Deng Jiang, Peng Zhe, Xia Zhiyuan, Mo Yixin, Guo Lijia, Wei Jintao, Sun Lvhui, Liu Meng
State Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Frontiers Science Center for Animal Breeding and Sustainable Production, Key Laboratory of Smart Farming Technology for Agricultural Animals of Ministry of Agriculture and Rural Affairs, College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
Hebei Panshuo Biotechnology Co., Ltd., Baoding, Hebei, 071500, China.
J Anim Sci Biotechnol. 2025 Apr 8;16(1):54. doi: 10.1186/s40104-025-01189-7.
AFB-8,9-exo-epoxide (AFBO) is the highly toxic product of Aflatoxin B (AFB). Glutathione S-transferases (GSTs) play pivotal roles in detoxifying AFB by catalyzing the conjugation of AFBO with glutathione (GSH). Although there are over 20 GST isozymes that have been identified in chicken, GST isozymes involved in the detoxification process of AFB have not been identified yet. The objective of this study was to determine which GST isozymes played key role in detoxification of AFB.
A total of 17 pcDNA3.1(+)-GST isozyme plasmids were constructed and the GST isozyme genes were overexpressed by 80-2,500,000 folds in the chicken Leghorn male hepatoma (LMH) cells. Compared to the AFB treatment, overexpression of GSTA2X, GSTA3, GSTT1L, GSTZ1-1, and GSTZ1-2 increased the cell viability by 6.5%-17.0% in LMH cells. Moreover, overexpression of five GST isozymes reduced the release of lactate dehydrogenase and reactive oxygen species by 8.8%-64.4%, and 57.2%-77.6%, respectively, as well as enhanced the production AFBO-GSH by 15.8%-19.6%, thus mitigating DNA damage induced by AFB. After comprehensive evaluation of various indicators, GSTA2X displayed the best detoxification effects against AFB. GSTA2X was expressed in Pichia pastoris X-33 and its enzymatic properties for catalyzing the conjugation of AFBO with GSH showed that the optimum temperature and pH were 20-25 °C and 7.6-8.6 as well as the enzymatic kinetic parameter V was 0.23 nmol/min/mg and the Michaelis constant was 86.05 μmol/L with the AFB as substrate.
In conclusion, GSTA2X, GSTA3, GSTT1L, GSTZ1-1, and GSTZ1-2 played key roles in AFB detoxification, which will provide new remediation strategies to prevent aflatoxicosis in chickens.
黄曲霉毒素B（AFB）的高毒性产物为AFB-8,9-外环氧物（AFBO）。谷胱甘肽S-转移酶（GSTs）通过催化AFBO与谷胱甘肽（GSH）结合，在AFB解毒过程中发挥关键作用。尽管在鸡体内已鉴定出20多种GST同工酶，但参与AFB解毒过程的GST同工酶尚未明确。本研究的目的是确定哪些GST同工酶在AFB解毒中起关键作用。
共构建了17个pcDNA3.1(+)-GST同工酶质粒，GST同工酶基因在鸡白来航雄性肝癌（LMH）细胞中过表达80 - 2500000倍。与AFB处理组相比，GSTA2X、GSTA3、GSTT1L、GSTZ1-1和GSTZ1-2的过表达使LMH细胞的活力提高了6.5% - 17.0%。此外，5种GST同工酶的过表达分别使乳酸脱氢酶和活性氧的释放量降低了8.8% - 64.4%和57.2% - 77.6%，同时使AFBO-GSH的生成量提高了15.8% - 19.6%，从而减轻了AFB诱导的DNA损伤。综合评估各项指标后，GSTA2X对AFB的解毒效果最佳。GSTA2X在毕赤酵母X-33中表达，其催化AFBO与GSH结合的酶学性质表明，以AFB为底物时，最适温度和pH分别为20 - 25℃和7.6 - 8.6，酶动力学参数V为0.23 nmol/min/mg，米氏常数为86.05 μmol/L。
总之，GSTA2X、GSTA3、GSTT1L、GSTZ1-1和GSTZ1-2在AFB解毒中起关键作用，这将为预防鸡的黄曲霉毒素中毒提供新的补救策略。
