White I N, De Matteis F, Gibbs A H, Lim C K, Wolf C R, Henderson C, Smith L L
MRC Toxicology Unit, University of Leicester, U.K.
Biochem Pharmacol. 1995 Apr 18;49(8):1035-42. doi: 10.1016/0006-2952(95)98498-x.
Species differences in the NADPH-dependent covalent binding of [14C]tamoxifen to liver microsomes have been studied using preparations from humans, female F344 rats and DBA/2 mice. Protein binding has been used as an index of metabolic activation and as a surrogate for DNA binding in order to establish which forms of cytochrome P450 are responsible for genotoxicity. A panel of 12 human liver microsomes has been characterized and immunoquantified for nine cytochrome P450 isoenzymes. Binding of tamoxifen (45 microM) (25 +/- 2.5 pmol/15 min/mg protein, mean +/- SE) correlated (P < 0.05) with CYP3A4 and CYP2B6 content. Covalent binding of [14C]tamoxifen to microsomal preparations from human breast tumour tissue could also be detected but at levels 7-fold lower than in liver. The covalent binding of tamoxifen to mice, rat or human liver microsomal preparations increased with increasing substrate concentration. Covalent binding of [14C]tamoxifen (45 microM) in rats was 3.8-fold and mice 17-fold higher than in human liver microsomal preparations. In mice, the apparent Km (9.6 +/- 1.9 microM) was very much lower than for rats (119 +/- 41 microM). Pretreatment of female rats with phenobarbitone or dexamethasone resulted in a 4- to 5-fold increase in [14C]tamoxifen binding, relative to controls, consistent with the involvement of CYP2B1 and CYP3A1 in the metabolic activation. It cannot be distinguished at present if the same reactive metabolites are involved in protein and DNA binding. The greater potential of mouse liver microsomes to activate tamoxifen, relative to rats, does not reflect DNA damage or hepatocarcinogenicity seen following dosing with tamoxifen in vivo. It is concluded that covalent binding of tamoxifen to protein in vitro cannot be directly related to the carcinogenic potential of this compound. However, in the three species investigated, results suggest that the rat is a better model than the mouse for human liver microsomal activation of tamoxifen both with respect to kinetic parameters and the pattern of metabolic products.
利用来自人类、雌性F344大鼠和DBA/2小鼠的肝脏微粒体制剂，研究了[14C]他莫昔芬与肝脏微粒体的NADPH依赖性共价结合的种属差异。蛋白质结合已被用作代谢活化的指标和DNA结合的替代指标，以确定哪些细胞色素P450形式与遗传毒性有关。对一组12个人类肝脏微粒体进行了表征，并对9种细胞色素P450同工酶进行了免疫定量。他莫昔芬（45微摩尔）的结合（25±2.5皮摩尔/15分钟/毫克蛋白质，平均值±标准误）与CYP3A4和CYP2B6含量相关（P<0.05）。也可以检测到[14C]他莫昔芬与人乳腺肿瘤组织微粒体制剂的共价结合，但水平比肝脏低7倍。他莫昔芬与小鼠、大鼠或人类肝脏微粒体制剂的共价结合随底物浓度增加而增加。[14C]他莫昔芬（45微摩尔）在大鼠中的共价结合比人类肝脏微粒体制剂高3.8倍，在小鼠中高17倍。在小鼠中，表观Km（9.6±1.9微摩尔）远低于大鼠（119±41微摩尔）。用苯巴比妥或地塞米松预处理雌性大鼠导致[14C]他莫昔芬结合相对于对照组增加4至5倍，这与CYP2B1和CYP3A1参与代谢活化一致。目前尚无法区分蛋白质和DNA结合是否涉及相同的反应性代谢产物。相对于大鼠，小鼠肝脏微粒体激活他莫昔芬的潜力更大，但这并未反映在体内给予他莫昔芬后所观察到的DNA损伤或肝癌发生情况。结论是，他莫昔芬在体外与蛋白质的共价结合不能直接与该化合物的致癌潜力相关。然而，在所研究的三个物种中，结果表明，就动力学参数和代谢产物模式而言，大鼠比小鼠更适合作为人类肝脏微粒体激活他莫昔芬的模型。
