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Memorandum To: Dr. J. F. Glenn and Staff From: D.H. Ford Re: Site visit with Dr. J. Weisz and Dr. Bui, The Milton Hershey Medical Center , Hershey, PA October 13, 1988 Grant No. 2339 entitled "Cytochrome P-450 and estrogen metabolism." Goals: To determine how xenobiotics influence the isozymes of cytochrome-P-450s in the conversion of estrogens to their 2 or 4-hydroxylated catechol metabolites (2-and 4-OH-CEs). This is based on her observation that estrogens may not only be metabolized by the established type of NADPH (OHP) dependent E-2-hydroxylation, but also by a peroxidative, organic hydroperoxide (OHP) dependent mechanism. To determine if different xenobiotics selectively influence which mechanism of CE formation occurs by either a E-2H (NADPH) or by a 2/4-H(OHP) pathway. This is important in that the 2-OH-CEs are weak estrogen agonists, while the 4-OH-CEs are potent long lasting estrogens. A further goal is to determine if the E-2-H (NADPH) and the E2/4-H (OHP) pathways of estrogen metabolism are related to activation of different isozymes of P-450. Finally she hopes to determine which of the two estrogen metabolic pathways are most active in estrogen target organs. (It is of further interest that the OHPs required for the E2/4-H (OHP) pathway promote conversion of catechol estrogens to quinones and semiquinones, which can form adducts and therefore, possibly act as mutagens.) The Investigator: Dr. Weisz is an elderly person of unbounded energy and enthusiam. Her current endeavors suggest that she is a biochemist. However, she is well grounded in biology, morphology and endocrinology as well. Her co-worker, Dr. Bui is a young Viet Namese MD ( a boat person), who like most Asian immigrants to this country is apparently devoted to hard work and learning. He is also very competent in delicate technological work. An interesting team where Dr. Weisz contributes the immagination and Dr. Bui contributes the criticism and the hands to undertake a great many of the technical aspects of the study. Results: Inasmuch as Dr. Weisz' support from CTR has only just commenced. There is little in the way of new completed work to evaluate. However, it is of interest to note her schematic derived from her work on the two mechanisms of CE biosynthesis in vitro. (See Fig. 1). One may note (and perhaps speculate) the predominance of 4-OH-CEs (long lasting estrogenic effect) in the hypothalamus and hippocampus where they might play an important role in behaviour.

-2- Her new observations: 2-OH-E2/(NADPH) inhibits 0-methylation of 4-OH-E2(OHP) by COMP. Microsomes obtained from human breast ductal epithelial cells express peroxidative activity consistently (while NADPH dependent activity was variable A dexamethasone) inducible isozyme of cytochrome P-450 was observed to induce some 2-OH-E(NADPH) activity in rat hamster liver microsomes obtained from human placenta. Prelimary studies with placental tissue from a chronic alcoholic suggest that this condition alters 17B-oxireductase activity in placental microsomes. Dr. Weisz, with the sure hands of Dr. Bui has been able to develop a rapid method for obtaining organoids (microsomes) from human breast tissue, which includes a reduction of the period of exposure to collagenase of from 36 to 4 hours. They have also been stockpiling microsomes from normal placentas for the isolation of cP-450s. These will be compared with those obtained from smokers, drinkers and those who indulge in both. Dr. Weisz is developing a questionaire to be used in the Hershey hospital as well as with all indivuals working for Hersey chocolate with an epidemiologist, Dr. M. Hatch, who has a major interest in cancer and in reproduction. Other collaborators include Dr. S. Wrighton, an expert on liver cP-450s, a Dr. J. Liehr who studies kidney carcinogensis in the hamster amd with Dr. Adesnik, with whom she plans to spend a 6 month sabatical at NYU to learn the techniques of molecular biology Comment: An enthusiastic well informed investigator pursuing a question of considerable interest in relation to estrogen dependent cancers. Question: Do xenobiotics alter the pathway of estrogen catechol metabolism to create products which might be more•or less mutagenic? If so, is there genetic variability between subjects which predispose toward the activation of one P-450 isozyme over another. Further, are there different responses in different organs? It will certainly be of interest to follow this program. If she succeeds in her objectives, she may well provide a considerable insight into the development of estrogen dependent cancer and their intiation by xenobiotics. DHF Publications: Bui and Weisz, "Identification of microsomal, organic hydroperoxide-dependent catechol estrogen formation: Comparison with NADPH-dependent mechanism," Pharm. 36:356-364, 1988. Bui and Weisz, "Monoxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase." Accepted for publication by J. Endocrinol. Both studies were completed prior to CTR support.

Fignre 1 TWO KgCUNISltS OF CA TECHOLESTROGEN • (CE) BIOSYNTSRSIS IN VITRO HXV~~ 0aaxmArl9 1ADP1A-DEQFHD0! OlaGFlNIC-Efl(DE0PERWCIDE-DEBPHD1W CytochLianes P-450 Certain Pezrmidases 2-OH-CEs in; P1aCenta Liver Blastocyst Ovai7f M 4-0H-CEB in; Anterior pituitary Nypothalamus Hippocanpus in; Placenta Liver Hypot2alamu3 HippocanVm KADPH GQNERATING SYSTFIt = PFMOSE PFUSPHATE SHUMr OEB'-GIINEtATING SYSTf2! = LIPID HYDROPE2tOXIDES FFORTED FRQH POLYUNSAI[JRATID FATfY ACIDS e.g., ARACNIDONIC ACID BY e.g.,. CYCUOOXYGFZIASE OR LIPOXYGFIiABE sos POINTS OF INPBZtACfiION WITH BNPIROKKEN'P • # Induction of P-450 isozTses by zenobiotics. Subject to GffiNSPIC variation Generation of organic hydroperoxides during cell dssnge. CSs AS INDQCSFS : ADDQ(.'T FdRlIATIOK WITH IKF+OAMA1'IONAI. NACBQHOI.BCAI.ffi Function of catechol structure Both 2- and 4-O6-LBa associated with generation of reactive, electrophilic species BUT Generation of reactive species proioted by perozidases CEa AS PRO?lDTF3S : YBCBp'POR MEDIATED lQTOGENIC ACTION Function of potency as estrogen agonists/antagoniats 11