Effects of Acute Alcohol Administration on Reproductive Endocrinology in the Male Rat Theodore J. Cicero, Ph.D., David Bernstein, and Thomas M. Badger The results of the current studies further document that acute alcohol administration markedly disrupts the function of the HPG in the male. Our results indicate that alcohol depresses serum testosterone levels and, thereby, produces clinical symptoms associated w i t h hypoandrogenization. Moreover, our studies suggest that acute alcohol administration also affects the hypothalamic-pituitary axis by reducing serum LH levels-an effect that may represent the primary action of alcohol on the HPG.
normal response to depressed serum T levels is a marked increase in serum LH levels,29 because of the release of negative feedback control of LH exerted by T. Since LH levels do not increase in response to the reduced serum T levels produced by alcohol, the function of the hypothalamic-pituitary axis must be inhibited.25.27.28 From the preceding studies with the chronic alcoholic, it is not possible to determine whether changes in serum T levels are contingent on changes in LH levels, whether alterations in the HRONIC alcoholic men and animals have two hormones occur simultaneously and by a many symptoms of demasculization'.' common mechanism, or whether they represent that appear to be related to low serum testostotally unrelated actions of the drug. Moreover, terone (T) levels.'-7 Much recent work has been conclusions regarding specific effects of alcohol directed towards an identification of t h e on the HPG have been impossible to make bemechanisms involved in alcohol's depressant cause of the intrusion of a number of confoundeffects on serum T levels. There are two general ing variables, such as liver damage, poor nutriways in which the drug could produce this tion, psychiatric disturbances, or other drug effect: (1) it could block the synthesis, release, abuse frequently found in chronic alcoholics. To or metabolism of T; or (2) it could depress the avoid some of these problems, several investigahypothalamic-pituitary aspect of the hypotors have examined the acute effects of alcohol thalamic-pituitary-gonadal (HPG) axis. There on LH and T levels in normal male volunteers is an impressive amount of data supporting the and animals. However, the results have genfirst of these possibilities. Specifically, it has erally been equivocal; some investigators been shown that the clearance and metabolism find decreases in serum L H and T lev- ~ ~others * " ~ *find ~ ' no significant of T are increased in the chronic a l c o h ~ l i c , ~ - ' ~ e l ~ , ~ . ~ ~while that its biosynthesis is inhibited,l8.I8and finally, change^.^*-^^ This confusion in the literature that the conversion of T to estrogens may be enmay be related to several pharmacologic probhanced in the chronic a l c ~ h o l i c . ~ ~ - ~ ~ lems in the design of these acute studies. Specifically, Cicero and badge^'^.'" have found that The principal question raised in the foregoing alcohol exerts biphasic effects on serum T and studies is whether alcohol exerts effects at any LH levels: low doses increase T and LH levels point in the HPG other than at the gonads or and high doses decrease the levels of both horliver. If alcohol does affect the function of the hypothalamus or pituitary, a significant drop in serum luteinizing hormone (LH) levels should From the Department o j Psychiatry, Washington be detectable in the chronic alcoholic. The reUniversity School of Medicine, St. Louis,Mo. sults of numerous studies with humans or aniSupported in part by USPHS Grants DA-01407 and DAmals chronically exposed to alcohol, however, 00259. T.J.C. is a recipient of Research Scientist Developindicate that gonadotropin levels are unaltered ment Award AA-70180; D.B. is a recipient of Grass relative to c o n t r ~ l s . ' ~Although ~ ~ ' ~ ~ these ~ ~ ~ ~ ~Neurobiology Fellowship. Reprint requests should be addressed to Theodore J . studies seem to discount a central effect of alcoCicero. Ph.D.. Department of Psychiatry, Washington hol, the fact that LH levels are not significantly University School of Medicine, 4940 Audubon Avenue, St. elevated in these individuals argues for an Louis, Mo. 631 10. alcohol-induced impairment in the hypo0 1978 by Grune 4 . t Stratton. Inc. 0145-6oos/78/0203-01$01 .00/0 thalamic-pituitary axis.25.27.28 That is, the
C
Alcoholism: Clinicaland Experimental Research, Vol. 2 . No. 3 (July). 1978
249
CICERO, BERNSTEIN. AND BADGER
2 50
mones. Moreover, these investigators also found that serum T and LH levels were initially unaffected by a single acute injection of alcohol, were markedly depressed 3-5 hr after the injection, and then returned to control levels 6-8 hr later.27*2sConsequently, it appears that two variables-when blood is sampled after acute alcohol injection and dose of alcohol employed-determine in which direction serum LH and T change after acute alcohol administration. When these variables are controlled, it appears that acute alcohol treatment can significantly alter the function of the HPG.'7-'X This acute model may thus prove to be extremely useful in examining the mechanisms underlying alcohol's acute effects on the HPG, since those variables that confound studies with human alcoholics are eliminated. The studies described in this paper were carried out to examine at what point in the HPG alcohol exerts its initial or primary effect and to assess the relative sensitivity of various levels in the axis to alcohol. To this end, we have conducted several experiments. First, we have examined the acute effects of alcohol on serum T and LH levels with particular emphasis on the temporal relationship between changes in the two and dose-response relationships. Second, we have determined whether alcohol blocks the castration-induced increase in serum LH levels. Since castration produces a near maximal activation of the hypothalamic-pituitary axis, an effect of alcohol on the HPG should be more readily discernible in this preparation than in the normal animal. Finally, the effects of alcohol on the ability of luteinizing hormone releasing hormone (LH-RH) to stimulate LH release by the pituitary were also evaluated. MATERIALS AND METHODS
Chemicals and Drugs Male Sprague-Dawley derived rats, 55-60 days of age (250-300 g), were employed in all studies. LH-RH was purchased from the Beckman Chemical Co. (Los Angeles, Calif.). Dr. Gordon Niswender (Colorado State University) generously provided antiovine LH serum (#IS) and Dr. Leo Reichert (Emory University) provided the LH suitable for radioiodination (LER-1056-C2). All other reagents necessary for the radioimmunoassay of LH were provided by the Rat Pituitary Hormone Distribution Program of the NIAMD. Dr. Walter G. Wiest (Washington University School of Medicine) kindly supplied the testosterone antibody that had been generated to 1 I-a-succinyl-testosteroneBSA.
Acute Alcohol Administration Rats were housed in groups of 3 under a 12-hr light-dark cycle. Rats were injected intraperitoneally with sufficient amounts of a 25% (v/v) solution of alcohol to yield the required doses. Control rats were injected with water. To examine changes in T and LH levels after a single injection, groups of rats (N = 6) were injected with alcohol (2.5 g/kg) or water intraperitoneally and were then killed at I , 2.4. or 6 hr after the injection. The time of injection was varied to insure the rats were always killed at the same time (=t60 min) to minimize any cyclic changes in serum LH or T levels. In the dose-response studies, rats were injected with various doses of alcohol or water and were then killed 3 or 4 hr later for LH and T, respectively. These intervals were selected as the peak depressions in both hormones occur at these time^.^'.^^ The rats were always killed at the same time of day as in the time-response studies to standardize conditions as much as possible.
Efects of Alcohol on the Increase in L H Levels Produced by Castration To determine whether alcohol would interfere with castration-induced increases in LH levels, groups of rats were castrated or sham operated and half of the animals in each group were then injected with water or alcohol (2.5 g/kg) intraperitoneally. Alcohol injections began 2 hr after castration and were given at 6 hr intervals thereafter. The four groups of animals-castrate plus water, castrate plus alcohol, sham plus water, and sham plus alcohol (N = 6 in each g r o u p t w e r e killed 24 hr after surgery. Their blood and pituitaries were obtained for radioimmunoassay, as described below.
Efects of Alcohol on L H- R H-Induced Secretion of LH by the Pituitary Gland To examine whether alcohol would interfere with LHRH-induced synthesis and/or secretion of LH by the pituitary, groups of rats (N = 4-6) were injected intraperitoneally with either alcohol (2.5 g/kg) or water. Thirty minutes later, the rats were injected subcutaneously with LH-RH at a dose of 150 ng/rat. They were killed 20 min after the injection of LH-RH. based on preliminary studies in which this interval was found to produce a maximal increase in LH levels produced by this dose of LH-RH.3s Thus, 50 min elapsed from the alcohol injection to the time the animals were killed. The rats were decapitated and their blood and pituitaries obtained as described below.
Radioimmunoassays and Blood Alcohol Determinations Blood was collected from the decapitated carcass, was allowed to stand at &4'C for 3-4 hr, and was centrifuged at 1600 g for 20 min. The sera were collected and stored at -2O'C until LH and T levels were measured. The pituitaries (both posterior and anterior lobes) were removed in situ and immediately frozen on dry ice. Each pituitary was homogenized in 2 ml of phosphate-buffered saline (PBS), pH 7.6. LH was assayed by a slight modification of the double antibody radioirnmunoassay originally described by Niswender et al." T levels were assayed by a specific and sensi-
ALCOHOL AND REPRODUCTIVE ENDOCRINOLOGY IN MALE RATS Table 1. The Acute Effects of a 2.5 g/kg Dose of Alcohol on the Mean (*SEMI L H and T Levels (ng/mll in Male Adult Spraaue-Dawlev Rats' Time
Ot 1 2 4 6
T
2.01 (4ZO.9) 2.35(+1.1) 1.34(*0.1) 0.84(*0.1)$ 0.93(=tO.l)*
LH
18.96(*3.1) 17.34(3~2.7) 3.33(+1.6)$ 4.75( *1.6)S 14.83(=t4.5)
' N = 6 at each time point. t All control values were pooled since there were no significant differences between groups killed at any of the postinjection time intervals. $p < 0.05when compared to controls (0time). tive radioimmunoassay, which has been described elsehere.^' Blood alcohol levels (BAL) were measured gas chromatographically as previously described.38
RESULTS
Acute Efects of Alcohol on T and L H Levels in Serum
As we have reported p r e v i o u ~ l y , ' ~acute . ~ ~ alcohol administration affects serum T and LH levels in a complex time-response and doseresponse fashion. Several representative experiments illustrating this phenomenon are shown in Tables 1 and 2. The effects of a single injection of 2.5 g/kg ethanol on serum T and LH levels at intervals after its injection are shown in Table 1. Alcohol significantly depleted both serum LH and T levels after its acute injection. As shown in this table, the decrease in LH levels appeared to precede the fall in serum T levels. Moreover, LH levels also returned to normal sooner than did testosterone levels. The data in Table 2 illustrate the biphasic dose effect of alcohol on serum T and LH levels. To illustrate the phenomenon, only three alcohol doses have been presented. As shown in this table, a low dose of alcohol (0.75 g / k g ) significantly increased T and LH and a moderate dose left
T
LH
Water
1.65(+0.3) 3.63(+0.9)* 1.96(h0.4) 0.44(+0.1 )t
18.9(k4.6) 32.76(*3.9)' 24.9(*3.3) 3.3(*1.6)t
0.75 1.25 2.5
them unchanged, whereas, a high (2.5 g/kg) dose significantly depressed T and LH. More complete dose-response and time-response curves can be found in earlier papers from our l a b o r a t ~ r y . The ~ ~ *dose ~ ~ required to depress serum T and LH levels by 50% was 1.75 g/kg and 1.9 g/kg, respectively. Eflects of Alcohol on Cast rat ion-Indue ed Increases in Serum T and L H Levels
The preceding data indicate that LH levels fall prior to any changes in T levels, suggesting that a depression in LH may represent the initial effect of alcohol on the HPG. To further document an action of alcohol a t the hypothalamus or pituitary, the effects of alcohol on the increase in serum LH levels produced by castration were examined (see Materials and Methods). The results of these studies are shown in Fig. I . As can be seen, a series of injections of 2.5 g/kg alcohol every 6 hr for 24 hr following castration reduced the rise in serum LH levels by approximately 50%. The same series of injections also markedly depressed LH levels in the sham-operated animals relative to controls (see Fig. I ) . There was no effect of castration on the levels of LH in the pituitary nor did alcohol change the pituitary LH content of castrated or sham-operated animals. 700
T
r
600 -
-E \
CQ C
500 400-
11
+t
300-
Table 2. The Dose-Response Relationships Between Alcohol and Serum LH and T Levels Dose
251
+
0
V
~
Six rats were utilized at each dose level. Data are means (+SEM). expressed as ng/ml for T and LH. Doses of alcohol are in g/kg. 'Significantly higher than control at t h e p < 0.05level. t Significantly lower than control at t h e p < 0.01level.
L Fig. 1. The effects of a series of alcohol injections (2.5 g/kg) or water given at 6-hr intervals for 24 hr o n the mean (*S E M ) serum LH levels in sham-operated or castrated animals. Four t o six rats were used in each group.
CICERO. BERNSTEIN. AND BADGER
2 52
300
T 250
*p
normal response to depressed serum T levels is a marked increase in serum LH levels,29 because of the release of negative feedback control of LH exerted by T. Since LH levels do not increase in response to the reduced serum T levels produced by alcohol, the function of the hypothalamic-pituitary axis must be inhibited.25.27.28 From the preceding studies with the chronic alcoholic, it is not possible to determine whether changes in serum T levels are contingent on changes in LH levels, whether alterations in the HRONIC alcoholic men and animals have two hormones occur simultaneously and by a many symptoms of demasculization'.' common mechanism, or whether they represent that appear to be related to low serum testostotally unrelated actions of the drug. Moreover, terone (T) levels.'-7 Much recent work has been conclusions regarding specific effects of alcohol directed towards an identification of t h e on the HPG have been impossible to make bemechanisms involved in alcohol's depressant cause of the intrusion of a number of confoundeffects on serum T levels. There are two general ing variables, such as liver damage, poor nutriways in which the drug could produce this tion, psychiatric disturbances, or other drug effect: (1) it could block the synthesis, release, abuse frequently found in chronic alcoholics. To or metabolism of T; or (2) it could depress the avoid some of these problems, several investigahypothalamic-pituitary aspect of the hypotors have examined the acute effects of alcohol thalamic-pituitary-gonadal (HPG) axis. There on LH and T levels in normal male volunteers is an impressive amount of data supporting the and animals. However, the results have genfirst of these possibilities. Specifically, it has erally been equivocal; some investigators been shown that the clearance and metabolism find decreases in serum L H and T lev- ~ ~others * " ~ *find ~ ' no significant of T are increased in the chronic a l c o h ~ l i c , ~ - ' ~ e l ~ , ~ . ~ ~while that its biosynthesis is inhibited,l8.I8and finally, change^.^*-^^ This confusion in the literature that the conversion of T to estrogens may be enmay be related to several pharmacologic probhanced in the chronic a l c ~ h o l i c . ~ ~ - ~ ~ lems in the design of these acute studies. Specifically, Cicero and badge^'^.'" have found that The principal question raised in the foregoing alcohol exerts biphasic effects on serum T and studies is whether alcohol exerts effects at any LH levels: low doses increase T and LH levels point in the HPG other than at the gonads or and high doses decrease the levels of both horliver. If alcohol does affect the function of the hypothalamus or pituitary, a significant drop in serum luteinizing hormone (LH) levels should From the Department o j Psychiatry, Washington be detectable in the chronic alcoholic. The reUniversity School of Medicine, St. Louis,Mo. sults of numerous studies with humans or aniSupported in part by USPHS Grants DA-01407 and DAmals chronically exposed to alcohol, however, 00259. T.J.C. is a recipient of Research Scientist Developindicate that gonadotropin levels are unaltered ment Award AA-70180; D.B. is a recipient of Grass relative to c o n t r ~ l s . ' ~Although ~ ~ ' ~ ~ these ~ ~ ~ ~ ~Neurobiology Fellowship. Reprint requests should be addressed to Theodore J . studies seem to discount a central effect of alcoCicero. Ph.D.. Department of Psychiatry, Washington hol, the fact that LH levels are not significantly University School of Medicine, 4940 Audubon Avenue, St. elevated in these individuals argues for an Louis, Mo. 631 10. alcohol-induced impairment in the hypo0 1978 by Grune 4 . t Stratton. Inc. 0145-6oos/78/0203-01$01 .00/0 thalamic-pituitary axis.25.27.28 That is, the
C
Alcoholism: Clinicaland Experimental Research, Vol. 2 . No. 3 (July). 1978
249
CICERO, BERNSTEIN. AND BADGER
2 50
mones. Moreover, these investigators also found that serum T and LH levels were initially unaffected by a single acute injection of alcohol, were markedly depressed 3-5 hr after the injection, and then returned to control levels 6-8 hr later.27*2sConsequently, it appears that two variables-when blood is sampled after acute alcohol injection and dose of alcohol employed-determine in which direction serum LH and T change after acute alcohol administration. When these variables are controlled, it appears that acute alcohol treatment can significantly alter the function of the HPG.'7-'X This acute model may thus prove to be extremely useful in examining the mechanisms underlying alcohol's acute effects on the HPG, since those variables that confound studies with human alcoholics are eliminated. The studies described in this paper were carried out to examine at what point in the HPG alcohol exerts its initial or primary effect and to assess the relative sensitivity of various levels in the axis to alcohol. To this end, we have conducted several experiments. First, we have examined the acute effects of alcohol on serum T and LH levels with particular emphasis on the temporal relationship between changes in the two and dose-response relationships. Second, we have determined whether alcohol blocks the castration-induced increase in serum LH levels. Since castration produces a near maximal activation of the hypothalamic-pituitary axis, an effect of alcohol on the HPG should be more readily discernible in this preparation than in the normal animal. Finally, the effects of alcohol on the ability of luteinizing hormone releasing hormone (LH-RH) to stimulate LH release by the pituitary were also evaluated. MATERIALS AND METHODS
Chemicals and Drugs Male Sprague-Dawley derived rats, 55-60 days of age (250-300 g), were employed in all studies. LH-RH was purchased from the Beckman Chemical Co. (Los Angeles, Calif.). Dr. Gordon Niswender (Colorado State University) generously provided antiovine LH serum (#IS) and Dr. Leo Reichert (Emory University) provided the LH suitable for radioiodination (LER-1056-C2). All other reagents necessary for the radioimmunoassay of LH were provided by the Rat Pituitary Hormone Distribution Program of the NIAMD. Dr. Walter G. Wiest (Washington University School of Medicine) kindly supplied the testosterone antibody that had been generated to 1 I-a-succinyl-testosteroneBSA.
Acute Alcohol Administration Rats were housed in groups of 3 under a 12-hr light-dark cycle. Rats were injected intraperitoneally with sufficient amounts of a 25% (v/v) solution of alcohol to yield the required doses. Control rats were injected with water. To examine changes in T and LH levels after a single injection, groups of rats (N = 6) were injected with alcohol (2.5 g/kg) or water intraperitoneally and were then killed at I , 2.4. or 6 hr after the injection. The time of injection was varied to insure the rats were always killed at the same time (=t60 min) to minimize any cyclic changes in serum LH or T levels. In the dose-response studies, rats were injected with various doses of alcohol or water and were then killed 3 or 4 hr later for LH and T, respectively. These intervals were selected as the peak depressions in both hormones occur at these time^.^'.^^ The rats were always killed at the same time of day as in the time-response studies to standardize conditions as much as possible.
Efects of Alcohol on the Increase in L H Levels Produced by Castration To determine whether alcohol would interfere with castration-induced increases in LH levels, groups of rats were castrated or sham operated and half of the animals in each group were then injected with water or alcohol (2.5 g/kg) intraperitoneally. Alcohol injections began 2 hr after castration and were given at 6 hr intervals thereafter. The four groups of animals-castrate plus water, castrate plus alcohol, sham plus water, and sham plus alcohol (N = 6 in each g r o u p t w e r e killed 24 hr after surgery. Their blood and pituitaries were obtained for radioimmunoassay, as described below.
Efects of Alcohol on L H- R H-Induced Secretion of LH by the Pituitary Gland To examine whether alcohol would interfere with LHRH-induced synthesis and/or secretion of LH by the pituitary, groups of rats (N = 4-6) were injected intraperitoneally with either alcohol (2.5 g/kg) or water. Thirty minutes later, the rats were injected subcutaneously with LH-RH at a dose of 150 ng/rat. They were killed 20 min after the injection of LH-RH. based on preliminary studies in which this interval was found to produce a maximal increase in LH levels produced by this dose of LH-RH.3s Thus, 50 min elapsed from the alcohol injection to the time the animals were killed. The rats were decapitated and their blood and pituitaries obtained as described below.
Radioimmunoassays and Blood Alcohol Determinations Blood was collected from the decapitated carcass, was allowed to stand at &4'C for 3-4 hr, and was centrifuged at 1600 g for 20 min. The sera were collected and stored at -2O'C until LH and T levels were measured. The pituitaries (both posterior and anterior lobes) were removed in situ and immediately frozen on dry ice. Each pituitary was homogenized in 2 ml of phosphate-buffered saline (PBS), pH 7.6. LH was assayed by a slight modification of the double antibody radioirnmunoassay originally described by Niswender et al." T levels were assayed by a specific and sensi-
ALCOHOL AND REPRODUCTIVE ENDOCRINOLOGY IN MALE RATS Table 1. The Acute Effects of a 2.5 g/kg Dose of Alcohol on the Mean (*SEMI L H and T Levels (ng/mll in Male Adult Spraaue-Dawlev Rats' Time
Ot 1 2 4 6
T
2.01 (4ZO.9) 2.35(+1.1) 1.34(*0.1) 0.84(*0.1)$ 0.93(=tO.l)*
LH
18.96(*3.1) 17.34(3~2.7) 3.33(+1.6)$ 4.75( *1.6)S 14.83(=t4.5)
' N = 6 at each time point. t All control values were pooled since there were no significant differences between groups killed at any of the postinjection time intervals. $p < 0.05when compared to controls (0time). tive radioimmunoassay, which has been described elsehere.^' Blood alcohol levels (BAL) were measured gas chromatographically as previously described.38
RESULTS
Acute Efects of Alcohol on T and L H Levels in Serum
As we have reported p r e v i o u ~ l y , ' ~acute . ~ ~ alcohol administration affects serum T and LH levels in a complex time-response and doseresponse fashion. Several representative experiments illustrating this phenomenon are shown in Tables 1 and 2. The effects of a single injection of 2.5 g/kg ethanol on serum T and LH levels at intervals after its injection are shown in Table 1. Alcohol significantly depleted both serum LH and T levels after its acute injection. As shown in this table, the decrease in LH levels appeared to precede the fall in serum T levels. Moreover, LH levels also returned to normal sooner than did testosterone levels. The data in Table 2 illustrate the biphasic dose effect of alcohol on serum T and LH levels. To illustrate the phenomenon, only three alcohol doses have been presented. As shown in this table, a low dose of alcohol (0.75 g / k g ) significantly increased T and LH and a moderate dose left
T
LH
Water
1.65(+0.3) 3.63(+0.9)* 1.96(h0.4) 0.44(+0.1 )t
18.9(k4.6) 32.76(*3.9)' 24.9(*3.3) 3.3(*1.6)t
0.75 1.25 2.5
them unchanged, whereas, a high (2.5 g/kg) dose significantly depressed T and LH. More complete dose-response and time-response curves can be found in earlier papers from our l a b o r a t ~ r y . The ~ ~ *dose ~ ~ required to depress serum T and LH levels by 50% was 1.75 g/kg and 1.9 g/kg, respectively. Eflects of Alcohol on Cast rat ion-Indue ed Increases in Serum T and L H Levels
The preceding data indicate that LH levels fall prior to any changes in T levels, suggesting that a depression in LH may represent the initial effect of alcohol on the HPG. To further document an action of alcohol a t the hypothalamus or pituitary, the effects of alcohol on the increase in serum LH levels produced by castration were examined (see Materials and Methods). The results of these studies are shown in Fig. I . As can be seen, a series of injections of 2.5 g/kg alcohol every 6 hr for 24 hr following castration reduced the rise in serum LH levels by approximately 50%. The same series of injections also markedly depressed LH levels in the sham-operated animals relative to controls (see Fig. I ) . There was no effect of castration on the levels of LH in the pituitary nor did alcohol change the pituitary LH content of castrated or sham-operated animals. 700
T
r
600 -
-E \
CQ C
500 400-
11
+t
300-
Table 2. The Dose-Response Relationships Between Alcohol and Serum LH and T Levels Dose
251
+
0
V
~
Six rats were utilized at each dose level. Data are means (+SEM). expressed as ng/ml for T and LH. Doses of alcohol are in g/kg. 'Significantly higher than control at t h e p < 0.05level. t Significantly lower than control at t h e p < 0.01level.
L Fig. 1. The effects of a series of alcohol injections (2.5 g/kg) or water given at 6-hr intervals for 24 hr o n the mean (*S E M ) serum LH levels in sham-operated or castrated animals. Four t o six rats were used in each group.
CICERO. BERNSTEIN. AND BADGER
2 52
300
T 250
*p
