137

IN VIVO ~T~OLISM -MALE RATS:

OF 3H-TESTOSTERONE IN ADULT

EFFECTS OF ESTROGEN ADMINISTRATION

D.R.H. Lee1 , C.E. Bird and A.F. Clark Departments of Biochemistry and Medicine Queen's University and Kingston General Hospital Kingston, Ontario, Canada

Receiued: b/i'/75

ABSTRACT The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney prostate, seminal vesicles and muscle were analysed for 5H-testosterone, 3H-5a-dihydrotestosterone (5orDHT)and 3H-5a-androstanediol (Adiolf. When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5aDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 ug/ day) for 4 days decreased the 3H-T levels in kidney and increased the 3H-T and 3H-5aDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 l/24 h/ 100 g body wt to 22.6 l/24 h/100 g body wt. INTRODUCTION Much information has been attained on the uptake of various androgens by tissues using a single injection of radioactive androgen

(21,

This type of approach does not

yield information on the relative levels of the various androgens in tissues and blood under normal and steady state conditions. Testosterone

(T) is quantitatively

the important

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TEIROID=

androgen in circulation of adult male rats (3). Dihydrotestosterone

5ct-

(5ctDHT) (4) is the important intra-

cellular androgen in terms of quantity and biological activity in target tissues such as the prostate

(5).

Oral administration of estrogen is known to influence the kinetics of T metabolism when studied by either the single injection or constant infusion technique in humans

(6,7).

We have previously shown (2) that intra-

peritoneal estrogen administration to normal adult rats increases the levels of

3H-androgens in prostate, seminal

vesicles and muscle at time intervals of up to 240 min following the subcutaneous administration of 'H-T. diol administration

Estra-

to castrated rats ten minutes prior to

intramuscular 3H-T injection led to an increased level of radioactivity in the prostate 1 h later (8). The constant infusion technique has been used previously

(9) to study the effects of castration on the

uptake and metabolism of

3 H-T by several target tissues

in adult male rats. Many of the reported studies on androgen metabolism in rats utilized castrated tectomized rats (5,121.

(10,ll) and functionally hepa-

This paper which reports studies

utilizing the constant infusion technique complements our previous study using the single injection technique 3H-T in normal adult male rats. Following the 3 intravenous administration of H-T to male rats by con3 stant infusion for 3 hour periods, the levels of H in (2) with

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139

TDItOXDrn

the form of T, 5aDHT and Sa-androstanediol

(Adiol) were

measured in plasma, liver, kidney, prostate, seminal vesicles and muscle.

The effects of estrogen adminis-

tration on these 'H-steroid patterns were studied. MATERIALS AND METHODS Materials [1,2-3H]testosterone (50 Ci/mmole) and [4-14C1 testosterone (50.5 mCi/mmole) and [4-14Cldihydrotestosterone (50.5 mCi/mmole) were obtained from New England Nuclear Corp. (Dorval, P.Q., Canada). [4-14ClSa-androstane-3a,l78-diol was prepared by the reduction of [4-14Cl 5a-dihydrotestosterone with partially purified 3a-hydroxysteroid dehydrogenase obtained from a dried extract of Pseudomonas testosteroni (13). All of these radioactive steroids were at least 97% pure as determined by paper chromatography and the reverse isotope dilution procedure. Non-radioactive steroids were obtained from Sigma Chemical Co. (St. Louis, MO., U.S.A.). Other chemicals were obtained from Fisher Scientific Co. (Ottawa, Ont., Canada). Adult male Sprague-Dawley rats (200-400 g) were purchased from Bio Breeding Laboratories (Ottawa, Ont., Canada). Estradiol valerate in sesame oil was obtained from E.R. Squibb & Sons Ltd. (Montreal, P.Q., Canada). Methods 3 The experimental protocol and H-T infusion prgcedure were as previously described for 3H-5aDHT and HAdiol (14). After the 3 h infusion period blood and tissues (liver, kidney, prostate, seminal vesicles and muscle) were removed. All procedures for counting, isolation of 3H-steroids, etc., were as previously described (2). Results are expressed as cpm/ml plasma/100 g body wt for plasma and cpm/g tissue/100 g body wt for the other tissues. Each result is the average of 3 experiments. The ranges of results for the individual 3Hsteroids are also shown in Table 2. For the in vivo administration of estrogen to the rats, 100 ug of?stradiol valerate in 0.1 ml sesame oil was injected intraperitoneally daily to each rat for 4 days. Control rats received the injection vehicle.

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TPIIEOXD6

RESULTS In the experimental protocol a priming dose of 2

x

lo6 cpm 3H-T (27 ng) was given intravenously followed by a constant infusion of 3H-T at an infusion rate of 6.5 X lo4 cpm (0.88 ng)/min.

Table 1 lists the total tissue

counts (corrected for quenching) and the total methylene chloride extractable counts for plasma (cpm/ml) and the tissues studied (cpm/g). TABLE 1 Distribution of total tissue counts (cpm/g/lOO g body wt) and total methylene chloride extractable radioactivity (cpm/g/lOO g body wt) in various tissues following a 3 h infusion (65,000 cpm/min) of 3H-T to 3 control rats (0) and 3 rats receiving estradiol valerate injections (E).

Total tissue counts Tissue

0

E

Methylene chloride extractable 3H 0

E

2750

6430

1020

1840

11600

40000

4370

21400

Kidney

4920

11400

2740

4430

Prostate

7030

15700

5680

14100

Seminal Vesicles

5300

10600

3350

9420

Muscle

1210

2000

828

Plasma Liver

1640

In each instance the total tissue count and the extractable counts were higher for the plasma and tissues from the rats receiving estrogen.

This increase was more pronounced for

liver, prostate and seminal vesicles. The levels of 'H in the form of T, 5ctDHT and Adiol for plasma, liver, kidney, prostate, seminal vesicles and

muscle are shown in Table 2. in a decrease in

Estrogen treatment resulted

3 H-T concentration in the kidneys and

increases in the prostate and seminal vesicles.

The

kidneys in control animals were the only tissues to 3 H-T above the levels in the

significantly concentrate circulation.

Since the

from the infused

3 H-5aDHT and 3H-Adiol are derived

3 H-T we have compared their tissue levels

to those of plasma 3H-T.

There are significantly higher

levels of 3H-5aDHT in both the prostate and seminal vesicles; these levels are further increased by estrogen administration.

In no instance was there a level of 3H-

Adiol above the plasma levels of

3 H-T.

The plasma level of 3H-T can be used to calculate its metabolic clearance rate (defined as the volume of plasma totally and irreversibly cleared of T per unit time) using the following expression Metabolic clearance rate

(15):

Infusion rate (cpm/24 h) X 100 =

(l/24 h/100 g body wt)

Plasma 3H-T Concentration

(cpm/l) ' Body wt (g)

The mean metabolic clearance rate for T was calculated to be 17.5 l/24 h/100 g body wt for control rats and 22.6 l/24 h/100 g body wt for rats which had received estradiol valerate for 4 days.

513 10300 798 3940 2200 648 (578,606, 760) (1070,1880,3660)(2390,2400,7030) (7500,8750,14500) (369,568,602) (455,818,1120)

Prostate

302)

798 322 857) (273,341,352) (455,818,1120)

Muscle

587 551 (554,551, 558) ( 428, 456, 877) ( 14,

82 82, 150) (

65,

78 79,

267 69 85) ( 68, 69, 70) ( 48,320, 433)

226 760 Seminal 7450 2630 1770 505 Vesicles (278,388, 849) (1000,2090,2210) (1520,1850,4530) (4220,6630,11500) (103,135,440) (134,676,1470)

117) ( 65, ;:,119) (148,%,

E

660 365 783 1030 (860,970,1250) ( 571, 849, 929) ( 214, 346, 535) ( 492, 531,

83 96,

Adiol

(E).

Kidney

64) ( 46,

0

injections

68 94 497) ( 61, 66, 77) ( 75, 91, 116)

32,

48 51,

E

valerate

220 169 83, 392) ( 47, 116,

29,

0

5a -DHT

estradiol

132 126 ( 78,134, 166) ( 62, 147, 187) (

E

receiving

Liver

T

(0) and 3 rats

537 782 (529,529, 553) ( 685, 687, 974) (

0

rats

Plasma

Tissue

to 3 untreated

3 Distribution of H in the form of T, 5aDHT, and Adiol in plasma (cpm/ml/lOO g body wt) and tissues (cpm/g/lOO g body wt) following a 3 h infusion (65,000 cpm/min) of 3H-T

TABLE 2

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143

DISCUSSION Current evidence indicates that T is the important circulating androgen in rats.

5aDHT appears to be the

important intracellular androgen in target tissues such as the prostate.

The interconversion of 5aDHT and Adiol

could play an important role in controlling the intracellular levels of 5aDHT (16).

Hence we decided to study

the relative levels of labelled T, 5aDHT and Adiol in plasma and several tissues in adult male rats after T had been infused to attain a state of equilibrium.

Kowarski

et al (17) have performed similar studies in dogs with infusions of up to 2 h and obtained a state of equilibrium in the blood.

However they were unable to determine

whether a state of equilibrium was obtained for the tissues because of the possibility that the steroids in a certain tissue exchanged slowly with the circulating compounds. of

Other studies in our laboratory where infusions

3 H-T were performed for varying time periods

(up to

5 h) indicated that a state of equilibrium in the plasma as well as the tissues studied was attained by a 3 h infusion. The data in Table 1 shows that 4 days of intraperitoneal estradiol valerate administration increased levels of 'H in each tissue.

lead to

There are signifi-

cant fractions of the radioactivity in plasma, liver and kidney which were not extracted by methylene chloride

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TIIROXDb

indicating the presence of water soluble conjugates in these tissues.

In androgen target tissues, the prostate

and seminal vesicles, the fractions of radioactivity not extracted by methylene

chloride were significantly less

indicating that the steroids were largely unconjugated in these tissues. When the radioactivity

in the tissue extracts

present as the three metabolites analysed

(T, 5ciDHT and Adiol) was

(Table 2), they accounted for approximately

50 -

80% of the 'H in plasma, kidney and muscle, less than 10% in liver and 90 - 95% in prostate and seminal vesicles for both control and estrogen treated rats.

In tissues

other than prostate and seminal vesicles there are obviously metabolites other than the three examined.

This

is particularly so in liver where one would expect the presence of hydroxylated T metabolites

(18).

The estrogen treated animals had a mean weight of 90 g less than the control animals.

The results have all

been corrected to a constant infusion rate of 65,000 cpm/ min.

Hence the difference in weight probably accounts for

the higher plasma level of

3 H-T in the estrogen treated

animals when the results are expressed on the basis of 100 g body wt. At equilibrium the plasma clearance of T by the tissues.

3 H-T level reflects the When the total metabolic

clearance rates were calculated the mean value for the

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145

control animals (55.2 l/24 h) was slightly higher than that for the animals receiving estrogen

(51.9 l/24 h).

However, MCR's are usually expressed per unit wt and when done this way the mean MCR for animals receiving estrogen is higher controls

(22.6 l/24 h/100 g body wt) than that for the (17.5 l/24 h/100 g body wt).

This is as antici-

pated on the basis of increased liver testosterone A4reductase levels which are found for rats which have 4 That A -reduction may

received estrogen for 4 days (19).

be rate limiting for T metabolism is supported by a significantly higher MCR (87.2 l/24 h/100 g body wt) for DHT (14).

A similar explanation has been suggested

(20) to

explain the decreased biologic effect of testosterone in rats receiving medroxyprogesterone

acetate which also

increases liver A 4-reductase levels.

Rats do not have

specific androgen binding B-globulin (21) to influence the MCR; in humans estrogen treatment increases the levels of this binding protein which at least in part decreases the MCR (7). value

Using a published

(3) plasma T

(250 ng/lOO ml) for adult rats gives a T production

rate of 43.8 ug/24 h/100 g body wt. The low level of 'H labelled T, 5aDHT and Adiol in the liver as compared to plasma reflects the rapid metabolism of these steroids by this tissue to other products. The high level of

3 H-T in the kidneys of control animals

as compared to plasma agrees with previous reports that

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SPPEOIDd

this organ concentrates T, not 5aDHT after T administration

(22,231.

The kidney has A 4-reductase activity

(22,231 which probably accounts for the higher level of 3 H-5aDHT in the kidney as compared to plasma.

Target

tissues such as the prostate and seminal vesicles had very high levels of

3 H-5aDHT for the control. animals

agreeing with previous reports that 5cxDHT is the important intracellular androgen in such tissues (5,121.

The

higher levels of both 3H-T and 3H-5aDHT in these two target tissues for the rats receiving estrogen is probably a result of a decrease in the levels of endogenous androgens due to inhibition of gonadotropin secretion and hence testicular steroid production.

The levels of

3 H-Adiol in

kidney, prostate and seminal vesicles are all higher than the levels of

3H-Adiol in plasma indicating that it is

produced in these tissues from

3 H-5aDHT, although we

cannot exclude the possibility that it was formed at other sites and concentrated in these tissues.

The

.

levels of all 'H-steroids in muscle are similar to those in plasma indicating that there is not specific accumulation or metabolism of these three androgens in this tissue.

This work was supported in part by grants from the National Cancer Institute of Canada, Medical Research Council of Canada (MT-2338 and MA-1852) and the Ontario Cancer Treatment and Research Foundation (grant no. 178).

REFERENCES 1.

2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22. 23.

Current address: Department of Endocrinology, Royal Victoria Hospital, Pine Avenue, Montreal, P.Q. Lee, D.K.H., Janikowsky,, A., Bird, C.E., and Clark, A.F. J. Ster. Biochem. 5: 27 (1974). Gupta, D., Zarzycki, J., and Rager, K. Steroids 25: 33 (1975). The following trivial names are used: 5a-dihydrotestosterone (5aDHT), 176-hydroxy-5a-androstan-3-one; 5a-androstanediol (Adiol), 5a-androstane-3a,l78-dial; estradiol valerate, 3-hydroxyestra-1,3,5(10)-triene178-yl valerate; medroxyprogesterone acetate, 17aacetoxy-6a-methyl-4-pregnene-3,20-dione. Bruchovsky, N. Endocrinology 89: 1212 (1971). Bird, C.E., Green, R.N., and Clark, A.F. J. Clin. Endocr. Metab. 29: 123 (1969). Clark, A.F., Carson, G.D., DeLory, B., Clemow, M.E., and Bird, C.E. Clin. Endocr. 2: 361 (1973). Tveter, K.J., and Aakvaag, A. Endocr. 85: 683 (1969). Prough, D., and Bullock, L. Proc. IV Int. Cong. Endocr., Wash., D.C., June 1972, Abstract 140. Tveter, K-J., and Attramadal, A. Acta endocr. (kbvn) 59: 218 (1968). Hansson, V., Tveter, K.J., and Attramadal, A. Acta endocr. (kbvn) 67: 384 (1971). Bruchovsky, N., and Wilson, J.D. J. Biol. Chem. 243: 2012 (1968). Delin, S., Squire, P.G., and Porath, J. Biochem. Biophys. Acta 89: 398 (1964). Van Doorn, E.J., Burns, B., Wood, D., Bird, C.E., and Clark, A.F. J. Ster. Biochem. (submitted for publication). Baird, D.T., Horton, R., Longcope, C., and Tait, J.F. Rec. Prog. Horm. Res. 25: 611 (1969). Clark, A.F., Van Doorn, E.J., and Bird, C.E. J. Ster. Biochem. 5: 346 (1974). Kowarski, A., Shalf, J., and Migeon, C.J. J. Biol. Chem. 244: 5269 (1969). Gustafsson, J.-A., and Lisboa, B.P. Eur. J. Biochem. 12: 369 (1970). Patterson, D.C., Clark, A.F., and Bird, C.E. J. Endocr. 63: 181 (1974). Gordon, G.G., Southren, A.L., Tochimoto, S., Olivo, J ., Altman, K., Rand, J., and Lemberger, L. J. Clin. Endocr. 30: 449 (1970). Corvol, P., and Bardin, C.W. Biol. Reprod. 8: 277 (1973). Wilson, J.D., and Gloyna, R.E. Rec. Prog. Norm. Res. 26: 309 (1970). Ritzen, E.M., Nayfeh, S.N., French, F.S., and Aronin, P.A. Endocr. 91: 116 (1972).

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein...
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