Studies on the metabolism and toxicity of drugs using mammalian cells expressed in drug metabolism enzymes

药物和天然产物通过各种药物代谢酶代谢和解毒。近年来，基因技术逐步发展，并用于药物代谢和毒性的研究。在这方面，我们尝试将药物代谢酶的基因整合到细胞中以表达其酶功能。本研究所用的药物代谢酶是羧酸酯酶、谷胱甘肽S-转移酶和细胞色素P-450，并已进行了这些酶的cDNA克隆。我们从大鼠、小鼠、猴和人肝脏中提取并纯化了RNA，并合成了每种酶的cDNA。然后，我们使用ramda gt 11、ramda gt 10和ramda gt Zap作为表达载体制备文库。接下来，我们使用针对每种酶的相应抗体作为探针来筛选cDNA文库，并确定每种酶的阳性克隆的DNA序列。作为这些实验的结果，我们获得了上述药物代谢酶的6个cDNA克隆。在这些cDNA中，我们使用哺乳动物细胞表达了酯酶的cDNA。实际上，我们用pCR 3作为表达载体处理了大鼠RL 1和RH 1以及小鼠MH 1的肝酯酶的cDNA，并将这些酯酶的基因整合到COS 7细胞中。作为这些实验的结果，酯酶同工酶的活性表示。我们在这里开发的具有几种药物代谢酶基因的新型哺乳动物细胞对未来的新药开发非常有用。

Drugs and naturally occurring products are metabolized and detoxified by various drug metabolizing enzymes. Recently, gene technology has been progressively developed and these techniques are used for the studies on the metabolism and toxicity of drugs. In this regard, we tried to incorporate the genes of the drug metabolizing enzymes into the cells to express their enzymatic functions. The drug metabolizing enxyme which we used in these studies are carboxylesterases, glutathione S-transferase a dn cytochrome P-450, and we had carried out the cDNA cloning of these enzymes. We had extracted and purified the RNA from rats, mice, monkeys and human livers, and synthesized cDNA of each enzyme. Then, we prepared the libraries using ramda gt 11, ramda gt 10 and ramda gt Zap as the expression vectors. Next, we have used the respective antibodies against each enzyme as the probes to screen the cDNA libraries, and determine the DNA sequences of the positive clones of each enzyme. As the results of these experiments, we had six cDNA clones of the drug metabolizing enzymes mentioned above. Among these cDNA,we have expressed the cDNA of esterases using mammalian cells. In fact, we treated the cDNAs of liver esterases of rat RL1 and RH1 and mouse MH1 with pCR3 as an expression vector, and incorporated the genes of these esterases into COS 7 cells. As the consequence of these experiments, the activities of esterase isozymes were expressed. The novel mammalian cells possessing the genes of several drug metabolizing enzymes we have developed here are extremely useful for new drug development in future.