Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity
Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity
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DOI：
10.1093/toxsci/kfh162
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发表时间：
2004-08-01
影响因子：
3.8
通讯作者：
Lehman-McKeeman, LD
中科院分区：
文献类型：
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作者：
Vassallo, JD;Hicks, SM;Lehman-McKeeman, LD
香豆素的肝毒性归因于对环氧化合物中间体香豆素3,4-环氧化合物（CE）的代谢激活。然而，尽管大鼠对香豆素诱导的肝毒性最敏感，但CE的形成在小鼠肝微粒体中最大，而小鼠肝微粒体几乎没有肝毒性的证据。因此，本研究旨在验证CE的解毒是香豆素肝毒性的主要决定因素这一假设。CE可以自发重排成邻羟基苯乙醛（o-HPA）或与谷胱甘肽（GSH）偶联。o-HPA具有肝毒性，可通过氧化生成o-羟基苯基乙酸（o-HPAA）进一步解毒。体外实验利用小鼠肝微粒体生成一定量的CE，并利用F344大鼠、B6C3F1小鼠和人肝脏的细胞质来表征CE的解毒作用。所有代谢产物均采用高效液相色谱法和紫外检测法进行定量分析。在大鼠和小鼠中，谷胱甘肽结合是通过谷胱甘肽- s转移酶（GSTs）进行的，并且大鼠和小鼠中谷胱甘肽结合的动力学相似。在大鼠肝细胞质中，o-HPA氧化为o-HPAA的特征是高亲和力K-m约为12 muM， V-max约为1.5 nmol/min/mg蛋白。相比之下，小鼠肝细胞质中o-HPAA氧化的K-m和V-max分别约为1.7 muM和5 nmol/min/mg蛋白，通过氧化产生的o-HPAA的总内在清除率在小鼠中比在大鼠中高20倍。人细胞质（两个单独的池）通过o-HPA氧化解毒CE，表观K-m为0.84 muM， V-max为5.7 nmol/min/mg蛋白，净内在清除率比大鼠高50倍以上。所有物种也将o-HPA还原为o-羟基苯乙醇（o-HPE），但这只是大鼠的主要反应。在充满所有必要辅因子的代谢反应中，谷胱甘肽偶联物占大鼠和小鼠中所有CE代谢物的近一半，而谷胱甘肽偶联物仅占人细胞质中代谢物的10%。在小鼠中，o-HPAA是主要的开环代谢物，占剩余代谢物的50%，在人细胞质中，o-HPAA是主要的代谢物，占所有CE代谢物的近90%。相比之下，在大鼠中未检测到o-HPAA，而o-HPE是主要代谢物。总的来说，这些体外数据表明，通过氧化生成o-HPAA的o-HPA解毒是香豆素诱导肝毒性的物种差异的主要决定因素。
Hepatotoxicity of coumarin is attributed to metabolic activation to an epoxide intermediate, coumarin 3,4-epoxide (CE). However, whereas rats are most susceptible to coumarin-induced hepatotoxicity, formation of CE is greatest in mouse liver microsomes, a species showing little evidence of hepatotoxicity. Therefore, the present work was designed to test the hypothesis that detoxification of CE is a major determinant of coumarin hepatotoxicity. CE can either rearrange spontaneously to o-hydroxyphenylacetaldehyde (o-HPA) or be conjugated with gluatathione (GSH). o-HPA is hepatotoxic and is further detoxified by oxidation to o-hydroxyphenylacetic acid (o-HPAA). In vitro experiments were conducted using mouse liver microsomes to generate a constant amount of CE, and cytosols from F344 rats, B6C3F1 mice, and human liver were used to characterize CE detoxification. All metabolites were quantified by HPLC methods with UV detection. In rats and mice, GSH conjugation occurred non-enzymatically and through glutathione-S-transferases (GSTs), and the kinetics of GSH conjugation were similar in rats and mice. In rat liver cytosol, oxidation of o-HPA to o-HPAA was characterized with a high affinity K-m of approximately 12 muM, and a V-max of approximately 1.5 nmol/min/mg protein. In contrast, the K-m and V-max for o-HPA oxidation in mouse liver cytosol were approximately 1.7 muM and 5 nmol/min/mg protein, respectively, yielding a total intrinsic clearance through oxidation to o-HPAA that was 20 times higher in mouse than in rats. Human cytosols (two separate pools) detoxified CE through o-HPA oxidation with an apparent K-m of 0.84 muM and a V-max of 5.7 nmol/min/mg protein, for a net intrinsic clearance that was more than 50 times higher than the rat. All species also reduced o-HPA to o-hydroxyphenylethanol (o-HPE), but this was only a major reaction in rats. In the presence of a metabolic reaction replete with all necessary cofactors, GSH conjugation accounted for nearly half of all CE metabolites in rat and mouse, whereas the GSH conjugate represented only 10% of the metabolites in human cytosol. In mouse, o-HPAA represented the major ring-opened metabolite, accounting for the remaining 50% of metabolites, and in human cytosol, o-HPAA was the major metabolite, representing nearly 90% of all CE metabolites. In contrast, no o-HPAA was detected in rats, whereas o-HPE represented a major metabolite. Collectively, these in vitro data implicate o-HPA detoxification through oxidation to o-HPAA as the major determinant of species differences in coumarin-induced hepatotoxicity.