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Role of the pituitary and the adrenals in mediating the effects of alcohol on testicular steroidogenesis in mice
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ROLE OF TEE PITUITARY AND THE ADRENALS IN MEDIATING THE EFFECTS OF ALCOHOL ON TESTICULAR STEROIDOGENESISIN MICE Fouad M. Badr,l M. Susan Smith', Susan L. Dalterio3, and Andrzej Bartke3 1 -Department of Zoology, University of Kuwait, P.O. Box 5969, State of Kuwait 2 -Department of Physiology, University of Massachusetts Medical School Worcester, MA 01601 3 -Department of Obstetrics & Gynecology, University of Texas Health Science Center, San Antonio, TX 78284 Received 7-2-79 ABSTRACT We have previously demonstrated that intragastric administration of alcohol (1.24 g/kg body wt) to adult male mice results in suppression of testosteroneproduction. We now report that the decline in peripheral testosterone levels in alcohol-treatedmice is not accompanied by changes in plasma levels of luteinizing hormone, follicle stimulating hormone or estradiol-178,and that it is markedly attenuated in adrenalectomizedor adrenalectomized-corticosterone treated males. INTRODUCTION Both chronic and acute administrationof ethyl alcohol can suppress plasma testosterone (T) levels in men and in experimental animals. Circumstantialevidence indicates that this could be due to a direct effect of alcohol, or its metabolite, on the gonad (l3), suppression of the release of pituitary luteinizing hormone, LB (4-6), increased secretory activity of the adrenal cortex (1,7) and/or medulla (8), or to altered peripheral metabolism of T (9-11). We have previously examined various parameters of testicular function in mice given 5 daily doses of 1.24 g alcohol per kg body weight (2,12). The aim of the present study was to determine whether the significant reduction in plasma T levels caused by this regimen of alcohol administration (2,12) is accompanied by changes in peripheral levels of LB, follicle-stimulatinghormone (FSH) or estradiol-178, and whether it will occur in the absence of the adrenals.
Volume 34, Nun6er 4
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1979
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MATERIALS AND METHODS Adult random-bred male mice were purchased from Charles River Breeding Laboratories (CD-l; Experiment 1) or raised in our own colony (Experiment 2). Ethyl alcohol, at a dose of 0.1 ml of 40% solution (approximately 1.24 g/kg body wt) was administered daily for 5 days by stomach tube. Control animals received an equivalent amount of distilled water. Blood was collected by heart puncture under ether anesthesia 1 hr after administration of the last dose of alcohol or water. Experiment 1. Twenty animals were treated with alcohol and 20 served as controls. Plasma levels of LH and FSH were measured using kits for rat LH and FSH radioimmunoassays which were kindly provided by NIAMDD, and expressed in terms of NIAMDD-rat LH-RP-1 and NIAMDD-rat-FSH-RP-1. The validity of these kits for determinations of mouse gonadotropins has already been demonstrated (13). Estradiol-176 was measured by a radioimmunoassay with a sensitivity of l-2 pg (14). To allow determination of the levels of all three hormones, equal amounts of blood from two animals were combined in every sample. Experiment 2. Thirty-two mice were adrenalectomized and given 0.9% NaCl in water as a drinking solution. Sixteen of these animals were injected SC daily for 12 days with 10 ng corticosterone in 0.05 ml of sesame oil, while the remaining males received injections of sesame oil alone. An additional sixteen mice were intact and not injected. Intragastric administration of alcohol was initiated one week after adrenalectomy in half of the animals in every group and continued daily for 5 days. Control mice received water. Blood samples were collected from individual mice 1 hr after the last dose of alcohol or water and saved for determination of T levels by radioimmunoassay (12). The significance of the differences was determined by a one-way single classification analysis of variance and Duncan's test. RESULTS Experiment 1.
Plasma concentrations of LH, FSH and estradiol-178 were
nearly identical in control and in alcohol-treated mice (Table 1). Experiment 2.
In intact mice, treatment with alcohol produced the ex-
pected significant decline in plasma T levels (Table 2).
Adrenalectomy
resulted in a significant decrease in plasma T levels which was not reversed by administration of corticosterone. and adrenalectomized-corticosterone
In both adrenalectomized
treated males, the apparent slight
reduction in the concentration of T in the plasma after alcohol administration was not statistically significant (Table 2).
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TABLE 1, Plasma levels of luteinizing hormone (LH), folliclestimulating hormone (PSH) and estradiol-17S in adult male mice 1 hr after intragastric administrationof the last of five daily doses of alcohol (1.24 g/kg body weight; means f SE).
Controls
20 number of animals 26.2 2 3.6 LH (ng NIAMDD-ratLH-RP-l/ml) FSH (ng NIAMDD-ratFSH-RP-l/ml) 1115.9 + 55.7 8.2 ? 0.7 Estradiol-176 (pg/ml)
Alcohol-treated
20 26.1 2 4.8 1065.7 f 59.9 7.2 f 0.7
The concentrationsof LH, FSH and estradiol-17S in control and alcoholtreated mice were not significantlydifferent. TABLE 2. Effects of adrenalectomy and replacement with corticosterone (10 pg/day) on the ability of alcohol to suppress plasma testosterone levels in adult male mice. (The animals were bled 1 hr after intragastric administrationof the last of five daily doses of 1.24 g alcohol/kg body weight; means f SE).
Treatment
Number of animals
Water Alcohol Adrenalectomy;water Adrenalectomy;alcohol Adrenalectomy; corticosterone;water Adrenalectomy; corticosterone;alcohol
8 8 8 8 8 8
Plasma testosterone (ng/ml) 5.00 0.88 1.60 1.04 2.30 1.42
+ f + * f f
1.70; o.53b 0.50 0.44b l.OSb 0.42b
a,b - values with the same superscript are not significantlydifferent from each other. DISCUSSION
Treatment of adult male mice with ethyl alcohol can drastically suppress testicular T production (2,12). In our earlier studies, plasma T levels measured 1 hr after intragastric administrationof 1.24 g alcohol per kg body weight were reduced to less than 10% of the pretreatment values (2,12). Administration of identical doses of alcohol to male mice in the present studies did not alter plasma levels of LH
or FSH.
The ability of alcohol to cause a reduction in peripheral T
levels without a concomitant decrease in circulating LH was already described in rats (1) and in men (15,16).
However, other investigators
reported reduced LH levels in the plasma after both acute and chronic exposure to alcohol (4-6).
The discrepancies between results obtained
in different laboratories are probably due to differences in schedules of alcohol administration and blood collection.
Under the conditions of
the present study, gonadotropin levels were not altered 1 hr after administration of alcohol at a dose which causes a significant reduction in plasma T levels (2,12) and requires some 2-3 hrs to be completely metabolized
(17).
It can therefore be concluded that the acute reduction
in plasma T levels observed in male mice after alcohol administration is not the result of suppression of LH and/or FSH release and must be due to other causes.
Since in alcohol-treated mice the concentrations of T
in the testis and in the peripheral circulation are reduced to a comparable extent (2), a major shift in peripheral T metabolism is unlikely to account for the observed changes in plasma T levels. Administration of alcohol to adrenalectomized mice failed to reduce the concentration of T in the plasma.
Therefore it can be suspected
that stimulation of adrenal activity by alcohol was responsible for the reduction in plasma T levels which was observed in intact alcoholtreated male mice.
In support of this possibility, alcohol has been
shown to increase peripheral levels of both medullary (8) and cortical (1,7) adrenal secretions.
However, the findings of Sze (18) suggest a
different interpretation of the present results.
From his studies of
alcohol effects on brain and liver function in adrenalectomized and adrenalectomized-corticosterone-treated
mice, Sze (18) concluded that
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corticoids play a permissive role in the action of alcohol. In his experiments, replacementwith corticosteronereversed the effect of adrenalectomy on the responsivenessof the brain and the liver to treated alcohol (18). In our adrenalectomized-corticosterone animals, alcohol failed to affect plasma T levels. This could be due to employing different dose of corticosteronefor replacement therapy, or to a differential involvement of the adrenal medulla and cortex in mediating the effects of alcohol on different tissues. However, it
should be indicated that the failure of alcohol treatment
to reduce plasma T levels in adrenalectomizedmice in the present study may have been related to a significant decrease in the concentration of T in the plasma after adrenalectomy. Against the background of subnormal T production, a further inhibitory effect of alcohol may be difficult if not impossible to demonstrate. In both adrenalectomized and adrenalectomized-corticosterone treated males, mean plasma T levels were numerically, although not significantly,lower than in respective controls. Even though stimulation of adrenal function by alcohol is very likely to contribute to its effects on testicular steroidogenesis,a direct effect of alcohol or its metabolite(s) on the testis is probably also important (l-3). The unaltered plasma estradiol-178 levels in alcohol treated male mice in the present study agree with the view that the elevation of peripheral estradiol level in alcoholic liver cirrhosis (19) reflects an abnormal pattern of hepatic steroid metabolism (10) rather than changes in testicular function.
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ACKNOWLEDGMENT This work was supported by NIH through grant HD-09584 (A.B.). We thank Ms. Carol Roberson for determinations of LH and FSH, NIAMDD for rat LH and FSH radioimmunoassay kits, Dr. B.V. Caldwell for antiserum to testosterone and Dr. D.C. Collins for antiserum to estradiol-178.
REFERENCES 1. 2. 3.
4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Van Thiel, D.H., Gavaler, J.S. and Lester, R., DRUG AND ALCOHOL DEPENDENCE 1, 373 (1977). Badr, F.M., Bartke, A., Dalterio, S. and Bulger, W., STEROIDS 30, 647 (1977). Farnsworth, W.E., Cavanaugh, A.H., Brown, J.R., Alvarez, I. and Lewandowski, L.M., Program 3rd Meeting Am. Sot. Andrology, p 25, 1978. Leppaluoto, J., Rapeli, M., Varis, R. and Ranta, T., ACTA PHYSIOL. SCAND. 2, 400 (1975). Symons, A.M. and Marks, V., BIOCHEM. PHARMACOL. 2, 955 (1975). Cicero, T.J. and Badger, T.M., J. PHARMACOL. EXP. THERAP. 201, 427 (1977). Ylikahri, R.H., Huttunen, M.O., Harkonen, M., Leino, T., Helenius, T ., Liewendahl, K. and Karonen, S.-L., J. CLIN. ENDOCR. METAB. 46, 715 (1978). Klingman, G.I. and Goodall, McC., J. PHARMACOL. EXP. THERAP. I&, 313 (1957). Williams, T.L., Cantarow, A., Paschkis, K.E. and Havens, W.P., Jr., ENDOCRINOLOGY 48, 651 (1951). Admirand, W.H., Cronholm, T. and Sjovall, J., BIOCHIM. BIOPHYS. ACTA 202, 343 (1970). Rubin, E., Lieber, C.S., Altman, K., Gordon, G.G. and Souther, A.L., SCIENCE 191, 563 (1976). Badr, F.M. and Bartke, A., STEROIDS 3, 921 (1974). Beamer, W.G., Murr, S.M. and Geschwind, I.I., ENDOCRINOLOGY 90, 823 (1972). Smith, M.S., Freeman, M.E. and Neill, J.D., ENDOCRINOLOGY 96, 219 (1975). Gordon, G.G., Altman, K., Southren, A.L., Rubin, E. and Lieber, C.S., N. ENGL. J. MED. 295, 793 (1976). Mendelson, J.H., Mello, N.K. and Ellingboe, J., J. PHARMACOL. EXP. THERAP. 202, 676 (1977). Erickson, C.K., Koch, K.I., Mehta, C.S. and McGinity, J.W., SCIENCE l99, 1457 (1978). Sze, P.Y., DRUG AND ALCOHOL DEPENDENCE 2, 381 (1977). Chopra, I.J., Tulchinsky, D. and Greenway, F., CLIN. RES. 21, 200 (1973).
477
ROLE OF TEE PITUITARY AND THE ADRENALS IN MEDIATING THE EFFECTS OF ALCOHOL ON TESTICULAR STEROIDOGENESISIN MICE Fouad M. Badr,l M. Susan Smith', Susan L. Dalterio3, and Andrzej Bartke3 1 -Department of Zoology, University of Kuwait, P.O. Box 5969, State of Kuwait 2 -Department of Physiology, University of Massachusetts Medical School Worcester, MA 01601 3 -Department of Obstetrics & Gynecology, University of Texas Health Science Center, San Antonio, TX 78284 Received 7-2-79 ABSTRACT We have previously demonstrated that intragastric administration of alcohol (1.24 g/kg body wt) to adult male mice results in suppression of testosteroneproduction. We now report that the decline in peripheral testosterone levels in alcohol-treatedmice is not accompanied by changes in plasma levels of luteinizing hormone, follicle stimulating hormone or estradiol-178,and that it is markedly attenuated in adrenalectomizedor adrenalectomized-corticosterone treated males. INTRODUCTION Both chronic and acute administrationof ethyl alcohol can suppress plasma testosterone (T) levels in men and in experimental animals. Circumstantialevidence indicates that this could be due to a direct effect of alcohol, or its metabolite, on the gonad (l3), suppression of the release of pituitary luteinizing hormone, LB (4-6), increased secretory activity of the adrenal cortex (1,7) and/or medulla (8), or to altered peripheral metabolism of T (9-11). We have previously examined various parameters of testicular function in mice given 5 daily doses of 1.24 g alcohol per kg body weight (2,12). The aim of the present study was to determine whether the significant reduction in plasma T levels caused by this regimen of alcohol administration (2,12) is accompanied by changes in peripheral levels of LB, follicle-stimulatinghormone (FSH) or estradiol-178, and whether it will occur in the absence of the adrenals.
Volume 34, Nun6er 4
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October,
1979
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MATERIALS AND METHODS Adult random-bred male mice were purchased from Charles River Breeding Laboratories (CD-l; Experiment 1) or raised in our own colony (Experiment 2). Ethyl alcohol, at a dose of 0.1 ml of 40% solution (approximately 1.24 g/kg body wt) was administered daily for 5 days by stomach tube. Control animals received an equivalent amount of distilled water. Blood was collected by heart puncture under ether anesthesia 1 hr after administration of the last dose of alcohol or water. Experiment 1. Twenty animals were treated with alcohol and 20 served as controls. Plasma levels of LH and FSH were measured using kits for rat LH and FSH radioimmunoassays which were kindly provided by NIAMDD, and expressed in terms of NIAMDD-rat LH-RP-1 and NIAMDD-rat-FSH-RP-1. The validity of these kits for determinations of mouse gonadotropins has already been demonstrated (13). Estradiol-176 was measured by a radioimmunoassay with a sensitivity of l-2 pg (14). To allow determination of the levels of all three hormones, equal amounts of blood from two animals were combined in every sample. Experiment 2. Thirty-two mice were adrenalectomized and given 0.9% NaCl in water as a drinking solution. Sixteen of these animals were injected SC daily for 12 days with 10 ng corticosterone in 0.05 ml of sesame oil, while the remaining males received injections of sesame oil alone. An additional sixteen mice were intact and not injected. Intragastric administration of alcohol was initiated one week after adrenalectomy in half of the animals in every group and continued daily for 5 days. Control mice received water. Blood samples were collected from individual mice 1 hr after the last dose of alcohol or water and saved for determination of T levels by radioimmunoassay (12). The significance of the differences was determined by a one-way single classification analysis of variance and Duncan's test. RESULTS Experiment 1.
Plasma concentrations of LH, FSH and estradiol-178 were
nearly identical in control and in alcohol-treated mice (Table 1). Experiment 2.
In intact mice, treatment with alcohol produced the ex-
pected significant decline in plasma T levels (Table 2).
Adrenalectomy
resulted in a significant decrease in plasma T levels which was not reversed by administration of corticosterone. and adrenalectomized-corticosterone
In both adrenalectomized
treated males, the apparent slight
reduction in the concentration of T in the plasma after alcohol administration was not statistically significant (Table 2).
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TABLE 1, Plasma levels of luteinizing hormone (LH), folliclestimulating hormone (PSH) and estradiol-17S in adult male mice 1 hr after intragastric administrationof the last of five daily doses of alcohol (1.24 g/kg body weight; means f SE).
Controls
20 number of animals 26.2 2 3.6 LH (ng NIAMDD-ratLH-RP-l/ml) FSH (ng NIAMDD-ratFSH-RP-l/ml) 1115.9 + 55.7 8.2 ? 0.7 Estradiol-176 (pg/ml)
Alcohol-treated
20 26.1 2 4.8 1065.7 f 59.9 7.2 f 0.7
The concentrationsof LH, FSH and estradiol-17S in control and alcoholtreated mice were not significantlydifferent. TABLE 2. Effects of adrenalectomy and replacement with corticosterone (10 pg/day) on the ability of alcohol to suppress plasma testosterone levels in adult male mice. (The animals were bled 1 hr after intragastric administrationof the last of five daily doses of 1.24 g alcohol/kg body weight; means f SE).
Treatment
Number of animals
Water Alcohol Adrenalectomy;water Adrenalectomy;alcohol Adrenalectomy; corticosterone;water Adrenalectomy; corticosterone;alcohol
8 8 8 8 8 8
Plasma testosterone (ng/ml) 5.00 0.88 1.60 1.04 2.30 1.42
+ f + * f f
1.70; o.53b 0.50 0.44b l.OSb 0.42b
a,b - values with the same superscript are not significantlydifferent from each other. DISCUSSION
Treatment of adult male mice with ethyl alcohol can drastically suppress testicular T production (2,12). In our earlier studies, plasma T levels measured 1 hr after intragastric administrationof 1.24 g alcohol per kg body weight were reduced to less than 10% of the pretreatment values (2,12). Administration of identical doses of alcohol to male mice in the present studies did not alter plasma levels of LH
or FSH.
The ability of alcohol to cause a reduction in peripheral T
levels without a concomitant decrease in circulating LH was already described in rats (1) and in men (15,16).
However, other investigators
reported reduced LH levels in the plasma after both acute and chronic exposure to alcohol (4-6).
The discrepancies between results obtained
in different laboratories are probably due to differences in schedules of alcohol administration and blood collection.
Under the conditions of
the present study, gonadotropin levels were not altered 1 hr after administration of alcohol at a dose which causes a significant reduction in plasma T levels (2,12) and requires some 2-3 hrs to be completely metabolized
(17).
It can therefore be concluded that the acute reduction
in plasma T levels observed in male mice after alcohol administration is not the result of suppression of LH and/or FSH release and must be due to other causes.
Since in alcohol-treated mice the concentrations of T
in the testis and in the peripheral circulation are reduced to a comparable extent (2), a major shift in peripheral T metabolism is unlikely to account for the observed changes in plasma T levels. Administration of alcohol to adrenalectomized mice failed to reduce the concentration of T in the plasma.
Therefore it can be suspected
that stimulation of adrenal activity by alcohol was responsible for the reduction in plasma T levels which was observed in intact alcoholtreated male mice.
In support of this possibility, alcohol has been
shown to increase peripheral levels of both medullary (8) and cortical (1,7) adrenal secretions.
However, the findings of Sze (18) suggest a
different interpretation of the present results.
From his studies of
alcohol effects on brain and liver function in adrenalectomized and adrenalectomized-corticosterone-treated
mice, Sze (18) concluded that
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481
corticoids play a permissive role in the action of alcohol. In his experiments, replacementwith corticosteronereversed the effect of adrenalectomy on the responsivenessof the brain and the liver to treated alcohol (18). In our adrenalectomized-corticosterone animals, alcohol failed to affect plasma T levels. This could be due to employing different dose of corticosteronefor replacement therapy, or to a differential involvement of the adrenal medulla and cortex in mediating the effects of alcohol on different tissues. However, it
should be indicated that the failure of alcohol treatment
to reduce plasma T levels in adrenalectomizedmice in the present study may have been related to a significant decrease in the concentration of T in the plasma after adrenalectomy. Against the background of subnormal T production, a further inhibitory effect of alcohol may be difficult if not impossible to demonstrate. In both adrenalectomized and adrenalectomized-corticosterone treated males, mean plasma T levels were numerically, although not significantly,lower than in respective controls. Even though stimulation of adrenal function by alcohol is very likely to contribute to its effects on testicular steroidogenesis,a direct effect of alcohol or its metabolite(s) on the testis is probably also important (l-3). The unaltered plasma estradiol-178 levels in alcohol treated male mice in the present study agree with the view that the elevation of peripheral estradiol level in alcoholic liver cirrhosis (19) reflects an abnormal pattern of hepatic steroid metabolism (10) rather than changes in testicular function.
482
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ACKNOWLEDGMENT This work was supported by NIH through grant HD-09584 (A.B.). We thank Ms. Carol Roberson for determinations of LH and FSH, NIAMDD for rat LH and FSH radioimmunoassay kits, Dr. B.V. Caldwell for antiserum to testosterone and Dr. D.C. Collins for antiserum to estradiol-178.
REFERENCES 1. 2. 3.
4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Van Thiel, D.H., Gavaler, J.S. and Lester, R., DRUG AND ALCOHOL DEPENDENCE 1, 373 (1977). Badr, F.M., Bartke, A., Dalterio, S. and Bulger, W., STEROIDS 30, 647 (1977). Farnsworth, W.E., Cavanaugh, A.H., Brown, J.R., Alvarez, I. and Lewandowski, L.M., Program 3rd Meeting Am. Sot. Andrology, p 25, 1978. Leppaluoto, J., Rapeli, M., Varis, R. and Ranta, T., ACTA PHYSIOL. SCAND. 2, 400 (1975). Symons, A.M. and Marks, V., BIOCHEM. PHARMACOL. 2, 955 (1975). Cicero, T.J. and Badger, T.M., J. PHARMACOL. EXP. THERAP. 201, 427 (1977). Ylikahri, R.H., Huttunen, M.O., Harkonen, M., Leino, T., Helenius, T ., Liewendahl, K. and Karonen, S.-L., J. CLIN. ENDOCR. METAB. 46, 715 (1978). Klingman, G.I. and Goodall, McC., J. PHARMACOL. EXP. THERAP. I&, 313 (1957). Williams, T.L., Cantarow, A., Paschkis, K.E. and Havens, W.P., Jr., ENDOCRINOLOGY 48, 651 (1951). Admirand, W.H., Cronholm, T. and Sjovall, J., BIOCHIM. BIOPHYS. ACTA 202, 343 (1970). Rubin, E., Lieber, C.S., Altman, K., Gordon, G.G. and Souther, A.L., SCIENCE 191, 563 (1976). Badr, F.M. and Bartke, A., STEROIDS 3, 921 (1974). Beamer, W.G., Murr, S.M. and Geschwind, I.I., ENDOCRINOLOGY 90, 823 (1972). Smith, M.S., Freeman, M.E. and Neill, J.D., ENDOCRINOLOGY 96, 219 (1975). Gordon, G.G., Altman, K., Southren, A.L., Rubin, E. and Lieber, C.S., N. ENGL. J. MED. 295, 793 (1976). Mendelson, J.H., Mello, N.K. and Ellingboe, J., J. PHARMACOL. EXP. THERAP. 202, 676 (1977). Erickson, C.K., Koch, K.I., Mehta, C.S. and McGinity, J.W., SCIENCE l99, 1457 (1978). Sze, P.Y., DRUG AND ALCOHOL DEPENDENCE 2, 381 (1977). Chopra, I.J., Tulchinsky, D. and Greenway, F., CLIN. RES. 21, 200 (1973).