Four functional genotoxic marker genes (Bax, Btg2, Ccng1, and Cdkn1a) discriminate genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens and non-genotoxic non-hepatocarcinogens in rat public toxicogenomics data, Open TG-GATEs.

Abstract

Background: Previously, Japanese Environmental Mutagen and Genome Society/Mammalian Mutagenicity Study Group/Toxicogenomics Study Group (JEMS/MMS toxicogenomic study group) proposed 12 genotoxic marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) to discriminate genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) and non-genotoxic non-hepatocarcinogens (NGTNHCs) in mouse and rat liver using qPCR and RNA-Seq and confirmed in public rat toxicogenomics data, Open TG-GATEs, by principal component analysis (PCA). On the other hand, the U.S. Environmental Protection Agency (US EPA) suggested seven genotoxic marker genes (Bax, Btg2, Ccng1, Cgrrf1, Cdkn1a, Mgmt, and Tmem47) with Open TG-GATEs data. Four genes (Bax, Btg2, Ccng1, and Cdkn1a) were common in these two studies. In the present study, we examined the performance of these four genes in Open TG-GATEs data using PCA.

Results: The study's findings are of paramount significance, as these four genes proved to be highly effective in distinguishing five typical GTHCs (2-acetylaminofluorene, aflatoxin B1, 2-nitrofluorene, N-nitrosodiethylamine and N-nitrosomorpholine) from seven typical NGTHCs (clofibrate, ethanol, fenofibrate, gemfibrozil, hexachlorobenzene, phenobarbital, and WY-14643) and 11 NGTNHCs (allyl alcohol, aspirin, caffeine, chlorpheniramine, chlorpropamide, dexamethasone, diazepam, indomethacin, phenylbutazone, theophylline, and tolbutamide) by PCA at 24 h after a single administration with 100% accuracy. These four genes also effectively distinguished two typical GTHCs (2-acetylaminofluorene and N-nitrosodiethylamine) from seven NGTHCs and ten NGTNHCs by PCA on 29 days after 28 days-repeated administrations, with a similar or even better performance compared to the previous 12 genes. Furthermore, the study's analysis revealed that the three intermediate GTHC/NGTHCs (methapyrilene, monocrotaline, and thioacetamide, which were negative in the Salmonella test but positive in the in vivo rat liver test) were located in the intermediate region between typical GTHCs and typical NGTHCs by PCA.

Conclusions: The present results unequivocally demonstrate the availability of four genotoxic marker genes ((Bax, Btg2, Ccng1, and Cdkn1a) and PCA in discriminating GTHCs from NGTHCs and NGTNHCs in Open TG-GATEs. These findings strongly support our recommendation that future rat liver in vivo toxicogenomics tests prioritize these four genotoxic marker genes, as they have proven to be highly effective in discriminating between different types of hepatocarcinogens.