METABOLISM OF 17α-ETHYNYLESTRADIOL BY INTACT LIVER PARENCHYMAL CELLS ISOLATED FROM MOUSE AND RAT Edward D. Helton, Daniel A. Casciano, Zelda R. Althaus, Harris D. Plant Department of Health, Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas

Liver parenchymal cells isolated by perfusion from female C3H/HeN-MTV+Nctr mice and Sprague-Dawley rats were incubated with [6,7- 3 H] 17α-ethynylestradiol (EE2). The incubates were individually fractionated into free steroid (organic phase), steroid conjugates (aqueous), and bound steroids (macromolecular pellet). The rat had significantly less total free radioactive steroid but significantly more total conjugated and irreversibly bound radioactivity than the mouse. However, when the metabolic conversion of EE2 was compared in the rat and the mouse on a cellular basis (metabolic clearance per 106 cells), the rat was found to be less efficient than the mouse. The two species were essentially equivalent in their covalent binding when expressed on a per 106 cell basis. Purification of the free radiolabeled steroids on LH-20 demonstrated the mouse to have the parent compound and an identifiable 2-OH-EE2 fraction. The rat had EE2 and an identifiable 2-methoxy-EE2 fraction. A major metabolite fraction for both species was very nonpolar and, although not identified, was found to be ethynylated as demonstrated by silver-sulfoethylcellulose chromatography. The conjugate fractions of the mouse were indicative of glucuronide conjugation, whereas the rat had additional conjugate fractions suggestive of sulfoconjugation.

INTRODUCTION Intact liver parenchymal cell suspensions have been used to study a variety of mammalian liver functions. Some of the biochemical parameters monitored have been reviewed by Seglen (1976) and include carbohydrate, lipid, protein, and nucleic acid metabolism. In addition, liver parenchymal cells have been presumed to be the major cell type involved in drug metabolism, and such cells have been shown to metabolize a variety of drugs (Holtzmann et al., 1972; Von Bahr et al., 1974). Although these types of cellular preparations have been used to study the kinetics of steroid transport through cell membrane (Rao et al., 1976), there is no The authors wish to thank Dr. Sydney Shain of the Southwest Foundation for Research and Education, San Antonio, Texas, for the reverse isotope recrystallization of the steroid metabolites. Requests for reprints should be sent to Edward D. Helton, Division of Molecular Biology, National Center for Toxicological Research, Jefferson, Arkansas 72079. 953 Journal of Toxicology and Environmental Health, 3:953-963,1977 Copyright © 1977 by Hemisphere Publishing Corporation

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information concerning the capacity of these cells to metabolize estrogen hormones. Similarly, there is no evidence demonstrating the capacity of isolated liver cells to oxidatively metabolize and covalently bind estrogen hormones to tissue nucleophiles. In a brief review, Hecker and Marks (1965) demonstrated that estrone binds to rat liver microsomal protein irreversibly via a thioether bond following 2-hydroxylation. This was postulated by Marks and Hecker (1969) to result from enzymatic rearrangement of the catechol estrogen to a reactive orf/jo-semiquinone. These studies were confirmed in vitro by Hoppen et al. (1974) and in vivo by Elce and Harris (1971). Kappus et al. (1973) reported 17a-ethynylestradiol covalently bound to rat liver microsomal proteins and found the reaction requirements identical to those for estrone and estradiol. Bolt et al. (1973, 1974) confirmed this work and also found that the covalent reaction phenomenon occurred with human liver microsomes. Numerous investigators have postulated that the covalent binding may be the cause of estrogen hepatotoxic syndromes. To this end, we used isolated intact liver parenchymal cells to determine their capacity to metabolize estrogen hormones and to determine their utility as a model system for the study of estrogen covalent binding.

MATERIALS AND METHODS Preparation of Radiolabeled Compounds [6,7-3H]17a-Ethynylestradiol (EE2) was prepared from [6,7- 3 H]estrone (Amersham/Searle; specific activity, 42 Ci/nmol) as previously described (Rao, 1971). The radiolabeled steroid was purified before use (Williams et al., 1975), giving a 98% pure substrate. Isolation of Intact Liver Parenchymal Cell Suspension and In vitro Incubation Adult female rats of the Sprague-Dawley (SD) strain (150-200 g) and adult female mice of the C3H/HeN-MTV+Nctr (C3H) strain (19-24 g), both with defined flora, were maintained on a Purina 5010M diet up to the time of perfusion. Isolated intact liver parenchymal cells were prepared individually from four rats and four mice under aseptic conditions by a modification of the liver perfusion technique described by Bonney (1974). This in situ technique involved cannulation of the inferior vena cava and preperfusion of the liver at 37°C with a calcium-free Hank's balanced salt solution (HBSS, Grand Island Biological), gassed with 95% O2 and 5% CO 2 , supplemented with 2 units/ml heparin, 1.0 mM MgSO4) 100 units/ml penicillin, 10 /xg/ml streptomycin, and 0.5% bovine serum albumin. The liver was then perfused for approximately 8-10 min with this solution minus heparin but supplemented with 0.5% collagenase (Sigma, type 1).

METABOLISM OF EEj BY INTACT LIVER CELLS

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After this, the liver was removed and placed in a beaker containing 10 ml sterile incubation medium, which consisted of HBSS containing vitamins, the essential and nonessential amino acids of Eagle's minimum essential medium (Grand Island Biological), and 0.5 //g/ml insulin. Individual intact cells were liberated from the connective and vascular tissue either by mild aspiration with a wide-bore pipette or by gentle bubbling with 95% O2 and 5% CO 2 . The suspension was filtered through 250 and 62 jum sterile nylon mesh (Small Parts, Inc.) to remove the liver stroma, aggregates, and tissue fragments. The cell suspension was centrifuged once at 300 Xg for 1 min at 4°C. The final cell suspension was counted in a hemacytometer and diluted to 1.5-3.0 X10 6 cells/ml in sterile incubation medium and placed in a sterile 50 ml screw-cap Erlenmeyer flask containing the tritiated EE2 (5 (id). The cell suspension was gassed with 95% O2 and 5% CO2 and incubated for 24 hr in a rotary shaker at 37 ± 1°C. Each experiment was terminated by centrifugation of the entire mixture at 500 Xg for 15 min at 4°C to obtain a cell pellet and an extracellular supernatant. Each fraction was then frozen at either —20 or —70°C prior to metabolic analysis. Control incubations were performed using radiolabeled substrate and incubation medium. Fractionation of the Metabolic Products The cellular pellet and extracellular supernatant of each experiment were recombined and homogenized, and an aliquot was taken to determine the total radioactivity. Five volumes of dimethoxymethane and methanol (1:1) was added to the homogenate to extract the unbound radioactivity and precipitate the macromolecular components. The precipitation and extraction was allowed to proceed for 18 hr at 2°C. The supernatant was removed and the pellet washed and centrifuged three times with organic mixture. Each wash was added to the original supernatant. The dimethoxymethane-methanol extract was taken to dryness in vacuo and the residue partitioned between benzene and H2O. Aliquots of the organic and aqueous phases were taken for scintillation counting. The macromolecular precipitate was solubilized in 1% sodium dodecyl sulfate and extracted three times with benzene. The pH was adjusted to 1.0 with 0.1 N HCI and the solubilized precipitate refluxed for 1 hr. The refluxed material was reextracted three times with benzene. The loss of radioactivity before and after refluxing was determined. Conjugate Purification and Hydrolysis

The aqueous phases (steroid conjugates) from the studies performed with the rat (or mouse) were pooled and chromatographed on a LH-20 (Pharmacia) column (300 X 22 mm ID) using chloroform and methanol (1:1) containing 0.1 M NaCI as the mobile phase. After chromatographic profiling of the conjugate pool for each species, the total conjugates for

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each species were hydrolyzed with Helix pomatia )3-glucuronidase and phenosulfatase in 0.1 M acetate buffer for 72 hr. The hydrolysate of each was collected by exhaustive chloroform extraction. Metabolite Purification and Identification The organic phase of each experiment was chromatographed on a LH-20 column (300 X 22 mm ID) using benzene and methanol (85:15) as the mobile phase. The individual profiles for each species were pooled and combined with the appropriate steroid conjugate hydrolysate and rechromatographed on the LH-20 column to provide a composite metabolite or aglycone profile for each species. The metabolites were subjected to reverse isotope recrystallization with authentic crystalline steroids. These recrystallizations were performed in accordance with published criteria (Axelrod et al., 1965). A silver-sulfoethylcellulose column (Pellizzari et al., 1973) was used or) one nonpolar metabolite peak to determine whether it was an ethynylated product. Methanol was used to elute nonethynyl metabolites, and methanol saturated with NaCI to elute ethynylated metabolites from the silver column. Liquid Scintillation Counting

All counting was performed on a Packard Tri-Carb 3380 liquid scintillation counter with an automatic external standard for quench monitoring. The scintillation fluid was Scintiverse (Fisher). RESULTS The fractionation of the cellular incubates of both species into the radiolabeled organic phase (aglycone), aqueous phase (steroid conjugates), and macromolecular pellet (bound steroid) suggested interspecies differences. The fractionation procedure resulted in a loss of radioactivity, as might be expected. These losses were similar to those of previous experiments (Helton et al., 1977). The data (Table 1), when presented on a total radioactivity basis, demonstrated that the rat had significantly less free radioactive steroid than the mouse, and that significantly greater amounts of radioactivity were present in the rat conjugate and bound fractions compared with those of the mouse. After acid reflux the amount remaining irreversibly associated was also significantly greater for the rat. However, an additional calculation was made that provided a cellular metabolic clearance rate. This value represents the total radioactive substrate metabolically converted per 106 cells incubated during the 24 hr period. The mean value (Table 1) for the rat experiments was significantly less than that for the mouse (p < 0.05). This suggests that the mouse (per 106 cells) was more efficient in the total metabolic conversion of EE2 than the rat. However, when the covalent binding after reflux in each

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METABOLISM OF EEa BY INTACT LIVER CELLS

TABLE 1. Ethynylestradiol Metabolic Fractions of Rat and Mouse Intact Liver Parenchymal Cell Incubates Percentage of total radioactivity analyzed Cellular MCR°

Organic phase

1 2 3 4

0.029 0.082 0.047 0.052

X

0.053 ± 0.022

Experiment

Bound after acid reflux

Bound activity per 106 cells

Aqueous phase

Bound

23.2 16.5 42.4 27.1

42.0 43.2 23.8 32.5

29.5 24.8 15.7 22.9

94.7 84.5 81.9 82.5

16.3 19.6 12.2 19.4

0.109 0.392 0.243 0.243

27.3 ± 10.9

35.4 ±9.0

23.2 ±5.7

85.9 ±5.9

19.4 ±3.5

0.227 ± 0.123

88.8 93.0 74.1 87.3

10.8 10.8 14.1 5.6

0.220 0.263 0.256 0.187

Total

Rat

±1SD

Mouse 5 6 7 8 X

± 1SD

0.087 0.075 0.059 0.080

64.1 63.6 36.2 65.4

14.3 16.4 22.0 13.4

10.3 13.0 15.9 8.5

0.075 6 + 0.012

57.3C ± 14.1

16.5C ±3.9

11.9C ±3.2

85.7 ( N S ^ ± 8.2

10.3c ±3.5

0.232 (NS)rf ± 0.035

"Metabolic clearance rate calculated as EE2 metabolically cleared per 106 cells per 24 hr. b P< 0.05. 0.01. "NS, not significant compared with value for the rat.

species was expressed with respect to the total cells incubated, the mouse and rat were found to have essentially equivalent binding. In the past (Williams et al., 1975; Helton et al., 1976), EE2 has been assigned a retention volume of 1.0 on LH-20 using benzene and methanol (85:15). A relative retention volume (RRV) was provided for other identified steroids: estrone (0.55), estradiol (0.83), estriol (1.60), 2methoxy-EE2 (0.71), and 2-hydroxy-EE2 (1.85). Metabolites with RRVs similar to those of estrone (0.49-0.55), 2-methoxy-EE2 (0.71-0.79), and EE2 and estriol (1.50-1.59) were seen in the individual rat organic phase (Fig. 1). In the mouse profiles, metabolites with RRVs similar to those of estrone (0.51-0.53), estradiol (0.80-0.84), and EE2 and 2-hydroxy-EE2 (1.75-1.88) were observed. In both species a significant metabolite with an RRV of 0.20-0.24 was observed. Chromatographic purification of the control incubations (EE2 plus medium) indicated only the parent compound. The conjugate products resulting from the purification of the aqueous phase of the rat and mouse indicated interspecies differences (Fig. 2). Earlier studies (Williams et al., 1975; Helton et al., 1976) of EE2 conjugates have demonstrated that the products eluted in the first 20-25

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Metabolism of 17alpha-ethynylestradiol by intact liver parenchymal cells isolated from mouse and rat.

METABOLISM OF 17α-ETHYNYLESTRADIOL BY INTACT LIVER PARENCHYMAL CELLS ISOLATED FROM MOUSE AND RAT Edward D. Helton, Daniel A. Casciano, Zelda R. Althau...
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