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

Chie Furihata,Kumiko Ogawa,Takeshi Toyoda,Xinyue You,Takayoshi Suzuki

doi:10.1186/s41021-020-00152-4

Abstract

IntroductionVarious 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.FindingsIn 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.ConclusionOur results suggest that FFPE RNA-Seq and PCA are useful for evaluating typical rat GTHCs and NGTHCs.

Highlights

Various challenges have been overcome with regard to applying ‘omics technologies for chemical risk assessments
Our results suggest that formalin-fixed paraffin-embedded (FFPE) results detailing targeted mRNA sequencing (RNA-Seq) and principal component analysis (PCA) are useful for evaluating typical rat Genotoxic hepatocarcinogen (GTHC) and Non-genotoxic hepatocarcinogen (NGTHC)
The general aims of the present study were to evaluate GTHC and NGTHC via the analysis of selected gene expression patterns within the liver as analyzed using FFPE RNA-Seq and PCA, to determine the usefulness of FFPE RNA-Seq for this analysis, and to compare the typical GTHC, 2-acetylaminofluorene (AAF)-induced gene expression profile to expression profiles that were induced by p-cresidine (CRE), which is equivocal for genotoxicity and carcinogenic in rat liver

Summary

Introduction

Various challenges have been overcome with regard to applying ‘omics technologies for chemical risk assessments. We published results detailing targeted mRNA sequencing (RNA-Seq) on a generation sequencer using intact RNA derived from freshly frozen rat liver tissues. Targeted mRNA sequencing (RNA-Seq) has become an important tool for examining the role of the transcriptome in biological processes [2]; few studies have examined the feasibility of chemical risk assessment using RNASeq. Previously, we published RNA-Seq data generated from a generation sequencer using intact RNA derived from freshly frozen rat liver tissues [3]. We successfully discriminated genotoxic hepatocarcinogens (GTHCs, N-nitrosodiethylamine and 3,3′-dimethylbennzidine·HCl) from a non-genotoxic hepatocarcinogen [NGTHC, di(2-ethylhexyl)phthalate] and a different intermediate hepatocarcinogen (1,4-dioxane) [3] using 11 selected marker genes that have been described previously [4]. We attempted to use FFPE pathology specimens for RNASeq. Currently, few papers have been published regarding FFPE RNA-Seq (non-targeted) in the rat liver [6]

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Conclusion