TOXICOLOGY

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APPLIED

PHARMACOLOGY

(I 990)

102,486-496

Metabolism of the Liver Tumor Promoter Ethinyl Estradiol by Primary Cultures of Rat Hepatocytes ANDREW

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M. STANDEVEN,*YUENIAN E. SHI,~ JACQUELINEF.SINCLAIR,$ PETER R. SINCLAIR,$ANDJAMES D. YAGER~' New’ Humpshirc

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Metabolism ofthe Liver Tumor Promoter Ethinyl Estradiol by Primary Cultures ofRat Hepatocytes. STANDEVEN. A. M., SHI. Y. E.. SINCLAIR. J. F., SINCLAIR, P. R.. AND YAGER. J. D. ( 1990). ?it.Yicxt/. &II/. ~~JUrtJlUd. 102, 486-496. Previously, we reported that relatively high micromolar concentrations of the liver tumor promoter l7n-ethinyl estradiol (EE2) stimulated DNA synthesis and enhanced the DNA synthetic response to epidermal growth factor (EGF) in primary cultures of female rat hepatocytes [J. D. Yager, B. D. Roebuck. T. L. Paluszcyk, and V. A. Memoli. C’llrcitzot~cnc,ti.s 7, 2007-2014 (1986): Y. E. Shi and J. D. Yager, Chnwr Rex 49, 3574-3580 ( 1989)]. In this study, our goal was to examine the metabolism of EE2 in cultured hepatocytes. After 4. 24, and 48 hr of culture. hepatocytes maintained their ability to convert up to 95%) of a 4 niw concentration of [‘H]EE? to polar conjugates within 4 hr. EE2 at 2 PM was also 95’b metabolized within 4 hr. HPLC analysis of the metabolites confirmed the rapid disappearance of [‘H]EEZ and the formation of polar conjugates as detected by organic extraction. HPLC separation of hydrolyzed conjugates indicated that the major aglycone was the parcnt compound. EE,. In general. the metabolites differed both qualitatively and quantitatively from those reported in vivrt in the rat. The rapid metabolism of EE2 by hepatocytes in culture may, at least in part. explain the high concentrations of EE2 required to stimulate DNA synthesis in cultured hepatocytes and to potentiate the response to EGF. CL IYW Academic Press. Inc.

A number of clinical and laboratory studies have linked oral contraceptives to the development of liver tumors (see Porter et al.. 1987, for review). 17a-Ethinyl estradiol (EE,), an ethinylated derivative of the natural estrogen 17/3-estradiol, is commonly used in oral contraceptive formulations since it undergoes a relatively small first-pass effect in the liver (Murad and Haynes, 1985). In the context of the multistage model of chemical carcinogenesis, EE1 is generally regarded as a promoter (Porter et al., 1987). Previous work in this laboratory has demonstrated that EE:! enhances y-glutamyl

’ To whom should be sent: Johns Hopkins Health. 615 N.

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transpeptidase-positive foci development and hepatocellular carcinoma incidence in diethylnitrosamine-initiated female rats (Yager et al., 1984). Additional studies (Yager and Fifield, 1982; Schuppler et al., 1983) indicated that the synthetic estrogens lack detectable genotoxic effects and initiating activity. In viva, EE1 treatment causes a transient increase in liver DNA synthesis in female rats (Yager er al., 1986: Ochs et al., 1986). In monolayer cultures of primary rat hepatocytes, EE? alone stimulates a modest increase in hepatocyte DNA synthesis, and EE2 pretreatment dramatically enhances the DNA synthetic response of hepatocytes to epidermal growth factor (EGF) (Shi and Yager, 1989). The goal of this study was to determine the extent of EE? metabolism under culture con-

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ditions where EE2 potentiates the DNA synthetic response of cultured rat hepatocytes to EGF. To our knowledge, this is the first report of EE2 metabolism in monolayer cultures of rat hepatocytes. The results show that EE2 is rapidly and extensively metabolized to various conjugated derivatives. METHODS C%cwical.s 17a-[6.7-“H (M]Ethinyl estradiol(60.0 Ci/mmol) and Glusulase were purchased from DuPont New England Nuclear (Boston, MA). 17a-Ethinyl estradiol. bovine serum albumin. insulin. ethinyl estradiol- I7-glucuronide (EEZ- 17glucuronide). EEZ-3-methyl ether. and gentamycin were obtained from Sigma Chemical Company (St. Louis, MO). 17&Estradiol, EE?-3-sulfate, EE2- 17sulfate, and EE,-3. I7-disulfate were generous gifts of Dr. W. Slikker (National Center for Toxicological Research, Jefferson. AR). Collagenase was purchased from Cooper Biomedical (Malvern. PA). Ham’s-F12 and Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco Laboratories (Grand Island. NY). ITS (insulintransferrin-selenium) wasa product ofCollaborative Research, Inc. (Bedford. MA). All other chemicals were of reagent or HPLC grade.

Female Lewis rats were obtained from Charles River Breeding Laboratories. Inc. (Wilmington. MA) and maintained on a I?-hr light/l2-hr dark photoperiod. Liver perfusions were always performed near the end of the dark cycle. Rats were fasted the night before perfusion; otherwise. they were provided food (AIN-76A diet. Dyets, Inc., Bethlehem. PA) and distilled water ad lihz[u?rr. The rats weighed between 1 10 and 220 g at the time of the experiments.

Hepatocytes were isolated by a modification of twostep perfusion technique of Seglen ( 1976) as described by Zurlo and Yager ( 1984). Cell viability, checked by trypan blue exclusion, was generally greater than 95%. The ceils were inoculated into rat tail collagen-coated culture dishes (Michalopoulos and Pitot. 1975) at a densityofl.3-1.4X IOJcells/cm’in2mlofl:l Ham’sF12/ DMEM. The cultures were maintained in an incubator at 37°C in 956 02/5% COz. The medium was changed after a 4-hr attachment period and again after 24 and 4X hr for the longer incubations. All buffers used for perfu-

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sion and the medium were adjusted to pH 7.4 and contained 10 pg/ml gentamycin. In addition, the buffers contained 10 fig/ml insulin, while the medium contained ITS (insulin. 5 @g/ml: transferrin. 5 @g/ml; selenium, 5 w/ml).

After the medium change at 4. 24. or 48 hr. 10 ~1 of [3H]EE, (0.05 &i/jd) in EtOH was added to each plate to give 4 nM EE2 and 22 mM EtOH. The plates were returned to the incubator and duplicate or triplicate plates were removed at the intervals indicated in the figure legends. At the time of harvest. the medium was collected. and the cells were washed once with 2 ml phosphate-buffered saline containing EDTA (Na?EDTA, 10 mM: NaCl. 136 mM: KC]. 2.7 mM: NaZHPO,, 8.1 mM; KHzP04. 1.5 mM). The wash and medium were combined and the samples were frozen at -20°C for no more than 1 week prior to analysis. The amount of ‘H collected from collagen-coated culture dishes immediately after [‘H]EE: addition was taken as the zero time control.

Unconjugated EE-, in medium was separated from polar EE2 conjugates by combining 1 ml of sample with 1 ml of ethyl acetate. The mixture was vortexed and then centrifuged briefly to separate the phases. One hundred microliters of the upper organic phase was counted in 3 ml of Econofluor or Biofluor scintillation fluid (DuPont New England Nuclear) on a Beckman LS 7000 scintillation counter (Beckman Instruments, Inc.. Palo Alto, CA).

In a study that compared rate of metabolism at 4 nM and 2 PM, the concentration of EE2 was made 2 FM in one set of dishes by the addition of unlabeled EE2 to the [‘H]EEZ. The final EtOH concentration was 44 mM for all cultures. including controls.

HPLC analysis of the metabolites was carried out on medium stored at -20°C for 9 months using the method of Slikker ef o/. ( 19g I ). The samples were thawed. combined 1: 1 with MeOH. and centrifuged to pellet protein. The supernatant was combined with reference standards dissolved in buffer A ( 10 mM ammonium acetate. pH 6.9) and injected onto a LiChrosorb RP- 18 reverse-phase column. 4.5 X 250 mm (Altex Scientific. Inc., Berkeley,

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Rc;. I. Comparison of ethinyl estradiol (EE:) metabolism after various times in culture. Cultures were prepared and maintained for various periods (4.24. or 48 hr) at 37°C before the addition of4 nM [‘H]EE>. Medium was harvested from duplicate or triplicate plates as indicated and extracted with ethyl acetate. Radioactivity was expressed as a percentage of time zero control. Values for replicate plates within an experiment were generally within 5’r. The values shown are the means of several separate experiments, with the number of experiments indicated in parentheses. Error bars show SD. except for the 48-hr incubation. where they indicate the range of duplicate experiments. The results of a control experiment conducted as above but in the absence of cells is also shown. Each control value represents the mean of triplicate cultures, with error bars showing SD. Where not shown. error bars were within the height of the symbols.

CA). A 40-min convex gradient began with 20% MeOH in buffer A and ended with 100% MeOH. The flow rate was I .O ml/min. and fractions for scintillation counting were collected every 30 sec. A lJV detector equipped with a X0-nm filter was used to determine elution times of the standards.

To hydrolyze conjugates, 200 PI of the thawed incubation medium was combined with 2 I ~1 of 0.3 M sodium acetate, pH 5.0 (final concentration, 40 tnM). Glusulase ( I ~1) was added and the mixture was acidiiied to pH 5.0 with acetic acid and incubated at 37°C for 22 hr. Controls for Glusulase activity consisted of EE: (0. I &PI), EE23.sulfate (4.0 pg/Ml). EEZ-I 7-sulfate (3 pg/J). EE?-3, I7disulfate (3.0 pg/jd). or EE:-17-glucuronide (0.4 wg/pl). each incubated separately with and without Glusulase. Following incubation. protein was precipitated with an equal volume of MeOH. and the resulting supernatant was combined with aglycone standards and injected onto the reverse-phase system.

HPLC to 100% radioactive purity on a 4.6 x 300-mm Chromegabond Diol column (ES Industries. Marlton. NJ). The normal phase system used was based on that of Slikkcr et al. ( I98 I ). A 30-min linear gradient was used. beginning with 100%1 hexane and concluding with 80:20 (v/v) hexane:isopropanol, with a flow rate of 2 ml/min. Fractions were collected every minute and counted in Biofluor on a Tracer Analytic Mark III 688 I liquid scinlillation system (Tracer Analytical. Inc., Elk Grove Village. IL). The elution time of EE2 was verified by injecting authentic EEZ and measuring UV absorbance at 280 nm. The EEZ fractions were pooled. evaporated to dryness with a nitrogen stream, frozen at -20°C for storage, and then resuspended in 100% EtOH just prior to use. The original [‘HIEEL was found to be greater than 96% pure.

Protein content was determined using the Bradford assay as modified by Bio-Rad (Bio-Rad Laboratories, Richmond, CA) with bovine serum albumin as the standard.

RESULTS Titne Course Studies

For the experiment comparing at 4 nM and 2 PM. the [‘H]EE?

the metabolites of EE2 used was repurified by

The time course of EE2 metabolism after various periods of culture is shown in Fig. 1.

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FIG. 2. Distribution of ‘H after addition of 4 nM [3H]EE, to cultures. A time course experiment was conducted as described in Fig. I using 24-hr cultures. The ‘H in the organic extract of the medium. in the aqueous phase of the medium after extraction, and in the cells was quantified by liquid scintillation. Values shown represent the mean dpm expressed as a percentage of time zero control. Error bars indicate the range of duplicate cultures. Where not shown. error bars were within the height of the symbols.

The hepatocytes retained the ability to metabolize 90-95% of the EE2 in 4 hr, regardless of whether the EE? was added 4,24, or 48 hr after inoculation of cells into culture. Somewhat slower initial metabolism was evident in cells incubated for 48 hr before addition of EE?. In a control study, [3H]EEZ was added to medium in culture dishes lacking cells, and the recovery of 3H in the ethyl acetate phase was determined after various times of incubation at 37°C. As shown in Fig. 1 (control, no cells), no loss in extractable 3H relative to time zero was observed; indeed, a small increase was seen. About 5- 10% of the [3H]EE2 initially became bound to the collagen-coated dishes, only to be released during incubation at 37°C thus allowing for slightly greater than 100% recovery of 3H relative to time zero. To account for the particularly rapid initial disappearance of [3H]EE2 from the organic extract of the medium, we counted the 3H remaining in the medium after extraction (Fig. 2, aqueous) as well as the 3H present in the cells (Fig. 2, intracellular). A significant portion (23.1%) of the 3H was present in the cells after 10 min, and ethyl acetate extraction of the TCA-precipitable and TCA-soluble fractions together recovered

over 80% of the radioactivity. Thus, the vast majority of the IO-mm cellular fraction of EE2 was not yet conjugated (data not shown). At later time points, however, the amount of 3H in the cells progressively decreased (Fig. 2). In contrast, the amount of 3H from [3H]EEz remaining in the aqueous phase following extraction of the medium increased linearly during the first hour and then leveled off, in parallel with the organic extract (Fig. 1). Comparison qf EEJ Metabolism trations sf 4 r.w and 2 pnr

at Concen-

Previously, we showed that pretreatment of hepatocytes with 2- 15 PM EEz enhanced their DNA synthetic response to EGF (Shi and Yager, 1989). Thus, in hepatocytes cultured for 4 hr, we compared the extent of EE2 metabolism at a concentration of 2 PM to the 4 nM concentration which was used in the experiments described above. As shown in Fig. 3, in the first hour, a greater percentage of the 4 nM EE2 was initially metabolized than the 2 PM EE2. However, considerably more EE2 was actually metabolized on a mole-per-mil-

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FIG. 3. Comparison of metabolism of EE2 at 4 nM and 2 pM. Four hours after inoculation of hepatocytes into culture, unlabeled EE2 dissolved in EtOH was added to one set ofculture dishes, while another set received EtOH atone. Immediately after. 4 nM [3H]EEZ was added to both, bringing final concentrations to 2 pM EEZ or 4 nM EE2. respectively, and 44 mM EtOH. The extent of metabolism with time was then determined as described in Fig. 1. Values represent the mean dpm expressed as a percentage of time zero control, with error bars indicating the range of duplicate cultures. Where not shown, error bars were within the height of the symbols

ligram of cellular protein basis at the higher EEz concentration. For example, considering the difference in specific activities, 22 nmol EE,/mg protein was conjugated at 4 ttM within 30 min, whereas 5000 nmol/mg protein was conjugated at 2 PM. However, almost the same percentage of [3H]EE2 (approximately 95%) was conjugated after 4 hr at both concentrations (Fig. 3). HPLC Analysis of Conjugates Duplicate samples obtained after 30 min and 4 hr of incubation from the experiment with cultured hepatocytes just described were separated by reverse-phase HPLC to identify the EE2 metabolites (Figs. 4a and b, respectively). The radiochromatograph of one 30min 4 nM EE1 sample showed a peak (F) representing 12% of the recovered radioactivity that coeluted with EE2 at 22 min (Fig. 4a). A series of other radiolabeled peaks (A-E) eluted from 4 to 15 min. In the 4-hr samples (Fig. 4b), no EE2 peak was detectable, and peaks B-D were consid-

erably larger than at 30 min. Thus, these data corroborated the time course data obtained by organic extraction of the same samples (Fig. 3), which indicated that only 20% unmetabolized EE2 remained after 30 min and 4% remained unmetabolized after 4 hr. With regard to identification of the radioactive metabolites, peak A eluted at the solvent front. Only one peak (B) coeluted with a standard and thus could be tentatively identified as EEz-3, 17-disulfate. Radiochromatographs of replicates suggested that peak E (Fig. 4a) was still present at 4 hr but hidden by peak D. The standard for EE?- 17-glucuronide coeluted with the standards for EE?-3sulfate and EEz-17-sulfate on this system (Slikker et al., 1984) so we did not inject EE217-glucuronide with samples. Since no sample peak coeluted with the monosulfate standards, we assume that no EE2- 17glucuronide was present either. The metabolic profile seen by HPLC separation of samples from the culture exposed to 2 /IM EEZ was identical to that seen above, except that considerably more of the radioac-

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tivity (53%) was present in the EE2 peak after 30 min and a small EE2 peak (2%) was still detectable after 4 hr (data not shown). Again, this agrees well with the results obtained by organic extraction, which showed that 52% remained unmetabolized after 30 min and 9% remained unmetabolized after 4 hr (Fig. 3). HPLC of Aglycones To hydrolyze the samples, we used Glusulase, a Helix pomatia intestinal extract containing /?-glucuronidase and sulfatase activities. Using a series of internal controls, we found that Glusulase completely hydrolyzed EEz-3-sulfate and EE2- 17glucuronide to EE?; EE2- 17-sulfate and EE2 itself were completely resistant to hydrolysis; and EE,-3,17disulfate was quantitatively converted to a peak coeluting with EE?- 17-sulfate (data not shown). These results indicated that Glusulase was unable to hydrolyze sulfate groups on the 17 position of EE2. This result was significant since it suggested that if peak B (Fig. 4) were the EE,-3,17-disulfate, it would be quantitatively converted by Glusulase to EE?-17-sulfate. Thus, one would expect this peak to decrease and another peak eluting between 12 and 14 min (the typical elution time of EE?- 17-sulfate) to increase following hydrolysis. An aliquot of the same 4-hr sample used to produce the radiochromatograph in Fig. 4b was hydrolyzed with Glusulase and analyzed by HPLC (Fig. 5). Several of the same peaks present in the unhydrolyzed 4-hr sample (Fig. 4b, peaks A-D) were still present. Peak A was not hydrolyzed by Glusulase. One possibility for the identity of peak A is that it represents 3Hz0. If so, this would suggest that hydroxylation of EE2 had occurred at the C-6 and/or C-7 position in the cultured hepatocytes. Another possibility is that peak A represents a highly polar EE, conjugate which was resistant to hydrolysis. The other peaks (B-D in Fig. 4b) were present in relatively smaller quantities after hydrolysis (Fig. 5). However, since peak B was sensitive to hydrolysis but no new peak appeared at 12 to 14 min, peak B is unlikely to be EE2-3,17-disulfate and

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thus is unidentified. Two new peaks (F and G), which eluted later than the others, appeared after hydrolysis. The larger of these (F) coeluted at 23.5 min with EE2. Peak G eluted at 25.5 min, which was distinctly later than the standard for 17B-estradiol, the putative deethynylation product of EE2. Thus, peak G remains unidentified. It should be noted that all peaks eluted 2 min later than those shown in Fig. 4b. DISCUSSION The liver tumor promoter EE2 and epiderma1 growth factor exert synergistic effects on DNA synthesis in rat hepatocyte cultures (Shi and Yager, 1989). However, since the synergism required micromolar concentrations of EE,, we were interested in determining the rate and extent of metabolism of EE2 under these culture conditions. We have presented both organic extraction and HPLC data to show that rat hepatocytes in monolayer culture can rapidly metabolize EE2, converting approximately 95% of 4 nM [3H]EE2 to polar conjugates by 4 hr. Similar to our results, S. A. Sundstrom, Z. Althaus, W. Slikker, Jr., and J. F. Sinclair (in preparation) found that chick embryo hepatocytes in monolayer culture metabolized 85-95% of EE2 present at 15 pM in 4 hr. Using fresh suspensions of rat hepatocytes, Schwenk et al. ( 1978) found 65% of 0.115 pM EE2 in the polar fraction after 20 min. At a higher concentration (10 PM), only 25% was conjugated, and they suggested that this difference was due to saturation of conjugating enzymes. In our system, the failure of hepatocytes to initially (in the first hour) metabolize the same percentage of radiolabeled EE2 in the presence of 2 PM unlabeled EE2 as in its absence suggested that 2 PM EE2 was a saturating concentration. This result, based on organic extraction of the medium, was supported by HPLC analysis, which revealed far more unchanged EE2 at 2 PM after 30 min than at 4 IIM. Nevertheless, nearly the same percentage of EE2 was conjugated after 4 hr at either concentration (Fig. 3).

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FIG. 4. High-performance liquid chromatograph of EE2 (4 nM) metabolites in cultured hepatocytes from the experiment whose results are shown in Fig. 3. Medium harvested after 30 min (a) or 4 hr (b) of incubation was deproteinized, mixed with available standards (EE2- I7-sulfate, EE2-3-sulfate. EEZ-3, I7-disulfate, and EE?), and then chromatographed on a reverse-phase column eluted with a gradient (broken line) of MeOH in 10 mM ammonium acetate buffer. Fractions for scintillation counting were collected every 30 sec. The shaded area represents the dpm in the fractions. and the solid line represents the ODz8”“,,, of authentic standards.

We observed less initial metabolism in cultures preincubated for 48 hr than in those preincubated for only 4 or 24 hr, suggesting that the relevant enzymes had decreased to a rate-limiting level. However, 4 hr after EE2 addition, the extent of metabolism was similar in 4-, 24-, and 4%hr-old cultures. The level of cytochrome P450 is known to decline rapidly in primary cultures (Sirica and Pitot, 1980) and Bolt et al. ( 1973) provided strong evidence that 2-hydroxylation, the major pathway of EE1 metabolism in rats, was cyto-

chrome P450 mediated. Although a decline in activity with time in culture has also been observed with phase II enzymes (Suolinna and Pitkaranta, 1986; Haake et al., 1989) our finding of at least one aglycone other than EE7 (Fig. 5) further suggested antecedent P450 reactions. HPLC analysis of the incubation medium provided inconclusive evidence regarding the metabolic profile of EE, in culture. At least three conjugate peaks were observed, including the major metabolite that had an elution

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/ loo /’ /’

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time similar to that of the 3,17-disulfate. More likely, it was another highly polar derivative since it did not yield the expected 17sulfate conjugate upon Glusulase hydrolysis. None of the conjugate peaks was completely hydrolyzed by treatment with the combined sulfatase/P-glucuronidase, similar to the result of Maggs et al. (1982) in the rat. Hydrolysis of the conjugates with Glusulase released two distinct aglycones, and the major one appeared, on the basis of coelution. to be EE2. The other peak did not coelute with available standards. However, Maggs et al. (1982) separated deconjugated rat biliary EE2 metabolites by HPLC and, us-

ing mass spectroscopy, positively identified a peak eluting immediately after EE2 on their reverse-phase system as 2-methoxy-EE? . Based on this study and others (Ball et al., 1973; Heiton et al., 1977a), we hypothesize that the unidentified aglycone (Fig. 5, peak G) was the 2-methoxy derivative. If this assumption is correct. then the cultured hepatocytes are capable of carrying out hydroxylation at the C-2 position since 2-hydroxy-EE2 is known to be an obligatory intermediate in 2-methoxy-EE, formation (Ball et al., 1973). As mentioned above, peak A may represent 3H20 resulting from hydroxylation at C-6 and/or C-7. If this is correct, it demonstrates

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FIG. 5. High-pertormance liquid chromatograph of deconjugated EE2 metabolites. Medium harvested after 4 hr in one time course experiment (same sample as in Fig. 4b) was incubated with Glusulase. an enzyme preparation with ~glucuronidase and sulfatase activities. for 22 hr at 37°C. The sample was deproteinized, mixed with available standards (EEZ. 17&estradiol. EE,-3-methyl ether), and then chromatographed as described in Fig. 4.

that the hepatocytes can also carry out hydroxylation at one or both of these positions. However, it is difficult to compare the extent of hydroxylation between the C-6/C-7 and C2 positions since hydrolysis by Glusulase did not appear to go to completion and therefore the actual height of peak G is not known. The literature in fact suggests that hydroxylation at the C-6 and/or C-7 positions represents only a very minor route of EE2 metabolism (Bolt et al., 1973).

Additional oxidative pathways of EE2 metabolism, D-homoannulation and deethynylation, have been reported in a number of species (Schmid et al., 1983; Williams et al., 1975; Helton et al., 1977b), but they were not found to occur in the rat in vivo (Maggs et al., 1982) or in vitro (Helton et al., 1977a). Similarly, we found no evidence of D-homoannulaton or deethynylation products. Minor hydroxylation at the C-6, C-7, and C-16 positions has been reported in the rat (Bolt et

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al., 1973; Maggs et al., 1982); however, we did not find these products. Furthermore, 2methoxy-EE2 was the major aglycone found after hydrolysis of biliary conjugates in the male rat (Maggs et al., 1982), whereas EE2 itself was the major aglycone in our cultured female rat hepatocytes. Thus, EE2 metabolism by primary cultures of rat hepatocytes may differ both qualitatively and quantitatively from EE2 metabolism in vivo. In conclusion, these data show that EE2 is rapidly and extensively metabolized to polar conjugates by hepatocytes under culture conditions where it markedly synergizes their DNA synthetic response to EGF. This may account for the relatively high micromolar concentrations required for the synergism. It is also possible that one or more metabolites could contribute to this potentiation effect, but determination of this requires further study. ACKNOWLEDGMENTS The authors thank Nadia Gorman and Dr. R. Lambrecht for expert HPLC guidance, Dr. J. Silverman for helpful advice, and Dr. W. Slikker and Dr. Joanne Zurlo for critical comments on the manuscript. This work was supported by NC1 Grant CA-3670 1 to J.D.Y. and Cancer Center Core Support Grant CA-23 108 to the Norris Cotton Cancer Center. A.M.S. received support from NIEHS Training Grant BS07 104 and a NSF Graduate Fellowship. Y.E.S. was supported by a Friends of the Norris Cotton Cancer Center Predoctoral Fellowship.

REFERENCES BALL, P., GELBI(E, H. P., HOUPT, 0.. AND KNUPPEN. R. (1973). Metabolism of 17a-ethynyl [4-“‘Cloestradiol and [4-‘4C]mestranol in rat liver slices and interaction between 17Lu-ethynyl-2-hydroxy-oestradiol and adrenalin. Hoppa-Seyler ‘.s Z. Phyyiol. Chem. 354, 1S671575. BOLT. H. M.. KAPPUS. H.. AND REMMER, H. (1973). Studies on the metabolism of ethinyl estradiol in vitro and in vivo: The significance of 2-hydroxylation and the formation of polar products. Xenohiotica 3, 773785.

HAAKE. J., LEAKEY. J.. SHADDOCK, J., AND CASCIANO, D. (1989). Effect of dexamethasone on drug metabolizing enzyme activities in rat hepatocyte cultures. To.uicologist 9, 69.

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HELTON, E. D., CASCIANO, D. A., ALTHAUS, Z. R., AND PLANT, H. D. (1977a). Metabolism of I7a-ethynyl estradiol by intact liver parenchymal cells isolated from mouse and rat. J. Toxicol. Environ. Health 3, 953963.

HELTON, E. D., WILLIAMS, M. C., AND GOLDZIEHER. J. W. (1977b). Oxidative metabolism and de-ethynylation of 17ol-ethinylestradiol by baboon liver microsomes. Steroids 30.7 l-83. MAGGS, J. L., GRABOWSKI, P. S., ROSE. M. E.. AND PARK, B. K. (1982). The biotransformation of 1701ethynyl [3H]estradiol in the rat: Irreversible binding and biliary metabolites. Xenohiotica 12,657-668. MICHALOPOULOS, G.. AND PITOT, H. C. (1975). Primary culture of parenchymal liver cells on collagen membranes. Exp. Cell Rex 94,70-78. MURAD, F., AND HAYNES, R. C. (1985). Estrogens and progestins. In Goodman and Gilmun’s the Pharmacological Basis I$ Therapeutics (A. G. Gilman. L. S. Goodman, and A. Gilman, Eds.). 7th ed., pp. 14121439. MacMillan, New York. OCHS, H., DUSTERBERG,B.. GUNZEL, P.. AND SCHULTEHERMANN, R. (1986). Effect of tumor promoting contraceptive steroids on growth and drug metabolising enzymes in rat liver. Cancer Rex 46, 1224- 1232. PORTER, L. E., VAN THIEL, D. H.. AND EAGON. P. K. (1987). Estrogens and progestins as tumor inducers. Semin. Liver Dis. 7,24-3 1. SCHMID, S. E., Au, W. Y., HILL, D. E.. KADLUBAR, F. F.. AND SLIKKER, W., JR. (1983). Cytochrome P450-dependent oxidation of the 17ol-ethynyl group of steroids: D-homoannulation or enzyme inactivation. Drug Metah. und Dispos. 11, 53 l-536. SCHUPPLER,J., DAMME. J., AND SCHULTE-HERMANN. R. ( 1983). Assay of some endogenous and synthetic sex steroids for tumor-initiating activity in rat liver using the Solt-Farber system.Curcinogene.yis4,239-24 1. SCHWENK, M.. LOPEZ DEL PINO, V., AND BOLT, H. M. ( 1978). Metabolism and disposition of 17a-ethinylestradiol and estrone sulfate in isolated rat liver cells. Acta Endocrinol. (Copenhagen) Suppl. 215,42-43. SEGLEN, P. 0. (1976). Preparation of isolated rat liver cells. In Methods in Cell Biology (D. M. Prescott. Ed.). pp. 29-83. Academic Press, New York. SHI. Y. E.. AND YAGER, J. D. ( 1989). The effects of the liver tumor promoter ethinyl estradiol on epidermal growth factor-induced DNA synthesis and epidermal growth factor receptor levels in cultured rat hepatocytes. Cancer Res. 49,3574-3580. SIRICA, A. E., AND PITOT, H. C. (1980). Drug metabolism and effects of carcinogens in cultured hepatic cells. Phurmacol. Rev. 31, X-228. SLIKKER. W.. JR., LIPE, G. W., AND NEWPORT, G. D. ( 198 I). High-performance liquid chromatographic analysis of estradiol-1713 and metabolites in biological media. J. Chromatogr. 224,205-2 19. SLIKKER. W.. JR., LIPE, G. W., SZISZAK, T. J., AND BAILEY, J. R. (1984). Changes in estrogen metabolism

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Metabolism of the liver tumor promoter ethinyl estradiol by primary cultures of rat hepatocytes.

Previously, we reported that relatively high micromolar concentrations of the liver tumor promoter 17 alpha-ethinyl estradiol (EE2) stimulated DNA syn...
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