The Effect Activity
of Alcohol Ingestion on Hepatic Aromatase and Plasma Steroid Hormones in the Rat
Gary G. Gordon, Chronic
alcohol
ingestion
in the
A. Louis
rat
Southren,
resulted
in
increased hepatic aromatase activity, elevation of plasma estradiol, and a decrease in plasma testosterone levels. Testicular incubation studies indicated that the source of the estrogen was not of gonadal origin but was, most likely, due to increased peripheral conversion. The failure of HCG in vitro to restore testicular secretion of testosterone to normal levels suggested a direct action of alcohol, or a metabolic product, on gonadal secretory processes, as distinct from trophic hormone effects. This study demonstrates that many of the hormonal alterations seen in cirrhosis of the liver in man may be produced directly by alcohol feeding without cirrhotic changes in the rat.
LIVER ALCOHOLIC frequently associated
in the male is with hypogonadism and feminization.’ Classically, this has been ascribed to failure of the diseased liver to metabolize (i.e., “detoxify”) estrogenic hormones resulting in their accumulation (“retention”) and increased manifestation of hormonal effect (i.e., gynecomastia, testicular atrophy, loss of potentia).2m4 However, recent studies by our group and others’-* have demonstrated that the increase in circulating estrogens (estradiol and estrone), in patients with alcoholic liver disease, is not due to defective metabolic clearance but is associated, in many patients, with an increase in the production rate of these hormones. Indeed, infusion studies in men with cirrhosis of the liver have suggested that increased peripheral conversion of androgenic precursor hormones (testosterone and androstenedione) is responsible for the increased circulating estrogens.’ Since alcohol is known to induce a variety of hepatic disease
Jozef
Vittek,
and
Medicine (CUNY), New York, N. Y. Received for publication March 3. I978 Supported in part by the Medical Research Service of the Veterans Administration and USPHS Grants AA-02300. AA-00224. and AA-03508. Address reprint requests to Gary C. Gordon, M.D., The New York Medical College, 1249 Fifth Avenue, New York, N. Y. 10029. c, 1979 by Grune & Stratton, Inc. 0026-0495/79/280/-0004~01.00/0
20
S. Lieber
microsomal enzyme systems, including those that metabolize steroid hormones, such as the A-ring reductases,“*” the microsomal mixedfunction oxidases” (a system with high affinity for steroid hormonesi3) and the microsomal ethanol-oxidizing system,14 we have studied the effects of chronic alcohol ingestion in the rat on another group of hepatic oxidative enzymes (aromatases) that are responsible for the conversion of androgens to estrogens. In addition, plasma sex-hormone levels were measured and testicular incubations were carried out to study possible gonadal effects of alcohol consumption on hormone secretion.
Forty-six
MATERfALS
AND
weanling
six adult
were maintained (average this
cirrhosis,
of alcohol
of alcohol
weanlings
and
identical
the alcohol
feeding
was given except
was discontinued
8:00 and
morning.
animals
were
(alcohol treated
immediately
at this
obtained
isocaloric
etherized
activity studies
carbohy-
and
in the blood of either
time).
The
plasma
and deep frozen for hormonal
for various
dietary
were maintained
into heparinized
a later date. The livers were removed, assayed for aromatase
of (23
10:00 a.m. the following
lightly
puncture
was not detectable animals
group
at 4:00 p.m., and the animals
between by aortic
Rats on
changes
36% of their
that
days
The day before the assay,
were sacrificed exsanguinated
develop
controls
for alcohol.
rats
for 34-54
was 46 days).
do not
and the pair-fed
conditions,
The
diet”
liver. I6 The experimental
3 adults)
drate was substituted
Sprague-Dawley
liquid
feeding
but only fatty
calories as ethanol under
and
METHODS
on a synthetic
duration
mode
control
or
was separated
measurements
frozen at -70°C
within (vida
then
syringes
at and
72 hr. The testes were
infra).
Hepatic Aromatase Activity (HAA} Hepatic
aromatase
formation
activity
was determined
of ‘H labeled estradiol
a modification
From the Endocrine Section, Department of Medicine. New York Medical College aad the Alcohol Research Center, Bronx V.A. Hospital and the Mount Sinai School of
Charles
of the method of Vittek
homogenized
were prepared
of Ragab et af.19 The microsomal mg of protein)
The assay mixture 3 mM
7n-‘H-testosterone
consisted
I
fraction
to method
(equivalent after
to 5
preparation.
of 0.05 A4 phosphate
buffer 5 mM
NADPH,
and appropriate
or 7a-3H-androstenedione) (equivalent
ml. Incubation
shaking
as previously
0.25 M sucrose, 5 mM of MgCI,,
tration of 3 X IO -*A4 Dubnoff
The livers were
according
was assayed immediately
(pH 7.4) containing
volume of
et al.”
in a glass and teflon homogenizer
described.18 Microsomes
of ATP,
by the rate of
(E2) and estrone (E,) using
incubator.
to IO&i
was carried Aliquots
substrate
(either
at a concen‘H)
in a total
out at 37O in a
for analysis were taken
at 0, 15, 30, 60, and I20 min and placed into 10 ml of ether containing substrate, estradiol, and estrone each labeled with
Mefc?bolism, Vol. 28, No. 1 (January),
1979
EFFECT
OF
ALCOHOL
INGESTION
ON
21
RATS
14C (5000 9500 7and 500 cpm, respectively) for measurement of recovery. Steroids were extracted with 2 X IO ml of ether, evaporated under a stream of N, and the residue redissolved in 200 pl of methanol. The recovery of the steroids was over 95%. Aliquots of the extract containing 20 rg of nonradioactive carrier steroids were then spotted on thin layer chromatographic glass plates (20 X 20 cm) and coated with silica gel 60,250 p thickness (Merk-Darmstadt). Two dimensional chromatography was carried out using chloroform and methanol (98:2 v/v) followed by chloroform and ethyl acetate (l6:4 v/v) after drying for 15 min and developed with a spray of 2% iodine in ethanol. This method separates all of the androgenic and estrogenic compounds.” The purity of all labeled compounds (i.e., substrates and isolated metabolites) was determined as previously described.” The areas corresponding to E, and E, were scraped into scintillation vials, extracted with I ml of methanol and after addition of IO ml of scintillation fluid (liqui-Ruor in toluene). counted in a Packard liquid scintillation spectrometer system (efhciency 59%, for ‘H and 86% for “C). The overall recovery for the method was at least 85%. However, because of the low percentage conversion to the estrogens the individual recovered counts of E, and E, were pooled for calculation of HAA activity after recovery correction. The results were expressed as pmole of E, + E, formed/hr/ IO mg microsomal protein.
Plasma
ETHANOL
. P 0.15); Ez (0.75 k 0.05 vs. 0.73 k 0.10 ng/g,p > 0.35) or E, (0.87 f 0.08 vs. 0.92 k 0.12 rig/g,, p > 0.35). This
El
T P
of Alcohol Ingestion on Hepatic Aromatase and Plasma Steroid Hormones in the Rat
Gary G. Gordon, Chronic
alcohol
ingestion
in the
A. Louis
rat
Southren,
resulted
in
increased hepatic aromatase activity, elevation of plasma estradiol, and a decrease in plasma testosterone levels. Testicular incubation studies indicated that the source of the estrogen was not of gonadal origin but was, most likely, due to increased peripheral conversion. The failure of HCG in vitro to restore testicular secretion of testosterone to normal levels suggested a direct action of alcohol, or a metabolic product, on gonadal secretory processes, as distinct from trophic hormone effects. This study demonstrates that many of the hormonal alterations seen in cirrhosis of the liver in man may be produced directly by alcohol feeding without cirrhotic changes in the rat.
LIVER ALCOHOLIC frequently associated
in the male is with hypogonadism and feminization.’ Classically, this has been ascribed to failure of the diseased liver to metabolize (i.e., “detoxify”) estrogenic hormones resulting in their accumulation (“retention”) and increased manifestation of hormonal effect (i.e., gynecomastia, testicular atrophy, loss of potentia).2m4 However, recent studies by our group and others’-* have demonstrated that the increase in circulating estrogens (estradiol and estrone), in patients with alcoholic liver disease, is not due to defective metabolic clearance but is associated, in many patients, with an increase in the production rate of these hormones. Indeed, infusion studies in men with cirrhosis of the liver have suggested that increased peripheral conversion of androgenic precursor hormones (testosterone and androstenedione) is responsible for the increased circulating estrogens.’ Since alcohol is known to induce a variety of hepatic disease
Jozef
Vittek,
and
Medicine (CUNY), New York, N. Y. Received for publication March 3. I978 Supported in part by the Medical Research Service of the Veterans Administration and USPHS Grants AA-02300. AA-00224. and AA-03508. Address reprint requests to Gary C. Gordon, M.D., The New York Medical College, 1249 Fifth Avenue, New York, N. Y. 10029. c, 1979 by Grune & Stratton, Inc. 0026-0495/79/280/-0004~01.00/0
20
S. Lieber
microsomal enzyme systems, including those that metabolize steroid hormones, such as the A-ring reductases,“*” the microsomal mixedfunction oxidases” (a system with high affinity for steroid hormonesi3) and the microsomal ethanol-oxidizing system,14 we have studied the effects of chronic alcohol ingestion in the rat on another group of hepatic oxidative enzymes (aromatases) that are responsible for the conversion of androgens to estrogens. In addition, plasma sex-hormone levels were measured and testicular incubations were carried out to study possible gonadal effects of alcohol consumption on hormone secretion.
Forty-six
MATERfALS
AND
weanling
six adult
were maintained (average this
cirrhosis,
of alcohol
of alcohol
weanlings
and
identical
the alcohol
feeding
was given except
was discontinued
8:00 and
morning.
animals
were
(alcohol treated
immediately
at this
obtained
isocaloric
etherized
activity studies
carbohy-
and
in the blood of either
time).
The
plasma
and deep frozen for hormonal
for various
dietary
were maintained
into heparinized
a later date. The livers were removed, assayed for aromatase
of (23
10:00 a.m. the following
lightly
puncture
was not detectable animals
group
at 4:00 p.m., and the animals
between by aortic
Rats on
changes
36% of their
that
days
The day before the assay,
were sacrificed exsanguinated
develop
controls
for alcohol.
rats
for 34-54
was 46 days).
do not
and the pair-fed
conditions,
The
diet”
liver. I6 The experimental
3 adults)
drate was substituted
Sprague-Dawley
liquid
feeding
but only fatty
calories as ethanol under
and
METHODS
on a synthetic
duration
mode
control
or
was separated
measurements
frozen at -70°C
within (vida
then
syringes
at and
72 hr. The testes were
infra).
Hepatic Aromatase Activity (HAA} Hepatic
aromatase
formation
activity
was determined
of ‘H labeled estradiol
a modification
From the Endocrine Section, Department of Medicine. New York Medical College aad the Alcohol Research Center, Bronx V.A. Hospital and the Mount Sinai School of
Charles
of the method of Vittek
homogenized
were prepared
of Ragab et af.19 The microsomal mg of protein)
The assay mixture 3 mM
7n-‘H-testosterone
consisted
I
fraction
to method
(equivalent after
to 5
preparation.
of 0.05 A4 phosphate
buffer 5 mM
NADPH,
and appropriate
or 7a-3H-androstenedione) (equivalent
ml. Incubation
shaking
as previously
0.25 M sucrose, 5 mM of MgCI,,
tration of 3 X IO -*A4 Dubnoff
The livers were
according
was assayed immediately
(pH 7.4) containing
volume of
et al.”
in a glass and teflon homogenizer
described.18 Microsomes
of ATP,
by the rate of
(E2) and estrone (E,) using
incubator.
to IO&i
was carried Aliquots
substrate
(either
at a concen‘H)
in a total
out at 37O in a
for analysis were taken
at 0, 15, 30, 60, and I20 min and placed into 10 ml of ether containing substrate, estradiol, and estrone each labeled with
Mefc?bolism, Vol. 28, No. 1 (January),
1979
EFFECT
OF
ALCOHOL
INGESTION
ON
21
RATS
14C (5000 9500 7and 500 cpm, respectively) for measurement of recovery. Steroids were extracted with 2 X IO ml of ether, evaporated under a stream of N, and the residue redissolved in 200 pl of methanol. The recovery of the steroids was over 95%. Aliquots of the extract containing 20 rg of nonradioactive carrier steroids were then spotted on thin layer chromatographic glass plates (20 X 20 cm) and coated with silica gel 60,250 p thickness (Merk-Darmstadt). Two dimensional chromatography was carried out using chloroform and methanol (98:2 v/v) followed by chloroform and ethyl acetate (l6:4 v/v) after drying for 15 min and developed with a spray of 2% iodine in ethanol. This method separates all of the androgenic and estrogenic compounds.” The purity of all labeled compounds (i.e., substrates and isolated metabolites) was determined as previously described.” The areas corresponding to E, and E, were scraped into scintillation vials, extracted with I ml of methanol and after addition of IO ml of scintillation fluid (liqui-Ruor in toluene). counted in a Packard liquid scintillation spectrometer system (efhciency 59%, for ‘H and 86% for “C). The overall recovery for the method was at least 85%. However, because of the low percentage conversion to the estrogens the individual recovered counts of E, and E, were pooled for calculation of HAA activity after recovery correction. The results were expressed as pmole of E, + E, formed/hr/ IO mg microsomal protein.
Plasma
ETHANOL
. P 0.15); Ez (0.75 k 0.05 vs. 0.73 k 0.10 ng/g,p > 0.35) or E, (0.87 f 0.08 vs. 0.92 k 0.12 rig/g,, p > 0.35). This
El
T P
