GLUTATHIONE S TRANSFERASE ACTIVITY TOWARD AFLATOXIN B1

黄曲霉毒素B1 （AFB）是一种真菌肝毒素和致癌物，在世界一些地区经常污染玉米和花生等食品。对afb诱导的肝癌的敏感性存在较大的物种差异。特定形式谷胱甘肽s -转移酶（GST）的表达在决定物种对AFB的敏感性中起重要作用。小鼠对AFB具有抗性，因为它们表达一种α类GST异构体（mGSTA3-3），该异构体对活性中间体AFB-8,9-环氧化物（AFBO）具有高活性。相比之下，大鼠不构成表达具有高AFBO活性的同源GST，因此对afb诱导的肝癌发生敏感。我们发现人类肝脏细胞质没有AFBO解毒活性，但非人灵长类动物Macaca fascicularis （MF）的肝脏具有显著的肝脏AFBO- gst活性。先前的研究利用MF肝cDNA文库鉴定了三种不同的α类gst，但没有一种具有可测量的AFBO活性。为了确定哪种形式的GST负责这种活性，我们使用了一种蛋白质纯化方案（谷胱甘肽琼脂糖亲和纯化，然后进行色谱和聚焦）。具有高AFBO活性的GST片段被分离并鉴定为mu-class GST。利用逆转录酶聚合酶链反应（RT-PCR） cDNA克隆技术鉴定了与该AFBO解毒相关的特定mu类GST。获得了两种不同的mu-class MF GST cdna。其中一个克隆与人类GSTM4同源，没有AFBO活性。我们已经将另一个mu-class cDNA亚克隆到一个表达系统中，该GST的蛋白表达和鉴定正在进行中。我们的研究结果表明，在这种非人灵长类动物中，一种多类GST负责肝细胞质中针对AFBO的GST活性。

Aflatoxin B1 (AFB) is a fungal liver toxin and carcinogen that frequently contaminates foodstuffs such as corn and peanuts in some regions of the world. There are large species differences in sensitivity to AFB-induced liver cancer. Expression of specific forms of glutathione S-transferases (GST) plays an important role in determining species sensitivity to AFB. Mice are resistant to AFB because they express an alpha-class GST isoform (mGSTA3-3) that has high activity toward the reactive intermediate, AFB-8,9-epoxide (AFBO). In contrast, rats do not constitutively express a homologous form of GST with high AFBO activity and are thus sensitive to AFB-induced hepatocarcinogenesis. We have found that human liver cytosol has no AFBO detoxifying activity, but liver from a nonhuman primate species, Macaca fascicularis (MF), has significant hepatic AFBO-GST activity. Previous studies that utilized a MF hepatic cDNA library identified three different alpha class GSTs, but none had measurable AFBO activity. To determine which form of GST is responsible for this activity, we used a protein purification scheme (glutathione agarose affinity purification followed by chromatography and chromatofocusing). A fraction of GST with high AFBO activity was isolated and identified as a mu-class GST. Reverse-transcriptase polymerase-chain-reaction (RT-PCR) cDNA cloning was used to identify the specific mu-class GST related to this AFBO detoxification. Two different mu-class MF GST cDNAs were obtained. One of these clones is homologous to human GSTM4 and has no AFBO activity. We have subcloned the other mu-class cDNA into an expression system, and the protein expression and characterization on this GST is in progress. Our results suggest that a mu-class GST is responsible for the hepatic cytosolic GST activity toward AFBO in this species of nonhuman primate.