Quinn B A, Crane T L, Kocal T E, Best S J, Cameron R G, Rushmore T H, Farber E, Hayes M A
Department of Pathology, University of Guelph, Ontario, Canada.
Toxicol Appl Pharmacol. 1990 Sep 15;105(3):351-63. doi: 10.1016/0041-008x(90)90139-l.
To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.
为了评估谷胱甘肽S-转移酶（GST）同工酶在诱导肝细胞对黄曲霉毒素B1（AFB1）抗性中的作用，我们比较了来自正常大鼠、暴露于不同多氯联苯（PCB）的大鼠以及致癌物诱导的肝细胞肿瘤大鼠的不同肝细胞溶质制剂和纯化的GST与来自小鼠、虹鳟鱼和人类肝脏的细胞溶质或纯化的GST的DNA保护活性。这些比较是在大鼠肝微粒体激活后进行的[3H]AFB1-DNA结合的体外测定中进行的。来自小鼠肝脏的细胞溶质和S-己基谷胱甘肽亲和纯化的GST制剂始终对AFB1-DNA结合具有很强的保护活性。这种活性的大部分依赖于还原型谷胱甘肽（GSH）的存在，但在小鼠肝细胞溶质中观察到一些不依赖GSH的保护作用，而在纯化的GST制剂中未观察到。我们发现，小鼠肝脏中所有依赖GSH的DNA保护活性通过羟基磷灰石色谱法作为单一的GST同工酶洗脱。来自正常大鼠肝脏、虹鳟鱼肝脏和人类肝脏的细胞溶质和纯化的GST制剂对AFB1特异性DNA的保护活性比在小鼠肝脏制剂中发现的GST低得多。致癌物诱导的肝肿瘤大鼠和用3,3',4,4'-四氯联苯和2,2',4,4',5,5'-六氯联苯诱导的肝脏的细胞溶质对1-氯-2,4-二硝基苯（CDNB）的GST活性比正常大鼠肝脏的细胞溶质高。当比较等量的GST活性单位（CDNB）时，在PCB诱导的和正常大鼠肝脏细胞溶质的DNA保护活性之间几乎没有观察到差异，但来自大鼠肝肿瘤的细胞溶质更具保护作用。纯化的GST-P（7-7），即致癌物诱导的大鼠肝肿瘤中诱导最多的GST同工酶，当以发现对从这些肿瘤中分离的总GST具有保护作用的蛋白质浓度添加时没有保护作用。这些研究表明，小鼠肝脏对AFB1的抗性主要可以由一种组成型GST同工酶（YaYa或4-4）来解释，该同工酶对AFB1的DNA结合代谢物具有相对较高的活性。在人类、大鼠或虹鳟鱼肝脏或PCB诱导的或肿瘤性大鼠肝脏制剂中，对AFB1代谢物具有如此高特异性DNA保护活性的GST同工酶并不明显。
