Using RNA-Seq with 11 marker genes to evaluate 1,4-dioxane compared with typical genotoxic and non-genotoxic rat hepatocarcinogens

Abstract

It has long been unclear whether 1,4-dioxane (DO) is a genotoxic hepatocarcinogen (GTHC). Therefore, the present study aimed to evaluate rat GTHCs and non-genotoxic hepatocarcinogens (NGTHCs) via selected gene expression patterns in the liver, as determined by next generation sequencing-targeted mRNA sequencing (RNA-Seq) and principal component analysis (PCA). Previously, we selected 11 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) to discriminate GTHCs and NGTHCs. In the present study, we quantified changes in the expression of these genes following DO treatment, and compared them with treatment with two typical rat GTHCs, N-nitrosodiethylamine (DEN) and 3,3'-dimethylbenzidine·2HCl (DMB), and a typical rat NGTHC, di(2-ethylhexyl)phthalate (DEHP). RNA-Seq was conducted on liver samples from groups of five male, 10-week-old F344 rats after 4 weeks' feeding of chemicals in the water or the food. Rats in the control group were given water and a basal diet. Significant changes in gene expression in experimental groups compared with the control group were observed in eight genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Phlda3 and Plk2), as shown by Tukey's test. Gene expression profiles of the 11 genes under DO treatment differed significantly from those with DEN and DMB, as well as DEHP. Gene expression profiles with DO treatment differed partially from those with typical GTHCs for five genes (Bax, Btg2, Cdkn1a, Lrp1 and Plk2) and were substantially different from treatment with a typical NGTHC (DEHP) for nine genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Mbd1, Phlda3 and Tubb4b) as determined by Tukey's test. Finally, PCA successfully differentiated GTHCs from DEHP and DO with the 11 genes. The present results suggest that RNA-Seq and PCA are useful to evaluate rat typical GTHCs and typical NGTHCs. DO was suggested to result in a different intermediate gene expression profile from typical GTHCs and NGTHC.

Keywords: 1,4-dioxane; Genotoxic hepatocarcinogens; Non-genotoxic hepatocarcinogen; PCA; RNA-Seq; Toxicogenomics.