Collaborative studies in toxicogenomics in rodent liver in JEMS·MMS; a useful application of principal component analysis on toxicogenomics

Chie Furihata, Takashi Watanabe, Takayoshi Suzuki, Shuichi Hamada, Madoka Nakajima

Toxicogenomics is a rapidly developing discipline focused on the elucidation of the molecular and cellular effects of chemicals on biological systems. As a collaborative study group of Toxicogenomics/JEMS·MMS, we conducted studies on hepatocarcinogens in rodent liver in which 100 candidate marker genes were selected to discriminate genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens. Differential gene expression induced by 13 chemicals were examined using DNA microarray and quantitative real-time PCR (qPCR), including eight genotoxic hepatocarcinogens [o-aminoazotoluene, chrysene, dibenzo[a,l]pyrene, diethylnitrosamine (DEN), 7,12-dimethylbenz[a]anthracene, dimethylnitrosamine, dipropylnitrosamine and ethylnitrosourea (ENU)], four non-genotoxic hepatocarcinogens [carbon tetrachloride, di(2-ethylhexyl)phthalate (DEHP), phenobarbital and trichloroethylene] and a non-genotoxic non-hepatocarcinogen [ethanol]. Using qPCR, 30 key genes were extracted from mouse livers at 4 h and 28 days following dose-dependent gene expression alteration induced by DEN and ENU: the most significant changes in gene expression were observed at 4 h. Next, we selected key point times at 4 and 48 h from changes in time-dependent gene expression during the acute phase following administration of chrysene by qPCR. We successfully showed discrimination of eight genotoxic hepatocarcinogens [2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitsosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane] from four non-genotoxic hepatocarcinogens [1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, DEHP and furan] using qPCR and principal component analysis. Additionally, we successfully identified two rat genotoxic hepatocarcinogens [DEN and 2,6-dinitrotoluene] from a nongenotoxic-hepatocarcinogen [DEHP] and a non-genotoxic non-hepatocarcinogen [phenacetin] at 4 and 48 h. The subsequent gene pathway analysis by Ingenuity Pathway Analysis extracted the DNA damage response, resulting from the signal transduction of a p53-class mediator leading to the induction of apoptosis. The present review of these studies suggests that application of principal component analysis on the gene expression profile in rodent liver during the acute phase is useful to predict genotoxic hepatocarcinogens in comparison to non-genotoxic hepatocarcinogens and/or non-carcinogenic hepatotoxins.