Taguchi Keiko, Takaku Misaki, Egner Patricia A, Morita Masanobu, Kaneko Takehito, Mashimo Tomoji, Kensler Thomas W, Yamamoto Masayuki
*Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba, Sendai 980-8575, Japan;
Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205;
Toxicol Sci. 2016 Jul;152(1):40-52. doi: 10.1093/toxsci/kfw065. Epub 2016 Apr 12.
THE TRANSCRIPTION FACTOR NRF2: (NF-E2-related-factor 2) REGULATES A BATTERY OF ANTIOXIDATIVE STRESS-RESPONSE GENES AND DETOXICATION GENES, AND NRF2 KNOCKOUT LINES OF MICE HAVE BEEN CONTRIBUTING CRITICALLY TO THE CLARIFICATION OF ROLES THAT NRF2 PLAYS FOR CELL PROTECTION HOWEVER, THERE ARE APPARENT LIMITATIONS IN USE OF THE MOUSE MODELS FOR INSTANCE, RATS EXHIBIT MORE SUITABLE FEATURES FOR TOXICOLOGICAL OR PHYSIOLOGICAL EXAMINATIONS THAN MICE IN THIS STUDY, WE GENERATED 2 LINES OF NRF2 KNOCKOUT RATS BY USING A GENOME EDITING TECHNOLOGY; 1 LINE HARBORS A 7-BP DELETION Δ7 AND THE OTHER LINE HARBORS A 1-BP INSERTION +1 IN THE NRF2 GENE IN THE LIVERS OF RATS HOMOZYGOUSLY DELETING THE NRF2 GENE, AN ACTIVATOR OF NRF2 SIGNALING, CDDO-IM, COULD NOT INDUCE EXPRESSION OF REPRESENTATIVE NRF2 TARGET GENES TO EXAMINE ALTERED TOXICOLOGICAL RESPONSE, WE TREATED THE NRF2 KNOCKOUT RATS WITH AFLATOXIN B1 AFB1, A CARCINOGENIC MYCOTOXIN THAT ELICITS GENE MUTATIONS THROUGH BINDING OF ITS METABOLITES TO DNA AND FOR WHICH THE RAT HAS BEEN PROPOSED AS A REASONABLE SURROGATE FOR HUMAN TOXICITY INDEED, IN THE NRF2 KNOCKOUT RAT LIVERS THE ENZYMES OF THE AFB1 DETOXICATION PATHWAY WERE SIGNIFICANTLY DOWNREGULATED SINGLE DOSE ADMINISTRATION OF AFB1 INCREASED HEPATOTOXICITY AND BINDING OF AFB1-N7-GUANINE TO HEPATIC DNA IN NRF2 KNOCKOUT RATS COMPARED WITH WILD-TYPE NRF2 KNOCKOUT RATS REPEATEDLY TREATED WITH AFB1 WERE PRONE TO LETHALITY AND CDDO-IM WAS NO LONGER PROTECTIVE THESE RESULTS DEMONSTRATE THAT NRF2 KNOCKOUT RATS ARE QUITE SENSITIVE TO AFB1 TOXICITIES AND THIS RAT GENOTYPE EMERGES AS A NEW MODEL ANIMAL IN TOXICOLOGY.
转录因子NRF2（NF-E2相关因子2）调控一系列抗氧化应激反应基因和解毒基因，NRF2基因敲除小鼠品系对阐明NRF2在细胞保护中所起的作用至关重要。然而，小鼠模型的应用存在明显局限性。例如，在毒理学或生理学研究方面，大鼠比小鼠展现出更合适的特性。在本研究中，我们利用基因组编辑技术构建了2个NRF2基因敲除大鼠品系；一个品系在NRF2基因中有一个7个碱基对的缺失（Δ7），另一个品系在NRF2基因中有一个1个碱基对的插入（+1）。在纯合缺失NRF2基因的大鼠肝脏中，NRF2信号通路的激活剂CDDO-IM不能诱导代表性NRF2靶基因的表达。为了检测毒理学反应的改变，我们用黄曲霉毒素B1（AFB1）处理NRF2基因敲除大鼠。AFB1是一种致癌霉菌毒素，其代谢产物与DNA结合会引发基因突变，并且大鼠被认为是人类毒性的合理替代模型。事实上，在NRF2基因敲除大鼠肝脏中，AFB1解毒途径的酶显著下调。与野生型相比，单次给予AFB1会增加NRF2基因敲除大鼠的肝毒性以及AFB1-N7-鸟嘌呤与肝DNA的结合。反复给予AFB1的NRF2基因敲除大鼠易发生致死性，且CDDO-IM不再具有保护作用。这些结果表明，NRF2基因敲除大鼠对AFB1毒性非常敏感，并且该大鼠基因型成为毒理学中的一种新型模式动物。
