Spink D C, Zhang F, Hussain M M, Katz B H, Liu X, Hilker D R, Bolton J L
Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Chem Res Toxicol. 2001 May;14(5):572-81. doi: 10.1021/tx000219r.
Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.
B环不饱和雌激素马萘雌酮、马萘雌甾酮和8-脱氢雌酮及其17α-和17β-二氢类似物的硫酸盐共轭物，约占普雷马林（惠氏-艾尔斯特公司生产）的54%，普雷马林是雌激素替代疗法中最常用的雌激素制剂。尽管普雷马林在临床上广泛应用，但关于B环不饱和雌激素在人体内代谢的研究却非常少，而且对于这些化合物在乳腺组织或肿瘤中的归宿也一无所知。在本研究中，我们调查了马萘雌甾酮在两株人乳腺癌细胞系MCF-7和MDA-MB-231中的代谢情况。MCF-7细胞在用芳烃受体激动剂处理后会诱导细胞色素P450 1A1和1B1的产生，而在MDA-MB-231细胞中，主要诱导产生P450 1B1。利用一系列合成代谢物标准品和氘标记类似物作为内标，通过气相色谱/质谱法对马萘雌甾酮的代谢物进行了鉴定和定量。在这两株细胞系中，观察到了相同的马萘雌甾酮代谢途径。马萘雌甾酮在C-17位被还原为17β-二氢形式，17α-二氢异构体的生成量极少。马萘雌甾酮和17β-二氢马萘雌甾酮在C-4位均被羟基化，生成的儿茶酚代谢物被甲基化形成4-甲氧基马萘雌甾酮和4-甲氧基-17β-二氢马萘雌甾酮。在用芳烃受体激动剂2,3,7,8-四氯二苯并-p-二恶英和3,4,4',5-四氯联苯处理的培养物中，马萘雌甾酮的代谢速率显著提高，这表明P450 1A1和1B1的活性参与了代谢过程。未观察到马萘雌甾酮和17β-二氢马萘雌甾酮的2-羟基化代谢途径。在与cDNA表达的人酶的微粒体反应中，P450 1A1和1B1均催化17β-二氢马萘雌甾酮的4-羟基化反应，而以雌二醇为底物时，P450 1A1主要催化2-羟基化反应，P450 1B1主要催化4-羟基化反应。由于P450 1B1是组成型表达且P450 1A1和1B1在包括乳腺上皮在内的许多肝外组织中均可被诱导，这些结果表明在肝外雌激素反应性组织中，马萘雌甾酮和17β-二氢马萘雌甾酮有发生4-羟基化的可能性。
