- Email: [email protected]
Effects of a high fat diet on in vivo mutagenicity induced by heterocyclic amine in the livers of GPT delta rats
Abstracts / Toxicology Letters 229S (2014) S40–S252
P-3.30 Effects of a high fat diet on in vivo mutagenicity induced by heterocyclic amine in the livers of GPT delta rats Shinji Takasu 1,∗ , Yuji Ishii 1 , Aki Kijima 1 , Yuh Yokoo 1 , Takehiko Nohmi 2 , Akiyoshi Nishikawa 2 , Kumiko Ogawa 1 , Takashi Umemura 1 1 Division of Pathology, National Institute of Health Sciences, Tokyo, Japan, 2 Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan
Epidemiologically, excess intake of fat has been suggested to be a risk factor for human cancer. In rodent models, high fat diets (HFDs) have been shown to exert tumor-promoting effects on the liver. However, the effects of an HFD on the initiation of carcinogenesis are not fully understood. To reveal the effects of an HFD on the initiation of carcinogenesis, particularly gene mutations induced by genotoxic carcinogens, we examined whether consumption of an HFD altered reporter gene mutations in the livers of rats treated with heterocyclic amines. Six-week-old male F344 gpt delta rats were treated for 28 days with 1.0 mg/kg/day 2-amino-3-methylimidazo[4,5-f]quinolone (IQ) or 5.0 mg/kg/day 2-amino-3,8-dimethylimadazo[4,5-f]quinoxaline (MeIQx) by gavage. The animals were fed an HFD or standard diet (STD) throughout the experimental period, after which reporter gene mutation assays were performed in liver tissues. gpt and Spi− mutant frequencies (MFs) in the livers of rats treated with IQ or MeIQx were significantly increased compared to those in corresponding control rats fed either diet. However, these increases were independent of the fat content in their diet. These results indicated that consumption of an HFD may not influence the in vivo mutagenicity induced by genotoxic carcinogens in rodent livers. Further data from gpt and Spi− mutation spectra analysis will be presented in order to analyze the effects of an HFD on in vivo mutagenicity induced by heterocyclic amines. http://dx.doi.org/10.1016/j.toxlet.2014.06.539 P-3.31 Comparison of genotoxic effects of major diesel exhaust components in human alveolar basal epithelial cells (A549) Jan Topinka 1,∗ , Pavel Rossner 1 , Jitka Stolcpartova 1 , Jana Schmuczerova 1 , Alena Milcova 1 , Eva Hruba 2 , Miroslav Machala 2 1 2
Institute of Experimental Medicine AS CR, Prague, Czech Republic, Veterinary Research Institute, Brno, Czech Republic
Internal combustion engines (ICE), including diesel engines, power most of the motorized road vehicles. ICE are a major source of air pollution in metropolitan areas. Among other compounds they emit polycyclic aromatic hydrocarbons (PAHs) and their nitro-derivatives. Genotoxicity of benzo[a]pyrene (B[a]P), a model PAH, and PAH nitro-derivatives (1-nitropyrene (1-NP) and 3nitrobenzantrone (3-NBA)), was studied in a model cell line A549. The cells were treated for 4 and 24 h with different concentrations of tested compounds (B[a]P: 0.1 and 1 M; 1-NP: 1 and 10 M; 3-NBA: 0.5 and 5 M) and several endpoints, including bulky DNA adducts and oxidative stress markers (lipid peroxidation, protein and DNA oxidation) were analyzed. All tested compounds increased bulky DNA adduct levels after both the 4-h and 24-h treatment, although the effect of 1-NP was relatively weak. A 24-h
S155
treatment resulted mostly in higher DNA adduct levels than the 4-h treatment with the exception of 3-NBA. 1-NP induced protein oxidation after both the 4-h and 24-h treatment. Interestingly, lipid peroxidation measured as levels of 15-F2t -isoprostane decreased after the B[a]P treatment, but was elevated after incubation of the cells with 1-NP and 3-NBA. 8-Oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, was not affected by either compound and/or treatment period. Our data highlight differences in genotoxic mechanisms of PAHs and their nitroderivatives, particularly for oxidative stress markers. Acknowledgements: Supported by the Czech Science Foundation (13-01438S) and by EU LIFE+ Program (LIFE-ENV-CZ-651). http://dx.doi.org/10.1016/j.toxlet.2014.06.540 P-3.32 Modulation of genotoxic effects of benzo[a]pyrene in colon cancer cells by inhibition of Wnt/beta-catenin signaling Jan Vondracek 1,∗ , Marketa Kabatkova 1,2 , Iva Jelinkova 1,2 , Miroslav Machala 3 , Jan Topinka 4 , Alena Milcova 4 , Alois Kozubik 1,2 1
Institute of Biophysics AS CR, Brno, Czech Republic, 2 Institute of Experimental Biology, Faculty of Sciences, Masaryk University, Brno, Czech Republic, 3 Veterinary Research Institute, Brno, Czech Republic, 4 Institute of Experimental Medicine AS CR, Prague, Czech Republic
Benzo[a]pyrene (BaP) is a potent carcinogenic polycyclic aromatic hydrocarbon found in various processed food, which has been suggested to pose a risk factor for the development of sporadic colon carcinomas. Our previous data have suggested that the expression of enzymes responsible for the bioactivation of BaP in colon epithelial cells, such as cytochrome P4501A1 may depend on activity of Wnt/beta-catenin pathway. Using HCT-116 cell line as a principal model derived from colon carcinoma, we further explored the effects of various experimental approaches leading to inhibition of beta-catenin-dependent signaling (including small molecule chemical inhibitor, transfection with anti-beta-catenin siRNA or with a dominant-negative TCF4 construct) on genotoxicity of BaP. We found that all treatments leading to inhibition of beta-catenin activity modulated levels of stable DNA adducts formed by ultimate BaP-7,8-diol-9,10-epoxide – DNA adducts. The siRNA-mediated beta-catenin knock-down significantly increased levels of DNA adducts and, in parallel, it modulated further genotoxic events, as indicated by an increased expression of genes associated with DNA damage response, including p53 tumor suppressor target genes (such as GADD45A, DDB2, or CDKN1A), changes in cell phenotype or results of Comet assay. These observations suggest that inhibition of Wnt/beta-catenin activity, a pathway frequently over-activated during colon cancer development, may have consequences for genotoxicity of food carcinogens within colon epithelium. Acknowledgement: This study was supported by the Czech Science Foundation grant no. 13-09766S. http://dx.doi.org/10.1016/j.toxlet.2014.06.541
P-3.30 Effects of a high fat diet on in vivo mutagenicity induced by heterocyclic amine in the livers of GPT delta rats Shinji Takasu 1,∗ , Yuji Ishii 1 , Aki Kijima 1 , Yuh Yokoo 1 , Takehiko Nohmi 2 , Akiyoshi Nishikawa 2 , Kumiko Ogawa 1 , Takashi Umemura 1 1 Division of Pathology, National Institute of Health Sciences, Tokyo, Japan, 2 Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan
Epidemiologically, excess intake of fat has been suggested to be a risk factor for human cancer. In rodent models, high fat diets (HFDs) have been shown to exert tumor-promoting effects on the liver. However, the effects of an HFD on the initiation of carcinogenesis are not fully understood. To reveal the effects of an HFD on the initiation of carcinogenesis, particularly gene mutations induced by genotoxic carcinogens, we examined whether consumption of an HFD altered reporter gene mutations in the livers of rats treated with heterocyclic amines. Six-week-old male F344 gpt delta rats were treated for 28 days with 1.0 mg/kg/day 2-amino-3-methylimidazo[4,5-f]quinolone (IQ) or 5.0 mg/kg/day 2-amino-3,8-dimethylimadazo[4,5-f]quinoxaline (MeIQx) by gavage. The animals were fed an HFD or standard diet (STD) throughout the experimental period, after which reporter gene mutation assays were performed in liver tissues. gpt and Spi− mutant frequencies (MFs) in the livers of rats treated with IQ or MeIQx were significantly increased compared to those in corresponding control rats fed either diet. However, these increases were independent of the fat content in their diet. These results indicated that consumption of an HFD may not influence the in vivo mutagenicity induced by genotoxic carcinogens in rodent livers. Further data from gpt and Spi− mutation spectra analysis will be presented in order to analyze the effects of an HFD on in vivo mutagenicity induced by heterocyclic amines. http://dx.doi.org/10.1016/j.toxlet.2014.06.539 P-3.31 Comparison of genotoxic effects of major diesel exhaust components in human alveolar basal epithelial cells (A549) Jan Topinka 1,∗ , Pavel Rossner 1 , Jitka Stolcpartova 1 , Jana Schmuczerova 1 , Alena Milcova 1 , Eva Hruba 2 , Miroslav Machala 2 1 2
Institute of Experimental Medicine AS CR, Prague, Czech Republic, Veterinary Research Institute, Brno, Czech Republic
Internal combustion engines (ICE), including diesel engines, power most of the motorized road vehicles. ICE are a major source of air pollution in metropolitan areas. Among other compounds they emit polycyclic aromatic hydrocarbons (PAHs) and their nitro-derivatives. Genotoxicity of benzo[a]pyrene (B[a]P), a model PAH, and PAH nitro-derivatives (1-nitropyrene (1-NP) and 3nitrobenzantrone (3-NBA)), was studied in a model cell line A549. The cells were treated for 4 and 24 h with different concentrations of tested compounds (B[a]P: 0.1 and 1 M; 1-NP: 1 and 10 M; 3-NBA: 0.5 and 5 M) and several endpoints, including bulky DNA adducts and oxidative stress markers (lipid peroxidation, protein and DNA oxidation) were analyzed. All tested compounds increased bulky DNA adduct levels after both the 4-h and 24-h treatment, although the effect of 1-NP was relatively weak. A 24-h
S155
treatment resulted mostly in higher DNA adduct levels than the 4-h treatment with the exception of 3-NBA. 1-NP induced protein oxidation after both the 4-h and 24-h treatment. Interestingly, lipid peroxidation measured as levels of 15-F2t -isoprostane decreased after the B[a]P treatment, but was elevated after incubation of the cells with 1-NP and 3-NBA. 8-Oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG), a marker of oxidative DNA damage, was not affected by either compound and/or treatment period. Our data highlight differences in genotoxic mechanisms of PAHs and their nitroderivatives, particularly for oxidative stress markers. Acknowledgements: Supported by the Czech Science Foundation (13-01438S) and by EU LIFE+ Program (LIFE-ENV-CZ-651). http://dx.doi.org/10.1016/j.toxlet.2014.06.540 P-3.32 Modulation of genotoxic effects of benzo[a]pyrene in colon cancer cells by inhibition of Wnt/beta-catenin signaling Jan Vondracek 1,∗ , Marketa Kabatkova 1,2 , Iva Jelinkova 1,2 , Miroslav Machala 3 , Jan Topinka 4 , Alena Milcova 4 , Alois Kozubik 1,2 1
Institute of Biophysics AS CR, Brno, Czech Republic, 2 Institute of Experimental Biology, Faculty of Sciences, Masaryk University, Brno, Czech Republic, 3 Veterinary Research Institute, Brno, Czech Republic, 4 Institute of Experimental Medicine AS CR, Prague, Czech Republic
Benzo[a]pyrene (BaP) is a potent carcinogenic polycyclic aromatic hydrocarbon found in various processed food, which has been suggested to pose a risk factor for the development of sporadic colon carcinomas. Our previous data have suggested that the expression of enzymes responsible for the bioactivation of BaP in colon epithelial cells, such as cytochrome P4501A1 may depend on activity of Wnt/beta-catenin pathway. Using HCT-116 cell line as a principal model derived from colon carcinoma, we further explored the effects of various experimental approaches leading to inhibition of beta-catenin-dependent signaling (including small molecule chemical inhibitor, transfection with anti-beta-catenin siRNA or with a dominant-negative TCF4 construct) on genotoxicity of BaP. We found that all treatments leading to inhibition of beta-catenin activity modulated levels of stable DNA adducts formed by ultimate BaP-7,8-diol-9,10-epoxide – DNA adducts. The siRNA-mediated beta-catenin knock-down significantly increased levels of DNA adducts and, in parallel, it modulated further genotoxic events, as indicated by an increased expression of genes associated with DNA damage response, including p53 tumor suppressor target genes (such as GADD45A, DDB2, or CDKN1A), changes in cell phenotype or results of Comet assay. These observations suggest that inhibition of Wnt/beta-catenin activity, a pathway frequently over-activated during colon cancer development, may have consequences for genotoxicity of food carcinogens within colon epithelium. Acknowledgement: This study was supported by the Czech Science Foundation grant no. 13-09766S. http://dx.doi.org/10.1016/j.toxlet.2014.06.541