Effects of ethanol and acetaldehyde on the biosynthesis of testosterone in the rodent testes

Vol.

94, No.

June

3. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 814.-819

16, 1980

EFFECTS OF ETHANOL AND ACETALDEHYDE ON THE BIOSYNTHESIS OF TESTOSTERONE IN THE RODENT TESTES Theodore

J.

Cicero

and Roy D. Bell

Department of Psychiatry, School of Medicine, Saint

Received

March

Washington University Louis, Missouri 63710

17,198O SUMMARY

The effects of ethanol and acetaldehyde on testicular steroidogenesis were examined in enzymatically dispersed cells of the rodent testes. Both drugs significantly inhibited gonadotropin-stimulated steroidogenesis, but acetaldehyde was considerably more potent (>lOOO times) than ethanol. To determine the step in testosterone's biosynthetic pathway which was inhibited by the two drugs, cells were incubated in the presence of [3H]pregnenolone and [3H]progesterone, and the amount of label incorporated into testosterone and its precursors was determined. Ethanol and acetaldehyde inhibited only the conversion of androstenedione to testosterone; none of the other precursors of testosterone was affected. INTRODUCTION Ethanol the male genesis

decreases

(l-4).

Ethanol

--in vitro,

but

which

mechanism

occurs

readily

support

of this

of testicular In the vitro isolate

formation

the step

in the

that

in vivo -__

biosynthesis

minimally

the effects

of ethanol

to acetal(9),

is

the

production.

In

to be a potent

inhibitor

concentrations

(6,9).

and acetaldehyde

precursors

of testosterone

affected

in an effort by the

ti to

two drugs.

AND METHODS

Enzymatically Dispersed Cell Preparations: were prepared as described previously (10). otropin-stimulated testosterone production --in Testosterone levels scribed previously (9). media were determined as described elsewhere 0006-291X/80/110814-06$01.00/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

steroido-

of ethanol

labeled

MATERIALS

in

at nonphysiological

of testosterone

at physiological

from

testicular

in vitro -___

has been found

of testosterone

administration

only

the metabolism

inhibition

--in vitro

we examined

its

occurred

but only

acetaldehyde

steroidogenesis studies

suggests

after

to suppress

inhibitions

ethanol's

conclusion

present on the

for

levels

been found

substantial This

responsible

testosterone

has also

(5-8).

concentrations dehyde,

serum

814

Enzymatically dispersed cells Studies of drug effects on gonadvitro were carried out as deand drug concentrations in the (9,101.

Vol. 94, No. 3, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Incubation with [3H]Progesterone and [3H]Pregnenolone: The isotopic purity of the LjHJprogesterone (56.5 Ci/mM) and L3HJpregnenolone (17.2 Ci/mM) was determined by TLC (11). Incubations were carried out as described above, but in these studies incubations contained [3H]pregnenolone or [3H]progesterone and ethanol or acetaldehyde. The concentrations of acetaldehyde and ethanol were selected on the basis of initial studies (see Figure 1) to provide a 30-50% inhibition of testicular steroidogenesis. This range was selected to provide significant decreases in testicular steroidogenesis without the possibility of nonspecific cellular toxicity which might occur at higher concentrations. Aliquots of the media were extracted with ethyl acetate and dried under a stream of N2. The residues were dissolved in methanol, containing carrier steroids, and were spotted on silica gel GF TLC plates. The plates were developed as described previously (11); the steroids were visualized with ultraviolet light or 5% phosphomolybdic acid. The spots were scraped from the plates and counted. The identity of the various steroids was determined by recrystallization to constant specific activity (12). Recoveries were found to be within 85-90% in all studies, and results are expressed as CPM incorporated into each product. Generation of Interferinq Compounds: To determine whether acetaldehyde or ethanol might spontaneously react with testosterone under our assay or extraction conditions to form a compound which would comigrate with one of the steroids examined, 50 nCi testosterone was incubated with 100 PM acetaldehyde or 200 mM ethanol under the incubation conditions described above, except that no cells were included. The media were extracted and subjected to TLC as described above. RESULTS Effects shown

in Figure

testicular whereas

of Ethanol

and Acetaldehvde

1, ethanol

steroidogenesis ethanol

and acetaldehyde ; however,

was effect

on Testicular

ive

inhibited

acetaldehyde

at mM0lar

Steroidoqenesis:

As

gonadotropin-stimulated was effective

at UMolar,

concentrations.

ACETALDEHYDE

ETHANOL

10

1000 1

10’

ooJ-----2 IO 20 ‘lo 60 PERCENT

2

10

20

40

60

INHIBITION

Figure

1. The effects of acetaldehyde and ethanol, expressed as percent inhibition, on human menopausal gonadotropin-stimulated (5 mIU) testosterone production in intact enzymatically dispersed cells of the rodent testes. Values arethemeans of three experiments carried out in triplicate. Standard errors averaged less than 52 of the mean at all points.

815

Vol.

94, No.

3, 1980

8lOCHEMlCAL

Effects

of

AND

8lOPHYSlCAL

RESEARCH

TABLE 1 on the incorporation

acetaldehyde

Precursor

of

[3H]pregnenolone

%a

Control

COMMUNICATIONS

Acetaldehyde

%a

Pregnenolone

2,858.0

(t114.8)

5.0

2,810.O

(t183.4)

4.7

Progesterone

5,802.g

(k308.4)

10.3

6,531.l

(k656.0)

10.9

17-a-hydroxyprogesterone

1,260.8

(2137.5)

2.2

1,379.5

(t194.8)

2.3

Androstenedione

21,775.l

(~568.2)

38.4

30,368.3

(k877.8)

51.1b

Testosterone

24,886.6

(t921.4)

43.9

18,303.5

(i472.3)

30.8'

Total

56,583.4

100

59,392.4

100

The effects of acetaldehyde (100 uM) on the incorporation of L3H]pregnenolone into the precursors of testosterone via the preferred route of metabolism. Dispersed cells were pr5pared as described in the Methods section and were incubated with 1.25 nCi [ H]pregnenolone with or without 100 UN acetaldehyde. Values are Imeans (?SEM) CPM representing three experiments carried out in tripaPercent of total CPM (botton line); bsignificantly higher (~~0.01) licate. when compared to control; csignificantly lower (~~0.01) when compared to control.

Effects

of

Acetaldehyde

Testosterone:

The

nenolone

into

"preferred" only

effects

in

conversion lation

of

the of

of

biosynthesis

in

and

a proportional

also

with

examined Effects

gesterone

into

of

testosterone

to

testosterone; in

preferred in

CPM

CPM

in

of in

in

in

of

in

the

Table

the

CPM

in

1 for

rodent

significantly

[3H]preg-

testes.

The

affected was

was

a significant cells,

accumu-

The

its

to

effects

biosynthesis and

the

relative

testosterone.

testosterone

the

and

precursors

of on

the

were

results. on

the

Incorporation The

testosterone

of

effects

of

and

its

Ethanol

biosynthesis). androstenedione,

testosterone.

shown

acetaldehyde-treated

into

Testosterone:

route

are

testosterone's

identical

into

incorporation

there

into

[3H]Pregnenolone

the

biosynthesis

drop

Ethanol

[3H]pregnenolone

in

precursors

[3H]progesterone

of

of on

onthenonpreferrodrouteof of

drop

its

androstenedione

incorporation

crease

Incorporation

testosterone's

androstenedione

acetaldehyde

the

acetaldehyde

and

CPM

controls,

of

testosterone route

step

on

when Similar

ethanol precursors selectively

compared results

816

[3H]Preqnenolone

to were

on

and

the

are

r3H]Pro-

incorporation shown

caused

in

of Table

2 (the

a significant

controls

and

a corresponding

obtained

when

C3H]progester-

in-

Vol.

94, No.

3, 1980

BIOCHEMICAL

Effects

of

ethanol

AND

TABLE 2 incorporation

on the

Precursor

BIOPHYSICAL

RESEARCH

of

Control

COMMUNICATIONS

[3H]pregnenolone

79

Ethanol

%a

Pregnenolone

4,829.7

(2

701.5)

11.1

4,782.4

(k

240.1)

10.5

Progesterone

6,853.Z

(k

640.4)

15.8

6,982.6

(+ 920.2)

15.3

17-a-hydroxyprogesterone

2,579.z

(k

171.4)

5.9

2,392.6

(k

Androstenedione

18,156.0

(k1440.6)

41.9

23,779.8

Testosterone

10,862.Z

(i- 998.3)

25.0

7,492.2

Total

43,280.3

The effects the precursors merits carried apercent of compared to

one

was

of

as the precursor

Spontaneous

cursor

(k1765.2)

52.3b

(t

16.4'

531.9)

45,429.6

100

ethanol (200 mM) on the incorporation of [3H]pre9nenolone into of testosterone. Values are means (GEM) CPM of three exoeriout in triplicate. (See le 8. end to . Table T for further deteils). the total CPM (bottom line); slgnlflcantly higher (~~0.01) when control; csignificantly lower (~~0.01) when compared to control.

testosterone

etaldehyde

5.3

of

employed

route

100

404.5)

and

biosynthesis

Generation

affected

when

was

either

the

preferred

or

nonpreferred

examined.

of Interfering

Compounds:

the CPM in testosterone,

Neither

ethanol

androstenedione,

nor ac-

or any other

pre-

of testosterone. DISCUSSION The

ular

present

results

steroidogenesis

tosterone.

Both

corresponding precursors incorporated

and the CPM in

an accumulation

was

testosterone

can

conversion

affected.

completely

- at

has been only for

of

account

steroidogenesis

pathway

inhibition

testosterone;

to

least

one other

testosterone.

for

testic-

to tes-

the

in

offset of

other in

CPM

gonadotropin-

radioimmunoassay

conversion and

reduction

reduction

by

and a

none of the

percent

completely

ethanol in

the

measured

the

block

of CPM in androstenedione

corresponded as

acetaldehyde

of androstenedione

Since

in CPM in androstenedione an

and

into

production,

testosterone,

There synthetic

caused

testosterone

tosterone ticular

the

testosterone

increase

ethanol

in CPM incorporated

into

stimulated

that

by inhibiting drugs

fall of

indicate

(Figure

the

reduction

androstenedione

acetaldehyde's

l),

effects

in to

tes-

on

tes-

vitro.

study

of the

Gordon

817

effects

et a1.(13)

of ethanol reported

on the that

rats,

bio-

Vol.

94, No.

3, 1980

chronically

maintained

nase activity. cit,

genesis

of its

whether one,

the enzyme

involved

that

in preliminary

competitively

shown

that

reverse

steroidogenesis

The possibility

that

ethanol

out

acetaldehyde, version

completely

in both the

to be best

and it

likelihood

explained

to the clearly

admin-

the --in vivo however,

of this

enzyme was

enzyme does not that

in the

In fact,

we have on tes-

Thus,

effects

re-

a change

or acetaldehyde

although

of ethanol

not required

that

favor blocking

studies. appears

acetaldehyde

to androstenedione simply

ethanol

about

observations).

than

in our

reactions

of testosterone

to testoster-

activity.

and acetaldehyde

cannot

involved

of ethanol

examine

for

and acet-

the drugs

to

the

However,

or ethanol

inhibition

reaction

forward

since

reaction,

the same enzyme

to be competitively

seems remote.

by a drug-induced

the reverse

inhibited

enhances Thus,

by

the con-

our

data

seem

of 17-B-hydroxysteroid

oxidoreductase. In the present potent

than

ethanol

and much earlier tions

both

studies

ethanol

we found

in inhibiting work

(5,6,9),

a

steroidogenesis.

to androstenedione,rather

is

effects

is

not

be noted,

this

on its

may contribute

of testosterone be ruled

as a re-

time

seems improbable

(unpublished

testicular

occurring

activity

effects

such an alteration

block

that

Moreover, it

the

--in vitro

in the NAD/NADH ratio

significantly

thus,

ethanol's

not

steroid0

by chronic

should

defi-

on testicular

did

present

the

explain

--in vivo,

at the

we have found

state

NAD+ did

was affected

by acetaldehyde.

this

of androstenedione

studies

redox

aldehyde

conversion

It

(14);

reversed

et a1.(13)

we have observed.

NAD+ as a cofactor

change

Gordon

COMMUNICATIONS

in A5-3B-dehydroge-

of ethanol

effects

quire

ticular

effect

can be drawn

inhibited

could

deficiency

NAD+ completely

oxidoreductase,

of the

RESEARCH

in the NAD+/NADH ratio,

in the

so no conclusions

significance

the

Unfortunately,

17-B-hydroxysteroid

istration

that

that

was due to a change

metabolism.

BIOPHYSICAL

had a marked

however,

them to conclude

in vivo

AND

on ethanol,

They found,

leading

sult

BIOCHEMICAL

that

testicular suggest

and acetaldehyde

that

actaldehyde

steroidogenesis. under

may be involved

818

was considerably

in vivo -__

These and in vitro

in inhibiting

more results, condi-

testicular

Vol.

94, No.

3, 1980

steroidogenesis these

BIOCHEMICAL

and that

acetaldehyde

AND

BIOPHYSICAL

may be the

RESEARCH

primary

agent

COMMUNICATIONS

responsible

for

effects. ACKNOWLEDGMENTS

This research was supported in part by USPHS grants AA-03242 and AA-03539. Cicero is a recipient of Research Scientist Development Award AA-70180.

Dr.

REFERENCES 1.

Cicero,

T. J.,

and Badger,

T. M. (1977)

J. Pharmacol.

Exp. Ther.

201,

427-433. 2. 3. 4. 5.

Gordon, G. G., Southren, A. L., and Lieber, C. S. (1978) Ale. Clin. Exp. Res. 2, 259-269. Mendelson, J. H., Mello, N. K., and Ellingboe, J. (1977) J. Pharmacol. Exp. Ther. 202, 676-682. Van Thiel, D. H., Gavaler, J., Lester, R., and Goodman, M. 0. (1975) Gastroenterology 69, 326-332. Badr, F. M., Bartke, A., Dalterio, S., and Bulger, M. (1977) Steroids 30,

647-655. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Cobb, C. F., Ennis, M. F., Van Thiel, D. H., Gavaler, J. S., and Lester, R. (1980) Metabolism 29, 71-79. Ellingboe, J., and Varanelli, C. E. (1979) Res. Commun. Chem. Pathol. Pharmacol. 24, 87-102. Gordon, G. G., Southren, A. L. Vittek, J., and Lieber, C. S. (1979) Metabolism 28, 20-24. Cicero, T. J., Bell, R. D., Meyer, E. R., and Badger, T. M. J. Pharmacol. Exp. Ther., in press. Dufau, M. L., Mendelson, C., and Catt, K. J. (1974) J. Clin. Endocrinol. Metab. 39, 610-613. Burstein, S., Hunter, S. A., Shoupe, T. S., and Taylor, P. (1978) Res. Commun. Chem. Pathol. Pharmacol. 19, 557-560. Axelrod, L. R., Matthyssen, C., Goldzieher, J. W., and Pulliam, J. E. (1965) Acta Endocrinol. 49, Suppl. 99. Gordon, G. G., Vittek, J., Southren, A. L., Munnangi, P., and Lieber, C. S Endocrinology, in press. Inano, H., and Tamaoki, B. (1974) Eur. J. Biochem. 44, 13-23.

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