Biological activity of hormonally active and non-active androgen derivatives

Int. J. Immunopharmac., Vol. 4, No. 5, pp. 469-474, 1982 Printed in Great Britain.

0192-0561/82/050469-06 $03.00/0 © 1982 International Society for Immunopharmacology.

BIOLOGICAL ACTIVITY OF HORMONALLY ACTIVE AND NON-ACTIVE ANDROGEN DERIVATIVES I~VIARTAVOJTI~KovA, PETR DRABER, KAREL VERES* and ZORA POKORNA Institute of Molecular Genetics, Czechoslovak Academy of Sciences, 142 20 Prague, Czechoslovakia (Received 12 May 1981 and in final form 16 November 1981)

Abstract--Androgen derivatives appeared to have different biological activities in vivo and in vitro. Testosterone-17-isobutyrate given in three doses of 50 or 200 pg increased significantly the weight of seminal vesicles and reduced thymus weight in castrated males, whereas testosterone-17-hemisuccinate, testosteroneD-fl-glucosideand testosterone-3-(O-carboxymethyl)-oximehad no such effects. Similarly, ten l-mg doses of testosterone- 17-isobutyrate, unlike testosterone- 17-hemisuccinate,resulted in a marked reduction of thymus weight in non-castrated males and in a significant inhibitory effect on the activity of spermatogenesis. On the other hand, all three androgen derivatives, which had appeared inactive in vivo, had similar effects in vitro (as had active testosterone) as demonstrated by inhibitionof Concanavalin A-induced lymphocyte activation expressed by 14C thymidine incorporation and inhibition of cell agglutination. These results seem to suggest that as for the regulation of androgen-dependent organs and functions (such as the size of seminal vesicles, activity of spermatogenesis, thymus size), hormonally active androgens are also involved in certain immunosuppressive effects in vivo. On the other hand, in vitro immunological effects are produced by both hormonally active and non-active androgen derivatives as well as by other steroid hormones, the common denominator being the steroid structure.

The immunosuppressive effects of glucocorticoids glucocorticoids have an inhibitory effect on mitogenhave been used therapeutically in clinical medicine induced lymphocyte activation (Nowell, 1961; Fauci, for several decades. In recent years steroid hormonal 1978), and findings indicating that steroid hormones therapy was extended by quite a new area of long- may cause an altered tissue distribution of labelled term administration of the sex steroids for the regu- lymph node and peritoneal exudate cells (Viklick~, et lation of male fertility (e.g. Neumann, 1977; Mauss, al., 1977; Viklick~, & Pol~Ekov~, 1980). BOrsch, Richter & Bohrmacher, 1978) without taking In the present work, we have attempted to deterinto account any possible immunosuppressive mine whether the immunosuppressive effect of the effects. Such effects are manifested by a degree of "sex steroids" is associated with their hormonal depletion of the lymphoid system, particularly the activity. thymus, and an associated reduced immune capacity as has been described for testosterone and, to a greater extent, for the more potent synthetic antiEXPERIMENTAL PROCEDURES androgenic steroid, cyproterone acetate (Viklick~,, Pol~kova, Vojti~kov~, Dr~tber & Khoda, 1977; A n i m a l s Vojti~kov~t, Pol~tEkov~, Viklick~, & Pokorngt, 1979). Mice of the inbred BI0.A strain from the The mechanisms underlying the immunosuppressive Institute's breeding colony were used at 2.5-4 effect of steroid hormones have not been adequately months of age. defined. Important contributions to research in this area include; the recognition that glucocorticoid and H o r m o n a l preparations sex steroid receptors are present in cells of the The following androgen derivatives were used: lymphoid organs (glucocorticoids: for review see testosterone-17-hemisuccinate (TE-HS) was prepared Trager, 1977; further Duval, Homo, Fournier & by the reaction of testosterone with succinic anDausse, 1979; Homo, Duval, Hatzfeld & Evrard, hydride in pyridine (4 h, 80 ° C), testosterone1980: sex steroids: Gillette & Gillette, 1979; Le 3-(O-carboxymethyl)-oxime (TE-OX) was prepared Douarin, Michel & Baulieu, 1980; Grossman, by the reaction of testosterone with aminoxyacetic Nathan, Taylor & Sholiton, 1979; VojtBkov~, acid in pyridine (12 h, 20°C). Sodium salts of TE-HS Hilgertova & Dniber, 1981), the observation that and TE-OX were prepared by neutralization of water * Institute of Nuclear Biology and Radiochemistry. 469

47O

M. VOJT|~KOV,~et al.

alcohol solutions with an equivalent amount of NaHCO 3 and by evaporation of the solvents under reduced pressure. Testosterone-D-/3-glucoside (TE-G) was kindly provided by Dr. P. Ko~ovsk~,, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Praha, Czechoslovakia. All the three derivatives were water- and oil-soluble and their physical constants corresponded to the data in the literature (Ruzicka & Wettstein, 1936; Lieberman, Erlanger, Beiser & Agate, 1959; Ko~ovsk~, Ko~oev & Prochfizka, 1973). In vivo, the steroid compounds were administered either in water or in oil solution in a volume of 0.2 ml; control animals were given saline. For in vitro tests, testosterone (testosteronum purum) was dissolved in ethanol, ethanol was evaporated, and testosterone was transferred to culture medium with vigorous agitation. TE-HS, TE-OX and TE-G were dissolved directly in the culture medium. Commercial testosterone-17-isobutyrate (TS-IS, testosteronum isobutyricum in suspensione microcrystall, aquosa, trade name Agovirin Depot, Biotika, Czechoslovakia) was used as standard preparation in vivo.

Determination of biological activity in vivo (a) Weight of seminal vesicles and thymuses and thymus morphology. Adult males were castrated through scrotal incision under Nembutal (Abbott, U.S.A) anaesthesia. Twenty-one days later, animals were given three doses of tested derivative subcutaneously at 3-day intervals; 3 days after the last dose, they were sacrificed and their thymuses and seminal vesicles weighed. The thymuses were submitted to routine histological analysis. The doses of the derivative were weight-matched to be testosterone-equivalent to the TE-IS dose. The intergroup differences in organ weight were tested by Student's t-test. (b) Spermatogenesis. Adult males were given 10 daily (with exception of week-ends) doses of 1 mg of TE-IS or TE-HS each, and their testes were histologically analysed 12 days after the last dose. The activity of spermatogenesis was expressed as the percentage of seminiferous tubules containing maturing spermatozoa. The intergroup differences were tested by the Wilcoxon test (Viklick~' et al., 1977).

Determination of biological activity in vitro Lymphocyte activation. Lymph node cell suspensions were prepared as described earlier (Dr~tber, Viklick~, & Lengerovfi, 1977) and cultured in serumfree Eagle's minimal essential medium (MEM) supplemented with non-essential amino acids, 1 mM pyruvate, 2 mM L-glutamine, 5 mM N-2-hydroxy-

e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e s u l p h o n i c acid (HEPES) buffer, penicillin (100 i.u./ml), streptomycin (100 tag/ml), and 5 x 10 -5 M 2-mercaptoethanol. One million living cells (determined by trypan blue exclusion) in 200 tal of medium were placed into each well of a flat-bottomed microtest II tissue culture plate (No. 3040, Falcon Plastics, Oxnard, CA). Twenty microliters of compounds tested diluted in MEM (final concentration shown in the Results) and 20 tal of concanavalin A (final concentration 0.5 tag/ml. Pharmacia Fine Chemicals, Uppsala, Sweden) or an equal volume of MEM in controls were added at the beginning of the culture. Cells were cultured for 72 h at 37°C in a humidified atmosphere of 5°70 CO 2 in air. Ten hours before the termination of the culture, 4 kBq of ~4C thymidine (Institute for Research, Production and Uses of Radioisotopes, Prague, Czechoslovakia, spec. act. 1.7 GBq/mM) was added to each well. The cells were then harvested on glass fibre filters (Whatman GF/c), using a multiple cell harvester, and unbound isotope was washed free with distilled water. The radioactivity retained on each dry filter was measured in 5 ml of scintillation liquid by means of Isocap 300 counter (Nuclear, Chicago, IL). In the results means and S.D.M. calculated from quadruplicates of a representative experiment are shown.

Morphological evaluation. Agglutination of lymph node cells during cultivation was examined after 24 and 48 h of culture under an inverse microscope at a 30-fold magnification.

RESULTS

Activity of androgen derivatives in vivo (a) Weight of seminal vesicles and thymuses in castrated males. The results of two independent experiments are shown in Fig. 1. In the first experiment (Fig. 1A), castrated males were given TE-HS, TE-OX or TE-IS. In comparison with the control castrated and non-castrated males which were not injected with the androgen, no increase in the weight of seminal vesicles was seen after 50 and 200 tag of TE-HS and TE-OX, while 50 tag and particularly 200 tag of TE-IS increased the weight significantly (P<0.005). In contrast to TE-IS, the first two derivatives had no significant hormonal effect. In the second experiment (Fig. 1B), TE-HS, TE-G or TEIS were administered in three doses of 200 tag each. The seminal vesicles weighed 126 mg after TE-IS (P<0.005). Pure testosterone had a similar effect as had TE-IS. TE-HS and TE-G respectively had a similar effect if given in water or oil solution.

Biological Activity of Hormonally Active and Non-Active Androgen Derivatives mg 250 -

A

rtl I

200

I

I I

150

471

weight in non-castrated males (Table 2, column 1). Histologically, a marked involution of the lymphoid and reticuloepithelial components of the thymus was seen, the medulla and cortex could not be distinguished. In addition, it was found that castration itself caused a very significant increase in thymus weight irrespective of the administration of the derivatives when thymus weights in castrated (Table 1) and normal males (Table 2) were compared.

TE-IS 200 50

8

(b) Spermatogenesis. Spermatogenesis in control males resembled that of males treated with TE-HS but it was inhibited significantly by TE-IS (Table 2). Thus TE-HS had no effect on seminal vesicle weight nor on spermatogenesis.

100-

50200 50 200 50

0

Activity of androgen derivatives in vitro

B

150 -

TE-IS 200

o 100

{

-

50-

o~ TE-HS 200 TE-G 200

0

Fig. 1. Hormonal activity of androgen derivatives expressed by seminal vesicle weight (means±S.D.M.) in castrated males• Numbers at the top of each bar indicate the size of the derivative dose used in gg. The thymus weights of castrated males from the second experiment in Fig. IB are shown in Table 1. Neither TE-HS nor TE-G influenced the thymic weight which, however, decreased after TE-IS (P<0.05). In contrast to TE-HS, ten 1-mg doses of TE-IS also reduced very significantly the thymus

The results of the experiment in which lymph node cells were cultured in the presence of androgen derivatives and with or without Con A are shown in Fig. 2. Con A alone induced lymphocyte activation as evident from increased incorporation of ~4C thymidine when compared to the control cells without Con A. This activation was not affected by any of the derivatives used up to a concentration of 1 ~ . With the exception of TE-G, a 10/aM concentration partially inhibited while a 100/aM concentration completely inhibited lymphocyte activation. When higher concentrations of the derivatives were used for the inhibition of Con A-blastogenesis, their effects on cells in culture were not due to toxicity as the living cell counts remained essentially unaltered throughout the culture period. In order to compare the inhibitory activity of individual androgen derivatives, we determined the concentrations responsible for 50°7o inhibition of J4C thymidine incorporation in cultures with Con A. The values for individual derivatives were similar: TE--14/aM, TE-HS--19/aM, TE-OX--25/aM and TE-G--29/aM (Fig. 2). At concentrations of 10taM and particularly 100/aM these compounds caused an inhibition of one

Table 1. Effect of various androgen derivatives (3 doses of 200 lag each) on thymus weight in castrated males

No. males

Thymus weight mean_+S.D.M. (mg)

TE-HS TE-G TE-IS

10 8 10

107__.6.0 1~ 1.6 91-*-4.4

Saline control

15

107±4.3

Androgen

M. VOJT|~KOV,~et al.

472 Table 2.

Thymus weight and spermatogenesis in males pretreated with 10 daily 1 mg doses of TE-HS or TE-IS

Thymus weight mean_+S.D.M. (mg)

Spermatogenesis % tubules with spermatozoa

TE-HS (16) TE-IS (15)

39±1.6 7±0.6*

45.7±0.9 21.2±1.8"

Saline control (14)

41+ 1.6

45.4~0.8

Androgen (no. of males)

* Values significantly decreased (P<0.005).

14C- thymidine incorporetion (cpm x 103} 30

1 20 _

-----

3

I

2__ I

10

II 0

0.01

0,1

1

10

100

i

1

I

I

i

1

0

0.01

0.1

I

10

100

drug concentrotion (~uM) Fig. 2. Effect of TE-OX (1), TE (2), TE-HS (3) and TE-G (4) on 14C thymidine incorporation (counts per rain) into lymph node cells cultured in the presence of Con A. The broken line (bottom left) indicates 14C thymidine incorporation in cultures with TE but without Con A. Arrows indicate the derivative concentrations at which 50% inhibition of 14C thymidine incorporation occurs. of the morphological manifestation of blastic transformation, namely, cell agglutination. However, small agglutinates did occur at a concentration of 100/aM when t4C thymidine incorporation was completely suppressed. DISCUSSION The results of this study show that the testosterone molecule is modified by the functional derivatives o f testosterone (O-carboxymethyloxime on C 3, hemisuccinate, or glucoside on C~7) such that its h o r m o n a l activity is practically abolished when measured by its effect on seminal vesicle weight in castrated mice, on thymic weight and the activity of

spermatogenesis. However, hormonal activity is not altered as measured by its effect on Con A induced lymphocyte transformation in vitro. In other words, modification of the testosterone molecule results in the loss of biological activity in vivo but not in vitro. Two explanations may be offered for changes in hormonal activity as a result o f modifications o f the testosterone molecule. Firstly, in the above derivatives the nature of the testosterone molecule is changed to an extent that it is no longer capable of binding to the respective receptors in cells o f target tissue. Secondly, the ability of testosterone to reach the target organs and tissues in effective amounts and at an appropriate time is reduced or abolished by these modifications.

Biological Activity of Hormonally Active and Non-Active Androgen Derivatives Thymus weight decreases after TE-IS, but not after the in vivo administration o f hormonally nonactive derivatives, both in castrated and (especially) in normal mice, and the difference in thymus weights between castrated and normal males (as has already been described by Chiodi, 1938) indicates that in addition to the seminal vesicles and kidneys (Kochakian, 1965; Marshall, 1966), the thymus may be a target organ of the androgens. That the thymus is androgen-responsive has been supported by the finding of androgen receptors in the t h y m u s - - i n its lymphocytic and reticuloepithelial c o m p o n e n t s (Vojtigkovfi et al., 1981). The presence of these receptors is evidently in keeping with the selectivity of the suppressive effect. It was found that different testosterone derivatives, characterized as either hormonally active or non-active in vivo, have a similar inhibitory effect on lymphocyte activation by Con A in vitro. This may mean that lymphocyte activation is generally inhibited by steroid compounds, independent of any

473

specific hormonally active structure. The fact that testosterone and its derivatives influence lymphocyte activation in the same manner as do cortisol, progesterone and estradiol (Nowell, 1961; Mendelsohn, Multer & Bernheim, 1977; Clemens, Siiteri & Stites, 1979) might support this interpretation. It seems that while hormonally active androgens are required for the regulation of androgen-dependent organs and functions, the in vitro biological effect is obtained by both hormonally active and non-active androgens and also by chemically related hormones the common denominator of which is the steroid structure. Acknowledgements--The authors are indebted to Dr. P. Ko~ovsk~', Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, for providing testosterone-D-/]-glucoside, to Dr. V. Viklick~' of this Institute for performing the histological analysis, and to Dr. V. Matou~ek for his help in the statistical evaluation of the data. This investigation received financial support from the World Health Organization.

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