Furihata Chie, Suzuki Takayoshi
Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-Ku, Kawasaki, Kanagawa, 210-9501, Japan.
School of Science and Engineering, Aoyama Gakuin University, Sagamihara, Sagamihara, Kanagawa, 252-5258, Japan.
Genes Environ. 2024 Dec 19;46(1):28. doi: 10.1186/s41021-024-00322-8.
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.
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.
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.
此前，日本环境诱变剂与基因组学会/哺乳动物诱变性研究组/毒理基因组学研究组（JEMS/MMS毒理基因组学研究组）提出了12个基因毒性标记基因（Aen、Bax、Btg2、Ccnf、Ccng1、Cdkn1a、Gdf15、Lrp1、Mbd1、Phlda3、Plk2和Tubb4b），用于通过qPCR和RNA测序区分小鼠和大鼠肝脏中的基因毒性肝癌致癌物（GTHC）与非基因毒性肝癌致癌物（NGTHC）以及非基因毒性非肝癌致癌物（NGTNHC），并通过主成分分析（PCA）在公开的大鼠毒理基因组学数据Open TG-GATEs中得到了证实。另一方面，美国环境保护局（US EPA）利用Open TG-GATEs数据提出了7个基因毒性标记基因（Bax、Btg2、Ccng1、Cgrrf1、Cdkn1a、Mgmt和Tmem47）。这两项研究中有4个基因（Bax、Btg2、Ccng1和Cdkn1a）是相同的。在本研究中，我们利用PCA在Open TG-GATEs数据中检验了这4个基因的性能。
该研究的发现具有至关重要的意义，因为这4个基因在通过PCA区分5种典型的GTHC（2-乙酰氨基芴、黄曲霉毒素B1、2-硝基芴、N-亚硝基二乙胺和N-亚硝基吗啡啉）与7种典型的NGTHC（氯贝丁酯、乙醇、非诺贝特、吉非贝齐、六氯苯、苯巴比妥和WY-14643）以及11种NGTNHC（烯丙醇、阿司匹林、咖啡因、氯苯那敏、氯磺丙脲、地塞米松、地西泮、吲哚美辛、保泰松、茶碱和甲苯磺丁脲）时被证明是非常有效的，在单次给予100%准确剂量后24小时即可实现。在28天重复给药后29天，这4个基因通过PCA也能有效地区分2种典型的GTHC（2-乙酰氨基芴和N-亚硝基二乙胺）与7种NGTHC和10种NGTNHC，其性能与之前的12个基因相似甚至更好。此外，该研究的分析表明，3种中间型GTHC/NGTHC（在沙门氏菌试验中为阴性但在大鼠肝脏体内试验中为阳性的美吡拉敏、野百合碱和硫代乙酰胺）通过PCA位于典型GTHC和典型NGTHC之间的中间区域。
目前的结果明确证明了4个基因毒性标记基因（Bax、Btg2、Ccng1和Cdkn1a）以及PCA在Open TG-GATEs中区分GTHC与NGTHC和NGTNHC的可用性。这些发现有力地支持了我们的建议，即未来大鼠肝脏体内毒理基因组学试验应优先考虑这4个基因毒性标记基因，因为它们已被证明在区分不同类型的肝癌致癌物方面非常有效。
