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590 DNA adducts of benzo[A]pyrene (B[A]P) in white blood cells DNA of workers occupationally exposed to different B[A]P concentrations
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Poster Session P30. Biomarkers and exposure assessment
soil. Many xenobiotics, such as polycyclic aromatic hydrocarbons (PHAs), heavy metals, pesticides, have been recognized as being DNA damage inducers. Aim of the present study was to develop a new biomonitoring methodology for assessing soil genotoxicity using Trifolium repens L. Plants can be used as sensibility biological indicators to measure the potential contaminant toxicity. In order to investigate responsibility to known genotoxic substances Trifolium repens L. plants were exposed to soil artificially contaminated with three different heavy metal (Cr, Ni, Cd) and two different PHAs (naphthalene and benzo[a]pyrene). After two weeks DNA from root and shoot was analyzed by molecular tools to detect the DNA damage induced by these genotoxic compounds. DNA sequence alteration was revealed by amplified fragment length polymorphism (AFLP) whereas content alteration was evaluated by flow cytometry (FCM) techniques. Root and shoot dry weight as index of plant growth was also measured. Results showed that the combination of the two techniques led to efficient detection of the genotoxic effect of contaminants in soil. All of the metal treatments induced damage to the genomic sequence, except for the lowest 25 Ni mg/Kg and 5,25 Cd mg/Kg concentrations, both in root and in shoot. PHAs induced DNA alteration only in root and not in shoot, probably because these compounds were not traslocated in plant. Genotoxic effect is also early toxicity index on vegetable able to determine damage without significative difference on clover development by contrast with the classical vegetative endpoints. Work is in progress to apply the same methodology to C.elegans for detecting genotoxicity. 589
PAH-DNA ADDUCTS IN ENVIRONMENTALLY EXPOSED POPULATION IN RELATION TO METABOLIC AND DNA REPAIR GENES POLYMORPHISMS
B. Binkova, Z. Smerhovsky, I. Chvatalova, E. Biros, Z. Stavkova, A. Milcova, R.J. Sram. Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair genes polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates mutual relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N= 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N=52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32P-postlabeling assay. Polymorphisms of metabolizing (GSTML, GSTPL, GSTTL, EPHX, CYP1A1-MpsI) and DNA repair genes (XRCCL, XPD) were determined by PCR-based RFLP assays. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 µg/m3, PM2.5 27–38 µg/m3, c-PAHs 18–22 ng/m3; personal exposure to c-PAHs: 12.0±11.1 ng/m3 and 6.2±3.5 ng/m3, for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92±0.28 vs. 0.82±0.23 adducts/108 nucleotides, p=0.065), whereas the level of the “like” B[a]P-derived adduct was significantly higher in exposed group (0.122±0.036 vs. 0.099±0.035 adducts/108 nucleotides, p=0.003). The significant difference in both the total (P<0.05) and the “like” B[a]P-DNA adduct (P<0.01) between smokers and nonsmokers within both groups was observed. The significant positive association between DNA adduct and cotinine levels (r=0.368, P<0.001) and negative association between DNA adduct and vitamin C levels (r=-0.290, P=0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and epoxide hydroxylase gene in exon 4 as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for “like” B[a]P-DNA adduct. This study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels
and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from PAHs exposure. Supported by the grant of czech ministry of environment vav/340/2/00, and by ec grant qlk4-ct-2000 0091. 590
DNA ADDUCTS OF BENZO[A]PYRENE (B[A]P) IN WHITE BLOOD CELLS DNA OF WORKERS OCCUPATIONALLY EXPOSED TO DIFFERENT B[A]P CONCENTRATIONS
B. Marczynski 1 , T. Mensing 1 , R. Preuss 2 , M. Wilhelm 3 , J. Müller 2 , C. Broding 2 , T. Merz 2 , J. Angerer 2 , F. Müller 4 , T. Brüning 1 . 1 Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin, Bochum, 2 Institut für Arbeits-, Sozial- und Umweltmedizin, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, 3 Abteilung für Hygiene, Sozial- und Umweltmedizin, Ruhr-Universität Bochum, Bochum, 4 Didier-Werke AG, Wiesbaden, Germany B[a]P is metabolized to (±)-anti- and (±)-syn-benzo[a]pyrenediolepoxide (BPDE) which can covalently bind to the DNA. anti-BPDEDNA adducts in white blood cells (WBC) of occupationally exposed workers in a fireproof material producing plant were determined and compared to the ambient B[a]P concentrations before and three months after the substitution of the binding pitch. 17 PAH-exposed workers were examined before and after the substitution of the production material. Additionally, seven workers were examined before and 16 workers after the substitution. The B[a]P-concentrations in the air were measured by personal air sampling and subsequent HPLC method (diode array detection). We used HLPC separation and fluorescence detection to determine the B[a]P-tetrol I-1 ((±)-r-7,c10,t-8,t-9-Tetrahydroxy-7,8,9,10-Tetrahydro-Benzo[a]pyren) arising after acid hydrolysis from anti-BPDE-DNA. The change in the production process led to a decrease in ambient B[a]P concentrations. For the 17 workers median concentration of B[a]P was determined to be 0.165 µg/m3 (range <0.07 - 0.54) before and <0.07 µg/m3 (range <0.07 - 16.43) three months after the substitution. In these 17 workers the rate ranged between 0.5 and 1.9 adducts/108 nucleotides before the conditions had changed and <0.5 to 0.9 adducts/108 nucleotides three months later. For all workers the median B[a]Pconcentration was 0.14 µg/m3 (range <0.07–0.85 µg/m3 ) before (n=26) and <0.07 µg/m3 (range <0.07–16.43) after the substitution (n=33). For these workers the adduct rate decreases from 0.9 adducts/108 nucleotides (0.2 – 0.9/108 ) (n=24) to <0.05 adducts/108 nucleotides (<0.5 – 3.2/108 ) (n=33) after the substitution. Although ambient B[a]P concentration and DNA adduct level were in a low range, a good correlation between the airborne B[a]P concentration and the adduct level in WBC DNA was observed. 591
EVALUATIONS OF CYP 3A 4/5 AND DNA DAMAGE IN RAT ENTEROCYTES AND IN HUMAN AND RAT LYMPHOCYTES BY FLUOROQUINOLONES.
M.E. Fracasso, P. Franceschetti, D. Doria, A. Benini, E. Bertazzoni Minelli. Department of Medicine and Public Health, Section of Pharmacology, University of Verona, Verona, Italy Fluoroquinolones have been considered to be relatively well-tolerated and safe drugs, but gastrointestinal disorders and central nervous system events are the most common adverse effects. The fluoroquinolones are subjected to extensive metabolism and active metabolites can increase the risk of adverse reactions. Many interactions among drugs are due to metabolism through CYP 3A4/5 enzymes. The most expression of this emoprotein is in liver and in the small intestine. The presence of these compounds at intestinal level, at high concentration and/or for prolonged periods of time, suggested a potential risk of inducing toxic effects. On the basis of correlation with adverse reactions at skin level (phototoxicity) we evaluated a possible DNA damage in the gut and in the peripheral blood lymphocytes. The aim of this work is to assess in animals and in human subjects cellular damage (enterocytes and lymphocytes) and to verify changes in CYPs after repeated treatments. The expression of CYPs was determined by W.B. Lymphocyte and intestinal mucosa DNA damage was evaluated by Comet assay: the parameters were tail moment (TM), tail intensity (TI) and tail length (TL).
Poster Session P30. Biomarkers and exposure assessment
soil. Many xenobiotics, such as polycyclic aromatic hydrocarbons (PHAs), heavy metals, pesticides, have been recognized as being DNA damage inducers. Aim of the present study was to develop a new biomonitoring methodology for assessing soil genotoxicity using Trifolium repens L. Plants can be used as sensibility biological indicators to measure the potential contaminant toxicity. In order to investigate responsibility to known genotoxic substances Trifolium repens L. plants were exposed to soil artificially contaminated with three different heavy metal (Cr, Ni, Cd) and two different PHAs (naphthalene and benzo[a]pyrene). After two weeks DNA from root and shoot was analyzed by molecular tools to detect the DNA damage induced by these genotoxic compounds. DNA sequence alteration was revealed by amplified fragment length polymorphism (AFLP) whereas content alteration was evaluated by flow cytometry (FCM) techniques. Root and shoot dry weight as index of plant growth was also measured. Results showed that the combination of the two techniques led to efficient detection of the genotoxic effect of contaminants in soil. All of the metal treatments induced damage to the genomic sequence, except for the lowest 25 Ni mg/Kg and 5,25 Cd mg/Kg concentrations, both in root and in shoot. PHAs induced DNA alteration only in root and not in shoot, probably because these compounds were not traslocated in plant. Genotoxic effect is also early toxicity index on vegetable able to determine damage without significative difference on clover development by contrast with the classical vegetative endpoints. Work is in progress to apply the same methodology to C.elegans for detecting genotoxicity. 589
PAH-DNA ADDUCTS IN ENVIRONMENTALLY EXPOSED POPULATION IN RELATION TO METABOLIC AND DNA REPAIR GENES POLYMORPHISMS
B. Binkova, Z. Smerhovsky, I. Chvatalova, E. Biros, Z. Stavkova, A. Milcova, R.J. Sram. Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair genes polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates mutual relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N= 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N=52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32P-postlabeling assay. Polymorphisms of metabolizing (GSTML, GSTPL, GSTTL, EPHX, CYP1A1-MpsI) and DNA repair genes (XRCCL, XPD) were determined by PCR-based RFLP assays. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 µg/m3, PM2.5 27–38 µg/m3, c-PAHs 18–22 ng/m3; personal exposure to c-PAHs: 12.0±11.1 ng/m3 and 6.2±3.5 ng/m3, for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92±0.28 vs. 0.82±0.23 adducts/108 nucleotides, p=0.065), whereas the level of the “like” B[a]P-derived adduct was significantly higher in exposed group (0.122±0.036 vs. 0.099±0.035 adducts/108 nucleotides, p=0.003). The significant difference in both the total (P<0.05) and the “like” B[a]P-DNA adduct (P<0.01) between smokers and nonsmokers within both groups was observed. The significant positive association between DNA adduct and cotinine levels (r=0.368, P<0.001) and negative association between DNA adduct and vitamin C levels (r=-0.290, P=0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and epoxide hydroxylase gene in exon 4 as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for “like” B[a]P-DNA adduct. This study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels
and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from PAHs exposure. Supported by the grant of czech ministry of environment vav/340/2/00, and by ec grant qlk4-ct-2000 0091. 590
DNA ADDUCTS OF BENZO[A]PYRENE (B[A]P) IN WHITE BLOOD CELLS DNA OF WORKERS OCCUPATIONALLY EXPOSED TO DIFFERENT B[A]P CONCENTRATIONS
B. Marczynski 1 , T. Mensing 1 , R. Preuss 2 , M. Wilhelm 3 , J. Müller 2 , C. Broding 2 , T. Merz 2 , J. Angerer 2 , F. Müller 4 , T. Brüning 1 . 1 Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin, Bochum, 2 Institut für Arbeits-, Sozial- und Umweltmedizin, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, 3 Abteilung für Hygiene, Sozial- und Umweltmedizin, Ruhr-Universität Bochum, Bochum, 4 Didier-Werke AG, Wiesbaden, Germany B[a]P is metabolized to (±)-anti- and (±)-syn-benzo[a]pyrenediolepoxide (BPDE) which can covalently bind to the DNA. anti-BPDEDNA adducts in white blood cells (WBC) of occupationally exposed workers in a fireproof material producing plant were determined and compared to the ambient B[a]P concentrations before and three months after the substitution of the binding pitch. 17 PAH-exposed workers were examined before and after the substitution of the production material. Additionally, seven workers were examined before and 16 workers after the substitution. The B[a]P-concentrations in the air were measured by personal air sampling and subsequent HPLC method (diode array detection). We used HLPC separation and fluorescence detection to determine the B[a]P-tetrol I-1 ((±)-r-7,c10,t-8,t-9-Tetrahydroxy-7,8,9,10-Tetrahydro-Benzo[a]pyren) arising after acid hydrolysis from anti-BPDE-DNA. The change in the production process led to a decrease in ambient B[a]P concentrations. For the 17 workers median concentration of B[a]P was determined to be 0.165 µg/m3 (range <0.07 - 0.54) before and <0.07 µg/m3 (range <0.07 - 16.43) three months after the substitution. In these 17 workers the rate ranged between 0.5 and 1.9 adducts/108 nucleotides before the conditions had changed and <0.5 to 0.9 adducts/108 nucleotides three months later. For all workers the median B[a]Pconcentration was 0.14 µg/m3 (range <0.07–0.85 µg/m3 ) before (n=26) and <0.07 µg/m3 (range <0.07–16.43) after the substitution (n=33). For these workers the adduct rate decreases from 0.9 adducts/108 nucleotides (0.2 – 0.9/108 ) (n=24) to <0.05 adducts/108 nucleotides (<0.5 – 3.2/108 ) (n=33) after the substitution. Although ambient B[a]P concentration and DNA adduct level were in a low range, a good correlation between the airborne B[a]P concentration and the adduct level in WBC DNA was observed. 591
EVALUATIONS OF CYP 3A 4/5 AND DNA DAMAGE IN RAT ENTEROCYTES AND IN HUMAN AND RAT LYMPHOCYTES BY FLUOROQUINOLONES.
M.E. Fracasso, P. Franceschetti, D. Doria, A. Benini, E. Bertazzoni Minelli. Department of Medicine and Public Health, Section of Pharmacology, University of Verona, Verona, Italy Fluoroquinolones have been considered to be relatively well-tolerated and safe drugs, but gastrointestinal disorders and central nervous system events are the most common adverse effects. The fluoroquinolones are subjected to extensive metabolism and active metabolites can increase the risk of adverse reactions. Many interactions among drugs are due to metabolism through CYP 3A4/5 enzymes. The most expression of this emoprotein is in liver and in the small intestine. The presence of these compounds at intestinal level, at high concentration and/or for prolonged periods of time, suggested a potential risk of inducing toxic effects. On the basis of correlation with adverse reactions at skin level (phototoxicity) we evaluated a possible DNA damage in the gut and in the peripheral blood lymphocytes. The aim of this work is to assess in animals and in human subjects cellular damage (enterocytes and lymphocytes) and to verify changes in CYPs after repeated treatments. The expression of CYPs was determined by W.B. Lymphocyte and intestinal mucosa DNA damage was evaluated by Comet assay: the parameters were tail moment (TM), tail intensity (TI) and tail length (TL).