The need for an efficient carcinogenicity test prompted this study, in which we used a microarray-based genomics approach, with a short-term in vivo model, in combination with insights from statistical and mechanistic analyses. We performed additional experiments to support the significance of the microarray results. Carcinogens were evaluated based on differences in the mechanisms involved in the response to genotoxic (GTX) carcinogens and non-genotoxic (NGTX) carcinogens. Microarray data were analyzed for 2 time points after treatment with the following 6 carcinogens. The analysis was performed using t-tests to compare the fold changes, and we selected differentially expressed genes (DEGs) and evaluated the reasons for differential expression in terms of cellular pathways and processes. We mapped the DEG-related pathways to analyze cellular processes, and we were able to uncover significant mechanisms that involve critical cellular components, such as CDKN1A (p21) and BAX. In addition, a comparison of the data from two time points showed that the repeated administration model was more effective than a single administration for carcinogen research. The classification analysis of selected DEGs was performed by setting microarray data of 4 carcinogens as test sets; these test sets were evaluated as classifiers. Microarray results were further supported using the Comet and micronucleus assays. It was found that gene expression profiling using microarrays, followed by pathway analysis, was effective in increasing the understanding of the characteristics of different carcinogens, and the efficiency of these methods was exemplified by the short-term (3 day) nature of the animal experiments.
Overall design
The following 10 hepatocarcinogens were used: 2-acetylaminofluorene (53-96-3), diethylnitrosamine (55-18-5), 4-dimethylamino-3'-methyl azobenzene (55-80-1), clofibrate (637-07-0), 1,4-dioxane (123-91-1), DL-ethionine (67-21-0), 1,3-dichloro-2'-propanol (96-23-1), urethane (51-79-6), sodium nitrite (7632-00-0), and methyleugenol (93-15-2). Forty-one data were obtained for the training compounds in this study. We treated rats with one of three compounds or a control for each GTX and NGTX experimental group and measured 2 time points for each group. All experimental procedures were repeated three times. We excluded 1 file that was found to be a failure. Each ‘.CEL’ file obtained as a raw data file was standardized using the RMA (Robust Multi-array Average) algorithm.
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