371
INFLUENCE OF ETHANOL NATAL DEVELOPMENT
ON HORMONAL
SYSTEMS
AND PRE-
Moderator: Dr. Carrie L. Randall Discussants : Dr. Gerhard
Freund
Dr. Dora B. Goldstein Dr. David Lester
Drug and Alcohol Dependence, 2 (1977) 373 - 380 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
ETHANOL:
A GONADAL
TOXIN
373
IN THE FEMALE
DAVID H. VAN THIEL*, JUDITH S. GAVALER
and ROGER LESTER
Division of Gastroenterology, Department of Medicine, of Medicine, Pittsburgh, Pennsylwnia 15261 (U.S.A.)
University of Pittsburgh School
Summary It has been estimated recently that there are at least two million female alcoholics presently within the United States. Even more disturbing is the fact that this number is steadily increasing. The number of female alcoholics has increased such that it has been estimated that one out of every three alcoholics is probably a woman. As distressing as these social facts are, even more disturbing are the clinical suggestions that the female liver is more readily injured by ethanol than is the male%. In addition to the expected hepatic consequences of alcohol abuse, problems relating to sexual functioning are also frequently present in alcoholic women. Specifically, abnormalities of menstrual function, reproductive difficulty, and loss of secondary sex characteristics are reported to occur not infrequently in female alcoholics. Elucidation of the pathophysiologic mechanisms responsible for these abnormalities of sexual functioning in alcoholic women require the development of an experimental animal model. We report such an animal model system. Animals fed a diet with ethanol accounting for 36% of total calories over a 56day period develop significant ovarian (30.6 f 2.2 mg) and uterine (39.0 f 4.1) atrophy. Age-matched control animals fed a similar diet with dextrimaltose isocalorically substituted for ethanol do not show gonadal atrophy (ovarian weight 75.5 f 3.9 mg; uterine weight 180.5 f 18.7 mg, both p < 0.01). When organ weights were corrected for differences in body mass, the differences between alcohol-fed and isocaloric pair-fed controls either do not change or increase further. The ovaries of the alcohol-fed animals contain primordial follicles only, while the ovaries of the isocaloric pair-fed controls contain follicles in alI stages of development as well as numerous corpora lutea and corpora hemorrhagica. The endometrium and ductular epithelium of the alcohol-fed animals is atrophic and lacks all signs of secretory activity. In contrast, the endometrium and fallopian tubes of the isocaloric pair-fed controls are well developed and clearly demonstrate secretory activity. As expected from the lack of sexual development observed in the alcohol-fed animals, their plasma estradiol (Es) levels and progesterone (P) levels are reduced (E,: 27.5 + 1.3 pg/ml; P: 25 f 5.0 ng/ml) compared with those of the isocaloric pair-fed controls (E 2: 33.4 rf:1.5 pg/ml; P: 60 + 5.1 ng/ml) *Address all communications
to David H. Van Thiel.
374
(both p < 0.01). These differences in gonadaland pelvic structures between alcohol-fed and isocaloric pair-fed controls occurred in the absence of significant differences in hepatic biochemical function. These findings suggest that: (1) ethanol is toxic for the female gonad, (2) sexual dysfunction can occur in female alcoholics in the absence of irreversible histologic and biochemical liver disease and (3) differences in nutrition alone can not account for the gonadal dysfunction reported to occur in female alcoholics.
1. Introduction During the last decade, both in the laboratory and on medical wards, considerable data have been accumulated to suggest that ethanol is a general tissue toxin [l - 61. Of considerable interest is the recent observation that ethanol is toxic to the male gonads, the testes. Studies in animals have shown that ethanol acutely suppresses plasma testosterone levels in mice [ 71 and chronic ethanol ingestion results in testicular atrophy, characterized by loss of normal spermatogenesis, and Leydig cell failure which is characterized by hypoandrogenization [8]. Studies in human male chronic alcoholics have documented gonadal failure manifested as infertility, hypoandrogenization and feminization [9, lo]. Not only is gonadal dysfunction present in chronic alcoholic men, but a second central defect in gonadotropin secretion also has been suggested to be present in these men based upon inadequate responses to clomiphene and luteinizing hormone releasing factor responses [9,11]. Moreover, studies performed in normal volunteers in different laboratories have demonstrated that both the gonadal and central (hypothalamic-pituitary) abnormalities can be produced by relatively short-term alcohol feeding [ll - 131. Despite our rapidly expanding understanding of the effect of ethanol on male gonadal function, little is known about the effect of ethanol ingestion on female gonadal function. Clinical studies report that chronic alcoholic women frequently have menstrual irregularities [ 14 - 161 and more recently the toxic effects of chronic alcohol abuse on fetal development during the course of pregnancy have become recognized [17 - 191. The present studies were performed to evaluate the effect of chronic alcohol ingestion on female gonadal structure and function in rats in order to increase our understanding of the gonadal consequences of alcohol abuse.
2. Methods 2.1. Animals One hundred female white Wistar rats, age 28 days, obtained from Charles River Breeding Laboratories, Wilmington, Massachusetts, and match-
375
ed for age and weight, were housed in individual cages in the Central Animal Facility of the University of Pittsburgh School of Medicine. They were divided into four groups of 25. The first group was fed a liquid diet with ethanol accounting for 36% of the total calories [S, 201. The second group was pair-fed a similar diet in which dextrimaltose was isocalorically substituted for ethanol with each animal being paired with an animal from the first group. The third group was fed a standard rat chow (Wayne lab blox F4 obtained from Best Feeds, Oakdale, Pennsylvania) ad libitum. The fourth group was oophorectomized and fed the standard rat chow diet ad libitum. All animals were sacrificed at age 77 days. 2.2. Gross and microscopic anatomy After exsanguination, all animals were examined at necropsy with the liver, ovaries, uterus and fallopian tubes being removed, trimmed of exogenous tissue, weighed, and then fixed in Bouin’s solution for histological examination. All tissues were studied with hematoxylin and eosin and graded on an arbitrary scale of one to four with one being normal and four being most abnormal. 2.3. Blood alcohol and liver function studies Blood alcohol was determined by the method of Bernt and Gutman [ 211. Serum alkaline phosphatase, gamma glutamyl transpeptidase, glutamic pyruvic and glutamic oxalacetic transaminase activities were measured by the Clinical Chemistry Laboratories of the Presbyterian-University Hospital, Pittsburgh, Pennsylvania. All samples were run in a single assay, thus eliminating interassay variation. 2.4. Hormone assays Plasma estrone, estradiol, progesterone and corticosterone were measured in duplicate by specific radioimmunoassay methods [22 - 251. To eliminate any interassay variation, each steroid was assayed in a single assay. 2.5. Statistical analysis The Student t test was used for all statistical analyses. Differences between groups were considered significant at p < 0.01.
3. Results All animals grew steadily throughout the study period. At the time of sacrifice, the alcohol-fed animals and their pair-fed controls weighed less (p < 0.01) than the intact control-fed animals. Alcohol-fed animals had smaller ovaries, uteri, and fallopian tubes than the isocaloric pair-fed animals and the intact ad libitum control animals. In addition to being smaller, the alcohol-fed animal ovaries lacked any recognizable corpora lutea
956
Alkaline phosphotase (III/ml)
4.3*
27.5 +
23.3 f
73.7 +
Progesterone (ng/ml)
Corticosterone (pg/dl)
***UD = undetectable.
*p < 0.01 **p < 0.05
1.2**
156.1 +
Estrone (pg/ml)
Bstradiol (pg/ml) 8.8
26.7
52
14* 20*
* r
65
0.6*
SGOT (III/ml)
2.3 +
SGPT (IU/ml)
y-GTP (III/ml)
+ 204*
5.0
110.0 +
Body mass (g)
Blood alcohol (mg/dl)
0.1*
0.3
2.2 _+
0.2*
5.3
7.6 ?
0.8
?
138.0 ?
49.5
Alcohol-fed
2.8 i
Uterus + fallopian tube (g)
OV~Y
m= W Body mm (g)
Body mass (g)
Liver mass (g)
Weight at sacrifice (g)
Weight at onset (g)
Biochemical parameters in female rats
TABLE 1
1.1
0.8
3.8
8
0.9
0.2
0.8
2.1
0.8
41.7 +
7.3 8.6
54.3 r 78.0 t
48.3 ;
80.5 +
+
f
7.0
6.7
1.4
6.3
9
8
I 34
48.4 f
20
25
UD
603
UD
16.7 f
5.0 +
4.5 +
33.3 _+ 1.5
+ 10
r
f 26
1.2
t
184.2 f
49.6
Intact ad Zibitum fed
114.9 t 13.9
21
23
UD
601
UD***
11.1 f
4.7 _+ 0.2
3.9+
161.6 f
50.7 +
Isocaloric pair-fed
0.3
3.2
0.1
c
+
0.6
1.6
5.2
46.0 +_13.7
18.0 +
29.8 2
9
7
? 28
48.0 +
16
24
UD
596
UD
2.6 +
Not applicable
4.4 +
187.0 +
50.2 _+ 1.1
Oophorectomized ad libitum fed
377
and corpora hemorrhagica, documenting ovulatory failure. Moreover, the few follicles recognizable in the ovaries of the alcohol-fed animals were small and failed to show evidence of follicular maturation, suggesting ovarian failure. The uterus and fallopian tubes of the alcohol-fed animals were small and histologically immature (flat, cuboidal epithelial cell lining) as were those obtained from the oophorectomized ad libitum controls. The uterus and fallopian tubes obtained from the isocaloric pair-fed controls and the intact ad libitum controls were identical and showed signs of active secretory activity with redundant columnar epithelium rich with secretory granules. The livers of the alcohol-fed animals were larger and heavier than those of all three other groups. Histologically, they showed moderate to marked fatty metamorphosis, but no evidence of cellular necrosis or fibrosis. Blood alcohol levels were easily measurable in the alcohol-fed animals but were undetectable in the three control groups. Liver function studies were mildly to moderately abnormal, based upon plasma enzymatic activities, only in the alcohol-fed animals. As might be expected from the anatomic data, the plasma levels of estradiol and progesterone in the alcohol-fed animals were reduced and essentially identical in the alcohol-fed and oophorectomized ad libitum control animals. In contrast, the plasma levels of estrone and corticosterone were increased in the alcohol-fed and their isocaloric pair-fed controls relative to those of the ad libitum control animals.
Discussion These results document alcohol-induced ovarian atrophy and functional ovulatory failure in rats fed ethanol for seven weeks. Grossly, the ovaries and sex steroid responsive tissues of the alcohol-fed rats were smaller and showed no signs of reproductive and endocrine function, lacking corpora lutea, corpora hemorrhagica, and visible secretory activity. In contrast, ovaries from isocaloric pair-fed control animals, although smaller than those of the intact ad libitum controls, contained numerous maturing follicles, corpora lutea and corpora hemorrhagica. Levels of the various sex steroids (estradiol and progesterone) which reflect normal ovarian function were reduced @ < 0.01) in the plasma of the alcohol-fed animals compared with those of the pair-fed isocaloric and the intact ad libitum control animals. In contrast to these steroids which arise primarily from ovarian secretion, those steroids that arise either directly or indirectly, in large part, from adrenal secretion (corticosterone and estrone) were increased in the plasma of the alcohol-fed animals and their isocaloric pair-fed controls, suggesting that both alcohol feeding and isocaloric pairfeeding are stressful situations associated with an increased adrenal cortical secretion. Taking together clinical observations reported by others [ 14 - 161 and our studies in the rat, it would appear that alcohol is a direct gonadal toxin
318
in the female. In males, chronic alcohol ingestion is associated with testicular atrophy, loss of libido, azoospermia, and hypoandrogenization [S, 9,11 13, 261. In females, chronic alcohol abuse is associated with ovarian atrophy, failure to ovulate and hypoestrogenization [14 - 161. In both men and women, the addition of alcohol-induced liver disease to these gonadal dysfunctions is associated with an increased peripheral (non-hepatic) conversion of weak adrenal androgens to estrone, and to a lesser degree estradiol, which results in feminization in males and probably reduces the absolute degree of hypoestrogenization observed in the female. Further compounding the gonadal injury induced by alcohol is a secondary central defect in hypothalamic pituitary secretion of gonadotropins which prevents the expected rise in gonadotropins associated with primary alcohol-induced gonadal failure [9, lo]. Whether alcohol-induced hepatic injury contributes to this secondary central defect in gonadotropin secretion as a consequence of the pre-hepatic retention of weak steroidal and nonsteroidal estrogens is as yet an unsettled question. Clearly, more research is indicated in this exciting new area of alcohol research in order to determine its reversibility, its dose responsiveness (both durations and degree of alcohol abuse needed to produce these injuries), and the contribution the associated problems of malnutrition, liver disease, neurologic disease, etc. play in the genesis and course of alcohol-induced hypogonadism.
Acknowledgments This work has been supported by the National Institutes of Health Grants AA01450 and HD08954.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
J. D. Beard and D. H. Knott, Amer. J. Med. Sci., 252 (1966) 518. L. W, Sullivan and V. Herbert, J. Clin. Invest., 43 (1964) 2048. T. J. Reogun et al., J. Clin. Invest., 45 (1966) 270. K. Ekbom et al., Arch. Neurol., 10 (1964) 449. C. S. Lieber et al., J. Clin. Invest., 41 (1962) 1863. D. H. Van Thiel et al., Metabolism, in press. F. Bard and A. Bartke, Steroids, 23 (1974) 921. D. H. Van Thiel et al., Gastroentetology, 69 (1975) 326. D. H. Van Thiel ef al., Gastroenterology, 67 (1974) 1188. D. H. Van Thiel et al., Metabolism, 24 (1974) 1015. D. H. Van Thiel et al., New England J. Med., submitted for publication. R. Ylikahri et al., J. Steroid Biochem., 5 (1974) 655. G. G. Gordan et al, New England J. Med., 295 (1976) 793. B. Kinsey, The Female Alcoholic: A Social Psychological Study, C. C. Thomas, Springfield, Illinois, 1966. S. C. Wilsnack, J. Abnormal Psychol., 82 (1973) 253. S. C. Cowley and J. B. Copeland, Newsweek, (Nov. 15) (1976) 73.. H. G. Green, Amer. J. Obstet. Gynecol., 118 (1974) 7313.
379
18 19 20 21 22 23 24 25 26
J. J. Mulvihill et al., Amer. J. Obstet. Gynecol., 125 (1976) 937. M. D. Manteuffel and J. H. Hofsteig, Drug Ale. Dependence, 2 (1977) 421. C. S. Lieber and L. M. DeCarli, Amer. J. Clin. Nutr., 23 (1970) 474. E. Bernt and I. Gutman, in H. U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Vol. 3, Academic Press, New York, 1974, p. 1499. D. L. Loriaux et al., Steroids, 18 (1971) 463. J. Hotchkiss et al., Endocrinology, 89 (1971) 177. G. P. Orczyk et al., in B. M. Jaffe and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 347. P. Vecseri, in B. M. Jaffe and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 393. D. H. Van Thiel and R. Lester, Gastroenterology, 71 (1976) 318.
Discussion D. LESTER - You seem to get effects in your pair-fed sucrose subjects that are quite different from those in ad libitum chow animals. I wonder if it wouldn’t be a good idea to use other kinds of controls, especially since sucrose is not a substrate for alcohol dehydrogenase and alcohol is. VAN THIEL that you use quately used store sucrose. Thus, I think
- We use dextrimaltose not sucrose. I don’t think that sucrose or any sugar isocalorically replaces ethanol in the diet because ethanol calories are inadeby the animal. That is, animals tend to burn up ethanol calories while they Even so, the isocaloric control animals show normal gonadal function. that it is an adequate control, but it is not an ideal one.
D. LESTER - I notice that your pair-fed controls actually weigh more than your alcohol animals. We certainly have not found that in our laboratory. Our pair-fed controls have exactly the same weight. Are you sure that you are pair-feeding correctly? VAN THIEL - Yes, but don’t forget that we started to study.our animals when they were pre-pubertal and still growing. The immature animal fed alcohol grows more slowly than its control and thus the difference in body weight between the two groups is magnified. We do not see this with our adult animals, however, probably because they are not growing as rapidly. FREUND - Once you have a difference in body weight between groups as you have, the question of nutritional factors comes up as possibly interacting with or independently causing the effects you are measuring. GOLDSTEIN - Even though the pair-feeding paradigm controls for what goes into the gut, what gets out may be affected by the presence of ethanol itself. Is anything known about whether the particular kind of hormonal and morphological changes that you see could be caused by a nutritional factor? VAN THIEL - Sodium or glucose absorption is significantly reduced by ethanol’s presence in the gut, so that even if you pair-feed what passes the lips, you do not necessarily pair-feed what passes the mucosa, and this certainly is a problem. FREUND - The age at which you started your animals on the alcohol-containing diets is important, for it is difficult to extrapolate data from an individual who starts to drink at an early age to one who begins to drink at full maturity, as is the usual case with human alcoholism.
380 VAN THIEL -We can produce gonadal failure in immature animals in a much shorter time than we can when the diet is first introduced to an adult, fully mature, animal. FREUND
-
VAN THIEL
Is this gonadal -
defect
reversible?
It is if you only give alcohol
for relatively
short
periods
of time.
MARCUS - Have you looked for reversibility of these gonadal effects in animals ing the paradigm employed in the present study, rather than in adult animals? VAN THIEL
-
No, we haven’t
follow-
as yet.
FREUND - I notice that your alcohol animals have impaired liver function. To what degree do you think the effects that you see are attributed to this fact, rather than to alcohol itself, since the liver is a central organ in steroid metabolism? VAN THIEL - I think that it depends upon what you are looking at. It takes a week or two to develop significant fatty liver, at least histologically, and yet alterations in plasma testosterone, for instance, can be demonstrated within three to four days. ELLIS
-
Do you know
VAN THIEL hours.
-
the blood
alcohol
The levels ranged
between
levels in your animals? 100 and 125 mg% when measured
at 1000
RANDALL - Many models of the fetal alcohol syndrome introduce alcohol-containing diets to the females prior to breeding and continue them throughout gestation. Your results suggest that these models may be complicating matters by producing alterations in the reproductive system of the mother, which may indirectly be affecting the developing fetus. These models may, however, better simulate the human condition. VAN THIEL - I agree. We have had difficulty mating chronically fed males as well as females, even when we tried to mate them with normal chow mates. In the case of the alcoholic females, we saw no successful progeny. STIJRTEVANT - Have you considered trying to sample corticosterone more about the time of your light to dark cycle change when the levels would be at their peak? The relationship between the two could be changed. VAN THIEL -We measured see if they were related.
them
at the time that we measured
the estrone
levels to
KALANT _- If you have animals on ad libitum feeding of alcohol-containing diets, you never get ethanol levels of the kind that will produce marked CNS effects. I wonder if the animals on these ad lib. diets are really the appropriate model for looking at the effects of intoxication in humans upon hypophyseal or gonadal hormones. It seems to me that one should be looking at the effects of graded levels of intoxication, rather than 24-hour intakes of alcohol in the diet. There is almost certainly a threshold concentration that must be exceeded before you get an effect on hormone release. For example, we found many years ago that the same total dose of alcohol if given as one single intubation would give very different effects on corticosterone release from the rat adrenal than would the same dose divided into three fractions at hourly intervals.
INFLUENCE OF ETHANOL NATAL DEVELOPMENT
ON HORMONAL
SYSTEMS
AND PRE-
Moderator: Dr. Carrie L. Randall Discussants : Dr. Gerhard
Freund
Dr. Dora B. Goldstein Dr. David Lester
Drug and Alcohol Dependence, 2 (1977) 373 - 380 @ Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
ETHANOL:
A GONADAL
TOXIN
373
IN THE FEMALE
DAVID H. VAN THIEL*, JUDITH S. GAVALER
and ROGER LESTER
Division of Gastroenterology, Department of Medicine, of Medicine, Pittsburgh, Pennsylwnia 15261 (U.S.A.)
University of Pittsburgh School
Summary It has been estimated recently that there are at least two million female alcoholics presently within the United States. Even more disturbing is the fact that this number is steadily increasing. The number of female alcoholics has increased such that it has been estimated that one out of every three alcoholics is probably a woman. As distressing as these social facts are, even more disturbing are the clinical suggestions that the female liver is more readily injured by ethanol than is the male%. In addition to the expected hepatic consequences of alcohol abuse, problems relating to sexual functioning are also frequently present in alcoholic women. Specifically, abnormalities of menstrual function, reproductive difficulty, and loss of secondary sex characteristics are reported to occur not infrequently in female alcoholics. Elucidation of the pathophysiologic mechanisms responsible for these abnormalities of sexual functioning in alcoholic women require the development of an experimental animal model. We report such an animal model system. Animals fed a diet with ethanol accounting for 36% of total calories over a 56day period develop significant ovarian (30.6 f 2.2 mg) and uterine (39.0 f 4.1) atrophy. Age-matched control animals fed a similar diet with dextrimaltose isocalorically substituted for ethanol do not show gonadal atrophy (ovarian weight 75.5 f 3.9 mg; uterine weight 180.5 f 18.7 mg, both p < 0.01). When organ weights were corrected for differences in body mass, the differences between alcohol-fed and isocaloric pair-fed controls either do not change or increase further. The ovaries of the alcohol-fed animals contain primordial follicles only, while the ovaries of the isocaloric pair-fed controls contain follicles in alI stages of development as well as numerous corpora lutea and corpora hemorrhagica. The endometrium and ductular epithelium of the alcohol-fed animals is atrophic and lacks all signs of secretory activity. In contrast, the endometrium and fallopian tubes of the isocaloric pair-fed controls are well developed and clearly demonstrate secretory activity. As expected from the lack of sexual development observed in the alcohol-fed animals, their plasma estradiol (Es) levels and progesterone (P) levels are reduced (E,: 27.5 + 1.3 pg/ml; P: 25 f 5.0 ng/ml) compared with those of the isocaloric pair-fed controls (E 2: 33.4 rf:1.5 pg/ml; P: 60 + 5.1 ng/ml) *Address all communications
to David H. Van Thiel.
374
(both p < 0.01). These differences in gonadaland pelvic structures between alcohol-fed and isocaloric pair-fed controls occurred in the absence of significant differences in hepatic biochemical function. These findings suggest that: (1) ethanol is toxic for the female gonad, (2) sexual dysfunction can occur in female alcoholics in the absence of irreversible histologic and biochemical liver disease and (3) differences in nutrition alone can not account for the gonadal dysfunction reported to occur in female alcoholics.
1. Introduction During the last decade, both in the laboratory and on medical wards, considerable data have been accumulated to suggest that ethanol is a general tissue toxin [l - 61. Of considerable interest is the recent observation that ethanol is toxic to the male gonads, the testes. Studies in animals have shown that ethanol acutely suppresses plasma testosterone levels in mice [ 71 and chronic ethanol ingestion results in testicular atrophy, characterized by loss of normal spermatogenesis, and Leydig cell failure which is characterized by hypoandrogenization [8]. Studies in human male chronic alcoholics have documented gonadal failure manifested as infertility, hypoandrogenization and feminization [9, lo]. Not only is gonadal dysfunction present in chronic alcoholic men, but a second central defect in gonadotropin secretion also has been suggested to be present in these men based upon inadequate responses to clomiphene and luteinizing hormone releasing factor responses [9,11]. Moreover, studies performed in normal volunteers in different laboratories have demonstrated that both the gonadal and central (hypothalamic-pituitary) abnormalities can be produced by relatively short-term alcohol feeding [ll - 131. Despite our rapidly expanding understanding of the effect of ethanol on male gonadal function, little is known about the effect of ethanol ingestion on female gonadal function. Clinical studies report that chronic alcoholic women frequently have menstrual irregularities [ 14 - 161 and more recently the toxic effects of chronic alcohol abuse on fetal development during the course of pregnancy have become recognized [17 - 191. The present studies were performed to evaluate the effect of chronic alcohol ingestion on female gonadal structure and function in rats in order to increase our understanding of the gonadal consequences of alcohol abuse.
2. Methods 2.1. Animals One hundred female white Wistar rats, age 28 days, obtained from Charles River Breeding Laboratories, Wilmington, Massachusetts, and match-
375
ed for age and weight, were housed in individual cages in the Central Animal Facility of the University of Pittsburgh School of Medicine. They were divided into four groups of 25. The first group was fed a liquid diet with ethanol accounting for 36% of the total calories [S, 201. The second group was pair-fed a similar diet in which dextrimaltose was isocalorically substituted for ethanol with each animal being paired with an animal from the first group. The third group was fed a standard rat chow (Wayne lab blox F4 obtained from Best Feeds, Oakdale, Pennsylvania) ad libitum. The fourth group was oophorectomized and fed the standard rat chow diet ad libitum. All animals were sacrificed at age 77 days. 2.2. Gross and microscopic anatomy After exsanguination, all animals were examined at necropsy with the liver, ovaries, uterus and fallopian tubes being removed, trimmed of exogenous tissue, weighed, and then fixed in Bouin’s solution for histological examination. All tissues were studied with hematoxylin and eosin and graded on an arbitrary scale of one to four with one being normal and four being most abnormal. 2.3. Blood alcohol and liver function studies Blood alcohol was determined by the method of Bernt and Gutman [ 211. Serum alkaline phosphatase, gamma glutamyl transpeptidase, glutamic pyruvic and glutamic oxalacetic transaminase activities were measured by the Clinical Chemistry Laboratories of the Presbyterian-University Hospital, Pittsburgh, Pennsylvania. All samples were run in a single assay, thus eliminating interassay variation. 2.4. Hormone assays Plasma estrone, estradiol, progesterone and corticosterone were measured in duplicate by specific radioimmunoassay methods [22 - 251. To eliminate any interassay variation, each steroid was assayed in a single assay. 2.5. Statistical analysis The Student t test was used for all statistical analyses. Differences between groups were considered significant at p < 0.01.
3. Results All animals grew steadily throughout the study period. At the time of sacrifice, the alcohol-fed animals and their pair-fed controls weighed less (p < 0.01) than the intact control-fed animals. Alcohol-fed animals had smaller ovaries, uteri, and fallopian tubes than the isocaloric pair-fed animals and the intact ad libitum control animals. In addition to being smaller, the alcohol-fed animal ovaries lacked any recognizable corpora lutea
956
Alkaline phosphotase (III/ml)
4.3*
27.5 +
23.3 f
73.7 +
Progesterone (ng/ml)
Corticosterone (pg/dl)
***UD = undetectable.
*p < 0.01 **p < 0.05
1.2**
156.1 +
Estrone (pg/ml)
Bstradiol (pg/ml) 8.8
26.7
52
14* 20*
* r
65
0.6*
SGOT (III/ml)
2.3 +
SGPT (IU/ml)
y-GTP (III/ml)
+ 204*
5.0
110.0 +
Body mass (g)
Blood alcohol (mg/dl)
0.1*
0.3
2.2 _+
0.2*
5.3
7.6 ?
0.8
?
138.0 ?
49.5
Alcohol-fed
2.8 i
Uterus + fallopian tube (g)
OV~Y
m= W Body mm (g)
Body mass (g)
Liver mass (g)
Weight at sacrifice (g)
Weight at onset (g)
Biochemical parameters in female rats
TABLE 1
1.1
0.8
3.8
8
0.9
0.2
0.8
2.1
0.8
41.7 +
7.3 8.6
54.3 r 78.0 t
48.3 ;
80.5 +
+
f
7.0
6.7
1.4
6.3
9
8
I 34
48.4 f
20
25
UD
603
UD
16.7 f
5.0 +
4.5 +
33.3 _+ 1.5
+ 10
r
f 26
1.2
t
184.2 f
49.6
Intact ad Zibitum fed
114.9 t 13.9
21
23
UD
601
UD***
11.1 f
4.7 _+ 0.2
3.9+
161.6 f
50.7 +
Isocaloric pair-fed
0.3
3.2
0.1
c
+
0.6
1.6
5.2
46.0 +_13.7
18.0 +
29.8 2
9
7
? 28
48.0 +
16
24
UD
596
UD
2.6 +
Not applicable
4.4 +
187.0 +
50.2 _+ 1.1
Oophorectomized ad libitum fed
377
and corpora hemorrhagica, documenting ovulatory failure. Moreover, the few follicles recognizable in the ovaries of the alcohol-fed animals were small and failed to show evidence of follicular maturation, suggesting ovarian failure. The uterus and fallopian tubes of the alcohol-fed animals were small and histologically immature (flat, cuboidal epithelial cell lining) as were those obtained from the oophorectomized ad libitum controls. The uterus and fallopian tubes obtained from the isocaloric pair-fed controls and the intact ad libitum controls were identical and showed signs of active secretory activity with redundant columnar epithelium rich with secretory granules. The livers of the alcohol-fed animals were larger and heavier than those of all three other groups. Histologically, they showed moderate to marked fatty metamorphosis, but no evidence of cellular necrosis or fibrosis. Blood alcohol levels were easily measurable in the alcohol-fed animals but were undetectable in the three control groups. Liver function studies were mildly to moderately abnormal, based upon plasma enzymatic activities, only in the alcohol-fed animals. As might be expected from the anatomic data, the plasma levels of estradiol and progesterone in the alcohol-fed animals were reduced and essentially identical in the alcohol-fed and oophorectomized ad libitum control animals. In contrast, the plasma levels of estrone and corticosterone were increased in the alcohol-fed and their isocaloric pair-fed controls relative to those of the ad libitum control animals.
Discussion These results document alcohol-induced ovarian atrophy and functional ovulatory failure in rats fed ethanol for seven weeks. Grossly, the ovaries and sex steroid responsive tissues of the alcohol-fed rats were smaller and showed no signs of reproductive and endocrine function, lacking corpora lutea, corpora hemorrhagica, and visible secretory activity. In contrast, ovaries from isocaloric pair-fed control animals, although smaller than those of the intact ad libitum controls, contained numerous maturing follicles, corpora lutea and corpora hemorrhagica. Levels of the various sex steroids (estradiol and progesterone) which reflect normal ovarian function were reduced @ < 0.01) in the plasma of the alcohol-fed animals compared with those of the pair-fed isocaloric and the intact ad libitum control animals. In contrast to these steroids which arise primarily from ovarian secretion, those steroids that arise either directly or indirectly, in large part, from adrenal secretion (corticosterone and estrone) were increased in the plasma of the alcohol-fed animals and their isocaloric pair-fed controls, suggesting that both alcohol feeding and isocaloric pairfeeding are stressful situations associated with an increased adrenal cortical secretion. Taking together clinical observations reported by others [ 14 - 161 and our studies in the rat, it would appear that alcohol is a direct gonadal toxin
318
in the female. In males, chronic alcohol ingestion is associated with testicular atrophy, loss of libido, azoospermia, and hypoandrogenization [S, 9,11 13, 261. In females, chronic alcohol abuse is associated with ovarian atrophy, failure to ovulate and hypoestrogenization [14 - 161. In both men and women, the addition of alcohol-induced liver disease to these gonadal dysfunctions is associated with an increased peripheral (non-hepatic) conversion of weak adrenal androgens to estrone, and to a lesser degree estradiol, which results in feminization in males and probably reduces the absolute degree of hypoestrogenization observed in the female. Further compounding the gonadal injury induced by alcohol is a secondary central defect in hypothalamic pituitary secretion of gonadotropins which prevents the expected rise in gonadotropins associated with primary alcohol-induced gonadal failure [9, lo]. Whether alcohol-induced hepatic injury contributes to this secondary central defect in gonadotropin secretion as a consequence of the pre-hepatic retention of weak steroidal and nonsteroidal estrogens is as yet an unsettled question. Clearly, more research is indicated in this exciting new area of alcohol research in order to determine its reversibility, its dose responsiveness (both durations and degree of alcohol abuse needed to produce these injuries), and the contribution the associated problems of malnutrition, liver disease, neurologic disease, etc. play in the genesis and course of alcohol-induced hypogonadism.
Acknowledgments This work has been supported by the National Institutes of Health Grants AA01450 and HD08954.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
J. D. Beard and D. H. Knott, Amer. J. Med. Sci., 252 (1966) 518. L. W, Sullivan and V. Herbert, J. Clin. Invest., 43 (1964) 2048. T. J. Reogun et al., J. Clin. Invest., 45 (1966) 270. K. Ekbom et al., Arch. Neurol., 10 (1964) 449. C. S. Lieber et al., J. Clin. Invest., 41 (1962) 1863. D. H. Van Thiel et al., Metabolism, in press. F. Bard and A. Bartke, Steroids, 23 (1974) 921. D. H. Van Thiel et al., Gastroentetology, 69 (1975) 326. D. H. Van Thiel ef al., Gastroenterology, 67 (1974) 1188. D. H. Van Thiel et al., Metabolism, 24 (1974) 1015. D. H. Van Thiel et al., New England J. Med., submitted for publication. R. Ylikahri et al., J. Steroid Biochem., 5 (1974) 655. G. G. Gordan et al, New England J. Med., 295 (1976) 793. B. Kinsey, The Female Alcoholic: A Social Psychological Study, C. C. Thomas, Springfield, Illinois, 1966. S. C. Wilsnack, J. Abnormal Psychol., 82 (1973) 253. S. C. Cowley and J. B. Copeland, Newsweek, (Nov. 15) (1976) 73.. H. G. Green, Amer. J. Obstet. Gynecol., 118 (1974) 7313.
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J. J. Mulvihill et al., Amer. J. Obstet. Gynecol., 125 (1976) 937. M. D. Manteuffel and J. H. Hofsteig, Drug Ale. Dependence, 2 (1977) 421. C. S. Lieber and L. M. DeCarli, Amer. J. Clin. Nutr., 23 (1970) 474. E. Bernt and I. Gutman, in H. U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Vol. 3, Academic Press, New York, 1974, p. 1499. D. L. Loriaux et al., Steroids, 18 (1971) 463. J. Hotchkiss et al., Endocrinology, 89 (1971) 177. G. P. Orczyk et al., in B. M. Jaffe and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 347. P. Vecseri, in B. M. Jaffe and H. R. Behrman (eds.), Methods of Hormone Radioimmunoassay, Academic Press, New York, 1974, p. 393. D. H. Van Thiel and R. Lester, Gastroenterology, 71 (1976) 318.
Discussion D. LESTER - You seem to get effects in your pair-fed sucrose subjects that are quite different from those in ad libitum chow animals. I wonder if it wouldn’t be a good idea to use other kinds of controls, especially since sucrose is not a substrate for alcohol dehydrogenase and alcohol is. VAN THIEL that you use quately used store sucrose. Thus, I think
- We use dextrimaltose not sucrose. I don’t think that sucrose or any sugar isocalorically replaces ethanol in the diet because ethanol calories are inadeby the animal. That is, animals tend to burn up ethanol calories while they Even so, the isocaloric control animals show normal gonadal function. that it is an adequate control, but it is not an ideal one.
D. LESTER - I notice that your pair-fed controls actually weigh more than your alcohol animals. We certainly have not found that in our laboratory. Our pair-fed controls have exactly the same weight. Are you sure that you are pair-feeding correctly? VAN THIEL - Yes, but don’t forget that we started to study.our animals when they were pre-pubertal and still growing. The immature animal fed alcohol grows more slowly than its control and thus the difference in body weight between the two groups is magnified. We do not see this with our adult animals, however, probably because they are not growing as rapidly. FREUND - Once you have a difference in body weight between groups as you have, the question of nutritional factors comes up as possibly interacting with or independently causing the effects you are measuring. GOLDSTEIN - Even though the pair-feeding paradigm controls for what goes into the gut, what gets out may be affected by the presence of ethanol itself. Is anything known about whether the particular kind of hormonal and morphological changes that you see could be caused by a nutritional factor? VAN THIEL - Sodium or glucose absorption is significantly reduced by ethanol’s presence in the gut, so that even if you pair-feed what passes the lips, you do not necessarily pair-feed what passes the mucosa, and this certainly is a problem. FREUND - The age at which you started your animals on the alcohol-containing diets is important, for it is difficult to extrapolate data from an individual who starts to drink at an early age to one who begins to drink at full maturity, as is the usual case with human alcoholism.
380 VAN THIEL -We can produce gonadal failure in immature animals in a much shorter time than we can when the diet is first introduced to an adult, fully mature, animal. FREUND
-
VAN THIEL
Is this gonadal -
defect
reversible?
It is if you only give alcohol
for relatively
short
periods
of time.
MARCUS - Have you looked for reversibility of these gonadal effects in animals ing the paradigm employed in the present study, rather than in adult animals? VAN THIEL
-
No, we haven’t
follow-
as yet.
FREUND - I notice that your alcohol animals have impaired liver function. To what degree do you think the effects that you see are attributed to this fact, rather than to alcohol itself, since the liver is a central organ in steroid metabolism? VAN THIEL - I think that it depends upon what you are looking at. It takes a week or two to develop significant fatty liver, at least histologically, and yet alterations in plasma testosterone, for instance, can be demonstrated within three to four days. ELLIS
-
Do you know
VAN THIEL hours.
-
the blood
alcohol
The levels ranged
between
levels in your animals? 100 and 125 mg% when measured
at 1000
RANDALL - Many models of the fetal alcohol syndrome introduce alcohol-containing diets to the females prior to breeding and continue them throughout gestation. Your results suggest that these models may be complicating matters by producing alterations in the reproductive system of the mother, which may indirectly be affecting the developing fetus. These models may, however, better simulate the human condition. VAN THIEL - I agree. We have had difficulty mating chronically fed males as well as females, even when we tried to mate them with normal chow mates. In the case of the alcoholic females, we saw no successful progeny. STIJRTEVANT - Have you considered trying to sample corticosterone more about the time of your light to dark cycle change when the levels would be at their peak? The relationship between the two could be changed. VAN THIEL -We measured see if they were related.
them
at the time that we measured
the estrone
levels to
KALANT _- If you have animals on ad libitum feeding of alcohol-containing diets, you never get ethanol levels of the kind that will produce marked CNS effects. I wonder if the animals on these ad lib. diets are really the appropriate model for looking at the effects of intoxication in humans upon hypophyseal or gonadal hormones. It seems to me that one should be looking at the effects of graded levels of intoxication, rather than 24-hour intakes of alcohol in the diet. There is almost certainly a threshold concentration that must be exceeded before you get an effect on hormone release. For example, we found many years ago that the same total dose of alcohol if given as one single intubation would give very different effects on corticosterone release from the rat adrenal than would the same dose divided into three fractions at hourly intervals.
