[Show abstract][Hide abstract] ABSTRACT: To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: The potential of carbon tetrachloride (CCl4) to induce pre-neoplastic lesions in rat liver using a medium-term liver assay (Ito method) for the prediction of carcinogenicity was examined by nose-only inhalation exposure of male rats (15/group) to CCl4 vapor at concentrations of 0, 1, 5, 25, 125 ppm for 6h/day, 6 day/week, for a period of 6 weeks. The numbers and area of glutathione S-transferase placental (GST-P) positive foci were then determined. Additionally, other histopathological observations on the livers were recorded and serum chemical parameters and CCl4 concentrations in blood were measured. The areas and numbers of GST-P positive foci significantly increased in the CCl4-exposed rats at 25 and 125 ppm; but not at concentrations of 1 and 5 ppm. CCl4 blood concentration 24h after initiation of exposure in the 125 ppm group remained at about 5% of the 6h maximum concentration. These data from CCl4-exposed rats clearly show that inhalation exposure can be used in the rat medium-term liver assay, the method is available for the screening of volatile chemicals and is therefore a useful tool in cancer risk assessment. This is the first report of the use of inhalation exposure in this medium-term predictive assay.
No preview · Article · Mar 2008 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: Toxicogenomics is a promising new tool for prediction of chemical toxicities including carcinogenicity in a relatively short period. However, it is important to develop a reliable animal test protocol for toxicogenomics studies. The preparation of RNA and tissues is also crucial, since it greatly influences outcomes of gene expression analysis. In the present study, we examined an animal test protocol by comparing gene expression data from different conditions and proposed a reliable animal test protocol for toxicogenomic studies. With regard to the preparation of tissues and RNA, here we present evidence that quality of RNA and tissues is well-preserved even after freezer storage for up to 2.5 years. Gene expression levels were compared using a GeneChip System (RGU34A, Affymetrix, Santa Clara, CA, USA) between RNA samples that were freshly prepared, stored at -80 degrees C or re-prepared from tissue kept at -20 degrees C. None showed degradation and no significant differences in expression were evident among the three sets of samples. The data demonstrate that gene expression analysis by DNA microarray is suitable for RNA or tissues that have been stored at an appropriate temperature.
No preview · Article · Mar 2007 · The Journal of Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: Toxicogenomics is a promising new tool for prediction of chemical toxicities including carcinogenicity in a relatively short period. However, it is important to develop a reliable animal test protocol for toxicogenomics studies. The preparation of RNA and tissues is also crucial, since it greatly influences outcomes of gene expression analysis. We proposed an animal test protocol for toxicogenomics studies. In the present study, we examined an animal test protocol by comparing biological and gene expression data from different laboratories running identical in vivo studies on the same microarray platform. The results gave good correspondence in all three laboratories at the level of biological responses and gene expression, especially for genes whose expression changes were quite large. As the fold change or the signal values become smaller, however, discrepancies occur in gene expression data. For example, one laboratory shows an opposite directional change to the other two or no change. The results of hierarchical clustering and principal component analysis (PCA) demonstrated all samples from the three laboratories being clearly divided between control and treatment. Examination of the reproducibility of gene expression data across laboratories using the proposed animal test protocol thus confirmed only minor differences, which was expected to present no problems for gene expression analysis.
No preview · Article · Mar 2007 · The Journal of Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: The long-term rodent bioassay is the standard method to predict the carcinogenic hazard of chemicals for humans. However, this assay is costly, and the results take at least two years to produce. In the present study, we conducted gene expression profiling of cultured cells exposed to carcinogenic chemicals with the aim of providing a basis for rapid and reliable prediction of carcinogenicity using microarray technology. We selected 39 chemicals, including 17 rat hepatocarcinogens and eight compounds demonstrating carcinogenicity in organs other than the liver. The remaining 14 were non-carcinogens. When rat hepatoma cells (MH1C1) were treated with the chemicals for 3 days at a non-toxic dose, analysis of gene expression changes with our in-house microarray allowed a set of genes to be identified differentiating hepatocarcinogens from non-carcinogens, and all carcinogens from non-carcinogens, by statistical methods. Moreover, optimization of the two gene sets for classification with an SVM and LOO-CV resulted in selection of 39 genes. The highest predictivity was achieved with 207 genes for differentiation between non-hepatocarcinogens and non-carcinogens. The overlap between the two selected gene sets encompassed 26 genes. This gene set contained significant genes for prediction of carcinogenicity, with a concordance of 84.6% by LOO-CV SVM. Using nine external samples, correct prediction of carcinogenicity by SVM was 88.9%. These results indicate that short-term bioassay systems for carcinogenicity using gene expression profiling in hepatoma cells have great promise.
[Show abstract][Hide abstract] ABSTRACT: Regulation of cell proliferation and apoptosis plays a key role in tumor development. To elucidate mechanisms underlying hepatocarcinogenesis, patterns of gene expression, including apoptosis-related genes, were compared between glutathione S-transferase placental form (GST-P)-positive preneoplastic foci and surrounding tissue in the rat. Lesions were induced with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg bw) and then 100 ppm 2-acetylaminofluorene (2-AAF)-containing diet combined with two-thirds partial hepatectomy. Frozen sections of the livers were applied for immunohistochemical staining of GST-P, and both positive foci and surrounding negative areas were harvested by laser microdissection. Total RNAs were extracted and amplified with T7 polymerase to allow gene expression analysis by cDNA microarray assays. In the GST-P-positive foci, altered levels were observed for many genes, mostly related to metabolism or catalysis, with increased expression of testosterone-repressive prostate message-2 (TRPM-2), which is reported to act as a protective factor against apoptosis, and decreased expression of thymus-expressed acidic protein (TEAP), which is considered to promote apoptosis. The results indicate that rat liver preneoplastic lesions might be protected against apoptosis and that the approach adopted is useful for clarification of mechanisms underlying hepatocarcinogenesis.
No preview · Article · Apr 2005 · Medical Molecular Morphology
[Show abstract][Hide abstract] ABSTRACT: Glutathione S-transferase placental form (GST-P), one of the glutathione S-transferases family of detoxification enzymes, is a very useful marker of rat liver pre-neoplastic lesions. We here investigated the gene expression profile in GST-P positive foci as compared with surrounding GST-P negative areas in the same liver of rats treated with diethylnitrosamine and then 2-acetylaminofluorene combined with partial hepatectomy. GST-P positive foci were harvested by laser microdissection and total RNAs were extracted to allow gene expression profiles to be assessed by cDNA microarray assays. Transaldolase, rat aflatoxin B1 aldehyde reductase and gamma-glutamylcysteine synthetase were found as up-regulated genes and regucalcin as a down-regulated gene, in line with findings for hepatocellular carcinomas. The results indicate that the approach adopted is useful for understanding mechanisms of hepatocarcinogenesis and identification of new markers for rat liver pre-neoplastic foci.
[Show abstract][Hide abstract] ABSTRACT: We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three-dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six-week-old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N-diethylnitrosamine (DEN), 0.01% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1'-acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip(\xa8) microarray A spotted for 28 genes of drug-metabolizing enzymes in duplicate, and PamChip microarray B spotted for 131 genes which are known to be up- or down-regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three-dimensional microarrays.