Biochemical studies on the roles of cytochrome P4501B1 in the causation and prevention of cancer.

摘要

The aims of this dissertation research are to investigate the roles of cytochrome P4501B1 (CYP1B1), a mixed function oxidase, in the etiology of cancer and cancer prevention in humans. This dissertation consists of three distinct projects: (i) characterization of 17β-estradiol (E2) hydroxylation catalyzed by rat and human CYP1B1 (Chapter 3); (ii) studies on the inhibition of human CYP1B1 by aromatase inhibitors (Chapter 4); and (iii) characterization of polymorphic CYP1B1 variants having greater frequency in African-Americans and their effects on the metabolism of (-)-benzo[a]pyrene-trans-7,8-dihydrodiol(B[a]P-7,8-diol) [Chapter 5].;Previous work demonstrated that human CYP1B1 forms predominantly 4-hydroxyestradiol (4-OHE2), a metabolite which is carcinogenic in animal models. Kinetic studies were performed to characterize the formation of 4-OHE2 and 2-hydroxyestradiol (2-OHE2) by rat CYP1B1 using E2 as a substrate. Km and Kcat values were estimated using the Michaelis-Menten equation. For rat CYP1B1, the apparent Km values for the formation of 4-OHE2 and 2-OHE2 were 0.61 ± 0.23 and 1.84 ± 0.73 μM; the turnover numbers were 0.23 ± 0.02 and 0.46 ± 0.05 pmol/min/pmol P450; and the catalytic efficiencies were 0.37 and 0.25, respectively. For human CYP1B1, the apparent Km values for 4-OHE2 and 2-OHE2 were 1.22 ± 0.25 and 1.10 ± 0.26; the turnover numbers were 1.23 ± 0.06 and 0.33 ± 0.02; and the catalytic efficiencies were 1.0 and 0.30, respectively. These results indicate that although rat CYPIB1 is a low K m E2 hydroxylase, its product ratio, unlike the human enzyme, favors 2-hydroxylation. The Ki values of the inhibitor 2,4,3',5'-tetramethoxystilbene (TMS) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.69 and 0.78 μM, respectively. The Ki values of 7,8-benzoflavone (α-NF) for E2 4- and 2-hydroxylation by rat CYP1B1 were 0.01 and 0.02 μM, respectively. The knowledge gained from this study will support the rational design of CYP1B1 inhibitors and clarify results of CYP1B1 related carcinogenesis studies performed in rats.;The effects of a series of aromatase inhibitors on human CYP1B1 were investigated. The inhibition properties of three steroidal inhibitors---formestane, exemestane, and androstenedione and five non-steroidal inhibitors---aminoglutethimide, fadrozole, anastrozole, letrozole, and vorozole were tested in an assay for E2 hydroxylation using microsomal fraction containing recombinant human CYP1B1 expressed in yeast. (Abstract shortened by UMI.).