ANTHONY J. LEE, JOSEPH W. KOSH, ALLAN H. CONNEY, 1 and BAO TING ZHU. ABSTRACT We characterized the NADPH-dependent metabolism of 17 estradiol

Transcription

1 /01/ $3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 2 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3751/ JPET 298: , 2001 Printed in U.S.A. Characterization of the NADPH-Dependent Metabolism of 17 -Estradiol to Multiple Metabolites by Human Liver Microsomes and Selectively Expressed Human Cytochrome P450 3A4 and 3A5 ANTHONY J. LEE, JOSEPH W. KOSH, ALLAN H. CONNEY, 1 and BAO TING ZHU Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina (A.J.L., J.W.K., B.T.Z.); and Department of Chemical Biology, College of Pharmacy, Rutgers The State University of New Jersey, Piscataway, New Jersey (A.H.C.) Received January 9, 2001; accepted April 17, 2001 This paper is available online at ABSTRACT We characterized the NADPH-dependent metabolism of 17 estradiol (E 2 ) by liver microsomes from 21 male and 12 female human subjects. A large number of radioactive estrogen metabolite peaks were detected following incubations of [ 3 H]E 2 with male or female human liver microsomes in the presence of NADPH. The structures of 18 hydroxylated or keto estrogen metabolites formed by these microsomes were identified by gas chromatography/mass spectrometry analysis. 2-Hydroxylation (the formation of 2-OH-E 2 and 2-OH-E 1 ) was the dominant metabolic pathway with all human liver microsomes tested. The average ratio of 4-OH-E 2 to 2-OH-E 2 formation was 1:6. A new monohydroxylated E 2 metabolite (chemical structure unidentified) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6 -OH-E 2 and 16 -OH-E 2 were also formed in significant quantities, but products of estrogen 16 -hydroxylation (16 -OH-E Endogenous estrogens [such as 17 -estradiol (E 2 ) and estrone (E 1 )] can be hydroxylated at multiple positions (as illustrated in Fig. 1) by drug-metabolizing enzymes present in liver as well as in extrahepatic organs (reviewed by Martucci and Fishman, 1993; Zhu and Conney, 1998a). Cytochrome P450 (CYP) family enzymes are the major enzymes that catalyze the NADPH-dependent oxidative metabolism of estrogens to various hydroxylated or keto metabolites (Martucci and Fishman, 1993; Zhu and Conney, 1998a). In most This study was supported by Grant CA from the National Institutes of Health. This study was presented in preliminary form at the 91st Annual Meeting of the American Association for Cancer Research, San Francisco, CA, April 2000 [Lee AJ, Conney AH and Zhu BT (2000) NADPH-Dependent metabolism of 17 -estradiol and estrone by microsomes from twenty-four human liver samples. Proc Am Assoc Cancer Res 41:743]. 1 William M. and Myrle W. Garbe Professor of Cancer and Leukemia Research. OH-E 1 ) were quantitatively minor metabolites. In addition, many other estrogen metabolites such as 6-keto-E 2,6 -OH-E 2,7 - OH-E 2,12 -OH-E 2,15 -OH-E 2,15 -OH-E 2,16 -OH-E 1, and 16-keto-E 2 were also formed in relatively small quantities. The overall profiles for the E 2 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. The activity of testosterone 6 -hydroxylation (a selective probe for CYP3A4/5 activity) strongly correlated with the rate of formation of 2-OH-E 2, 4-OH- E 2, and several other hydroxyestrogen metabolites by both male and female liver microsomes. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of selectively expressed human CYP3A4 or CYP3A5 with [ 3 H]E 2 and NADPH. animals as well as in humans, the hepatic tissues contain the highest levels of total CYP-dependent drug-metabolizing enzymes and possibly also the largest numbers of different CYP isoforms. By using a versatile HPLC separation method coupled with radioactivity detection, investigators recently showed that incubations of radiolabeled E 2 with rat or mouse liver microsomes (a crude preparation containing many different CYP isoforms) resulted in the formation of at least 15 estrogen metabolites (Suchar et al., 1995, 1996; Zhu et al., 1998). Several earlier studies have also examined the NADPHdependent metabolism of E 2 and E 1 by microsomal preparations from human liver (Ball et al., 1990; Kerlan et al., 1992; Shou et al., 1997; Huang et al., 1998; Yamazaki et al., 1998). In these studies, however, only a few hydroxylated estrogen metabolites (i.e., products of estrogen 2-, 4-, and 16 -hydroxy- ABBREVIATIONS: E 2,17 -estradiol; E 1, estrone; E 3, estriol; OH, hydroxy; CYP, cytochrome P450; NADPH, -nicotinamide adenine dinucleotide phosphate (reduced form); HPLC, high-performance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; TMS, trimethylsilyl; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane. 420

2 Fig. 1. Structures of E 2 and estrone E 1. The R is a -OH group for E 2 and is a keto group for E 1. The possible positions for the NADPH-dependent - or -hydroxylation or keto formation catalyzed by CYP enzymes are indicated by arrowheads. The filled arrowheads indicate the C-positions where either - or -hydroxylation or keto formation may take place, whereas the unfilled arrowheads indicate the C-positions where only a hydroxylation (single configuration) may occur. For the C-18 position, either hydroxylation or aldehyde formation may occur. lation) were determined. As discussed in a recent review article (Zhu and Conney, 1998a), a large number of hydroxylated or keto metabolites of E 2 and E 1 are known to be present in the biological samples (e.g., tissues, blood, or urine) from animals or humans. For years, researchers have postulated that 4-OH-E 2 and 16 -OH-E 1 may play an important role in estrogen-induced hormonal carcinogenesis in animal models and also humans, and there is growing evidence in support of such a role for these bioactive estrogen metabolites, in particular the 4-hydroxylated estrogen metabolites (Fishman et al., 1984; Bradlow et al., 1986, 1995; Cavalieri et al., 2000; Liehr, 2000; Newbold and Liehr, 2000). Recently, it has been suggested that some of the other estrogen metabolites (such as 2-methoxyestradiol and 15 -OH-E 2 ) may also have unique biological actions that are different from the parent hormones E 2 and E 1 (Zhu and Conney, 1998a,b). As part of a continuing effort to identify the multiple endogenous estrogen metabolites formed by human tissues, we characterized in this study the NADPH-dependent metabolism of [ 3 H]E 2 to various hydroxylated or keto metabolites by male and female human liver microsomes. Since it is known that human CYP3A family enzymes account for up to 60% of the total hepatic CYP enzymes present (Shimada and Guengerich, 1989; Guengerich and Kim, 1990), we also studied the NADPH-dependent metabolism of [ 3 H]E 2 by selectively expressed human CYP3A4 and CYP3A5, and their profiles for E 2 metabolism were compared with the metabolic profiles obtained with human liver microsomes. Detailed knowledge of the CYP-dependent formation of multiple estrogen metabolites, in particular the bioactive estrogen metabolites, in human liver as well as in extrahepatic target tissues may greatly enhance our understanding of the potential diverse biological actions of endogenous estrogens in the body. Materials and Methods Chemicals. E 2, NADPH, and ascorbic acid were purchased from the Sigma Chemical Co. (St. Louis, MO). 7 -OH-E 2 was a generous gift from Dr. I. Yoshizawa of Hokkaido College of Pharmacy in 17 -Estradiol Metabolism by Human Liver Microsomes 421 Japan. The sources of 6 -OH-E 1,7 -OH-E 2,12 -OH-E 2, 12-keto-E 2, 14-OH-E 1, 14-OH-E 2,15 -OH-E 2, and 15 -OH-E 2 were described in an earlier paper (Suchar et al., 1995). Several hydroxy-e 1 metabolites, including 6 -OH-E 1,7 -OH-E 1,7 -OH-E 1,12 -OH-E 1,15 - OH-E 1,15 -OH-E 1, and 16 -OH-E 1, were metabolically formed from their respective hydroxy-e 2 metabolites by human liver microsomes in the presence of NAD as a cofactor. The products formed were extracted with ethyl acetate and then isolated by HPLC. The reference compounds for all the other estrogen metabolites used in this study were obtained from Steraloids, Inc. (Newport, RI). BSTFA containing 1% TMCS was obtained from Pierce Chemical Co. (Rockford, IL). All the organic solvents used in this study were of HPLC grade and obtained from Fisher Scientific (Atlanta, GA). The radiolabeled E 2 used in this study, [2,4,6,7,16,17-3 H]E 2 (numerically labeled, specific radioactivity Ci/mmol), was purchased from the PerkinElmer Life Science Products (Boston, MA). There is no published information available on whether each of the designated positions was evenly labeled. A comparison of several tritium-labeled E 2 products such as [6,7-3 H]E 2, [2,4,6,7,- 3 H]E 2, and [2,4,6,7,16,17-3 H]E 2 prepared by the same company showed that their highest specific activities (Ci/mmol) increased almost proportionally with increasing positions labeled with tritium, suggesting that each position likely was quite evenly labeled. In addition, earlier we conducted a comparative analysis by using 50 M [2,4,6,7,16,17-3 H]E 2 or 50 M [4-14 C]E 2 as substrate and the rat liver microsomes as the enzyme source. The profile of the multiple E 2 metabolites formed and their average rates were found to be very similar with either [2,4,6,7,16,17-3 H]E 2 or [4-14 C]E 2 as substrate. This data suggested that using the [2,4,6,7,16,17-3 H]E 2 as substrate would not markedly skew the rates of formation for certain metabolites as a result of tritium loss during CYP-mediated hydroxylation of E 2 at its radiolabeled positions. Human Liver Microsomes and Selectively Expressed Human CYP3A4 and CYP3A5. Liver microsomes of 33 human subjects (21 males and 12 females) were obtained from Human Biologics International (Scottsdale, AZ). The average age of the donors was years (mean S.D.). According to the supplier, the liver tissues were the autopsy samples from the donors, and the average cold ischemia time was h (mean S.D.). The main causes of death of these donors included head trauma, intracerebral bleeding, and/or intracranial hemorrhage. The protein content of each microsomal preparation was adjusted to 20 mg/ml by the supplier. The catalytic activities for several CYP isoforms in human liver microsomes (summarized in Table 1) were also determined by the supplier by analyzing the metabolism of selective probe substrates. Note that although the drug probes utilized are useful for estimating the levels of certain CYP isoforms in different liver microsomal preparations, the probes may not be entirely specific for a single CYP isoform. The selectively expressed human CYP3A4 and CYP3A5 were purchased from GENTEST Co. (Woburn, MA). According to the supplier, these two human CYP isoforms were expressed in insect cells selectively transfected with a baculovirus expression system containing the cdna for human CYP3A4 or CYP3A5. The total CYP content and the microsomal protein concentration were 2000 pmol/ml and 2.5 mg/ml, respectively, for CYP3A4, and were 2000 pmol/ml and 3.6 mg/ml, respectively, for CYP3A5. The catalytic activities for testosterone 6 -hydroxylation by the expressed CYP3A4 and CYP3A5 were 7.0 and 3.6 pmol of product formed/pmol of P450/min, respectively. Assay of the NADPH-Dependent Metabolism of [ 3 H]E 2 by Human Liver Microsomes or Human CYP Isoforms. The reaction mixture consisted of 1 mg/ml of human liver microsomal protein or 140 pmol/ml of human CYP3A4 or CYP3A5, a desired concentration of E 2 (containing 2 Ci of [ 3 H]E 2 ), 2 mm NADPH, and 5 mm ascorbic acid in a final volume of 0.5 ml of Tris-HCl (0.1 M)/HEPES (0.05 M) buffer, ph 7.4. The presence of 5 mm ascorbic acid in the incubation mixture has previously been shown to protect catechol

3 422 Lee et al. TABLE 1 Information on human liver microsomal preparations used in the study The contents of total CYP, b 5, and cytochrome c reductase and the activities for various CYP isoforms were determined according methods described earlier (Pearce et al., 1996). Parameter Measured Male Donors Female Donors Number of donors Age of donors (yr) Adjusted protein content (mg/ml) Total CYP content a Cytochrome b 5 content b NADPH-cytochrome c reductase b Caffeine N3-demethylation (1A2) c Coumarin 7-hydroxylation (2A6) c S-Mephenytoin N-demethylation (2B6) c Tolbutamide methyl-hydroxylation (2C9) c S-Mephenytoin 4 -hydroxylation (2C19) c Dextromethorphan O-demethylation (2D6) c Chlorzoxazone 6-hydroxylation (2E1) c Dextromethorphan N-demethylation (3A4) c Testosterone 6 -hydroxylation (3A4/5) c Lauric acid 12-hydroxylation (4A11) c a Measured in nmol/mg of protein. b Measured in nmol/mg of protein/min. c All of these CYP-dependent activities are measured in pmol/mg of protein/min. estrogen metabolites from oxidative degradation without significantly altering the enzyme activity (Hersey et al., 1981). The enzymatic reaction was initiated by addition of microsomal protein, and the incubations were carried out for 20 min at 37 C with mild shaking. The reaction was arrested by placing test tubes on ice followed by addition of 10 l of100 M nonradiolabeled E 2. The mixture was then immediately extracted with 8 ml of ethyl acetate, and the supernatants were transferred to another set of test tubes and dried under a stream of nitrogen. The resulting residues were redissolved in 100 l of methanol, and an aliquot (50 l) was injected into HPLC for analysis of estrogen metabolite composition. Note that all the glass test tubes used in our study were silanized with 5% (v/v) dimethyldichlorosilane in toluene for 10 min followed by rinses in pure toluene twice and pure methanol three times. The test tubes were allowed to dry at room temperature and then thoroughly rinsed with distilled water. Our trial analyses of the NADPHdependent [ 3 H]E 2 metabolism by human and rat liver microsomes using seven different types of unsilanized glass test tubes obtained from three different manufacturers showed that even under exactly the same incubation, extraction, and HPLC assay conditions, the results were very different for each of the hydroxylated [ 3 H]E 2 metabolites detected. Based on measuring the radioactivity associated with [ 3 H]2-OH-E 2 and [ 3 H]4-OH-E 2 peaks, their overall recoveries with unsilanized test tubes were only 30 to 67% of the recoveries with the silanized test tubes. The increased recoveries of hydroxyestrogen metabolites with silanized glass tubes probably is because pretreatment of the glassware surface with dimethyldichlorosilane deactivates active chemical groups, thereby reducing physical adsorption of the hydroxylated estrogen metabolites to the test tubes. HPLC Analysis of [ 3 H]E 2 Metabolites. Analysis of [ 3 H]E 2 metabolites was performed with an HPLC system coupled with in-line UV and radioactivity detection as described previously (Suchar et al., 1995). The HPLC system consisted of a Waters 2690 separation module, a Waters UV detector (model 484), an IN/US -RAM radioactivity detector, and an Ultracarb 5 ODS column ( mm, Phenomenex, Torrance, CA). The solvent system for separation of E 2 and their metabolites consisted of acetonitrile (solvent A), 0.1% acetic acid in water (solvent B), and 0.1% acetic acid in methanol (solvent C). The solvent gradient (solvent A/solvent B/solvent C) used for eluting estrogen metabolites was as follows: 8 min of isocratic at 16:68:16, 7 min of a concave gradient (curve number 9) to 18:64:18, 13 min of a concave gradient (curve number 8) to 20:59:21, 10 min of a convex gradient (curve number 2) to 22:57:21, 13 min of a concave gradient (curve number 8) to 58:21:21, followed by a 0.1-min step to 92:5:3 and an 8.9-min isocratic period at 92:5:3. The gradient was then returned to the initial condition (16:68:16) and held for 10 min before analysis of the next sample. The HPLC retention times for all authentic estrogen metabolites were determined by in-line UV detection, whereas the [ 3 H]E 2 metabolite peaks formed with human liver microsomes were determined by in-line radioactivity detection. The calculation of the amount of each estrogen metabolite formed was based on the amount of radioactivity detected for each corresponding metabolite peak. Structural Identification of E 2 Metabolites Formed by Human Liver Microsomes or Selectively Expressed Human CYP3A4 and CYP3A5. The identity of each of the major E 2 metabolites formed by human liver microsomes was confirmed through comparison of its HPLC retention time, its GC/MS retention time, and its mass fragmentation spectrum with each of the 41 authentic reference compounds (listed in Table 2). For the purpose of comparison, the mass spectrum for each trimethylsilylated reference compound was obtained with our GC/MS system under the same analytical conditions. For GC/MS analysis, the collected HPLC fractions were first evaporated to dryness under a stream of nitrogen gas and then incubated at C for 30 min in the presence of 50 l of BSTFA containing 1% TMCS. A Hewlett Packard 5890 gas chromatograph and a 5970 mass spectrometer (GC/MS) were used with an RTX-5 MS capillary column (0.25 mm 30 m; m film thickness, Restek Corporation, Bellefonte, PA) with helium as the carrier gas. The mass spectrometer was operated in the electron impact mode (70 ev), and the mass abundance was determined by scanning masses from 50 to 600 m/z at 1.4 times/s. The injector and detector temperatures were 260 and 280 C, respectively. During analysis, the column temperature was increased from C at a rate of 4 C/min and then maintained isothermal at 260 C for the remainder of the run. The retention time and the mass spectrum for each of the major E 2 metabolite peaks were compared against a library of 41 authentic standards compiled in this study. We used the built-in library search function of our GC/MS system for spectrum match-up between the E 2 metabolites enzymatically formed and the known standards. Results Structural Identification of E 2 Metabolites Formed by Human Liver Microsomes A representative HPLC profile for the multiple [ 3 H]E 2 metabolite peaks detected after incubation of [ 3 H]E 2 with human liver microsomes and NADPH is shown in Fig. 2A.

4 17 -Estradiol Metabolism by Human Liver Microsomes 423 TABLE 2 Structural identification of E 2 metabolites Human liver microsomes were incubated with E 2 under conditions as described under Materials and Methods. Each peak was collected from the HPLC, derivatized with BSTFA containing 1% TMCS, and then analyzed by GC/MS as described under Materials and Methods. E 2 Standards and Metabolites Retention Times on HPLC Retention Times on GC Characteristic Mass Fragments (m/z) of the Trimethylsilylated E 2 Standard Metabolite Standard Metabolite Metabolites b Quality of Match min (RRT a ) min % E (1.038) (1.039) ,257,218, OH-E (0.833) (0.842) , OH-E (0.862) (0.871) , OH-E (0.453) N.D N.D. 430,340, OH-E (0.491) (0.509) ,415,340,280, keto-E (0.664) N.D N.D. 356,341,299, OH-E (0.428) N.D N.D. 430,340,312,283, OH-E (0.407) N.D N.D. 430,414,340,312, OH-E (0.469) N.D N.D. 430,415,340,242,218, OH-E (0.614) N.D N.D. 430,415,340,313,242,73 11-keto-E (0.651) N.D N.D. 356,340,312,218, OH-E (0.603) N.D N.D. 430,415,323,218, OH-E (0.439) N.D N.D. 340,325,312,297,284, OH-E (0.424) N.D N.D. 430,415,286,217, OH-E (0.546) N.D N.D. 430,286,218, OH-E (0.522) (0.509) ,415,286, OH-E (0.516) (0.509) ,415,286, keto-E (0.731) N.D N.D. 356,341,286,232, 73 E (1.00) (1.00) ,401,326,298,285, OH-E (0.765) (0.762) , OH-E (0.716) (0.716) , OH-E (0.232) (0.217) ,414,324,309,283, OH-E (0.403) (0.398) ,414,324,283,229, keto-E (0.510) (0.509) ,415,340,299,129, OH-E (0.215) (0.217) ,414,324,309,283, OH-E (0.241) N.D N.D. 504,414,324,309,283, OH-E (0.426) N.D N.D. 504,489,414,324,217, OH-E (0.597) N.D N.D. 504,414,324,297,244, OH-E (0.623) (0.640) ,399,324,283, keto-E (0.584) N.D N.D. 430,415,340,323, OH-E (0.259) N.D N.D. 414,324,309,283, OH-E (0.195) 9.33 (0.193) ,324,298,245,217, OH-E (0.361) (0.355) ,325,298,283,217, OH-E 2 (E 3 ) (0.321) (0.323) ,386,345,324,311, OH-E (0.575) (0.574) ,489,386,345,311, keto-E (0.487) (0.509) ,415,312,285,258, OH-17 -E (0.625) N.D N.D. 504,489,414,386,345, OH-17 -E (0.325) N.D N.D. 504,489,386,345,311,73 2-OH-E (0.146) N.D N.D. 592,147,129, 73 6-keto-E (0.152) N.D N.D. 518,325,245, OH-E (0.149) N.D N.D. 592,502,191, 73 2-MeO-E (1.045) N.D N.D. 446,431,416,315, 73 N.D., Not detected. a Relative retention time in relation to E 2. b The major fragment (base peak) for each metabolite is underlined. The identity of each of the major E 2 metabolites formed was confirmed through comparison of its HPLC retention time, its GC/MS retention time, and its mass fragmentation spectrum with each of the 41 authentic reference compounds (results are summarized in Table 2). For the purpose of comparison, the mass spectrum for each of the known standards was determined with our GC/MS system under the same analytical conditions. Most of the identified estrogen metabolites matched closely with the HPLC retention time for the authentic reference compounds (Table 2). Also, the GC/MS retention time of the TMS-derivative of each isolated metabolite matched well with that of the authentic reference compound. The final definitive verification of each isolated metabolite was based on the match-up between the mass fragmentation spectrum of its TMS-derivative with that of the authentic standard, which was done by using the built-in mass spectrum library search function of the instrument alongside with manual comparison of their mass spectra. In this study, we confirmed with high degrees of confidence the structural identities of the following E 2 metabolite peaks: E 1, 2-OH-E 2, 2-OH-E 1, 4-OH-E 2, 4-OH-E 1, 6 -OH-E 2, 6 - OH-E 2, 6 -OH-E 1, 6-keto-E 2, 7 -OH-E 2, 12 -OH-E 2, 15 - OH-E 2, 15 -OH-E 2, 16 -OH-E 2, 16 -OH-E 2, 16 -OH-E 1, 16 -OH-E 1, and 16-keto-E 2 (summarized in Table 2). As shown in Fig. 2, B and C, for example, a representative mass spectrum for the TMS-derivative of 16 -OH-E 2 (an uncommon hydroxy-e 2 metabolite formed by human liver microsomes) matched almost perfectly with the mass spectrum for the authentic reference compound. The chemical structure for one of the quantitatively major metabolites (peak 3 in Fig. 2A) was not fully identified. To determine its structure, we collected this estrogen metabolite from HPLC and examined the mass spectrum of its TMSderivative (shown in Fig. 2D) by GC/MS. The mass spectrum showed that its TMS-derivative has a molecular ion (m/z) of 504, suggesting that it is a monohydroxy-e 2 metabolite (designated as y-oh-e 2 for convenience). However, careful com-

5 424 Lee et al. Fig. 2. A representative HPLC profile for the multiple [ 3 H]E 2 metabolites formed by human liver microsomes (A) and the mass fragmentation spectra (B E) for the TMS derivatives of the authentic 16 -OH-E 2 as well as the metabolically formed 16 -OH-E 2, y-oh-e 2, and y-oh-e 1. The methods for the NADPH-dependent metabolism of E 2 by human liver microsomes, for the HPLC separation of the estrogen metabolites, and for the GC/MS analysis of the trimethylsilylated estrogen metabolites were described under Materials and Methods. Peak 3 was designated as y-oh-e 2 because our GC/MS data (D) indicated that this metabolite is a monohydroxy-e 2, but its structure did not match any of the known standards. Peak 5 was suggested to be y-oh-e 1 (the 17-dehydrogenated product of y-oh-e 2 ) based on the evidence discussed in the text. Peaks U1, U2, and U3 are the unidentified radioactive metabolite peaks formed from [ 3 H]E 2. Our GC/MS analysis indicated that they were not the monohydroxylated E 2 or E 1 metabolites. parison of its HPLC and GC/MS retention times as well as its mass fragmentation spectrum did not show a consistent match with any of the 41 authentic estrogen standards listed in Table 2. Since the monohydroxy-e 2 metabolites that were not studied here include only 1-OH-E 2, 8-OH-E 2, 9-OH-E 2, 12 -OH-E 2, and 18-OH-E 2, it is thus likely that y-oh-e 2 is one of them. The GC/MS analysis of the radioactive metabolite peak 5 showed that its TMS-derivative has a molecular ion (m/z) of 430 (Fig. 2E), suggesting that it is a monohydroxy-e 1 metabolite. Furthermore, this compound was formed as one of the major metabolites when [ 3 H]E 1 was used as the substrate (data not shown), providing additional support for this metabolite as a monohydroxy-e 1. However, its HPLC and GC/MS retention times as well as its mass spectrum did not consistently match any of the 41 authentic estrogen standards listed in Table 2. We noted that the ratio of this hydroxy-e 1 metabolite to y-oh-e 2 formed at different E 2 substrate concentrations was almost the same as the ratio of 2-OH-E 1 to 2-OH-E 2. This finding led us to suggest that the metabolite peak 5 very likely is y-oh-e 1. For the radioactive peaks U1, U2, and U3 shown in Fig. 2A, the mass spectra of their TMS-derivatives did not match any of the 41 reference compounds, and no characteristic estrogen-related mass fragments (m/z of 504 or 414 for hydroxy-e 2 metabolites; 430 or 340 for keto-e 2 or hydroxy-e 1 metabolites) were found in their mass spectra. Also, small amounts of these metabolite peaks were formed nonenzymatically

6 17 -Estradiol Metabolism by Human Liver Microsomes 425 when 20 M[ 3 H]E 2 was incubated with 2 mm NADPH in the absence of liver microsomes (data not shown). It is likely that these radioactive peaks may not be the hydroxylated or keto estrogen metabolites. Optimization of Assay Conditions and ph Dependence Extraction Efficiency and Reproducibility. An average of % (mean S.D.) of the total radioactivity was recovered when the incubation mixture was extracted once with 8 ml of ethyl acetate. Statistical analysis of triplicate determinations of several major [ 3 H]E 2 metabolites (2- OH-E 2, 2-OH-E 1, 4-OH-E 2, y-oh-e 2, and E 1 ) and the rate of overall [ 3 H]E 2 metabolism by three representative human male liver microsomes (HBI-101, HBI-102, and HBI-105) and three representative female liver microsomes (HBI-112, HBI- 217, and HBI-226) showed highly consistent rates, with average intra-assay variations 5%. Effect of Incubation Time and Microsomal Protein Concentration. When 50 M [ 3 H]E 2 was used as substrate, the rate of formation of 2-OH-E 2, 2-OH-E 1, 4-OH-E 2, y-oh- E 2, and E 1, as well as the rate of overall E 2 metabolism by a representative human liver microsomal preparation, was dependent on the incubation time (roughly linear up to 20 min; data not shown). The rate of formation of most hydroxy-e 2 metabolites essentially remained constant after 20 min of incubation. However, the rate of formation of E 1 and 2-OH-E 1 from [ 3 H]E 2 increased almost linearly for at least 60 min. When different concentrations (from mg/ml) of microsomal protein were incubated with 50 M [ 3 H]E 2 for 20 min, the rate of formation of most hydroxy-e 2 metabolites decreased at 1.0 mg/ml of microsomal protein. However, the rate of formation of E 1 and 2-OH-E 1 from [ 3 H]E 2 showed a linear increase up to the highest microsomal protein concentration (2.0 mg/ml) tested (data not shown). Effect of Incubation ph. We examined the effect of ph (from ) on the NADPH-dependent metabolism of [ 3 H]E 2 by human liver microsomes. The formation of most E 2 metabolites (such as 2-OH-E 2, 4-OH-E 2, and y-oh-e 2 ) showed the highest velocity at ph 7 to 8, but the conversion of E 2 to E 1 and the formation of some E 1 metabolites such as 2-OH-E 1 was optimal at a more basic ph (Fig. 3). Effect of Different E 2 Concentrations on Metabolite Formation The effect of different [ 3 H]E 2 concentrations on the rate of metabolite formation by three representative human liver microsomes (HBI-112, HBI-115, and HBI-229) is shown in Figs. 4 and 5. When the [ 3 H]E 2 concentration increased from 25 to 200 M, the overall profile of radioactive E 2 metabolites formed looked very similar, with 2-OH-E 2 and y-oh-e 2 as the quantitatively major hydroxyestrogen metabolites (representative HPLC traces for 25 and 200 M [ 3 H]E 2 as substrate are shown in Fig. 4, middle and bottom panels). However, when a relatively low concentration (3.1 M) of [ 3 H]E 2 was used as substrate, the overall estrogen metabolite profile looked very different, and 2-OH-E 1 and y-oh-e 1 were formed at higher rates than 2-OH-E 2 and y-oh-e 2, respectively (Fig. 4, top panel). These data suggest that the 17 -hydroxysteroid dehydrogenase contained in human liver microsomes is highly active during incubations with 3.1 M E 2. Double reciprocal analysis of the conversion of [ 3 H]E 2 to E 1 by three Fig. 3. Effect of the incubation ph on the formation of several [ 3 H]E 2 metabolites by HBI-115 human liver microsomes. The incubation mixtures consisted of human liver microsomes (1 mg/ml of microsomal protein), 20 M E 2 (containing 2 Ci [ 3 H]E 2 ), 2 mm NADPH, and 5 mm ascorbic acid in a final volume of 0.5 ml of Tris-HCl/HEPES (0.1:0.05 M) buffer with incubation ph as indicated on the x-axis. The incubations were carried out for 20 min at 37 C, and the formation of estrogen metabolites was determined by HPLC analysis as described under Materials and Methods. Each point is the mean of duplicate determinations. human liver microsomal preparations suggested an apparent K M value of 11.5 to 20.0 M for this catalytic activity. For the three human liver microsomes tested, the rate of overall E 2 metabolism increased continuously with increasing E 2 concentrations (Fig. 5). The rate of conversion of [ 3 H]E 2 to most oxidative metabolites by HBI-112 liver microsomes didn t show saturation at the highest substrate concentration (200 M) tested. In comparison, the formation of 2-OH-E 2, 4-OH-E 2, and 16 -OH-E 2 by HBI-115 and HBI-229 liver microsomes plateaued abruptly at 100 M E 2, while the formation of some other metabolites showed a linear increase up to the highest E 2 concentration tested. Note that the ratios of y-oh-e 1 to y-oh-e 2 formed with these three liver microsomes at different E 2 substrate concentrations are almost the same as the ratios of 2-OH-E 1 to 2-OH-E 2 (data not shown). This observation suggests that

7 426 Lee et al. Fig. 4. Representative HPLC profiles for the multiple estrogen metabolites formed by human liver microsomes at different [ 3 H]E 2 substrate concentrations. The incubation mixtures consisted of HBI-115 human liver microsomes (1 mg/ml of microsomal protein), [ 3 H]E 2, 2 mm NADPH, and 5 mm ascorbic acid in a final volume of 0.5 ml of Tris-HCl/HEPES (0.1:0.05 M) buffer, ph 7.4. The incubations were carried out for 20 min at 37 C, and the formation of estrogen metabolites was determined by HPLC analysis as described under Materials and Methods. Although a different final concentration of E 2 was in each test tube, the same amount of radioactive [ 3 H]E 2 (2 Ci) was present. The total radioactivities detected in the top, middle, and bottom HPLC profiles were 698,120, 727,751, and 707,805 cpm, respectively. the same 17 -hydroxysteroid dehydrogenase present in human liver microsomes may catalyze both the conversion of 2-OH-E 2 to 2-OH-E 1 and the conversion of y-oh-e 2 to y-oh-e 1. Fig. 5. Effect of different substrate concentrations on the rate of NADPHdependent metabolism of [ 3 H]E 2 by human liver microsomes. The incubation mixtures consisted of human liver microsomes (0.5 mg of protein), a different concentration of E 2 (containing 2 Ci of [ 3 H]E 2 ), 2 mm NADPH, and 5 mm ascorbic acid in a final volume of 0.5 ml of Tris-HCl/ HEPES (0.1:0.05 M) buffer at ph 7.4. The final substrate concentration was 3.1, 6.3, 12.5, 25, 50, 100, or 200 M. The incubations were carried out for 20 min at 37 C, and the formation of metabolites or overall E 2 metabolism was determined by HPLC analysis as described under Materials and Methods. Each point is the mean of duplicate determinations. NADPH-Dependent Metabolism of [ 3 H]E 2 by Male and Female Human Liver Microsomes The results for the NADPH-dependent metabolism of 20 M [ 3 H]E 2 by human male and female liver microsomes are summarized in Table 3. The average rates of formation of several hydroxy-e 2 metabolites by female liver microsomes were somewhat faster than the rates by male liver microsomes. However, these differences were not statistically significant. In both male and female liver microsomes, 2-OH-E 2 was the major hydroxyestrogen metabolite detected, followed by y-oh-e 2 and 2-OH-E 1. The average rate of estrogen 2-hydroxylation (formation of 2-OH-E 2 plus 2-OH-E 1 ) by all 33

8 17 -Estradiol Metabolism by Human Liver Microsomes 427 TABLE 3 The rate of metabolic conversion of [ 3 H]E 2 to major estrogen metabolites catalyzed by male and female liver microsomes The incubation mixtures consisted of human liver microsomes (1 mg/ml microsomal protein), 20 M E 2 (containing 2 Ci of [ 3 H]E 2 ), 2 mm NADPH, and 5 mm ascorbic acid in a final volume of 0.5 ml of Tris HCl/HEPES (0.1:0.05 M) buffer, ph 7.4. Incubations were carried out for 20 min at 37 C, and the formation of estrogen metabolites was determined by HPLC analysis as described under Materials and Methods. Each value is the mean S.D. from 33 human samples, and the values in parentheses indicate the range from the lowest to the highest rate. E 2 Metabolites Formed Average Rate S.D. (Range) of E 2 Metabolite Formation Male Subjects (n 21) Female Subjects (n 12) Total (n 33) pmol/mg of protein/min y-oh-e ( ) ( ) ( ) 16 -OH-E 2 (E 3 ) ( ) ( ) ( ) y-oh-e ( ) ( ) ( ) 6 -OH-E ( ) ( ) ( ) 6 -OH-E 1 6-keto-E OH-E OH-E ( ) a ( ) a ( ) a 16-keto-E OH-E ( ) ( ) ( ) 4-OH-E ( ) ( ) ( ) 2-OH-E ( ) ( ) ( ) 2-OH-E ( ) ( ) ( ) 4-OH-E ( ) ( ) ( ) E ( ) ( ) ( ) X ( ) ( ) ( ) Total E 2 metabolized ( ) ( ) ( ) a The ratio between 6 -OH-E 1, 6-keto-E 2,16 -OH-E 1,16 -OH-E 1, and 16-keto-E 2 was approximately 2:1:4:4:1 as determined by GC/MS analysis. liver microsomes was pmol/mg of protein/min, and the average rate of estrogen 4-hydroxylation (the formation of 4-OH-E 2 plus 4-OH-E 1 ) was pmol/mg of protein/min. The average ratio of 4-OH-E 2 to 2-OH-E 2 was 1:6. The average rate of y-oh-e 2 formation was pmol/mg of protein/min. Several other hydroxylated metabolites such as 6 -OH-E 2 and 16 -OH-E 2 were also formed in significant quantities. The combined rate of formation of 6 -OH-E 1, 6-keto-E 2,16 -OH-E 1,16 -OH-E 1, and 16-keto- E 2 (five coeluted metabolites) was only 7% of the rate of 2-OH-E 2 formation (Table 3). Additional GC/MS analysis of the collected radioactive HPLC peak (from min) corresponding to these five estrogen metabolites showed a ratio of approximately 2:1:4:4:1 for 6 -OH-E 1, 6-keto-E 2,16 - OH-E 1,16 -OH-E 1, and 16-keto-E 2. The formation of 6 -OH- E 2,7 -OH-E 2,12 -OH-E 2,15 -OH-E 2, and 15 -OH-E 2 was also detected with our HPLC system, but their rate of formation was not precisely quantified because these metabolites were formed at very small quantities ( 2.0 pmol/mg of protein/min). The formation of large amounts of E 1 from E 2 was also observed. Several nonpolar estrogen metabolites (collectively labeled as X) were consistently detected. Quantitatively, these nonpolar metabolites constitute a very significant fraction of the total amount of [ 3 H]E 2 substrate metabolized (Table 3). The structures of these nonpolar estrogen metabolites were not identified. In summary, the major hydroxyestrogen metabolites formed were 2-OH-E 2, y-oh-e 2, and 2-OH-E 1 when [ 3 H]E 2 was incubated with either male or female human liver microsomes and NADPH. Several other E 2 metabolites, including 4-OH-E 2, 4-OH-E 1,6 -OH-E 2,16 -OH-E 2 (E 3 ), and 16 - OH-E 2, were formed in substantial quantities. Small amounts of 6 -OH-E 2,6 -OH-E 1, 6-keto-E 2,7 -OH-E 2,12 - OH-E 2,15 -OH-E 2,15 -OH-E 2,16 -OH-E 1,16 -OH-E 1, and 16-keto-E 2 were also formed. The overall profiles of E 2 metabolites formed by male or female human liver microsomes were not significantly different. Correlation between the Activity of CYP Isoforms and the Rate of [ 3 H]E 2 Metabolite Formation To probe which CYP isoform(s) may be responsible for E 2 hydroxylation at specific positions, we determined in all 33 human liver microsomes the correlation coefficients between the rate of formation of each E 2 metabolite and the rate of metabolism of the selective probe substrate by the targeted CYP isoform. These data are summarized in Table 4 and Fig. 6. The total CYP content in human liver microsomes showed a high degree of correlation with the rate of formation of most hydroxyestrogen metabolites, suggesting that CYP-dependent enzymes constitute the major catalytic activity in human liver microsomes for the NADPH-dependent metabolism of E 2. Among the CYP isoforms examined, the catalytic activity of CYP3A4 (according to dextromethorphan N-demethylation) or CYP3A4/5 (according to testosterone 6 -hydroxylation) showed high degrees of correlation with the rate of formation of several E 2 metabolites (Table 4; Fig. 6). In addition, the catalytic activity of CYP2B6 (according to S- mephenytoin N-demethylation) also showed a good correlation with the rate of formation of y-oh-e 2,E 3, and 2-OH-E 2. Overall, liver microsomes from female human subjects showed a somewhat higher degree of correlation between the rate of formation of most E 2 metabolites and the catalytic activity of the corresponding CYP isoform as compared with male liver microsomes. NADPH-Dependent Metabolism of [ 3 H]E 2 by Human CYP3A4 and CYP3A5 Incubations of 20 or 50 M [ 3 H]E 2 with selectively expressed human CYP3A4 or CYP3A5 in the presence of NADPH resulted in the formation of multiple hydroxylated estrogen metabolites. Figure 7 shows the representative HPLC profiles for the radioactive estrogen metabolites formed by human CYP3A4 and CYP3A5 at a 20 M [ 3 H]E 2 substrate concentration. The overall profiles formed with these two CYP isoforms were very similar. 2-OH-E 2 was the

9 428 Lee et al. TABLE 4 Correlation coefficients (r) for comparing the formation of various E 2 metabolites with the activities of CYP1A2, 2B6, 3A4, or 3A4/5 by measuring the metabolism of probe substrates in 33 human liver microsomes (21 male and 12 female) The microsomal formation of various estrogen metabolites was as described in the legend to Table 3. The CYP1A2, 2B6, 3A4, or 3A4/5 activites in human liver microsomes were determined by the supplier by measuring the activities for caffeine N3-demethylation, S-mephenytoin N-demethylation, dextromethorphan N-demethylation, or testosterone 6 -hydroxylation, respectively, according to published methods (Pearce et al., 1996). CYP3A4/5 (Testosterone 6 -Hydroxylation) CYP3A4 (Dextromethorphan N-Demethylation) CYP2B6 (S-Mephenytoin N-Demethylation) CYP1A2 (Caffeine N3-Demethylation) Total CYP Content E 2 Metabolite Formed Male Female Total Male Female Total Male Female Total Male Female Total Male Female Total y-oh-e *** 0.90*** 0.79*** * 0.66** 0.94*** 0.80*** 0.80*** 0.90*** 0.87*** 0.91*** 0.94*** 0.94*** 16 -OH-E ** 0.81** 0.72*** * 0.71*** 0.87*** 0.80*** 0.72*** 0.81** 0.78*** 0.74*** 0.88*** 0.82*** y-oh-e *** 0.90*** 0.82*** * 0.86*** 0.72*** 0.59** 0.78** 0.73*** 0.80*** 0.96*** 0.92*** 6 -OH-E ** 0.81** 0.74*** * *** 0.67*** 0.45* 0.82** 0.71*** 0.62** 0.84*** 0.78*** 6 -OH-E 1 6-keto-E OH-E * 0.86*** 0.73*** 0.49* * 0.71*** 0.73** 0.72*** 0.70*** 0.73** 0.72*** 0.55* 0.92*** 0.77*** 16 -OH-E 1 16-keto-E OH-E ** 0.82** 0.71*** 0.45* * 0.70*** 0.87*** 0.76*** 0.76*** 0.87*** 0.84*** 0.70*** 0.87*** 0.84*** 4-OH-E *** 0.87*** 0.82*** * 0.65** 0.90*** 0.79*** 0.75*** 0.90*** 0.85*** 0.86*** 0.92*** 0.90*** 2-OH-E *** 0.92*** 0.83*** * 0.52** 0.65** 0.95*** 0.82*** 0.75*** 0.94*** 0.86*** 0.86*** 0.93*** 0.88*** 2-OH-E ** 0.87*** 0.76*** ** 0.58*** * 0.48** 0.67** 0.91*** 0.77*** 4-OH-E * 0.92*** 0.77*** * 0.55** * 0.54** 0.66** 0.92*** 0.83*** E * * X * 0.49** * 0.72*** 0.59** ** 0.45* 0.66* 0.63*** Total E 2 metabolized 0.71*** 0.86*** 0.79*** *** 0.79*** 0.70*** 0.45* 0.67* 0.61*** 0.77*** 0.90*** 0.86*** * P 0.05; ** P 0.01; *** P quantitatively major hydroxy-e 2 metabolite formed by either CYP3A4 or CYP3A5, followed by y-oh-e 2 and 4-OH-E 2. Several other hydroxy-e 2 metabolites (6 -OH-E 2, 16 -OH-E 2, and 16 -OH-E 2 ) were also formed in substantial quantities by CYP3A4 and CYP3A5. In addition, large amounts of nonpolar metabolites (collectively labeled as X in Fig. 7) were formed during incubations of E 2 with CYP3A4- or CYP3A5- expressed microsomes. The overall profiles for the hydroxylated and nonpolar E 2 metabolites formed by CYP3A4 or CYP3A5 looked quite similar to those formed by male or female human liver microsomes (compare Fig. 7 with Fig. 2). In contrast, the CYP3A4 or CYP3A5-expressed microsomes contained much lower activity for the formation of E 1 and several hydroxy-e 1 metabolites when compared with human liver microsomes. Quantitatively, the rates for 2-OH-E 2 formation (mean S.D. of triplicate determinations) by CYP3A4 and CYP3A5 were and pmol/nmol of P450/min, respectively, and the rates for 4-OH-E 2 formation were 53 9 and 43 4 pmol/nmol of P450/min, respectively. Therefore, the ratio of 4-OH-E 2 formation to 2-OH-E 2 formation was 1:5 with CYP3A4, and it was 1:2 with CYP3A5. The structural identities for the major hydroxy-e 2 metabolites (2-OH-E 2, 4-OH-E 2,6 -OH-E 2,16 -OH-E 2,16 -OH- E 2, and y-oh-e 2 ) formed by CYP3A4 and CYP3A5 were also confirmed by GC/MS analysis (data not presented). In summary, multiple hydroxy-e 2 metabolites were formed by selectively expressed human CYP3A4 and CYP3A5. The overall profiles for hydroxylated and nonpolar E 2 metabolites formed by human CYP3A4 and CYP3A5 were similar to the metabolites formed by either male or female human liver microsomes. It is of great interest to note that the ratio of 4-OH-E 2 to 2-OH-E 2 formation by CYP3A5 (a polymorphic hepatic CYP isoform) was much higher than the ratio for CYP3A4. Discussion Liver Microsomal Hydroxylation of E 2 at Various Positions. In the present study, we characterized the NADPH-dependent metabolism of [ 3 H]E 2 by 33 human liver microsomal preparations. By using a versatile HPLC separation method coupled with radioactivity detection, we identified over 18 hydroxylated or keto metabolites that were formed with [ 3 H]E 2 as substrate. The structural identities of all major [ 3 H]E 2 metabolites formed by human liver microsomes were confirmed by our triple match-up approach according to their HPLC retention times, the GC/MS retention times of their TMS-derivatives, and the mass fragmentation spectra of their TMS-derivatives (data summarized in Table 2). Earlier studies reported that 2-hydroxylation of E 2 to a catechol is a major metabolic pathway in rodent and human livers, whereas 4-hydroxylation to a different catechol represents a quantitatively minor pathway (usually 15% of 2-hydroxylation) in this organ (Dannan et al., 1986; Kerlan et al., 1992; Zhu et al., 1993; Suchar et al., 1995). The data from our present study also showed that E 2 2-hydroxylation is by far the major hydroxylation pathway in human liver microsomes, and the rate of E 2 4-hydroxylation was 1:4 to 1:6 of that for E 2 2-hydroxylation. The potential importance of 4-hydroxylated estrogen metabolites (4-OH-E 2 and 4-OH-E 1 )

10 17 -Estradiol Metabolism by Human Liver Microsomes 429 in hormonal carcinogenesis has recently received considerable attention (reviewed recently by Liehr, 2000). These catechol estrogens not only serve as intermediates for the generation of reactive chemical species (Liehr and Roy, 1990; Cavalieri et al., 2000; Liehr, 2000), but 4-OH-E 2 may also have its own signal transduction pathway that is refractory to E 2 (Das et al., 1997). The NADPH-dependent 6 - and 6 -hydroxylations of E 2 are known metabolic pathways in both animals and humans (Mueller and Rumney, 1957; Breuer et al., 1966). A partially purified CYP preparation from rat brain showed significant catalytic activity for E and 6 -hydroxylation (Sugita et al., 1987), and larger amounts of 6 -OH-E 2 were detected than 6 -OH-E 2 based on thin-layer chromatographic analysis. Similarly, an earlier study showed that 6 - and 6 - OH-E 1 were the major metabolites of E 1 formed by porcine uterine endometrial tissues (Maschler et al., 1983), and 6 - OH-E 2 was found to be a major metabolite of E 2 formed by human placental microsomes (B. T. Zhu, M. X. Cai, and A. H. Conney, unpublished observations). The results of our present study showed that 6 - and 6 -OH-E 2 were very minor metabolites formed by either male or female human liver microsomes. The formation of 6 -OH-E 2 was quantitatively less than the formation of its isomer. It has been suggested for many years that the 16 -hydroxylation of E 2 and E 1 plays an important role in mammary carcinogenesis (Fishman et al., 1984; Bradlow et al., 1986, 1995). The 16 -hydroxylated estrogens (i.e., 16 -OH- E 1 and 16 -OH-E 2 ) are not only hormonally active, but 16 - OH-E 1 is also chemically reactive and may bind covalently to Fig. 6. Analysis of the correlation coefficiency between the formation of three major hydroxy-e 2 metabolites (2-OH-E 2, 4-OH-E 2, and y-oh-e 2 ) and the activities for hepatic CYP3A4/5, CYP3A4, and CYP2B6. The rate of formation of 2-OH-E 2, 4-OH-E 2, and y- OH-E 2 was determined by incubation of human liver microsomes with 20 M [ 3 H]E 2 and 2 mm NADPH as described under Materials and Methods. The Pearson correlation coefficients (r) for liver microsomes from all 21 male subjects (M), from all 12 female subjects (F), and from all male and female subjects combined (T) were determined by using the SAS software (SAS Institute Inc., Cary, NC). the estrogen receptor possibly resulting in sustained hormonal stimulation (Swaneck and Fishman, 1988; Hsu et al., 1991). Because of the unique biological properties of 16 hydroxylated estrogens, several earlier studies have examined the NADPH-dependent 16 -hydroxylation of E 2 and E 1 in human subjects (Osborne et al., 1993) or by human hepatic and extrahepatic tissues or cells in vitro (Aoyama et al., 1990; Shou et al., 1997; Huang et al., 1998). Human liver homogenates were reported to metabolically convert E 2 to 16 - OH-E 2 at a relatively fast rate. A recent study by Yamazaki et al. (1998) estimated that the average rate of 16 -OH-E 2 formation from E 2 by human liver microsomes was 100 pmol/nmol of P450/min, which was much faster than the rate of 4-OH-E 2 formation. However, the results of our present study showed that only very minute amounts of 16 -OH-E 2 and 16 -OH-E 1 were formed by human liver microsomes when [ 3 H]E 2 was used as substrate (Fig. 2). In our study, the average rate of 16 -OH-E 2 formation by 33 human liver microsomes was pmol/mg of protein/min (Table 3); the rate of 16 -OH-E 1 formation was even lower ( 2.0 pmol/mg of protein/min) and could not be precisely quantified. The combined average rate of 16 -OH-E 2 and 16 -OH- E 1 formation from [ 3 H]E 2 substrate was much less than that of 4-OH-E 2 and 4-OH-E 1 formation. In additional studies, we found that 16 -hydroxylation of E 2 by human placental microsomes was also a very minor metabolic pathway (B. T. Zhu, M. X. Cai, and A. H. Conney, unpublished observations). It should be noted that the identification of the estrogen metabolites in several earlier studies was only based on comparison of their TLC or HPLC retention times against a very