Showa Pharmaceutical University.
J Toxicol Sci. 2021;46(8):371-378. doi: 10.2131/jts.46.371.
Dietary-derived coumarin is of clinical interest for its potential hepatotoxicity in humans because such toxicity is especially evident in rats. In this study, the oxidative metabolism of coumarin to active coumarin 3,4-epoxide (as judged by the formation rates of o-hydroxyphenylacetic acid) and excretable 7-hydroxycoumarin was investigated in liver fractions from rats and humans. In rat liver microsomes, the formation rate of o-hydroxyphenylacetic acid (6 pmol/min/mg microsomal protein) from coumarin at 10 μM was dependent on the presence of liver cytosolic fractions. Rat hepatocytes mediated similar formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin (0.1 nmol/hr/10 cells) at 0.20-20 μM coumarin. Human hepatocytes mediated the biotransformation of coumarin to o-hydroxyphenylacetic acid at roughly similar rates to those of rat hepatocytes. In contrast, the formation rates of 7-hydroxycoumarin by human hepatocytes were around 10-fold higher at 1 nmol/hr/10 cells. In the presence of human liver cytosolic fractions, the oxidative formation rate of o-hydroxyphenylacetic acid was relatively high in cytochrome P450 (P450) 1A2-rich human liver microsomes. The inhibitory effects of furafylline/α-naphthoflavone and 8-methoxypsoralen, P450 1A2 and 2A6 inhibitors, respectively, were seen on the rates of o-hydroxyphenylacetic and 7-hydroxylation formations, respectively, in pooled human liver microsomes. Human liver microsomes selectively inactivated for P450 1A2 and 2A6 showed low rates of o-hydroxyphenylacetic acid and 7-hydroxylation formation (20-30% of control), respectively. Among the P450 isoforms tested, recombinant human P450 1A2 predominantly mediated o-hydroxyphenylacetic formation. These results suggested that the metabolic activation and deactivation of coumarin were mediated mainly by P450 1A2 and 2A6 enzymes, respectively. The metabolic oxidation of coumarin via 3,4-epoxidation forming o-hydroxyphenylacetic acid could inform individual human risk assessments of dietary-derived coumarin, for which hepatotoxicity is especially evident in rats.
膳食衍生香豆素因其在人类中的潜在肝毒性而具有临床意义，因为这种毒性在大鼠中尤为明显。在这项研究中，研究了香豆素在大鼠和人体肝匀浆中的氧化代谢，以生成活性香豆素 3,4-环氧化物（通过形成邻羟苯乙酸的速率来判断）和可排泄的 7-羟基香豆素。在大鼠肝微粒体中，10μM 香豆素生成邻羟苯乙酸的速率（6 pmol/min/mg 微粒体蛋白）依赖于肝胞质部分的存在。大鼠肝细胞介导类似的邻羟苯乙酸和 7-羟基香豆素形成速率（0.1 nmol/hr/10 个细胞），香豆素浓度为 0.20-20μM。人肝细胞介导香豆素向邻羟苯乙酸的生物转化，其速率与人肝细胞大致相似。相比之下，人肝细胞生成 7-羟基香豆素的速率约高 10 倍，为1 nmol/hr/10 个细胞。在存在人肝胞质部分的情况下，富含细胞色素 P450（P450）1A2 的人肝微粒体中氧化生成邻羟苯乙酸的速率相对较高。呋喃西林/α-萘黄酮和 8-甲氧基补骨脂素，分别为 P450 1A2 和 2A6 抑制剂，对人肝微粒体中邻羟苯乙酸和 7-羟化形成速率的抑制作用分别可见，在人肝微粒体的混合物中。选择性失活 P450 1A2 和 2A6 的人肝微粒体显示邻羟苯乙酸和 7-羟化形成速率较低（对照的 20-30%）。在所测试的 P450 同工酶中，重组人 P450 1A2 主要介导邻羟苯乙酸的形成。这些结果表明，香豆素的代谢激活和失活主要分别由 P450 1A2 和 2A6 酶介导。香豆素通过 3,4-环氧化形成邻羟苯乙酸的代谢氧化可能为个体的膳食衍生香豆素风险评估提供信息，因为这种毒性在大鼠中尤为明显。
