Larsen M C, Angus W G, Brake P B, Eltom S E, Sukow K A, Jefcoate C R
Environmental Toxicology Center, University of Wisconsin, Madison 53706, USA.
Cancer Res. 1998 Jun 1;58(11):2366-74.
CYP1B1 and CYP1A1 expression and metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) have been characterized in early-passage human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue of seven individual donors. The level of constitutive microsomal CYP1B1 protein expression was donor dependent (<0.01-1.4 pmol/mg microsomal protein). CYP1B1 expression was substantially induced by exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to levels ranging from 2.3 to 16.6 pmol/mg among the seven donors. Extremely low, reproducible levels of constitutive CYP1A1 expression were detectable in three donors (0.03-0.16 pmol/mg microsomal protein). TCDD inductions were larger for CYP1A1, as compared to CYP1B1, demonstrating substantial variability in the induced levels among the donors (0.8-16.5 pmol/mg). Northern and reverse transcriptase PCR analyses corroborate the donor-dependent differences in protein expression, whereby CYP1B1 mRNA (5.2 kb) was constitutively expressed and was highly induced by TCDD (33-fold). The contributions of CYP1B1 and CYP1A1 to the metabolism of DMBA were analyzed using recombinant human CYP1B1 and CYP1A1, as references, in conjunction with antibody-specific inhibition analyses (anti-CYP1B1 and anti-CYP1A1). Constitutive microsomal activity exhibited a profile of regioselective DMBA metabolism that was characteristic of human CYP1B1 (increased proportions of 5,6- and 10,11-DMBA-dihydrodiols), which was inhibited by anti-CYP1B1 (84%) but not by anti-CYP1A1. TCDD-induced HMEC microsomal DMBA metabolism generated the 8,9-dihydrodiol of DMBA as the predominant metabolite, with a regioselectivity similar to that of recombinant human CYP1A1, which was subsequently inhibited by anti-CYP1A1 (79%). A CYP1B1 contribution was indicated by the regioselectivity of residual metabolism and by anti-CYP1B1 inhibition (25%). DMBA metabolism analyses of one of three donors expressing measurable basal expression of CYP1A1 confirmed DMBA metabolism levels equivalent to that from CYP1B1. The HMECs of all donors expressed similar, very high levels of the aryl hydrocarbon receptor and the aryl hydrocarbon nuclear translocator protein, suggesting that aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator protein expression are not responsible for differences in cytochrome P450 expression. This study indicates that CYP1B1 is an important activator of polycyclic aromatic hydrocarbons in the mammary gland when environmental chemical exposures minimally induce CYP1A1. Additionally, certain individuals express low levels of basal CYP1A1 in HMECs, representing a potential risk factor of mammary carcinogenesis through enhanced polycyclic aromatic hydrocarbon bioactivation.
在从7名个体供体的缩乳术组织中分离出的早期传代人乳腺上皮细胞（HMECs）中，对CYP1B1和CYP1A1的表达以及7,12 - 二甲基苯并(a)蒽（DMBA）的代谢进行了表征。组成型微粒体CYP1B1蛋白表达水平取决于供体（<0.01 - 1.4 pmol/mg微粒体蛋白）。通过将细胞暴露于2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD），CYP1B1表达在7名供体中被大幅诱导至2.3至16.6 pmol/mg的水平。在三名供体中可检测到极低且可重复的组成型CYP1A1表达水平（0.03 - 0.16 pmol/mg微粒体蛋白）。与CYP1B1相比，CYP1A1的TCDD诱导作用更大，表明供体之间诱导水平存在显著差异（0.8 - 16.5 pmol/mg）。Northern印迹和逆转录酶PCR分析证实了蛋白表达中存在供体依赖性差异，其中CYP1B1 mRNA（5.2 kb）组成型表达并被TCDD高度诱导（33倍）。以重组人CYP1B1和CYP1A1为参照，结合抗体特异性抑制分析（抗CYP1B1和抗CYP1A1），分析了CYP1B1和CYP1A1对DMBA代谢的贡献。组成型微粒体活性呈现出具有人CYP1B1特征的区域选择性DMBA代谢谱（5,6 - 和10,11 - DMBA - 二氢二醇比例增加），其被抗CYP1B1抑制（84%），但不被抗CYP1A1抑制。TCDD诱导的HMEC微粒体DMBA代谢产生DMBA的8,9 - 二氢二醇作为主要代谢物，其区域选择性类似于重组人CYP1A1，随后被抗CYP1A1抑制（79%）。残余代谢的区域选择性和抗CYP1B1抑制（25%）表明存在CYP1B1的贡献。对三名表达可测量基础水平CYP1A1的供体之一进行的DMBA代谢分析证实，DMBA代谢水平与CYP1B1相当。所有供体的HMECs表达相似且非常高水平的芳烃受体和芳烃核转运蛋白，表明芳烃受体和芳烃核转运蛋白表达并非细胞色素P450表达差异的原因。这项研究表明，当环境化学暴露对CYP1A1的诱导作用极小时，CYP1B1是乳腺中多环芳烃的重要激活剂。此外，某些个体在HMECs中表达低水平的基础CYP1A1，这代表了通过增强多环芳烃生物激活作用而导致乳腺癌发生的潜在风险因素。
