Microsomal ethanol-oxidizing system in purified rat Leydig cells

Biochimica et Biophysics Acru 918 (1987) 136-140 Elsevier

136

BBA 52508

Microsomal ethanol-oxidizing Eisuke P. Murono

system in purified rat Leydig cells and Vi&i Fish&-Simpson

Medical Research Service, WJB Darn Veierans’ Hospital and Deportments of Medicine and Physiology, University of SouthCarolina School of Medicine, Columbia, SC 29201 (U.S.A.) (Received

Key words:

Ethanol

metabolism;

1 August

1986)

Ethanol-oxidation;

Microsome;

(Rat Leydig cell)

These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 f 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 f 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent K, for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent K, for NADPH was 11 PM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.

Introduction Ethanol has been reported to acutely reduce circulating testosterone levels in rodents [1,2] and to directly inhibit testosterone accumulation in isolated testes [3] or interstitial cells [4-61. This decrease has been ascribed, in part, to the presence of NAD+-dependent alcohol dehydrogenase activity in Leydig cells which metabolizes ethanol to acetaldehyde [4-71, and thereby reduces the NAD+/NADH ratio. This is proposed to inhibit NAD+-dependent A5-3jShydroxysteroid dehydrogenase-isomerase activity (3j%hydroxy-AS-steroid : NAD+ 3-oxidoreductase, EC 1.1.1.145) [4-81, which converts pregnenolone to progesterone. The increased availability of NADH also is proposed to stimulate &androstan-3/3-hydroxysteroid deCorrespondence: E.P. Murono, Department of Medicine, Bldg 28, Dom Veterans’ Hospital, Columbia, SC 29201, U.S.A.

0005-2760/87/$03.50

0 1987 Elsevier Science Publishers

hydrogenase activity which converts 5cy-dihydrotestosterone to Sa-androstan-3fi,17fi-diol[9]. Other studies suggest that generation of acetaldehyde by alcohol dehydrogenase (alcohol : NAD+ oxidoreductase, EC 1.1.1.1) is the key step because acetaldehyde mimics the effects of ethanol in reducing testosterone [5,10], and the effects of alcohol can be prevented by adding, an alcohol dehydrogenase inhibitor, 4-methylpyrazole [ll]. However, others question the role of acetaldehyde in mediating the effects of ethanol [12,13]. Previous studies have shown that ethanol inhibits progesterone-stimulated testosterone accumulation in rat testis interstitial cells [6,14]. Therefore, a step(s) subsequent to progesterone formation also must be affected by ethanol. Elevation of testosterone precursors when interstitial cells are incubated with ethanol or acetaldehyde using progesterone as substrate suggested that 17-ketosteroid reductase activity (17/Shydroxysteroid:

B.V. (Biomedical

Division)

137

NADP+ 17_oxidoreductase, EC 1.1.1.64) which converts androstenedione to testosterone is inhibited by ethanol or acetaldehyde [14]. We confirmed these effects of ethanol, and further showed that the decrease in testosterone was due to direct inhibition of NADPH-dependent 17-ketosteroid reductase activity and/or to enhanced conversion of testosterone to androstenedione, which is catalyzed by NADP+-dependent 17/3-hydroxysteroid dehydrogenase activity [15]. This reciprocal change in enzyme activities by ethanol suggested that reduced testosterone might be due to an ethanol-induced increase in the NADP+/ NADPH ratio. A microsomal ethanol-oxidizing system has been identified in liver which metabolizes ethanol to acetaldehyde and is dependent on NADPH [16]. The presence of a similar system in Leydig cells might, in part, explain the pattern of steroid accumulation effected by ethanol. These studies were conducted to determine whether a similar microsomal system involved in ethanol metabolism is present in Leydig cells. Methods Animals and materials Sprague-Dawley rats (50-70 days old) were obtained from Zivic-Miller Laboratories, Allison Park, PA. Collagenase (Type I), nicotinamide, sodium azide, semicarbazide, bovine serum albumin, 4-methylpyrazole, NADPH, NADP+, DNA (calf thymus, Type I), cytochrome c (horse heart, Type III), and rotenone were from Sigma Chemical Co. (St. Louis, MO). NADf and NADH were from ICN Nutritional Biochemicals. Potassium cyanide, acetaldehyde and diphenylamine were from Fisher Scientific Company (Atlanta, GA). Metrizamide was from Accurate Chemical and Scientific Corp. (Westbury, NY). Medium 199 was from Grand Island Biological Company (Grand Island, NY). Isolation of Leydig Cells Interstitial cells were dispersed from decapsulated testes using Medium 199 containing 0.1% bovine serum albumin and 0.025% collagenase [17]. Leydig cells were isolated by centrifugation of these cells over a O-32% continuous metrizamide gradient as described by Conn et al. [18] and

Payne et al. [19], but with modifications [20]. Routinely, 60-80% of the third band of cells isolated using this procedure stained positively for 3P-hydroxysteroid dehydrogenase activity, a marker for Leydig cells [21]. Depending on the experiment, Leydig cells were pooled from testes of 3-12 rats. Fractionation of Leydig Cells Leydig cells were suspended in 2-3 ml 0.05 M sodium phosphate buffer (pH 7.4) containing 0.25 M sucrose (Medium A) and homogenized at 4“ C using a 10 ml capacity glass-Teflon Potter-Elvehjem tissue grinder. The homogenate was centrifuged at 500 x g for 15 min in a Sorvall superspeed RC2-B refrigerated centrifuge. The pellet was resuspended in Medium A and recentrifuged at 500 x g. The 500 X g supernatants were pooled and centrifuged at 10000 X g for 15 min. The 10 000 x g pellet was resuspended in Medium A and recentrifuged at 10000 x g. The 10000 X g supernatants were pooled and centrifuged at 100000 x g for 1 h in a Beckman L5-50B ultracentrifuge using an SW 50.1 rotor to obtain the microsomal pellet and cytosol. To obtain purified nuclei, the washed 500 X g pellet was resuspended in 0.5 ml Medium A and layered over a 5 ml cushion of 0.05 M sodium phosphate buffer (pH 7.4) containing 2.1 M sucrose, 25 mM KC1 and 2 mM MgCl,, and centrifuged for 1 h at 100 000 X g [22]. The nuclear pellet was washed in Medium A and resuspended in Medium A. The purity of the isolated nuclei was monitored by assaying for DNA [23]. To obtain purified mitochondria, the washed 10 000 x g pellet was suspended in 0.5 ml Medium A and layered over a discontinuous gradient containing 1 ml each of 60-, 50-, 40-, 30- and 20% sucrose prepared in 0.05 M sodium phosphate buffer (pH 7.4). Samples were centrifuged at 100000 X g for 1 h. Intact mitochondria which localized at the 40-50% sucrose interface [24], were washed and resuspended in Medium A. Mitochondrial purity was monitored by assaying for cytochrome oxidase activity [25]. Enzyme assays Activity for microsomal ethanol-oxidizing system was estimated using a method similar to that

138

described by Lieber and DeCarli for rat liver [16]. Incubations were performed in 10 ml Erlenmeyer flasks, which were sealed with rubber stoppers, and were initiated by adding 50 mM ethanol (or as otherwise designated). Reactions were for 20 min (or as otherwise designated) at 37°C and were terminated by adding 0.25 ml 50% trichloroacetic acid. The samples were equilibrated overnight and the generated acetaldehyde was quantitated by the presence of 0.5 ml 0.15 M semicarbazide in the center well which was suspended through the rubber stopper. The semicarbazone was read spectrophotometrically at 224 nm [26]. Alcohol dehydrogenase activity was estimated by using the procedure of Bonnichsen and Brink [27] but modified for Leydig cells [28]. Lactate dehydrogenase activity was estimated by monitoring the conversion of pyruvate (0.3 mM) to lactate in the presence of NADH (0.05 mM) [29]. Cellular protein was quantitated by the method of Lowry et al. [30] following precipitation with trichloroacetic acid [31]. Statistical analysis The results are presented as the mean f standard error (S.E.). The data was evaluated using Student’s t-test. A P value of < 0.05 was considered significant. Results and Discussion Activity of microsomal ethanol-oxidizing system was linear for 0.035-0.105 mg microsomal protein and for 30 min. Therefore, all subsequent

experiments utilized 0.05-0.10 mg protein and were for 20 min. Activities of microsomal ethanol-oxidizing system in Leydig cell washed 500 x g and 10000 x g pellets and cytosol were 16.5 f 2.0, 33.3 + 5.0 and 6.5 k 2.1 nmol acetaldehyde per 20 min per mg protein, respectively. The highest activity was observed in the microsomal fraction, 59.0 k 5.0 nmol. Because significant activity was localized in 500 x g and 10000 X g pellets, nuclei and mitochondria were purified further. The activities in purified nuclei and mitochondria decreased to 2.2 + 1.5 nmol and 8.7 k 3.5 nmol, respectively. The high activity in the original 10000 X g pellet may represent peroxisomal catalase localization in this fraction [32] and/or microsomal contamination. The latter is suggested because potassium cyanide, a catalase inhibitor [16], had minimal effect on enzyme activity in this fraction. The localization of microsomal ethanol-oxidizing system in Leydig cell fractions was compared to that of alcohol dehydrogenase activity and another soluble enzyme, lactate dehydrogenase. Essentially all of both enzyme activities was concentrated in the soluble fraction (Table I). Activity of microsomal ethanol-oxidizing system in interstitial cells was 26.0 f 5.4 nmol/20 min/mg protein, while activity in purified Leydig cells was 47.4 + 4.1 nmol. This suggests that among cells comprising interstitial cells, this enzyme system is concentrated in Leydig cells. However, the about 2-fold increase of enzyme activity of metrizamide gradient-purified Leydig cells over crude interstitial cells may suggest that this en-

TABLE I LOCALIZATION OF ALCOHOL CELL FRACTIONS Each value represents

DEHYDROGENASE

the mean + S.E. of four separate

Fractions

Washed 500 x g pellet Washed 10000 x g pellet Microsomes Cytosol

Alcohol

AND LACTATE

experiments.

nd.,

DEHYDROGENASE

nondetectable

under

Lactate

dehydrogenase

ACTIVITIES

the experimental

IN LEYDIG

conditions.

dehydrogenase

% of total activity

nmol NADH/ 10 min per mg protein

% of total activity

nmol NAD’/ min per mg protein

n.d. n.d. 2.3 f 2.0 91.1 f 6.5

n.d. n.d. 1.6 f 5.8 53.2 f 2.5

9.6 f 3.6 2.8 f 0.8 1.8f0.3 85.8k4.6

113.2 115.8 224.5 1312.9

+ f f f

20.3 28.0 89.6 94.1

139

zyme system is not exclusively localized in Leydig cells because we generally observe a 6-lo-fold enrichment of testosterone formation following purification on metrizamide gradients. Microsomal ethanol-oxidizing system in liver was 278.8 * 22.5 nmol/20 min per mg protein. Leydig cell microsomes were incubated with increasing ethanol concentrations (l-50 mM). Lineweaver-Burk analysis of the data demonstrated a single linear line with an apparent K, of 4 mM and an estimated V of 56 nmol/20 min per mg protein. This K, closely approximates values (8 and 15 mM) reported for the rat liver enzyme [16,33], and suggests that the Leydig cell enzyme could metabolize ethanol at concentrations commonly found in the blood of individuals who drink. Cofactor specificity of the microsomal ethanoloxidizing system in Leydig cells was next examined. Activity with 0.3 mM NADPH, NADH or NAD+ was 68.3 k 8.2 nmol, 22.2 k 8.9 nmol and 2.6 + 2.6 nmol acetaldehyde per 20 min per mg protein, respectively. There was no measurable activity with NADP+ or in the absence of cofactors. These results demonstrate that the most effective cofactor for microsomal ethanol-oxidizing system in Leydig cells is NADPH, which is similar to the liver enzyme [16]. This contrasts with Leydig cell alcohol dehydrogenase activity where NADf is the preferred cofactor [28]. TABLE

II

EFFECT OF 4-METHYLPYRAZOLE, POTASSIUM CYANIDE OR SODIUM AZIDE ON ACTIVITY OF MICROSOMAL ETHANOL-OXIDIZING SYSTEM IN LEYDIG CELLS Each value represents ments, each performed

the mean * S.E. of at least four experiin duplicate.

Treatment

Control 4-Methylpyrazole 4-Methylpyrazole Potassium cyanide Potassium cyanide Sodium azide (0.1 Sodium azide (1.0

Acetaldehyde (nmol/20 min per mg protein)

(0.1 mM) (1.0 mM) (0.01 mM) (0.1 mM) mM) mM)

* P < 0.05, when compared

52.2* 4.8 44.1* 3.7 54.1 f 9.5 46.9* 5.3 48.1+ 8.9 44.0+ 13.9 36.3* 8.0 * with control

The effect of increasing concentrations of NADPH (0.025-0.3 mM) on activity of microsomal ethanol-oxidizing system in Leydig cells was examined. A Lineweaver-Burk analysis of the data demonstrated a single linear plot, with an apparent K, for NADPH of 11 PM. This value is similar to the K, of 44 mM for NADPH of the hepatic enzyme [34]. Activity of microsomal ethanol-oxidizing system in control Leydig cells was 52.2 k 4.8 nmol/20 min per mg protein (Table II). Activity was unaffected by 4-methylpyrazole (0.1 or 1.0 mM) or by potassium cyanide (0.01 or 0.1 mM). However, 1.0 mM sodium azide, another catalase inhibitor [33], reduced activity to 36.3 k 7.80 nmol (P < O.OS), representing a drop of about 30%. It should be noted that sodium cyanide and sodium azide at the same or lower concentrations reduced ethanol metabolism by liver microsomes by about 12 and 37%, respectively [16]. Heretofore, testicular metabolism of ethanol was thought to be effected solely by alcohol dehydrogenase activity which has been identified in whole rat testes [7], in crude rat interstitial cells [35] and purified rat Leydig cells [8]. As a consequence of ethanol metabolism by Leydig cell alcohol dehydrogenase activity the NAD+/NADH ratio is proposed to decrease, resulting in inhibition of NAD+-dependent A5-3P-hydroxysteroid dehydrogenase-isomerase activity [4,7,8]. However, because progesterone-stimulated testosterone accumulation is inhibited by ethanol, an enzymic step(s) following progesterone formation also must be affected by ethanol [6,14]. Because the decrease in testosterone was associated with a build up of androstenedione, it was proposed that ethanol directly inhibits 17-ketosteroid reductase activity [14]. More recently, we have shown that reduced testosterone following progesterone formation may be due to decreased 17-ketosteroid reductase activity and/or enhanced 17fi-hydroxysteroid dehydrogenase activity [20]. Because these enzymes are dependent on NADPH and NADP+ as cofactors, respectively, altered activities may be due to an increase in NADP+/NADPH ratio, due to Leydig cell microsomal ethanol-oxidizing system. In conclusion, the present studies for the first time provide evidence for a microsomal ethanoloxidizing system in rat Leydig cells which exhibits

140

characteristics similar to the hepatic enzyme. Activity is concentrated in the microsomal fraction, shows a preference for NADPH as cofactor, and is not inhibited by 4-methylpyrazole or potassium cyanide, inhibitors of alcohol dehydrogenase and catalase activities, respectively. Acknowledgements These studies were supported by the Medical Research Service of the Veterans Administration. Our appreciation to Ms. Dorothea Barwick and Barbara Sibert for typing this manuscript. References 1 Badr, F.M. and Bartke, A. (1974) Steroids 23, 921-928 2 Cicero, T.J., Bernstein, D. and Badger, T.M. (1978) Ale. Clin. Exp. Res. 2, 249-254 3 Van Thiel, D.H., Cobb, C.G., Herman, C.B., Perez, H.A., Estes, L. and Gavaler, J.S. (1981) Endocrinology 109, 2009-2015 J. and Varanelli, C.C. (1979) Res. Commun. 4 Ellingboe, Chem. Pathol. Pharmacol. 24, 87-102 5 Cicero, T.J., Bell, R.D., Meyer, E.R. and Badger, T.M. (1980) J. Pharmacol. Exp. Ther. 213, 228-233 E.P., Lin, T., Osterman, J. and Nankin, H.R. 6 Murono, (1980) Steroids 36, 619-631 7 Van Thiel, D.H., Gavaler, J. and Lester, R. (1974) Science 186, 941-942 8 Murono, E.P. (1983) Steroids 42, 457-468 V. (1985) Life Sci. 36, 9 Murono, E.P. and Fisher-Simpson, 1381-1387 10 Badr, F.M., Bartke, A., Dalterio, S. and Bulger, W. (1977) Steroids 30, 647-655 11 Santucci, L., Graham, T.J. and Van Thiel, D.H. (1983) AC. Clin. Exp. Res. 7, 135-139 T.V., Ylikahri, R.H., Harkonen, 12 Eriksson, C.J.P., Widen&, M. and Leinonen, P. (1983) Biochem. J. 210, 29-36 R.A., Quigg, J.M., Oswald, C. and Zaneveld, 13 Anderson, L.J.D. (1985) Biochem. Pharmacol. 34, 685-695

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