Using FFPE RNA-Seq with 12 marker genes to evaluate genotoxic and non-genotoxic rat hepatocarcinogens

INTRODUCTION: Various challenges have been overcome with regard to applying ‘omics technologies for chemical risk assessments. Previously we published results detailing targeted mRNA sequencing (RNA-Seq) on a next generation sequencer using intact RNA derived from freshly frozen rat liver tissues. We successfully discriminated genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) using 11 selected marker genes. Based on this, we next attempted to use formalin-fixed paraffin-embedded (FFPE) pathology specimens for RNA-Seq analyses. FINDINGS: In this study we performed FFPE RNA-Seq to compare a typical GTHC, 2-acetylaminofluorene (AAF) to genotoxicity equivocal p-cresidine (CRE). CRE is used as a synthetic chemical intermediate, and this compound is classified as an IARC 2B carcinogen and is mutagenic in S. typhimurium, which is non-genotoxic to rat livers as assessed by single strand DNA damage analysis. RNA-Seq was used to examine liver FFPE samples obtained from groups of five 10-week-old male F344 rats that were fed with chemicals (AAF: 0.025% and CRE: 1% in food) for 4 weeks or from controls that were fed a basal diet. We extracted RNAs from FFPE samples and RNA-Seq was performed on a MiniSeq (Illumina) using the TruSeq custom RNA panel. AAF induced remarkable differences in the expression of eight genes (Aen, Bax, Btg2, Ccng1, Gdf15, Mbd1, Phlda3 and Tubb4b) from that in the control group, while CRE only induced expression changes in Gdf15, as shown using Tukey’s test. Gene expression profiles for nine genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Mbd1, Phlda3, and Plk2) differed. between samples treated with AAF and CRE. Finally, principal component analysis (PCA) of 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) using our previous Open TG-GATE data plus FFPE-AAF and FFPE-CRE successfully differentiated FFPE-AAF, as GTHC, from FFPE-CRE, as NGHTC. CONCLUSION: Our results suggest that FFPE RNA-Seq and PCA are useful for evaluating typical rat GTHCs and NGTHCs.