We characterized the NADPH-dependent metabolism of estrone (E 1 ) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E 1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E 1 was the dominant metabolic pathway with all human liver microsomes tested. E 1 is more prone to form catechol estrogens (particularly 4-OH-E 1 ) than 17␤-estradiol (E 2 ) and the average ratio of E 1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E 2 4-to 2-hydroxylation (0.20, P Ͻ 0.001). An unidentified monohydroxylated E 1 metabolite (y-OH-E 1 ) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6␤-OH-E 1 , 16␣-OH-E 1 , and 16␤-OH-E 1 were also formed in significant quantities. 16␣-Hydroxylation was not a major pathway for E 1 metabolism. The overall profiles for the E 1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [ 3 H]E 1 and NADPH. Notably, human CYP3A5 has very high relative activity for E 1 4-hydroxylation, exceeding its activity for E 1 2-hydroxylation by ϳ100%. It will be of interest to determine the potential biological functions associated with any of the E 1 metabolites identified in our present study.The endogenous estrogens 17␤-estradiol (E 2 ) and estrone (E 1 ) undergo extensive metabolism in the body (their structures and possible sites for oxidative metabolism are illustrated in Fig.

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In animal models, estrogens are complete carcinogens in certain target sites. 4-Hydroxyestradiol (4-OH-E), an endogenous metabolite of 17β-estradiol (E), is known to have prominent estrogenic activity plus potential genotoxicity and mutagenicity. We report here our finding that 4-OH-E does not induce pituitary tumors in ACI female rats, whereas E produces 100% pituitary tumor incidence. To probe the mechanism, we conducted a short-term animal experiment to compare the proliferative effect of 4-OH-E in several organs. We found that, whereas 4-OH-E had little ability to stimulate pituitary cell proliferation in ovariectomized female rats, it strongly stimulates cell proliferation in certain brain regions of these animals. Further, when we used in vitro cultured rat pituitary tumor cells as models, we found that 4-OH-E has similar efficacy as E in stimulating cell proliferation, but its potency is approximately 3 orders of magnitude lower than that of E. Moreover, we found that the pituitary tumor cells have the ability to selectively metabolize 4-OH-E (but not E) with ultrahigh efficiency. Additional analysis revealed that the rat pituitary expresses a membrane-bound catechol-O-methyltransferase that has an ultralow K value (in nM range) for catechol estrogens. On the basis of these observations, it is concluded that rapid metabolic disposition of 4-OH-E through enzymatic O-methylation in rat anterior pituitary cells largely contributes to its apparent lack of cell proliferative and tumorigenic effects in this target site.

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Wa^ington Headquarters Senrfces, Directorate for Infomiation Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Ariington, VA 22202-4302, and PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)University of South Carolina Research Foundation Columbia, South Carolina 29208 E-Mail: lmills@cop. sc. edu My studies have compared the carcinogenic effects of an Ei-ivp-fatty acid ester preparation, E2 and 4-OH-E2 in the breast, pituitary and uterus of ACI rats. Results showed that chronic administration of an Ea-lVP-fatty acid ester preparation to these rats preferentially induces the development of mammary tumors while chronic administration of E2 results in the preferential formation of pituitary tumors. The chronic administration of 4-OH-E2 to intact female ACI rats did not induce the formation of mammary or pituitary tumors, although there was hyperplasia present in the mammary glands. These results are the first report demonstrating that chronic administration of an estrogen fatty acid ester selectively induces the development of mammary tumors in this animal model. In order to study the carcinogenic activity of other estrogen fatty acid esters, such as those of 4-OH-E2, they must first be synthesized. Therefore, a facile method for the chemical synthesis of large amounts of 4-hydroxyestradiol-17p-stearate, a representative fatty acid ester of the strongly-procarcinogenic estrogen metabolite 4-hydroxyestradiol, has been developed with estrone as the starting material. The ready availability of large amounts of chemically-synthesized 4-hydroxyestradiol-IVP-fatty acid esters make it possible for future studies to systematically characterize their hormonal and carcinogenic potency and efficacy. Appendices 11 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U SUBJECT TERMS INTRODUCTIONExogenous administration of estrogens has been shown to induce tumor formation in animals (Nandi et al, 1995;Clifton and Meyer, 1956;Kirkman, 1959;Cutts and Noble, 1964;Newbold et al, 1990; and Li and Li, 1996). In humans, there is strong evidence showing that chronic estrogen administration more readily correlates with an increase in uterine cancer risk than breast cancer risk (Ziel and Finkle, 1975; Sitteri, et al., 1976;. Although the difference is not fully understood, it is possible that some of the biologically active estrogen derivatives, such the estrogen fatty acid esters, may play a more significant role than estradiol-17|32 in the induction of breast cancer.Recently it was suggested that the mammary adipocytes may serve as a storage site for the lipoidal estrogen fatty acid este...

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