Weisz J, Bui Q D, Roy D, Liehr J G
Department of Obstetrics and Gynecology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Endocrinology. 1992 Aug;131(2):655-61. doi: 10.1210/endo.131.2.1386303.
Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.
由于儿茶酚雌激素（CE）在雌激素诱导的仓鼠肾癌中所起的作用，对介导仓鼠肾脏中CE形成的酶进行表征具有重要意义。鉴于之前研究中所使用检测方法存在局限性，尤其是在测量雌激素的4-羟基化方面，我们重新研究了仓鼠肾脏将雌二醇（E2）转化为2-和4-羟基化CE的潜力。在针对NADPH依赖性活性优化的条件下，仓鼠肾脏微粒体表现出高水平的E2-2-和E2-4-羟化酶活性。通过以下两点证明了这两种活性依赖于不同形式的细胞色素P-450：1）用E2长期处理仓鼠以及2）使用盐酸法倔唑（一种选择性细胞色素P-450抑制剂），结果显示E2的2-和4-羟基化受到不同影响。通过直接产物分离测定法测得，对照仓鼠和E2处理仓鼠中NADPH依赖性的E2 2-羟基化水平比之前使用放射酶测定法报道的高1个数量级（对照仓鼠和E2处理仓鼠的表观最大速度分别为24 - 32和6 - 12.5 pmol/mg蛋白质·分钟）。对照仓鼠中NADPH依赖性的E2 4-羟基化水平接近且在E2处理仓鼠中超过了E2的2-羟基化水平（对照仓鼠和E2处理仓鼠的表观最大速度分别为17 - 21和7.5 - 19 pmol/mg蛋白质·分钟）。因此，雌激素处理通过使E2的2-羟基化比4-羟基化下降幅度更大，从而逆转了NADPH依赖性E2-2-/4-羟化酶活性的比例（方差分析，P小于0.007）。与对4-羟基化的小的非剂量依赖性抑制相反，盐酸法倔唑导致E2的2-羟基化显著剂量依赖性降低。在针对过氧化物酶有机氢过氧化物依赖性活性优化的条件下，仓鼠肾脏微粒体产生的2-和4-羟基化CE量相似。这两种CE的量以及相应的比例不受雌激素处理的影响（对照仓鼠和E2处理仓鼠中分别为1:0.9和1:1.0）。因此，本研究证实CE可在同一组织中通过三种不同途径生成，即NADPH依赖性E2-2-羟化酶、NADPH依赖性E2-4-羟化酶以及有机氢过氧化物依赖性E2-2/4-羟化酶活性。我们还表明这三种活性可被不同调节，因此可能由不同形式的细胞色素P-450介导。在仓鼠肾脏中，生成具有不同特性的4-羟基化CE代谢物的潜力可能是该组织易受雌激素诱导致癌作用影响的一个因素。
