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Effect of ethyl alcohol on plasma testosterone level in mice
921
EFFECT
OF ETHYL
TESTOSTERONE
ALCOHOL LEVEL
ON PLASMA IN MICE
Fouad M. Badr, Ph.D., and Andrzej Bartke, Ph.D. The Worcester Foundation for Experimental Biology Shrewsbury, Received:
Massachusetts
01545
315174
ABSTRACT The effect of ethanol ingestion on testicular steroidogenesis in mice was evaluated by measuring plasma testosFour groups of CBA/J male mice were treated terone level. 1,240, 0.620, with one of the following doses of ethanol: 0,310 or 0,155 g ethanol/Kg body weight. A control group The data showed no effect of the treatwas given water. ment on testicular weight, The concentration of testosterone in the plasma was significantly reduced in animals treated with alcohol. There was also a significant relationship between the dose of alcohol and the plasma testosterone level, with the decrease in testosterone being from 2 to 18 fold in various groups. INTRODUCTION Demonstration ethyl
alcohol
effect
in male mice
on several
Surprisingly, aspect
very
of alcohol
Testicular (3-5). dogs
with
VoZwne 23, Number 6
aspects
action
mutagenic
(1) prompted of gonadal
few data
atrophy
Similarly,
treated
of a significant
effect
us to study
structure
are available
of its
and function.
on this particular
(2). has been
a significant alcohol
reported drop
has been
STEROIDS
in alcoholics
in sperm
reported
count
(6) o
in
Alcohol
June,
2974
STEROIDS
922
was also
found
structural viduals
to decrease
changes
conflicting
of alcohol
of low doses no effect
of alcoholic
from studies Ln this
to normal levels
or prolactin caused
indi-
report,
Indirect
metabolism
testosterone
on the
The administration found
of testosterone,
(7).
the effect
reported
men was
by alcohol
on androgen
on plasma
been
on steroidogenesis,
of alcohol
imbalance
alcohol
cells
data have
on circulating
izing hormone crine
in the sperm
and to cause
(4).
However, effect
sperm motility
of endo-
was obtained
in alcoholic
levels
lutein-
evidence
ingestion
of several
to produce
doses
men
(8,9)
of ethyl
in male mice
is
described: MATERIAL Animals: Adult Laboratory, Bar same strain was effect of ethyl under controlled hours and food.
AND METHODS
CBA/J male mice, obtained from the Jackson Harbor, Maine, were used in the study. The also used in our work on the mutagenic alcohol (1). The animals were maintained conditions of temperature, humidity, light
Four groups of 12 mice each, were treated with the following doses of ethyl alcohol: 1.24, 0.62, 0.31 or The alcohol dilutions were prepared from com0.155 g/Kg. A fifth group received water and was mercial 95% ethanol. The treatment was given daily for 5 used as the control. consecutive days in 0.1 ml solution by stomach tubing. One hour after the last treatment, the mice were etherized, and the blood was collected from the jugular vein in heparinized tubes, Bleeding of the animals took place The blood was centrifuged and between 1400 and 1500 hour, Plasma testosterone levels the plasma stored at -2OOC. were measured by radioimmunoassay (lo), without chromatographic separation of the testosterone fraction. Excellent
STEROIDS
923
agreement between testosterone concentrations determined by radioimmunoassay with and without chromatography has been reported for the human plasma (11,12) and for the plasma of male mice of 19 different strains (13), We measured the testosterone concentration in 10 samples of rat plasma with and without chromatography and obtained nearly identical results (7.30 f 2.42 and 7.36 f 2-34 ng/ml, respectively). Apparently in the peripheral plasma of males of these species dihydrotestosterone is present in amounts too small to interfere with testosterone determinations. Body weight and testicular weight were also recorded for all groups. RESULTS Comparison of the relative testicular weight in the control and the alcohol treated groups shows no effect of the treatment over this relatively short period of exposure
(table 1)0
The effect of alcohol on plasma testosterone was striking.
The control group had a mean of 8.48 ng of tes-
tosterone per 1 ml of plasma, whereas the means of the alcohol treated groups were 2-18 times less than that of the control.
The significance of these notable differences in
plasma testosterone levels has been tested by the analysis of variance and Duncans' test,
The groups treated with
1.24, 0,62 or 0.31 g alcohol/Kg body weight showed significant differences from.the control (P < O.OS), Another striking feature of the data is the existence of a dose-response
relationship,
The form of the produced
curve is an exponential one (Pig0 I_)_ However, logarithmic
STEROIDS
924
transformation
of the data
The regression
of plasma
alcohol with
is significantly
a regression
resulted
in linear
testosterone
level
different
coefficient
regression.
on the dose
from zero
of
(P < O.Ol),
of -0.746.
DISCUSSION The effect noticeable. period
This
atrophy
longer
times
Nevertheless, was
sufficient
testosterone
have
drinking
of excreted
androgen
of androgen
metabolites
The role of alcohol
finding
indirectly
maintained Though
for consider-
suspected
studies
cirrhosis
period
of treatment in plasma
is in agreement
with
the
from the estimation
(8,9) and from estimation
in the liver
per se on plasma
found when
types of liver
short
reported
decreases
by others
metabolites
from studies
(5) and on alcoholic
levels were
with
alcohol
short
significant
This
levels.
In these
was not
due to the very
subjects
the relatively
derived
(16).
been
weight
(5,14).
contentions
cirrhosis
largely
All human
to produce
has also been
on testicular
is probably
of treatment.
testicular ably
of alcohol
in alcoholics
(15).
testosterone
levels
on alcoholic
liver
methadone-maintained
significant comparisons and with
reduction were
in testosterone
made with
non-alcoholic
patients
other
methadone-
patients. it seems difficult
to find a satisfactory
ex-
STEROIDS
planation
for the conflicting
experimental
animals
needed.
However,
tigators
in as much
metabolism
there
in man,
controlled
as alcohol
which
levels
reports
is general
in the liver
tive pathways steroid
under
925
conditions
agreement
tends
blood
on
are still inves-
androgen
pathways
then be reflected
in the circulating
data
between
to change
from oxidative
might
more
to reduc-
in the altered
and or in the urine
(8,9,17) The effect man
and in animals
tration
not alcohol through
an effect
concluded needed
in adrenocortical
by plasma
found
alcohol
releasing
study
epididymis,
level
(2).
on the gonads
or the pituitary
be is
involved.
reported
to affect
level of ethanol
given affect
vas deferens,
or
experimentation
to the peak
or severly
or
cannot
increase
when
(9).
Whether
a mani-fold
Epinephrine,
to damage
factor,
and further
has been
Adminis-
acts on the pituitary
directly
the mechanism
alcohol
corresponding (18).
corticosteroid
on the brain
and to cause
tubules,
that
its effect
to elucidate
medulla
blood
an increase
from this
Further,
phrine
produced
postulated
exerts
in
studied.
corticotrophin
via CRF,
steroidogenesis
extensively
as measured
It has been
on adrenal
has been
of alcohol
activity
was
of alcohol
the adrenal
in urinary
to both
epine-
in the
rats and humans,
the seminiferous
seminal
vesicles
and
STEROIDS
926
prostates as well as to diminish significantly the production rate and plasma level of testosterone
(19,20).
Sim-
ilar effects have been reported in rabbits (21), and in bulls
(22).
The role of the adrenal medulla as a mediator
of the gonadal response to other forms of stress has been demonstrated in mice (23),
The above mentioned findings
suggest the possibility that the effect of alcohol on the testes is similarly mediated by the adrenal via an increased production rate of epinephrine.
It is also.pos-
sible that alcohol produces its effect either by acting directly on the gonads to reduce the steroid synthesis or indirectly by altering the rate of hepatic metabolism of testosterone and renal clearence. In both adrenals and testes, the response of steroidogenesis to alcohol is dose-dependent but alcohol stimulates the adrenals and inhibits the testes (24, 25).
Acknowledgments:
This work was supported by AID grant
(csd/2837) and NIH grants SK04 HD70369 and 1 ROl Hd006867.
REFERENCES 1. 2.
3. 4.
Badr. F.M. and Badr, R,S., MUTATION RES. 2l, 45 (1973). Stock, P.E., THE BIOLOGY OF ALCOHOLISM Vol. l., Editors: Kissin, B. and Begleiter, H., Plenum Press. New York (1971) p. 397. Silvestrini, R,, RIF. MED. 142, 701 (1926), Doepfmer, R. and Hinckers, H-J., Z, HAUT- u, GESCHLECHTSKR 39, 94 (1965) (Abst. QUART. J. STUDIES ALC, 28, 364, 1967).
STEROIDS
5.
6. 7.
8. 9.
10. 1l. 12. 13. 14.
15. 16, 17" 18. 19,
20. 21. 22. 23. 24. 25"
927
Galvao-Teles, A,, Anderson, D.C., Burke, C.W., Marshall, J-C., Corker, C.S,, Bown, R.L. and Clark, M.L., LANCET 1, 173 (1973). Teitelbaum, H.A. and Gantt, W.H., QUART. J. STUDIES ALC. 2, 271 (1958). Toro, G,, Kolodny, R-C., Jacobs, L.S., Masters, W-H, and Daughaday, W.H. CLIN. RES. 21, 505 (1973). Cronholm, T. and Sjovall, J. BIOCHIM. BIOPHYS. ACTA. 152, 233 (1968). Fabre, L.F,, Pasco, P-J., Liegel, J-M. and Farmer, R.W. QUART. J. STUDIES ALC, %A, 57 (1973). Bartke, A., Steele, R,E., Musto, N, and Caldwell, B.V. ENDOCRINOL~Y 92, 1223 (1973). Geisthovel, W, and Morgner, K.D., ACTA ENDOCR, (Kbh) supp1. 177, 170 (1973). Pirke, K-Ma, ACTA ENDOCR, (Kbh) 2, 168 (1973). Lucas, L-A. and Abraham, G.E., ANALYT. LETT. 2, 773 (1972), Summerskill, W.H.J., Davidson, C-S,, Dible, J.H-, Mallory, G-K., Sherlock, S., Turner, M.S. and Wolf, S.J. NEW ENG, J. MED. 262, 1 (1960). Admirand, W-H., Cronholm, T. and Sjovall, J,, BIOCHIM. BIOPHYS. ACTA 202, 343 (1970). Cushman, Jr, P., AMER. J, MED. 55, 452 (1973). Williams, T.L_, Cantarow, A., Paschkis, K.E. and Havens, W,P, Jr., ENDOCRINOLOGY 48, 651 (1951). Klingman, G,I. and Goodall, McC., J. PHARMACOL. EXP, THERAP, 121, 313 (195710 Levin, J., Lloyd, C.W,, Lobotsky, J, and Friedrich, E-H., ACTA ENDOCR. (Kbh) 55, 184 (1967) o Chatterjee, A. and Paul, B.S,, ENDOCRINOLOGIE 52, 406 (1968). VanDemark, N-L, and Boyd, L-J., INTERN. J. FERTILITY I_, 245 (1956). VanDemark, N.L., and Baker, F,N., J. ANIMAL SCI. 12, 956 (1953). Christian, J.J., Lloyd, J-A. and Davis, D.E,, RECENT PROG, HORMONE RES, 21, 501 (1965). Ellis, F.W., J. P~~COL. 153, 121 (1966) u Fazekas, I.G., QUART. J. STUDIES ALC, 27, 439 (1966)p
STEROIDS
928
Table
(I).
Relative testicular weight and plasma testosterone level. in mice treated with ethyl alcohol. Treatment Control.
Testicular weight (mg/lOO g body weight) Means f S.E. Testosterone level (ng/ml plasma) Means f S.E. * Significantly
different
(g ethanol/Kg
body weight)
0.155
0.310
0.620
1.240
534 f 39
558 f 35
592 f 46
518 iz 28
550 f 24
8.48 f2.40
4.50 f1.14
1.27* f0.56
from the control
O&O* f0.17
O-49* f0.15
(P c 0.05).
7.5
5.0
2.5
0
0.5
0 DOSE
Fig.
1.
OF
Effect of ethyl in mice.
1.0
ALCOHOL alcohol
(g/kg)
on plasma
testosterone
level
EFFECT
OF ETHYL
TESTOSTERONE
ALCOHOL LEVEL
ON PLASMA IN MICE
Fouad M. Badr, Ph.D., and Andrzej Bartke, Ph.D. The Worcester Foundation for Experimental Biology Shrewsbury, Received:
Massachusetts
01545
315174
ABSTRACT The effect of ethanol ingestion on testicular steroidogenesis in mice was evaluated by measuring plasma testosFour groups of CBA/J male mice were treated terone level. 1,240, 0.620, with one of the following doses of ethanol: 0,310 or 0,155 g ethanol/Kg body weight. A control group The data showed no effect of the treatwas given water. ment on testicular weight, The concentration of testosterone in the plasma was significantly reduced in animals treated with alcohol. There was also a significant relationship between the dose of alcohol and the plasma testosterone level, with the decrease in testosterone being from 2 to 18 fold in various groups. INTRODUCTION Demonstration ethyl
alcohol
effect
in male mice
on several
Surprisingly, aspect
very
of alcohol
Testicular (3-5). dogs
with
VoZwne 23, Number 6
aspects
action
mutagenic
(1) prompted of gonadal
few data
atrophy
Similarly,
treated
of a significant
effect
us to study
structure
are available
of its
and function.
on this particular
(2). has been
a significant alcohol
reported drop
has been
STEROIDS
in alcoholics
in sperm
reported
count
(6) o
in
Alcohol
June,
2974
STEROIDS
922
was also
found
structural viduals
to decrease
changes
conflicting
of alcohol
of low doses no effect
of alcoholic
from studies Ln this
to normal levels
or prolactin caused
indi-
report,
Indirect
metabolism
testosterone
on the
The administration found
of testosterone,
(7).
the effect
reported
men was
by alcohol
on androgen
on plasma
been
on steroidogenesis,
of alcohol
imbalance
alcohol
cells
data have
on circulating
izing hormone crine
in the sperm
and to cause
(4).
However, effect
sperm motility
of endo-
was obtained
in alcoholic
levels
lutein-
evidence
ingestion
of several
to produce
doses
men
(8,9)
of ethyl
in male mice
is
described: MATERIAL Animals: Adult Laboratory, Bar same strain was effect of ethyl under controlled hours and food.
AND METHODS
CBA/J male mice, obtained from the Jackson Harbor, Maine, were used in the study. The also used in our work on the mutagenic alcohol (1). The animals were maintained conditions of temperature, humidity, light
Four groups of 12 mice each, were treated with the following doses of ethyl alcohol: 1.24, 0.62, 0.31 or The alcohol dilutions were prepared from com0.155 g/Kg. A fifth group received water and was mercial 95% ethanol. The treatment was given daily for 5 used as the control. consecutive days in 0.1 ml solution by stomach tubing. One hour after the last treatment, the mice were etherized, and the blood was collected from the jugular vein in heparinized tubes, Bleeding of the animals took place The blood was centrifuged and between 1400 and 1500 hour, Plasma testosterone levels the plasma stored at -2OOC. were measured by radioimmunoassay (lo), without chromatographic separation of the testosterone fraction. Excellent
STEROIDS
923
agreement between testosterone concentrations determined by radioimmunoassay with and without chromatography has been reported for the human plasma (11,12) and for the plasma of male mice of 19 different strains (13), We measured the testosterone concentration in 10 samples of rat plasma with and without chromatography and obtained nearly identical results (7.30 f 2.42 and 7.36 f 2-34 ng/ml, respectively). Apparently in the peripheral plasma of males of these species dihydrotestosterone is present in amounts too small to interfere with testosterone determinations. Body weight and testicular weight were also recorded for all groups. RESULTS Comparison of the relative testicular weight in the control and the alcohol treated groups shows no effect of the treatment over this relatively short period of exposure
(table 1)0
The effect of alcohol on plasma testosterone was striking.
The control group had a mean of 8.48 ng of tes-
tosterone per 1 ml of plasma, whereas the means of the alcohol treated groups were 2-18 times less than that of the control.
The significance of these notable differences in
plasma testosterone levels has been tested by the analysis of variance and Duncans' test,
The groups treated with
1.24, 0,62 or 0.31 g alcohol/Kg body weight showed significant differences from.the control (P < O.OS), Another striking feature of the data is the existence of a dose-response
relationship,
The form of the produced
curve is an exponential one (Pig0 I_)_ However, logarithmic
STEROIDS
924
transformation
of the data
The regression
of plasma
alcohol with
is significantly
a regression
resulted
in linear
testosterone
level
different
coefficient
regression.
on the dose
from zero
of
(P < O.Ol),
of -0.746.
DISCUSSION The effect noticeable. period
This
atrophy
longer
times
Nevertheless, was
sufficient
testosterone
have
drinking
of excreted
androgen
of androgen
metabolites
The role of alcohol
finding
indirectly
maintained Though
for consider-
suspected
studies
cirrhosis
period
of treatment in plasma
is in agreement
with
the
from the estimation
(8,9) and from estimation
in the liver
per se on plasma
found when
types of liver
short
reported
decreases
by others
metabolites
from studies
(5) and on alcoholic
levels were
with
alcohol
short
significant
This
levels.
In these
was not
due to the very
subjects
the relatively
derived
(16).
been
weight
(5,14).
contentions
cirrhosis
largely
All human
to produce
has also been
on testicular
is probably
of treatment.
testicular ably
of alcohol
in alcoholics
(15).
testosterone
levels
on alcoholic
liver
methadone-maintained
significant comparisons and with
reduction were
in testosterone
made with
non-alcoholic
patients
other
methadone-
patients. it seems difficult
to find a satisfactory
ex-
STEROIDS
planation
for the conflicting
experimental
animals
needed.
However,
tigators
in as much
metabolism
there
in man,
controlled
as alcohol
which
levels
reports
is general
in the liver
tive pathways steroid
under
925
conditions
agreement
tends
blood
on
are still inves-
androgen
pathways
then be reflected
in the circulating
data
between
to change
from oxidative
might
more
to reduc-
in the altered
and or in the urine
(8,9,17) The effect man
and in animals
tration
not alcohol through
an effect
concluded needed
in adrenocortical
by plasma
found
alcohol
releasing
study
epididymis,
level
(2).
on the gonads
or the pituitary
be is
involved.
reported
to affect
level of ethanol
given affect
vas deferens,
or
experimentation
to the peak
or severly
or
cannot
increase
when
(9).
Whether
a mani-fold
Epinephrine,
to damage
factor,
and further
has been
Adminis-
acts on the pituitary
directly
the mechanism
alcohol
corresponding (18).
corticosteroid
on the brain
and to cause
tubules,
that
its effect
to elucidate
medulla
blood
an increase
from this
Further,
phrine
produced
postulated
exerts
in
studied.
corticotrophin
via CRF,
steroidogenesis
extensively
as measured
It has been
on adrenal
has been
of alcohol
activity
was
of alcohol
the adrenal
in urinary
to both
epine-
in the
rats and humans,
the seminiferous
seminal
vesicles
and
STEROIDS
926
prostates as well as to diminish significantly the production rate and plasma level of testosterone
(19,20).
Sim-
ilar effects have been reported in rabbits (21), and in bulls
(22).
The role of the adrenal medulla as a mediator
of the gonadal response to other forms of stress has been demonstrated in mice (23),
The above mentioned findings
suggest the possibility that the effect of alcohol on the testes is similarly mediated by the adrenal via an increased production rate of epinephrine.
It is also.pos-
sible that alcohol produces its effect either by acting directly on the gonads to reduce the steroid synthesis or indirectly by altering the rate of hepatic metabolism of testosterone and renal clearence. In both adrenals and testes, the response of steroidogenesis to alcohol is dose-dependent but alcohol stimulates the adrenals and inhibits the testes (24, 25).
Acknowledgments:
This work was supported by AID grant
(csd/2837) and NIH grants SK04 HD70369 and 1 ROl Hd006867.
REFERENCES 1. 2.
3. 4.
Badr. F.M. and Badr, R,S., MUTATION RES. 2l, 45 (1973). Stock, P.E., THE BIOLOGY OF ALCOHOLISM Vol. l., Editors: Kissin, B. and Begleiter, H., Plenum Press. New York (1971) p. 397. Silvestrini, R,, RIF. MED. 142, 701 (1926), Doepfmer, R. and Hinckers, H-J., Z, HAUT- u, GESCHLECHTSKR 39, 94 (1965) (Abst. QUART. J. STUDIES ALC, 28, 364, 1967).
STEROIDS
5.
6. 7.
8. 9.
10. 1l. 12. 13. 14.
15. 16, 17" 18. 19,
20. 21. 22. 23. 24. 25"
927
Galvao-Teles, A,, Anderson, D.C., Burke, C.W., Marshall, J-C., Corker, C.S,, Bown, R.L. and Clark, M.L., LANCET 1, 173 (1973). Teitelbaum, H.A. and Gantt, W.H., QUART. J. STUDIES ALC. 2, 271 (1958). Toro, G,, Kolodny, R-C., Jacobs, L.S., Masters, W-H, and Daughaday, W.H. CLIN. RES. 21, 505 (1973). Cronholm, T. and Sjovall, J. BIOCHIM. BIOPHYS. ACTA. 152, 233 (1968). Fabre, L.F,, Pasco, P-J., Liegel, J-M. and Farmer, R.W. QUART. J. STUDIES ALC, %A, 57 (1973). Bartke, A., Steele, R,E., Musto, N, and Caldwell, B.V. ENDOCRINOL~Y 92, 1223 (1973). Geisthovel, W, and Morgner, K.D., ACTA ENDOCR, (Kbh) supp1. 177, 170 (1973). Pirke, K-Ma, ACTA ENDOCR, (Kbh) 2, 168 (1973). Lucas, L-A. and Abraham, G.E., ANALYT. LETT. 2, 773 (1972), Summerskill, W.H.J., Davidson, C-S,, Dible, J.H-, Mallory, G-K., Sherlock, S., Turner, M.S. and Wolf, S.J. NEW ENG, J. MED. 262, 1 (1960). Admirand, W-H., Cronholm, T. and Sjovall, J,, BIOCHIM. BIOPHYS. ACTA 202, 343 (1970). Cushman, Jr, P., AMER. J, MED. 55, 452 (1973). Williams, T.L_, Cantarow, A., Paschkis, K.E. and Havens, W,P, Jr., ENDOCRINOLOGY 48, 651 (1951). Klingman, G,I. and Goodall, McC., J. PHARMACOL. EXP, THERAP, 121, 313 (195710 Levin, J., Lloyd, C.W,, Lobotsky, J, and Friedrich, E-H., ACTA ENDOCR. (Kbh) 55, 184 (1967) o Chatterjee, A. and Paul, B.S,, ENDOCRINOLOGIE 52, 406 (1968). VanDemark, N-L, and Boyd, L-J., INTERN. J. FERTILITY I_, 245 (1956). VanDemark, N.L., and Baker, F,N., J. ANIMAL SCI. 12, 956 (1953). Christian, J.J., Lloyd, J-A. and Davis, D.E,, RECENT PROG, HORMONE RES, 21, 501 (1965). Ellis, F.W., J. P~~COL. 153, 121 (1966) u Fazekas, I.G., QUART. J. STUDIES ALC, 27, 439 (1966)p
STEROIDS
928
Table
(I).
Relative testicular weight and plasma testosterone level. in mice treated with ethyl alcohol. Treatment Control.
Testicular weight (mg/lOO g body weight) Means f S.E. Testosterone level (ng/ml plasma) Means f S.E. * Significantly
different
(g ethanol/Kg
body weight)
0.155
0.310
0.620
1.240
534 f 39
558 f 35
592 f 46
518 iz 28
550 f 24
8.48 f2.40
4.50 f1.14
1.27* f0.56
from the control
O&O* f0.17
O-49* f0.15
(P c 0.05).
7.5
5.0
2.5
0
0.5
0 DOSE
Fig.
1.
OF
Effect of ethyl in mice.
1.0
ALCOHOL alcohol
(g/kg)
on plasma
testosterone
level