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Prediction of carcinogenicity in vitro based on genotoxicity and toxicogenomic data
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Abstracts / Toxicology Letters 196S (2010) S37–S351
be regulated by PARP-1. To uncover the role of PARP-1 on B(a)P induced DNA methylation variation in vitro, DNA methylation alterations of 16HBE and its PARP-1-deficient cells exposed to B(a)P were investigated, and simultaneously, the expression level of PARP-1 and DNMT-1 was observed dynamically. Immunofluorescence assay and HPCE showed that genomic methylation in 16HBE-shPARP-1 increased significantly comparing with 16HBE cells. Treatment of higher dose B(a)P decreased DNA methylation of both cell lines significantly, notwithstanding increasing trend was showed for low dose groups. For higher dose groups, western blotting showed that PARP-1 expression level decreased whereas DNMT-1 expression level increased significantly, which was coincident with the results of Q-PCR. In addition, the expression of DNMT-1 in 16HBE-shPARP-1 increased significantly in comparison with 16HBE. According to these results, we come to the conclusion that 16HBE cells became genomic hypomethylation after being exposed to high dose B(a)P for 72 h, while the changes of PARP-1 and DNMT-1 were exactly opposite. It suggests that PARP-1 could prevent DNA methylation through inhibiting the expression of DNMT-1, and this effect can be reversed by PARP-1-deficiency. doi:10.1016/j.toxlet.2010.03.606
P203-017 Transcriptome analysis identifies the carcinogenic effects of aristolochic acid in rat kidney T. Chen, Z. Li, Z. Su, L. Shi US Food and Drug Administration/National Center for Toxicological Research, United States Aristolochic acid (AA) is the active component of herbal drugs derived from Aristolochia species that have been used for medicinal purposes since antiquity. AA, however, induced nephropathy and urothelial cancer in humans and malignant tumors in the kidney, urinary tract and other tissues in rodents. In this study, gene expression profiles in kidneys of the rats treated with carcinogenic doses (10 mg/kg) of AA for 3 months were examined using microarray and next generation sequencing (NGS) technologies. Microarray analysis was performed using the Affymetrix Rat Genome 230 2.0 Microarray and RNA-Seq by NGS was conducted using Illumina Genome Analyzer II platform. The gene expression profiles generated via the two types of transcriptome analysis platforms showed that AA treatment significantly altered expressions of a large number of genes that were related to carcinogenesis. Functional analysis demonstrated that the biological processes related to tumorigenesis and apoptosis were the top dysregulated functional pathways. Comparison of data from the microarray and NGS analyses indicates that alteration of gene expression determined by NGS is comparable to that by microarray in terms of the percentage of overlapping biological functions (over 70%). Our results suggest that analysis of the gene expression profiles can define the carcinogenicity of AA in rat kidneys using microarray or NGS transcriptome analysis. doi:10.1016/j.toxlet.2010.03.607
P203-018 Prediction of carcinogenicity in vitro based on genotoxicity and toxicogenomic data Y. Sheen 1 , E. Yoon 1 , J. Son 1 , C. Keum 2 , K. No 2 1
Ewha Womans University, Republic of Korea, 2 Yonsei University, Republic of Korea This study has been designed to establish the prediction model for chemical carcinogens via the use of genomics technology along with conventional genotoxicity testing, bioinformatics, chemoinformatics, eventually systems toxicology. We have collected genotoxic and carcinogenic toxicity data from FDA, IARC, IPCS, NTP and use them to build toxophore as well as algorithm to select test chemicals. Twenty chemicals, ten for genotoxic carcinogens, and ten for non-genotoxic carcinogens were tested, for the conventional genotoxicity test such as Ames test, in vitro MN assay, COMET assay. Also, we carried out microarray analysis of these twenty chemicals, and did MAS 5.0/RMA analysis along with global and quantile normalization. We have identified five specific gene expression profiles for genotoxic carcinogens and nine specific gene expression profiles for non-genotoxic carcinogens based on each class of chemicals. In addition, we have found out genotoxic carcinogen specific candidate molecular gene, Prkacb, and Caskin/nol3 gene pair. Also, we have identified genotoxic carcinogens specific network markers. It might be possible to hypothesize that carcinogenic genotoxins act with different mechanism from that of non-carcinogenic genotoxins. Thus, with the help of microarray technology, we tried to discriminate the gene expression patterns between the two genotoxin classes with and without carcinogenicity in L5178Y mouse lymphoma cell. Gene expression profiles of cells treated with two genotoxic carcinogens and two genotoxic non-carcinogens for 2 h followed by 22 h of growth were analyzed. 16 genotoxic carcinogen specific and 41 genotoxic non-carcinogen specific differentially expressed genes were extracted using at least twofold change and two sample t-test p-value less than 0.05. The gene sets found in this work might serve as valuable expression signature to characterize the carcinogenic toxicity among genotoxins and might be a feature gene set to classify the gene expression profiles of genotoxic carcinogen from those of genotoxic noncarcinogen. doi:10.1016/j.toxlet.2010.03.608
P203-019 A novel mechanism on modulation of APE1 activity via protein–protein interactions of Gadd45a and PCNA H.L. Kim 1 , S.U. Kim 2 , Y.R. Seo 3 1 Department of Pharmacology, Institute for Basic Medical Science (IBMS), School of Medicine, Kyung Hee University, Republic of Korea, 2 Department of Life Science, School of Interdisciplinary Bioscience and Bioengineering, Republic of Korea, 3 Department of Pharmacology, School of Medicine, Kyung Hee University, Republic of Korea
Base excision repair (BER) is a critical protective mechanism that maintains genomic integrity by correcting DNA base modifications. Our earlier study suggested a potential model of the interaction among Gadd45a, PCNA and APE1. Here, we investigated whether the interaction of Gadd45a and PCNA enhances the APE1 activity in BER pathway through the mutation of predicted PCNA binding site on Gadd45a. We found the suppression of APE1 activity in
Abstracts / Toxicology Letters 196S (2010) S37–S351
be regulated by PARP-1. To uncover the role of PARP-1 on B(a)P induced DNA methylation variation in vitro, DNA methylation alterations of 16HBE and its PARP-1-deficient cells exposed to B(a)P were investigated, and simultaneously, the expression level of PARP-1 and DNMT-1 was observed dynamically. Immunofluorescence assay and HPCE showed that genomic methylation in 16HBE-shPARP-1 increased significantly comparing with 16HBE cells. Treatment of higher dose B(a)P decreased DNA methylation of both cell lines significantly, notwithstanding increasing trend was showed for low dose groups. For higher dose groups, western blotting showed that PARP-1 expression level decreased whereas DNMT-1 expression level increased significantly, which was coincident with the results of Q-PCR. In addition, the expression of DNMT-1 in 16HBE-shPARP-1 increased significantly in comparison with 16HBE. According to these results, we come to the conclusion that 16HBE cells became genomic hypomethylation after being exposed to high dose B(a)P for 72 h, while the changes of PARP-1 and DNMT-1 were exactly opposite. It suggests that PARP-1 could prevent DNA methylation through inhibiting the expression of DNMT-1, and this effect can be reversed by PARP-1-deficiency. doi:10.1016/j.toxlet.2010.03.606
P203-017 Transcriptome analysis identifies the carcinogenic effects of aristolochic acid in rat kidney T. Chen, Z. Li, Z. Su, L. Shi US Food and Drug Administration/National Center for Toxicological Research, United States Aristolochic acid (AA) is the active component of herbal drugs derived from Aristolochia species that have been used for medicinal purposes since antiquity. AA, however, induced nephropathy and urothelial cancer in humans and malignant tumors in the kidney, urinary tract and other tissues in rodents. In this study, gene expression profiles in kidneys of the rats treated with carcinogenic doses (10 mg/kg) of AA for 3 months were examined using microarray and next generation sequencing (NGS) technologies. Microarray analysis was performed using the Affymetrix Rat Genome 230 2.0 Microarray and RNA-Seq by NGS was conducted using Illumina Genome Analyzer II platform. The gene expression profiles generated via the two types of transcriptome analysis platforms showed that AA treatment significantly altered expressions of a large number of genes that were related to carcinogenesis. Functional analysis demonstrated that the biological processes related to tumorigenesis and apoptosis were the top dysregulated functional pathways. Comparison of data from the microarray and NGS analyses indicates that alteration of gene expression determined by NGS is comparable to that by microarray in terms of the percentage of overlapping biological functions (over 70%). Our results suggest that analysis of the gene expression profiles can define the carcinogenicity of AA in rat kidneys using microarray or NGS transcriptome analysis. doi:10.1016/j.toxlet.2010.03.607
P203-018 Prediction of carcinogenicity in vitro based on genotoxicity and toxicogenomic data Y. Sheen 1 , E. Yoon 1 , J. Son 1 , C. Keum 2 , K. No 2 1
Ewha Womans University, Republic of Korea, 2 Yonsei University, Republic of Korea This study has been designed to establish the prediction model for chemical carcinogens via the use of genomics technology along with conventional genotoxicity testing, bioinformatics, chemoinformatics, eventually systems toxicology. We have collected genotoxic and carcinogenic toxicity data from FDA, IARC, IPCS, NTP and use them to build toxophore as well as algorithm to select test chemicals. Twenty chemicals, ten for genotoxic carcinogens, and ten for non-genotoxic carcinogens were tested, for the conventional genotoxicity test such as Ames test, in vitro MN assay, COMET assay. Also, we carried out microarray analysis of these twenty chemicals, and did MAS 5.0/RMA analysis along with global and quantile normalization. We have identified five specific gene expression profiles for genotoxic carcinogens and nine specific gene expression profiles for non-genotoxic carcinogens based on each class of chemicals. In addition, we have found out genotoxic carcinogen specific candidate molecular gene, Prkacb, and Caskin/nol3 gene pair. Also, we have identified genotoxic carcinogens specific network markers. It might be possible to hypothesize that carcinogenic genotoxins act with different mechanism from that of non-carcinogenic genotoxins. Thus, with the help of microarray technology, we tried to discriminate the gene expression patterns between the two genotoxin classes with and without carcinogenicity in L5178Y mouse lymphoma cell. Gene expression profiles of cells treated with two genotoxic carcinogens and two genotoxic non-carcinogens for 2 h followed by 22 h of growth were analyzed. 16 genotoxic carcinogen specific and 41 genotoxic non-carcinogen specific differentially expressed genes were extracted using at least twofold change and two sample t-test p-value less than 0.05. The gene sets found in this work might serve as valuable expression signature to characterize the carcinogenic toxicity among genotoxins and might be a feature gene set to classify the gene expression profiles of genotoxic carcinogen from those of genotoxic noncarcinogen. doi:10.1016/j.toxlet.2010.03.608
P203-019 A novel mechanism on modulation of APE1 activity via protein–protein interactions of Gadd45a and PCNA H.L. Kim 1 , S.U. Kim 2 , Y.R. Seo 3 1 Department of Pharmacology, Institute for Basic Medical Science (IBMS), School of Medicine, Kyung Hee University, Republic of Korea, 2 Department of Life Science, School of Interdisciplinary Bioscience and Bioengineering, Republic of Korea, 3 Department of Pharmacology, School of Medicine, Kyung Hee University, Republic of Korea
Base excision repair (BER) is a critical protective mechanism that maintains genomic integrity by correcting DNA base modifications. Our earlier study suggested a potential model of the interaction among Gadd45a, PCNA and APE1. Here, we investigated whether the interaction of Gadd45a and PCNA enhances the APE1 activity in BER pathway through the mutation of predicted PCNA binding site on Gadd45a. We found the suppression of APE1 activity in