Androgen-responsive functions of male rat liver

GASTROENTEROLOCY

LIVER

AND

BILIARY

1987;93:1162-9

TRACT

Androgen-Responsive Functions of Male Rat Liver Effect of Chronic Alcohol

Ingestion

PATRICIA K. EAGON, JOAN E. WILLETT, SANDRA M. SEGUITI, MARK L. APPLER, JUDITH S. GAVALER, and DAVID H. VAN THIEL Veterans Administration Medical Center and Department School of Medicine, Pittsburgh, Pennsylvania

Many liver processes are sexually dimorphic, and in rats, testosterone is the major steroid hormone determinant of the differing patterns of hepatic function. The microsomal content of specific enzymes and the syntheses of specific proteins are dependent on serum testosterone to maintain this dimorphism. Because the liver of male rats is strikingly androgen responsive, and because chronic alcohol ingestion decreases serum testosterone, we sought to determine whether chronic alcohol feeding would alter the masculine pattern of hepatic liver function in male rats. We quantitated both the cytosolic and nuclear forms of the hepatic androgen receptor. Alcohol feeding of male rats results in a significant loss of both types of androgen receptor sites; the specific binding capacity of both cytosolic and nuclear receptor in alcohol-fed rats is reduced to about 30% of that in either isocalorically fed rats or rats fed ad libitum. This reduction in hepatic androgen receptor activity is concomitant with a 50% reduction in serum testosterone content in the alcohol-fed animals. In addition, the activities of two hepatic androgen-responsive proteins, namely a cytosolic Received March 27, 1986. Accepted June 22, 1987. Address requests for reprints to: Patricia K. Eagon, Ph.D., Veterans Administration Medical Center, University Drive C, Pittsburgh, Pennsylvania 15240. This work was supported in part by the Veterans Administration and grants AM 30001, AM 31577, and AA06971 (P.K.E.) and AA 04425 and AA 06601 (D.H.V.T.) from the National Institutes of Health. The authors thank Ingrid Kuo for the preparation of the art work and Elaine Rosenblum for her expertise in generation of graphs on the DEC-10. The authors are especially grateful to Dr. Clifford Pohl for his assistance in the determinations of serum testosterone and luteinizing hormone content. They also thank Elizabeth JahnkeSpinnenweber for editorial assistance and Patricia Stafford for preparation of the manuscript. 0 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

of Medicine,

University

of Pittsburgh

estrogen binder and a microsomal enzyme, estrogen &hydroxylase, demonstrate a decrease in activity that parallels the decreases in both forms of the androgen receptor. Administration of testosterone to the alcohol-fed animals normalized both the hepatic androgen receptor and the androgen-responsive protein activities. From these results, we conclude that chronic alcohol feeding results in a decreased androgen responsiveness of the liver, a condition that most likely results from the decreased serum testosterone levels in the alcohol-fed animals.

The microsomal content of hepatic steroid and drug metabolizing enzymes is distinctly sexually dimorphic (l-4). Male rats have higher levels of many oxidative microsomal enzymes than do females. For example, the liver of male rats has about seven times more microsomal estrogen 2hydroxylase (E2OHase) activity than does that of females (5). The high activity of this enzyme in male rat liver may be required to metabolize estrogens rapidly, inasmuch as excess estrogens could compromise the sexual integrity of the male. In contrast, female rats have higher levels of 5a-reductase, a major enzyme in testosterone clearance (3). Androgen appears to be the major steroid hormone determinant of the “masculine” pattern of hepatic function (reviewed in Reference 2). Most of the activities elevated in the liver of male rats require androgen imprinting, i.e., a brief surge of androgen during the prenatal or neonatal period, to realize full expression in adulthood (5-7). Some of these imprinted functions require the constant presence of serum androgen after puberty, Abbreviations used in this paper: AF, alcohol-fed; libitum; EZ-OHase, estrogen 2-hydroxylase; IC, isocaloric MEB, male-specific estrogen binder.

AL, ad control;

December 1987

whereas others retain partial activity even after castration of the adult male rat (6). The liver also produces male-specific proteins in response to androgen. One of these, an unusual cytosolic male-specific estrogen binder (MEB), has been described by several groups (8-10)as well as our own (11-14). This protein has moderate affinity (Ko = 30 nM) for estradiol (E,), high binding capacity, and specificity for steroidal estrogens. Its exact function is unknown, although we have hypothesized that it serves to sequester estrogens and estrogenie metabolites. Male-specific estrogen binder is virtually undetectable in liver cytosol prepared from female rats (5,111. A second male-specific protein, cY,,-globulin, has been characterized extensively (15,16); this protein of unknown function is synthesized by the liver in response to androgen, secreted into the blood, and excreted by the kidneys. In spite of these very pronounced androgen effects on this tissue, very little is known about the androgen receptor in male rat liver. A recent report has documented the presence of androgen receptor in human liver (17). In contrast, hepatic estrogen receptor is well characterized in both male and female rats (11,12,18-21) as well as in humans (22). We have reported recently the presence of an androgen binder in both cytosolic and nuclear fractions of male rat liver that has the high affinity, low capacity, and steroid specificity characteristic of an androgen receptor (23-25). Preliminary evidence suggests that the activity of this receptor in the liver and that of two androgen-responsive hepatic proteins, MEB and E2-OHase, are maintained by serum androgen, as castration results in virtually complete loss of these activities (5,24,25). Chronic alcohol ingestion in men produces significant endocrine dysfunction. Alcoholic men are frequently hypogonadal, with low serum testosterone, reduced testicular mass, decreased libido, and impotence as common findings (reviewed in Reference 26). This hypogonadism can be explained to a great extent by the action of alcohol as a testicular toxin (27). In addition, these men are often feminized, in spite of relatively normal serum levels of Ez. However, serum levels of the weaker estrogens, estrone and estriol, are usually elevated in such men (26). Because of the observation of reduced serum testosterone in these men and because the androgen receptor in male rat liver is androgen-dependent, we wished to determine whether chronic alcohol ingestion, using a rat model of alcohol feeding, would result in alterations in hepatic androgen receptor content and in the hepatic content of androgenresponsive proteins. In this report we demonstrate that chronic alcohol feeding of male rats results in a significant reduction in both cytosolic and nuclear

ALCOHOL AND HEPATIC ANDROGEN RECEPTORS

androgen receptor, as well as a reduction cytosolic MEB and microsomal E2-OHase.

1163

in both

Materials and Methods Animals Thirty male Wistar rats, 28 days of age, were obtained from Charles River Breeding Laboratories, Wilmington, Mass. The rats were weighed upon arrival and paired according to weight. Starting at 30 days of age, the rats were pair-fed either a liquid diet containing 5% ethanol (vol/vol), equivalent to 36% of total calories, [alcohol-fed (AF) group] or a diet containing dextrimaltose and isocalorically matched to the alcohol-containing diet [isocaloric control (IC) group] (12,13).The animals were killed after 60-75 days of feeding. This time span before death was necessary because of the number of animals and the number of assays to be performed on each animal; no differences in results could be assigned to the order of death. The paired animals from the AF and IC groups were always killed on the same day. In addition, six pairs of AF and IC animals received subcutaneous implants containing crystalline testosterone (5) 2 wk before death. These animals so treated remained on their respective diet regimens. Any animals appearing sick or otherwise abnormal were excluded from the study. Other age-matched rats were allowed water and a standard rat diet ad libitum (AL group). All animals were maintained on a normal 12 h light/dark cycle.

Materials Radioactive 17a-methyl-[3H]methyltrienolone ([3H]R1881), 87 Ci/mmol, nonradioactive R1881, and Econofluor scintillation fluid were purchased from New England Nuclear, Boston, Mass. Radioactive [3H-methyl] S-adenosyl methionine, 15 Ciimmol, and Aqueous Counting Scintillant were purchased from Amersham, Arlington Heights, Ill. The purity of radiolabeled compounds was assessed periodically by thin-layer chromatography (11,22). The liquid diets were obtained from BioServe. Sources of other substances were as previously described (11,221.

Buffers Unless otherwise stated, all experiments were performed at 0”-4”C using the following buffers: 0.01 M Tris HCl, 1.5 mM ethylenediaminetetraacetic acid, pH 7.4 (TE buffer); TE buffer with 5 mM dithiothreitol (TED buffer); TE buffer with 20 mM sodium molybdate (TEM buffer); TE buffer with 0.25 M sucrose (TES buffer); TE buffer with both of the latter additions (TEMS buffer); 0.25 M sucrose, 3 mM MgC12, 10 mM HEPES, pH 7.4 (SMgH buffer); and SMgH with 20 mM sodium molybdate, pH 7.4 (SMgHM buffer). Leupeptin (0.15 mM) and benzamidine (1.0 mM) were added to all buffers used in preparation of nuclei and cytosol, and to those used for gel filtration chromatography.

1164

EAGON ET AL.

Preparation

GASTROENTEROLOGY

of Subcellular

Fractions

To prepare all subcellular fractions from the same liver, the liver was homogenized in 3 volumes of TES. The nuclei were sedimented by centrifugation of the homogenate at 800 g for 15 min. The crude nuclear pellet was washed five times by resuspension of the pellet in SMgHM buffer and recentrifugation. The final pellet was suspended in SMgHM buffer to a volume equal to that of the original homogenate. Nuclei so prepared appeared rounded under light microscopy and stained blue with Mayer’s hematoxylin and eosin. The final nuclear preparation contained no detectable cytosolic contamination as judged by lack of specific cytosolic staining (above) and by assay for alcohol dehydrogenase activity (28). In addition, microsomal contamination of the nuclear preparation was assessed by measuring glucose-6-phosphatase activity (29) in the homogenate and the nuclear preparation, and was found to be on average 6%-9%. Plasma membrane contamination of the nuclear preparation was assessed by measuring 5’-nucleotidase activity (30) in the homogenate and the nuclear preparation, and was found to be on average 7%-10%.

The supernate from the crude nuclear pellet was used for the preparation of cytosol and microsomes. The supernate was centrifuged at 27,000 g for 15 min; the pellet was discarded. The decanted supernate was centrifuged at 150,000 g for 30 min. The supernate from this step was considered to be the cytosolic fraction. Sodium molybdate (20 mM) was added immediately after the final centrifugation to the portion of cytosol to be used for receptor determinations because this salt is critical for maintenance of androgen binding activity. The microsomal pellet was washed by resuspension in SMgH, centrifuged again as above, and resuspended and subsequently assayed in SMgH containing 0.1 mM dithiothreitol. The final protein concentration of the microsomal suspension was typically 68 mgiml. Steroid

Binding

Assays

in Cytosol

Cytosol prepared as described above was diluted with one volume of TEM buffer before use. All incubations for determination of androgen receptor included 500 nM triamcinolone acetonide to block any contribution of the glucocorticoid receptor to [3H]R1881 binding. For quantitation of the cytosolic receptors, aliquots (200 ~1) of cytosol were incubated overnight at 4°C with 0.2-5.0 nM [3H]R1881 in the absence (total binding) and presence (nonspecific binding) of a 100-fold excess of unlabeled Rl881. The difference between these two values was considered specific binding. Bound steroid was separated from free steroid at the end of the incubation period using dextran-coated charcoal as described previously (11,22). Equilibrium dissociation constants and the concentration of binding sites were calculated by the method of Scatchard (31). The ligand commonly used in other tissues, [3H]dihydrotestosterone, could not be used in liver because of extensive metabolism of this ligand by the liver; however, no metabolism of [3H]R1881 could be detected under the conditions of our assays. R1881 has been used as a ligand for androgen receptors in classical sex hormone-

Vol. 93, No. 6

dependent tissues such as prostate (32,331. The specificity of R1881 as an androgenic ligand in rat liver has been determined (23). Nuclear

Binding

Assays

Because of the large number of animals in the alcohol feeding study, it was impossible to perform saturation curve analysis of nuclear steroid binding for each animal. To determine the best concentration of steroid for a one-point assay, aliquots (200 ~1) of nuclear suspensions from livers of several normal male and castrated male rats were incubated with 0.2-10 nM [3H]R1881 and 1 PM triamcinolone acetonide at 4°C overnight in the absence (total binding) and presence (nonspecific binding) of a 100-fold excess of unlabeled R1881. Both normal and castrated males were used to provide a range of high and low values for nuclear androgen receptor. Specific binding was calculated as the difference between total and nonspecific binding. The assays were terminated by centrifugation at 800 g, followed by washing the nuclear pellet five times with cold SMgHM buffer to remove unbound steroid. The washed pellet was then extracted with 2 ml of ethanol for 2 h at 30°C; the entire pellet and extract were transferred to a 20-ml scintillation vial and 8 ml of Aqueous Counting Scintillant was added. The single concentration at which the specific binding best agreed with the binding value calculated from the saturation curve analysis data using all points was 5 nM [3H]R1881 (r = 0.92); these conditions minimized nonspecific binding while specific binding was saturating. All multipoint determinations demonstrated equilibrium dissociation constants that are consistent with that of a receptor (0.5-0.8 nM). Thus, all nuclear androgen receptor assays in this study in preparations from AF, IC, or AL animals employed a triplicate one-point determination using 5 nM [3H]R1881 with and without a 100-fold excess of unlabeled ligand and an incubation of 16 h at 0°C. Assay

of Androgen-Responsive

Hepatic

Proteins Assays for the determination of cytosolic activity of MEB and of microsomal E2-OHase activity have been described previously (5). Briefly, MEB was separated from other cytosolic estrogen binding proteins by gel filtration chromatography in TED buffer on Sephadex G-100, followed by incubation of the fractions with a saturating dose of [3H]EZ (5 nM) at 4°C overnight. This assay is quantitative for MEB, and is linear with protein concentration over a broad range including that of the column fractions. Microsomal E2-OHase was assayed by the method of Paul et al. (34) with modifications described previously (5). Briefly, estrone was used as substrate for E2-OHase. The product of E2-OHase activity on estrone, 1,3,5(10)estrien-17-one-2,3-diol, was labeled in a second reaction at the &position with a tritiated methyl group. The tritiated methyl group was transferred from [3H]methyl Sadenosylmethionine by partially purified catechol-O(35,361 to form [3H]-2-methoxymethyltransferase 1,3,5(10)-estrien-17-one-3-01 ([3H]-2-Me0-E1). The activity of EZ-OHase is proportional to the rate of formation of the

ALCOHOL AND HEPATIC ANDROGEN RECEPTORS

December 1987

Figure

1. Specific binding of [3H]R1881 in male rat liver cytosol. A. Cytosol was prepared from an alcohol-fed animal (0) and an isocalorically fed control (01, and incubated with various concentrations of [3H]R1881 in the absence and presence of a loo-fold excess of unlabeled R1881 for 16 h at 4°C. Each point is the average of two determinations. The unbound steroid was removed by dextran-coated charcoal treatment. B. A Scatchard plot of the specific binding values displayed in A.

activity is lower in cytosol from the AF animal (3.5 fmol/mg protein) than that from the IC animal (13.0 fmol/mg protein). The observed difference between the AF and IC animals persists when similar results are displayed for a larger group of animals (Figure 2A). For nine such pairs of animals, the specific [3H]R1881 binding activities were 3.2 + 1.2 vs. 9.5 + 1.7 fmol/mg cytosolic protein (mean -+ SEM, p < 0.01) for the AF and the IC groups, respectively. The dissociation constants were not different between the two groups (1.9 k 0.9 vs. 1.9 k 0.8 nM, respectively). Age-matched rats fed ad libitum had cytosolic androgen receptor content comparable to the IC animals, having a specific binding activity of 9.8 k 1.9 fmol/mg protein and equilibrium dissociation constants of 0.5 nM ‘_ 0.1 nM.

Androgen tritiated termined

was deproduct. The quantity of [3H]-2-Me0-E, by two-phase scintillation counting (5).

Other Methods Protein concentrations were determined by the method of Lowry et al. (37) or Bradford (38) as modified by Seeley et al. (39). Deoxyribonucleic acid concentrations of homogenates and nuclear preparations were determined by the method of Kissane and Robins (40). Radioimmunoassays for serum Ez and testosterone determinations were described previously (12,13). Unweighted linear regression analyses of Scatchard plots and standard curves were performed on a Texas Instrument T155 calculator (Texas Instruments, Dallas, Tex.). Statistical analyses were performed using Student’s t-test program available on the Hewlett Packard 98158 (Hewlett-Packard, Palo Alto, Calif.). Radioactivity content of samples was determined using a Packard TriCarb 4530 with automatic disintegrations per minute conversion. Aqueous Counting Scintillant was used for single-phase scintillation counting. Econofluor scintillant with an acidified aqueous phase (5) was used for two-phase scintillation counting.

1165

Receptor

in Nuclei

The binding of [3H]R1881 in liver nuclei prepared from these groups of animals was assayed by an exchange assay employing incubation of washed nuclei with 5 nM [3H]R1881 for 16 h at O”C, as described in Methods. Figure 2B indicates that the hepatic nuclei prepared from the AF group have a significantly lower content of specific androgen binding, 8.6 k 3.6 vs. 27 t 5.9 fmol/lOO pg deoxyribonucleic acid (p < 0.05) for the AF and IC groups, respectively. Again, the AL group was comparable to, but somewhat lower than, the IC group (20 2 11 fmol/lOO E.cgdeoxyribonucleic acid).

B

1

t

Results Androgen

Receptor

in Cytosol

When cytosolic fractions prepared from the liver of an AF rat and its pair-fed IC rat were incubated with varying concentrations of [3H]R1881, saturable binding of this androgen to cytosolic protein was observed, as shown in Figure 1A. The Scatchard plot of specific binding values in Figure 1B indicates that the affinity of binding is similar in these two preparations, with an equilibrium dissociation constant of 0.96 nM and 0.46 nM for the AF and the IC animal, respectively. The specific binding

IC

IC

Figure 2. Effect of chronic alcohol feeding on the level of cytosolic and nuclear androgen receptors in rat liver. Male rats were fed either an alcohol containing (AF) or an isocalorically matched (IC) diet. Androgen receptors in cytosol (A) were quantitated by the assays shown in Figure 1, and nuclei (B) using the exchange assay described in Methods.

1166

EAGON

Table

GASTROENTEROLOGY

ET AL.

of Alcohol Feeding on the Total Hepatic Content of Androgen Receptor

Effect of Androgen

1. Effect

Androgen receptor content lfmol [‘H]R1881 bound/g liver) Animal

treatment

Alcohol fed (n = 9) Isocalorically fed (n = 9) Ad libitum fed (n = 6) Alcohol fed, testosterone treated (n = 6) Isocalorically fed, testosterone treated (n = 6) ‘Different from isocaloric from isocaloric control isocaloric control group, control group, p < 0.05.

Cytosolic

Nuclear

Total

230 2 85” 53’2 rt 101

141 t 54" 457 2 126

371 + 80" 1095 ?I 172

401 f 120 130 k 33”

294 2 142 528 k 134

695 " 136" 658 + 122"

167 ‘- 66”

582 2 259

749 + 161"

control group, p < 0.01. ‘Different group, p < 0.025. ‘Different from p < 0.005. d Different from isocaloric

Table 1 compares the content of cytosolic and nuclear androgen receptor in these groups expressed as receptor content per gram of liver. It is obvious that livers of the AF group have considerably less androgen receptor activity than livers from either the IC or AL groups. Because androgen is active in maintaining its own receptor, we measured the serum testosterone in these three groups of animals. As shown in Table 2, the AF animals have a 50% reduction in serum testosterone as compared with either the IC (p < 0.05) or AL (p < 0.01) animals. The AF animals also demonstrate reduced testicular weights (p < 0.005) as compared with either the IC or AL animals (Table 2). In general, the AL animals were larger with respect to body and liver weight, as these animals were not restricted in food intake as were the IC animals. These animals also have the highest serum testosterone levels of the three groups (Table 21, perhaps also as a result of unrestricted food intake. However, these animals have total hepatic AR values somewhat lower than those of the IC group (Table 1). The reason for this latter finding is unknown.

Table 2. Effect of Chronic Alcohol

lngestion

on Serum

Androgen-Responsive

Testosterone

and Weights Body weight

(ng/mU

Alcohol fed (n = 9) Isocalorically fed (n = 9) Ad libitum fed (n = 6) Alcohol fed, testosterone treated (n = 6) Isocalorically fed, testosterone treated (n = 6) ’ Different from isocaloric group, p < 0.025.

control

group,

1.02 C 2.02 + 3.34 f 5.74 2 6.63 t

p < 0.05. b Different

Proteins

In addition to the effect on the cytosolic and nuclear androgen binding activities described above, the effect of chronic alcohol feeding on the activities of two androgen-responsive hepatic proteins was determined. Figure 3A displays the results of the quantitation of the cytosolic content of MEB; Figure 3B shows the activity of the microsomal enzyme E2-OHase. In both cases, chronic alcohol feeding results in a significant decrease in the hepatic content of both of these androgen-responsive proteins. The values for these activities in the AL group, 403 t 66 fmolimg protein for MEB activity, and 0.49 + 0.03 nmol/min . mg protein for E2-OHase activity, are virtually identical to those of the IC group. Androgen repletion also resulted in a normalization of the activities of these two proteins in the AF animals. After 2 wk of testosterone administration, MEB activity in the AF group was 337 t 70 fmol/mg protein, and in the IC group was 313 + 44 fmol/mg

Serum treatment

Repletion

The importance of androgen in maintaining hepatic activity of these proteins was addressed by determining whether androgen administration to the AF animals would result in a reversal of these effects. Six pairs of AF and IC animals were given testosterone implants 2 wk before death. These animals achieved serum testosterone levels comparable to, but slightly higher than, AL animals (Table 2). Total androgen receptor activity in both the AF and IC animals was identical to that of the AL animals. However, the nuclear form of the receptor predominated in both groups of animals, a finding consistent with the increased nuclear retention usually observed after administration of a steroid hormone. Testosterone treatment did not affect liver or body weights, but did result in decreased testicular weights in both groups (Table 2). The reduction in testes weights most likely results from reduced or absent hypothalamic/pituitary stimulation of the testes; luteinizing hormone levels in these animals were undetectable (data not shown).

testosterone Animal

Vol. 93, No. 6

0.26” 0.42 0.49" 0.91b 1.04”

from isocaloric

(gl 294 312 403 299 317 control

+ -e + 2 +

of Body, Liver, and

Testes

Liver weight

Testes weight

(&!I 10” 14 lib 14 13

group,

(g)

11.6 -t 0.5b 9.1 -t 0.6 14.6 t 0.7b 11.3 -t 0.9" 9.2 r+ 0.5 p i 0.005. ’ Different

2.80 k 3.36 + 3.32 k 2.57 k 3.01 +

from isocaloric

O.lb 0.1 0.1 O.lb O.lb

control

December 1987

ALCOHOL AND HEPATIC ANDROGEN RECEPTORS

t

AF Figure

3.

IC

AF

Effect of chronic alcohol feeding on the of two androgen-responsive proteins, OHase. The activity of cytosolic MEB somal EZ-OHase (B) were determined male rats fed alcohol (AF) or a control the assays described in Methods.

IC hepatic activity MEB and EZ(A) and microin the livers of diet (IC) using

protein. Estrogen 2-hydroxylase activity was 0.40 k and 0.63 ? 0.01 nmobmin . mg protein for the testosterone-treated AF and IC groups, respectively. 0.01

Discussion The observation that chronic alcoholic men are frequently hypogonadal prompted us to determine whether the low serum testosterone levels common to this condition might be accompanied by a decreased androgen response in the liver. To this end, we used a rat model of chronic alcohol feeding. Because androgenic effects in the liver may be mediated by specific androgen receptors, we determined the effect of alcohol feeding on hepatic content of androgen receptor. This study demonstrates that alcohol ingestion results in a significant decrease in both cytosolic and nuclear forms of the androgen receptor in male rat liver. Likewise, the activities of two androgen-responsive hepatic proteins, cytosolic MEB and the microsomal enzyme EZ-OHase, are reduced similarly by chronic alcohol feeding. These findings, coupled with our previous observation of increased hepatic estrogen receptor activity in such rats (l2), demonstrate that chronic alcohol ingestion results in a feminization of hepatic sexually dimorphic liver function. The reduction in androgen receptor content of the liver is paralleled by a reduction in serum testosterone in the AF group. The low serum testosterone levels observed in the AF animals are likely to be a primary factor in the reductions in the androgen

1167

receptor, MEB, and E2-OHase. The activity of these proteins appears to be androgen-dependent, as castration of male rats results in a time-dependent loss of these proteins (5,24,25, and unpublished results). Furthermore, treatment of the AF animals with testosterone restores the level of these androgenresponsive proteins to that of control animals. Our results do not distinguish whether the reduction of hepatic androgen receptors is the cause of the reduction in the androgen-responsive proteins MEB and E2-OHase, or whether the receptor merely decreases in parallel with the decrease in the activity of these proteins. It is difficult to determine which is the case, because evidence to date suggests that both the receptor and these proteins probably are controlled by similar mechanisms. However, it seems likely that the hepatic androgen receptor plays some role in mediation and modulation of those hepatic processes requiring the presence of steroid for expression or enhancement of activity. Reduction in hepatic activity of E2-OHase and MEB should contribute to an increase in serum E2 in the AF animals. Indeed, previous work has shown that the AF group has both higher serum Ez and elevated estrogen receptor activity (12). Another factor that might contribute to the elevated serum estrogens in these animals is an increased conversion of androgens to estrogens by aromatase, as has been shown in cirrhotic men (41). Therefore, the increased serum estrogens may also play an important role in the observed hepatic feminization in the AF male rats. It is unknown at this time whether the alcoholinduced alterations in hepatic receptor and enzyme levels occur as an effect of the imbalance of androgens and estrogens on the liver directly. Alternately, or perhaps additionally, the effects may be on the hypothalamus or pituitary gland, or both. Evidence is accumulating that the sexually dimorphic pattern of growth hormone release by the pituitary (42,43) is a primary factor regulating maintenance of sexually dimorphic aspects of liver function (44). It is well established that alcohol is a testicular toxin and chronic ingestion results in reduction of serum testosterone. It is possible that these animals are incapable of maintaining the masculine pattern of growth hormone secretion as a result of low serum testosterone, and thus cannot maintain the masculine pattern of hepatic function. It is also possible that the liver alone cannot maintain the sexually dimorphic pattern in the absence of sufficient testosterone. An additional factor in the feminization of hepatic function in the AF animals may be the observed increase in serum Ez in this group (12), as administration of Ez to male rats can feminize both growth hormone release (42)and liver function

1168

EAGON

ET AL.

Wherever the defect lies, the inability of the AF male rat to metabolize estrogens and other steroids rapidly may compound the sexual dysfunction. Not only does the animal remain hypogonadal because of the testicular toxicity of alcohol, but it may also become progressively more feminized because of the inability to metabolize estrogens quickly. Although liver injury may play a role in the alcohol-induced reduction in these proteins, it is unlikely that liver injury is the predominant cause, inasmuch as AF animals display histologically normal or only mildly fatty livers (45). Further, alcohol feeding does not produce a generalized reduction in protein synthesis in the liver, but rather a decrease in secretion of certain serum glycoproteins (46,47). The imbalance of serum androgens and estrogens in the AF animals is likely to be the most important factor in the feminization of hepatic function, although it is certainly possible that undetectable damage to the liver may help compound the biochemical changes observed in these animals. (7).

References 1. Colby HD. Regulation of hepatic drug and steroid metabolism by androgens and estrogens. In: Thomas JA, Singal RL, eds. Advances in sex hormone research. Baltimore; Urban and Schwartzenberg, 1980:27-71. 2. Bardin CW, Catteral JF. Testosterone: a major determinant of extragenital sexual dimorphism. Science 1981;211:1285-94. 3. Gustafsson JA, Mode A, Norstedt G, et al. The hypothalamopituitary-liver axis: a new hormonal system in the control of hepatic steroid and drug metabolism. In: Litwack G, ed. Biochemical actions of hormones. Vol VII. 1980:47-89. 4. Eagon PK, Porter LE, Francavilla A, DiLeo A, Van Thiel DH. Estrogen and androgen receptors in liver: their role in liver disease and regeneration. Semin Liver Dis 1985;5:59-69. 5. Turocy JF, Chiang AN, Seeley DH, Eagon PK. Effects of H, antagonists on androgen imprinting of male hepatic functions. Endocrinology 1985;117:1953-62, 6. Einarsson K, Gustafsson J-A, Stenberg A. Neonatal inprinting of liver microsomal hydroxylation and reduction of steroids. J Biol Chem 1973;248:4987-97. 7. Gustafsson J-A, Mode A, Norstedt G, Skett P. Sex steroid induced changes in hepatic enzymes. Ann Rev Physiol1983: 45:51-60. 8. Dickson RB, Aten RF, Eisenfeld AJ. An unusual sex steroidbinding protein in mature male rat liver cytosol. Endocrinology 1978;103:1636&46. 9. Miroshnichenko ML, Smirnova OV, Smirnov AN, Rozen VB. The unusual estrogen binding protein (UEBP) of male rat liver: structural determinants of ligands. J Steroid Biochem 1983;18:403-9. 10. Thompson C, Lucier GW. Hepatic estrogen responsiveness. Possible mechanisms for sexual dimorphism. Mol Pharmacol 1983;24:69-76. 11. Eagon PK, Fisher SE, Imhoff AF, et al. Estrogen-binding proteins of male rat liver: influences of hormonal changes. Arch Biochem Biophys 1980;201:486-99. 12. Eagon PK, Zdunek JR, Van Thiel DH, et al. Alcohol induced changes in hepatic estrogen-binding proteins: a mechanism explaining feminization in alcoholics. Arch Biochem Biophys 1981;211:48-54.

GASTKOENTEKOLOGY

Vol. 93, No. 6

13. Eagon PK, Porter LE, Gavaler JS, Egler KM, Van Thiel DH. Effect of ethanol feeding upon levels of a male specific hepatic estrogen-binding protein: a possible mechanism for feminization. Alcoholism: Clin Exp Kes 1981;5:183-7. 14. Rogerson BJ, Eagon PK. A male specific hepatic estrogen binding protein: characteristics and binding properties. Arch Biochem Biophys 1986;250:70-85. 15. Kurtz DT, Feigelson P. Multihormonal regulation of the messenger RNA for the hepatic protein a,,,-globulin. In: Litwack G, ed. Biochemical actions of hormones. Vol. V. 1978:433-55. 16. Roy AK. Hormonal regulation of a,,,-globulin in liver. In: Litwack G, ed. Biochemical actions of hormones. Vol. VI. 1979:481-517. 17. Bannister P, Sheridan P, Losowsky MS. Identification and characterisation of the human hepatic androgen receptor. Clin Endocrinol 1985;23:495-502. 18. Powell-Jones W, Thompson C, Nayfeh SN, Lucier GW. Sex differences in estrogen binding by cytosolic and nuclear components of rat liver. J Steroid Biochem 1980;13:219-29. 19. Norstedt G, Wrange 0, Gustafsson JA. Multihormonal regulation of the estrogen receptor in rat liver. Endocrinology 1981;108:1190-6. 20. Eriksson HA. Estrogen binding sites of mammalian liver: endocrine regulation of estrogen receptor synthesis in the regenerating rat liver. J Steroid Biochem 1982;17:471-7. 21. Kneifel R, Katzenellenbogen BS. Comparative effects of estrogen and antiestrogen on plasma renin substrate levels and hepatic estrogen receptors in rats. Endocrinology 1981;108: 545-52. 22. Porter LE, Elm MS, Van Thiel DH, Dugas MC, Eagon PK. Characterization and quantitation of human hepatic estrogen receptor. Gastroenterology 1983:84:704-12. A, Eagon PK, DiLeo A, et al. Circadian rhythm of 23. Francavilla hepatic cytosolic and nuclear estrogen and androgen receptors. Gastroenterology 1986:91:182-8. BJ. Androgen 24. Eagon PK. Willett JE, Seguiti SM, Kogerson receptor in male rat liver (abstr). Presented at the Seventh International Congress of Endocrinology, July l-7, 1984, Quebec City, Quebec (abstract 777). SM, Willett JE, Rogerson BJ. Hepatic 25. Eagon PK, Seguiti androgen receptor: effects of hormonal alteration (abstr). Hepatology 1985;5:1046. 26. Eagon PK, Porter LE, Van Thiel DH. The role of estrogens and androgens Alcoholism: 27. Chiao

Thiel

DH.

Biochemical

alcoholic

male.

mechanisms

to alcohol-induced hypogonadism Clin Exp Kes 1983;7:131-9.

BL, Hoch

dehydrogenase. 29. Swanson

of the chronic

Clin Exp Res 1983;7:140-3.

Y-B, Van

contribute Alcoholism: 28. Vallee

in the feminization

FL. Zinc,

a component

that

in the of yeast

male. alcohol

Proc Nat1 Acad Sci USA 1955;41:327-32.

MA. Glucose-6-phosphatase

from liver.

Methods

Enzymol 1956;2:541-6. 30. Widnell CC, Unkeless JC. Partial purification of a lipoprotein with 5’-nucleotidase activity from membranes of rat liver cells. Proc Nat Acad Sci USA 1968;61:1050-7. 31. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949;46:660-72. 32. Bonne C, Raynaud JP. Methyltrienelone, a specific ligand for cellular androgen receptors. Steroids 1975;26:227-32. 33. Bonne C, Raynaud JP. Assay of androgen binding exchange 497-507.

with methyltrienelone

(R1881).

Steroids

sites by 1976;27:

34. Paul SM, Axelrod J, DiLiberto EJ. Catechol estrogen-forming enzyme of brain: demonstration of a cytochrome P450 monooyygenase. Endocrinology 1977;101:1604-10.

1169

December 1987

ALCOHOL AND HEPATIC ANDROGEN RECEPTORS

35. Axelrod J, Tomchick R. Enzymatic 0-methylation of epinephrine and other catechols. J Biol Chem 1954;233:702-5. 36. Nikodejevic B, Senoh S, Daly JW, Creveling CR. Catechol-Omethyltransferase. II. A new class of inhibitors of catechol0-methyltransferase; 3,5-dihydroxy-4-methoxybenzoic acid and related compounds. J Pharmacol Exp Ther 1970;174:83-

androgens to estrogens in cirrhosis of the liver. J Clin Endocrinol Metab 1975;40:1018-26. Eden S. Age and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 1979;105: 555-64. Jansson J-O, Eden S, Isaksson 0. Sexual dimorphism in the control of growth hormone secretion. Endocr Rev 1985;6:12950. Norstedt G, Palmiter R. Secretory rhythm of growth hormone regulates sexual differentiation of mouse liver. Cell 1984;36: 805-12. Van Thiel DH, Gavaler JS, Cobb CF, Sherins RJ. Alcohol induced testicular atrophy in adult male rats. Endocrinology 1979;105:888-95. Sorrel1 MF, Nauss JM, Donohue TM, Tuma DJ. Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat. Gastroenterology 1983;84:560-6. Volentine GD, Tuma DJ, Sorrel1 MF. Subcellular location of secretory proteins retained in the liver during the ethanolinduced inhibition of hepatic protein secretion in the rat. Gastroenterology 1986;90:158-65.

42.

43.

93. 37. Lowry OH, Rosenbrough

38.

39.

40.

41.

NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. Seeley DH, Wang WY, Salhanick HA. Molecular interactions of progesterone analogues with rabbit uterine cytoplasmic recept0r.J Biol Chem 1982;257:13359-66. Kissane JM, Robins E. A fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J Biol Chem 1958;233:184-8. Gordon GG, Olivo J, Rafii F, Southren AL. Conversion of

44.

45.

46.

47.