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

Abstract

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.

Keywords: 2-Acetylaminofluorene; FFPE RNA-Seq; Genotoxic; Non-genotoxic; PCA; Rat hepatocarcinogen; p-Cresidine.

Fig. 1

Changes in gene expression in 12 marker genes in the AAF and CRE groups. The mean of each control group was calculated as 0 (log₂) and ratio (exp/cont) log₂ was presented. The results of individual rats of control group (C), AAF and CRE were presented. The significance was analyzed using the Tukey test; *, P < 0.05; **, P < 0.01(each experimental group against the control group). The Tukey test: #, P < 0.05, ##, P < 0.01 (AAF against CRE)

Fig. 2

Discrimination of FFPE-AAF from FFPE-CRE together with previous rat GTHCs, NGTHCs and NGTNHCs calculated from public Open TG-GATEs data [4] using PCA. FFPE data show individual results and TG-GATEs data show mean of three rats in each point. Red: FFPE-AAF, brown: AAF at 24 h from Open TG-GATEs, light brown: AAF on 29 days from Open TG-GATEs, black: GTHCs from Open TG-GATEs. Yellow: FFPE-CRE, blue: NGTHCs from Open TG-GATEs, light blue: NGTNHCs from Open TG-GATEs. Two points of FFPE-CRE (− 0.042/− 3.26 and − 0.08/− 3.26) overlapped in Fig. 2. PCA was conducted on data of the ratio (exp/cont) as in Additional file 4 (FFPE-AAF and FFPE-CRE together with previously calculated TG-GATEs data [4]). PC1 and PC2 of the results are shown in Additional file 5. Five typical GTHCs (AAF, AFL, DEN, 2NF and NNM at 24 h and AAF and DEN on 29 days in Open TG-GATEs data) were clearly separated from the seven typical NGTHCs (CLO, ETH, FEN, GEM, HEX, PHE and WY at 24 h and 29 days in TG-GATEs data) and eleven NGTNHCs (AA, ASP, CAF, CPA, CPP, DEX, DIA, IND, PBZ, THE and TOL at 24 h and 29 days in Open TG-GATEs data) using PCA. Two groups of GTHCs and (NGTHCs and NGTNHCs) were separated using PC1 (− 0.637 for DEN24L against − 0.159 for FEN24M. Dashed line is border line of the two groups. FFPE-AAF in GTHCs group was clearly separated from FFPE-CRE grouped in NGTHCs

Fig. 3

Calculated approximate PC1 of FFPE-AAF and FFPE-CRE with previous rat GTHCs, NGTHCs and NGTNHCs calculated from public Open TG-GATEs data [4] in Additional file 6. Border line between GTHCs and (NGTHCs + NGTNHCs) is − 0.710. Red: FFPE-AAF, black: GTHCs from Open TG-GTAEs [4], yellow: FFPE-CRE (two points of − 0.273 and − 0.304 overlapped in Fig. 3) and blue: NGTHCs and NGTNHCs from Open TG-GTAEs [4]