Friedberg T, Becker R, Oesch F, Glatt H
Institute of Toxicology, University of Mainz, Germany.
Carcinogenesis. 1994 Feb;15(2):171-5. doi: 10.1093/carcin/15.2.171.
In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from the cell line BHK21-mEH/Mz1 had a specific mEH activity of 123 pmol/min/mg protein, as determined with benzo[a]pyrene 4,5-oxide (B[a]P 4,5-oxide), which was 60 times higher than the activity in the parental cell line and eight times lower than the activity found in rat hepatocytes. However, BHK21-mEH/Mz1 cell homogenates were found to catalyze the conjugation of B[a]P 4,5-oxide to glutathione extremely well. The ratio of the GST enzyme activity to the mEH enzyme activity towards this substrate was 23 in the BHK21-mEH/Mz1 cell line. For hepatocytes this ratio was only six. Despite their already high potential to inactivate B[a]P 4,5-oxide by conjugation to glutathione, BHK21-mEH/Mz1 cells were better protected against the toxic and mutagenic effects of B[a]P 4,5-oxide than the parental cell line due to the expression of the mEH. The mEH, however, failed to protect the cells from the toxic and mutagenic effects of the bay region epoxide anti-7-methylbenz[a]anthracene-3,4-diol 1,2-oxide.
为了研究微粒体环氧化物水解酶（mEH）在存在高内源性谷胱甘肽S-转移酶（GST）活性（这是在多个器官中发现的一种情况）时对芳烃氧化物解毒作用中的角色，我们在叙利亚仓鼠BHK21细胞中表达了大鼠mEH cDNA。这些细胞具有高GST活性，但内源性mEH酶活性极低。通过免疫印迹法确定，我们获得了几个异源表达mEH的细胞克隆。细胞克隆BHK21-mEH/Mz1的mEH蛋白水平最高。免疫荧光显示，整个细胞群体中的表达水平几乎是均匀的。从细胞系BHK21-mEH/Mz1中分离的总蛋白，用苯并[a]芘4,5-氧化物（B[a]P 4,5-氧化物）测定，其特异性mEH活性为123 pmol/分钟/毫克蛋白，这比亲代细胞系中的活性高60倍，比大鼠肝细胞中的活性低8倍。然而，发现BHK21-mEH/Mz1细胞匀浆能很好地催化B[a]P 4,5-氧化物与谷胱甘肽的结合。在BHK21-mEH/Mz1细胞系中，针对该底物的GST酶活性与mEH酶活性之比为23。对于肝细胞，该比例仅为6。尽管BHK21-mEH/Mz1细胞通过与谷胱甘肽结合使B[a]P 4,5-氧化物失活的潜力已经很高，但由于mEH的表达，它们比亲代细胞系更能抵御B[a]P 4,5-氧化物的毒性和诱变作用。然而，mEH未能保护细胞免受湾区环氧化物反式-7-甲基苯并[a]蒽-3,4-二醇1,2-氧化物的毒性和诱变作用。
