Alpha-Class Glutathione S-Transferases in Wild Turkeys (Meleagris gallopavo): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B1

Domestic turkeys (Meleagris gallopavo) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B1 (AFB1), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite exo-AFB1-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB1-resistant, possess hepatic GST-mediated AFBO conjugating activity. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (GSTA) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All GSTAs contained single nucleotide polymorphisms (SNPs) in their coding regions: GSTA1.1 (5 SNPs), GSTA1.2 (7), GSTA1.3 (3), GSTA2 (3), GSTA3 (1) and GSTA4 (2). E. coli-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB1-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB1, and may also have the benefit of identifying genetic markers which could be used to enhance AFB1 resistance in modern domestic strains.家养火鸡（Meleagris gallopavo）是已知对真菌毒素黄曲霉毒素B1（aflatoxin B1, AFB1）毒性最易感的动物之一，AFB1是一种强效人类肝癌致癌物，也是玉米中普遍存在的污染物。本研究已证实，这种易感性与肝谷胱甘肽S-转移酶（hepatic glutathione S-transferases, GSTs）无法解毒活性亲电代谢产物外型-AFB1-8,9-环氧化物（exo-AFB1-8,9-epoxide, AFBO）有关。与其家养同类不同，相对抗AFB1的野生火鸡具备肝GST介导的AFBO结合活性。本研究对野生和家养火鸡的肝α类谷胱甘肽S-转移酶（alpha-class GSTs, GSTAs）的分子与功能特性进行了表征，以阐明这两种近缘类群之间的抗性差异机制。我们从野生火鸡（东部亚种和里奥格兰德亚种）、传统品种火鸡（皇家棕榈品种）以及现代家养火鸡（尼古拉斯品系）中扩增得到6个α类GST基因（GSTA）并完成测序，同时测定了异源表达的重组酶的催化活性。通过保守的GST结构域与4个特征基序，确认了这些酶属于α类GST家族。所有GSTA的编码区均存在单核苷酸多态性（single nucleotide polymorphisms, SNPs）：其中GSTA1.1存在5个SNPs，GSTA1.2存在7个，GSTA1.3存在3个，GSTA2存在3个，GSTA3存在1个，GSTA4存在2个。经大肠杆菌（E. coli）异源表达的GSTAs，对谷胱甘肽S-转移酶常用底物1-氯-2,4-二硝基苯（1-chloro-2,4-dinitrobenzene, CDNB）、1,2-二氯-4-硝基苯（1,2-dichloro-4-nitrobenzene, DCNB）、依他尼酸（ethacrynic acid, ECA）以及异丙苯过氧化氢（cumene hydroperoxide, CHP）均表现出不同的催化活性。正如其相对抗性水平所预测的那样，家养火鸡的肝脏中无法检测到GST介导的AFBO解毒活性，而野生火鸡与传统品种火鸡的肝脏则具备这一关键活性，这表明在驯化过程中的集约化育种与选育，导致了AFB1保护性等位基因的丢失。本研究观察到重组火鸡GSTAs可解毒AFBO，但这些酶在肝脏内的天然形式却无法解毒，这提示肝脏内的这些酶存在表达下调、基因沉默或通过一种或多种机制发生了修饰。本研究数据可为阐明AFB1抗性的潜在分子机制提供参考，同时还有助于鉴定可用于增强现代家养火鸡品系AFB1抗性的遗传标记。