DICLOFENAC COVALENT PROTEIN-BINDING IS DEPENDENT ON ACYL GLUCURONIDE FORMATION AND IS INVERSELY RELATED TO P450-MEDIATED ACUTE CELL INJURY IN CULTURED RAT HEPATOCYTES
DICLOFENAC COVALENT PROTEIN-BINDING IS DEPENDENT ON ACYL GLUCURONIDE FORMATION AND IS INVERSELY RELATED TO P450-MEDIATED ACUTE CELL INJURY IN CULTURED RAT HEPATOCYTES
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DOI：
10.1006/taap.1993.1097
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发表时间：
1993-05-01
影响因子：
3.8
通讯作者：
BOELSTERLI, UA
中科院分区：
文献类型：
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作者：
KRETZROMMEL, A;BOELSTERLI, UA
少数患者双氯芬酸可使血清转氨酶活性轻度升高，少数病例可能与暴发性肝坏死的发生有关。双氯芬酸代谢物的直接毒性作用和超敏反应被认为可能是肝损伤的分子机制。我们研究了双氯芬酸在短期培养的大鼠肝细胞中的生物活化途径和细胞毒性，双氯芬酸经历芳香羟基化和酰基葡萄糖醛酸化。双氯芬酸（>500 μM）孵育4小时后LDH首次明显释放。此外，双氯芬酸[14C]与肝细胞蛋白发生了时间和浓度依赖的共价结合，表明存在一种活性中间体。为了明确探讨酰基葡萄糖醛酸化途径在诱导细胞毒性和共价药物-蛋白加合物中的作用，我们使用了两种UDPGT -葡萄糖醛酸转移酶抑制剂，冰片和7,7,7- n-苯基庚基- udp。在UDPGT抑制剂存在的情况下，LDH释放明显增加。另外，当葡糖苷形成被选择性阻断时，与肝细胞蛋白的共价结合大大减少。此外，CYP2C亚家族选择性抑制剂磺胺苯唑或西咪替丁对p450依赖性氧化生物转化的体外抑制显著降低了细胞毒性程度，而共价加合物的形成程度保持不变。同样，用苯巴比妥（80 mg/kg/天，连续4天）预处理大鼠可延迟发作，降低双氯芬酸诱导的LDH释放程度。总之，这些结果表明，肝细胞中由P4502C催化的有毒双氯芬酸代谢物的形成导致急性致死性细胞损伤，而双氯芬酸酰基葡萄糖醛酸盐的形成与活性代谢物与肝细胞蛋白的共价结合有关，而与急性细胞毒性无关。然而，蛋白加合物的形成及其UDPGT的调节可能与双氯芬酸肝炎的表达具有毒理学相关性。
In a few patients diclofenac produces mild increases in serum aminotransferase activity and in rare cases may be associated with the occurrence of fulminant hepatic necrosis. Both direct toxic effects of a diclofenac metabolite and hypersensitivity reactions have been suggested as possible molecular mechanisms of liver injury. We investigated the pathways of bioactivation and cytotoxicity of diclofenac, which undergoes both aromatic hydroxylation and acyl glucuronidation, in short-term cultured rat hepatocytes. LDH release was first evident after 4 hr of incubation with diclofenac (>500 μM). In addition, time- and concentration-dependent covalent binding of [14C]diclofenac to hepatocellular proteins occurred, indicating the presence of a reactive intermediate. To specifically explore the role of the acyl glucuronidation pathway in the induction of cytotoxicity and covalent drug-protein adducts, we used two inhibitors of the UDP-glucuronosyltransferase (UDPGT), borneol and 7,7,7-tn-phenylheptyl-UDP. LDH release was markedly increased in the presence of either UDPGT inhibitor. Alternatively, covalent binding to hepatocellular proteins was greatly reduced when the glucuronide formation was selectively blocked. Furthermore, in vitro inhibition of P450-dependent oxidative biotransformation with the selective inhibitor of the CYP2C subfamily sulfaphenazole or with cimetidine markedly reduced the extent of cytotoxicity, whereas the degree of covalent adduct formation remained unchanged. Similarly, pretreatment of the rats with phenobarbital (80 mg/kg/day for 4 days) delayed the onset and reduced the extent of diclofenac-induced LDH release. Collectively, these results indicate that the formation of a toxic diclofenac metabolite(s) catalyzed by P4502C in hepatocytes leads to acute lethal cell injury, whereas diclofenac acyl glucuronide formation is associated with covalent binding of a reactive metabolite to hepatocellular proteins that is not related to the acute cytotoxicity. The protein adduct formation and its modulation by UDPGT may, however, be toxicologically relevant for the expression of diclofenac hepatitis.