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

Abstract

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.

Mechanism-based risk assessment strategy for drug-induced cholestasis using the transcriptional benchmark dose derived by toxicogenomics

“…Our use of a combination of PCA and cluster analysis was shown to be useful for this classification. Although the application of PCA alone could also reveal the relationship between substance groups (Watanabe et al, 2012;Furihata et al, 2016), this approach requires expert judgment to achieve accurate classification. On the other hand, use of cluster analysis alone can discriminate substance groups without expert judgmente; however, it often produces less separation of the sub-groups such as was observed for Groups 2 and 3.…”

Short-term in vivo testing to discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens using next-generation RNA sequencing, DNA microarray, and qPCR

“…In the form of collaborative studies of the Toxicogenomics/The Japanese Environmental Mutagen Society •Mammalian Mutagenicity Study Group (JEMS•MMS), Furihata et al conducted research to discriminate GTHCs from NGTHCs and/or NGTNHCs using rodent liver [3,[39][40][41][42][43]. They selected 50 candidate marker genes and Gapdh as a control gene for normalization based on their preliminary results with nine chemicals using an original DNA microarray and Affymetrix GeneChip Mu74AV2.…”

“…He described the new approach of toxicogenomics, in which DNA microarrays are used to profile gene expression in cells exposed to test compounds [1]. Quantitative real-time PCR (qPCR) has been used independently or to confirm DNA microarray results [2,3]. However, RNA-Seq is now an important tool for examining the role of the transcriptome in biological processes [4], which could surpass DNA microarray and qPCR [5,6].…”

Screening Strategies and Methods for Better Off-Target Liability Prediction and Identification of Small-Molecule Pharmaceuticals

“…183 For example, Ingenuity Pathway Analysis software was used to differentiate genotoxic and nongenotoxic hepatocarcinogens and link back to pathways involved with DNA damage response and apoptosis. 184 Additionally, multiple pathway analysis tools were employed to study lipopolysaccharide-induced signaling from both in vivo and in vitro studies. 185…”