCR SSCP was done for the Exon 11 region of c-kit gene. Amplified fragment was sized for 256 bp with commercial ladder. The results reveal statistically significantly of higher concentration of Cr in human subjects suffering from lung ailments than normal ones. PCR, SSCP showed no shift in banding pattern in all human subjects, indicating that exon 11 is possibly not involved in lung ailments in these patients; and some other site(s) is (are) affected in Cr intoxication. Exon 11 of c-kit gene is supposed to be not involved in the pulmonary problems. doi:10.1016/j.toxlet.2007.05.439
General frequency of aberrant cells, average number of aberrations per aberrant cell, number of multiaberrant cells, and intercellular distribution of aberrations in aberrant cells were determined. The results demonstrate that in dosage studied cytogenetic damage under gammairradiation was much higher when compared with those under thiotepa. At the same time thiotepa demonstrated higher cytotoxicity (by mitotic index). Results obtained under seedlings growing in 241 Am solutions (calculated internal doses of alpha-irradiation were 0.37–37 cGy) demonstrated any visual cytogenetic effects. It is supposed to be due to unequal distribution of 241 Am in cells and its concentration on cell wall. Thus our results revealed particularities of cytogenetic effects of genotoxic factors studied in Allium-assay. doi:10.1016/j.toxlet.2007.05.440 G22 Risk assessment of workers exposed to fumes of bitumen: genotoxic effects and urinary metabolites of naphthalene, phenanthrene, and pyrene Boleslaw Marczynski 1 , Monika Raulf-Heimsoth 1 , Heiko Udo K¨afferlein 1 , Katrin F¨orster 2 , Anne Spickenheuer 1 , Beate Pesch 1 , Dietmar Breuer 3 , J¨urgen Angerer 2 , Peter Welge 1 , Rainer Bramer 1 , Jens-Uwe Hahn 3 , Thomas Mensing 1 , Thomas Br¨uning 1 1 Berufsgenossenschaftliches
G21 Comparative analysis of genotoxicity induced by irradiation and thiotepa in Allium assay Nataliya Kutsokon, Namik Rashydov Institute of Cell Biology and Genetic Engineering, Kyiv, Ukraine It is supposed the different mutagenic factors should induce the different ratios of chromosome aberrations types. This is of particularly importance in the cases of retrospective analysis of damaging agents, for example for assessment of environmental pollutions. We studied several cytogenetic characteristics to evaluate particularities of genotoxic effects of gamma-, alphairradiation and triethylenethiophosphoramide (thiotepa). Allium cepa L. air-dry seeds were irradiated by 60 Co gamma-rays and grown in distilled water during 3 days. Cytogenetic analysis of root tip cells was also performed after 72 h of seedlings growing in 241 AmCl3 and thiotepa solutions. The mitotic index and chromosome aberrations in each anaphase cell were analysed with in details.
Forschungsinstitut f¨ur Arbeitsmedizin (BGFA), Ruhr-Universit¨at Bochum, Bochum, Germany; 2 Institut und Poliklinik f¨ur Arbeits-, Soziales- und Umweltmedizin, Universit¨at Erlangen-N¨urnberg, Erlangen, Germany; 3 Berufsgenossenschaftliches Institut f¨ ur Arbeitsschutz (BGIA), St. Augustin, Germany We conducted a cross-shift study to determine the associations between biomarkers of external and internal exposure and genotoxic effects in 202 bitumen-exposed workers and 55 construction workers without exposure to bitumen. Fumes of bitumen during the shift were determined by personal air sampling. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine. 8-Oxo-7,8-dihydro2 -deoxyguanosine (8-oxodGuo) adducts and DNA strand breaks (Comet assay) were analysed in white blood cells. Personal air monitoring of exposed persons revealed a median shift concentration of fumes of bitumen of 3.7 mg/m3 . Concentrations of fumes of bitumen showed a moderate association with 1- and 2-naphthol (NOL, rs = 0.18, P < 0.01,), 1-,2 + 9-,3- and
Abstracts / Toxicology Letters 172S (2007) S1–S240
4-hydroxyphenanthrene (rs = 0.36, P < 0.001) and 1hydroxypyrene (1-OHP, rs = 0.25, P < 0.001) after shift. Significantly increased 8-oxodGuo adduct levels were observed after shift in exposed and non-exposed persons (P < 0.0001). However, decreased DNA strand break frequencies were observed after shift in both groups (P < 0.05). Significantly higher levels of DNA damage were found in exposed workers compared with reference subjects. DNA strand break frequencies after shift were only moderately associated with NOL (rs = 0.20, P = 0.01) and 1-OHP (rs = 0.19, P = 0.01) in urine after shift at the group level but not on the individual level. In conclusion, mastic asphalt workers show higher levels of oxidative DNA damage and DNA strand breaks frequencies. However, the observed effects are not dependent on the dose of fumes of bitumen. Additionally, associations between DNA strand break frequency and PAH metabolites of naphthalene and pyrene in urine after shift were observed. doi:10.1016/j.toxlet.2007.05.441 G23 See W9, abstract 51. doi:10.1016/j.toxlet.2007.05.083 G24 Mechanistic study on fenofibrate-induced hepatocarcinogenesis in a 2-stage hepatocarcinogenesis model of rats: Involvement of oxidative stress Jihei Nishimura 1 , Jihei Nishimura 2 , Yasuaki Dewa 1 , Yasuaki Dewa 2 , Toshiya Okamura 2 , Toshiya Okamura 4 , Jin Mailan 1 , Jin Mailan 3 , Masaomi Kawai 1 , Masaomi Kawai 2 , Yukie Saegusa 1 , Yukie Saegusa 2 , Takashi Umemura 4 , Kunitoshi Mitsumori 1 1 Laboratory
of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo, Japan; 2 Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan; 3 Biochemistry and Biotechnology, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, Tokyo, Japan; 4 Division of Pathology, National Institute of Health Sciences, Tokyo, Japan To clarify whether oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by fenofibrate (FF), PPAR alpha agonist, male F344/N rats were fed diet containing 6000, 3000 or 0 ppm of FF for 13 weeks after DEN initiation. Two-
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third partial hepatectomy was applied one week after treatment of FF. The number of hepatocellular foci was increased in FF-treated groups, but the number and area of GST-P positive foci were decreased in these groups. There was a significant increase in hepatocytes positive for anti-Ki-67 antibody in FF-treated groups. In gene expression analysis, significant up-regulations of Aco and Cyp4a1 related to lipid metabolism, Gpx2, Cat, Cyp2b15 and Ugt1a6 related to metabolic oxidative stress, Apex1, Mgmt, Xrcc5 and Gadd45a related to DNA repair, and CyclinD1 related to cell cycle were observed in FF-treated groups, while the expression levels of Cyp1a2, Gsta2, Gstm2 and Gstm3 related to phase I or II metabolism, Mlh1 and Top1 related to DNA repair, and p21, p27, chek2 and Gadd45b related to cell cycle/apoptosis were significantly decreased in these rats. In addition, microsomal ROS formation in the liver, 8-OHdG in the liver DNA and lipofuscin deposition in the liver were significantly increased in FF-treated groups. These results suggest the possibility that oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by FF. doi:10.1016/j.toxlet.2007.05.442 G25 Emblica officinalis prevents oxidative stress and genotoxicity against arsenic induced toxicity in Swiss albino mice Ambika Sharma, Mukesh Kumar Sharma, Madhu Kumar Department of Zoology, University of Rajasthan, Jaipur 302004, Rajasthan, India Health hazards caused by heavy metals have become a great concern to the population. All human populations are exposed to arsenic and its compounds through occupational or environmental processes. DNA damage and genetic instability induced by the inorganic arsenicals present in water are thought to be prerequisites for the initiation of carcinogenesis. The present study was undertaken to evaluate the protective efficacy of Emblica officinalis fruit extract in arsenic induced toxicity. Arsenic treated animals showed clastogenicity in form of chromatids and chromosomal breaks, centric rings, dicentrics and acentric fragments and exchanges. Highly significant (P < 0.001) enhancement in micronuclei (MN) frequency and highly significant depletion in various antioxidant/detoxification enzymes was also observed following arsenic intoxication. Pre and post treatment of Emblica fruit extract is effec-