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Effect of PCBs on androgen production by suspension of adult rat Leydig cells in vitro
J. Steroid Biochem. Molec. Biol. Vol. 52, No. 6, pp. 595-597, 1995 Copyright © 1995 Elsevier Science Ltd 0960-0760(95)00060-7 Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00
Pergamon
Effect of P C B s on A n d r o g e n P r o d u c t i o n by S u s p e n s i o n of A d u l t Rat Leydig Cells in v i t r o R. K o v a ~ e v i d , 1. M . V o j i n o v i ~ - M i l o r a d o v ,
2 I. T e o d o r o v i C
a n d S. A n d r i C
;Institute of Biology and 2Institute of Chemistry, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia The effects of P C B s ( m i x t u r e of 2, 3, 4, 5-tetra; 2, 2', 4, 5, 5'-penta; 2, 2', 3, 3', 6, 6"-hexa and 2, 2', 3, 3', 4, 4', 5, 5'-octa congeners) on a n d r o g e n p r o d u c t i o n were investigated by suspension of Leydig cells f r o m adult rat testis, h C G - s t i m u l a t e d a n d r o g e n p r o d u c t i o n was significantly inhibited by P C B s while p r o g e s t e r o n e level was not affected. P r o g e s t e r o n e s u p p o r t e d testosterone p r o d u c t i o n was also decreased by P C B s , while c o n v e r s i o n o f a n d r o s t e n e d i o n e to testosterone was unchanged. These results suggest that the activity of m i c r o s o m a l e n z y m e C21 side-chain cleveage P450 was decreased by P C B t r e a t m e n t of Leydig cells in vitro.
37. Steroid Biochem. Moh~c. Biol., Vol. 52, No. 6, pp. 595-597, 1995
EXPERIMENTAL
INTRODUCTION T h e polychlorinated biphenyls (PCBs) have been widely identified as environmental pollutants. T h e i r industrial use lasted from the early '30s until the 1980s when they were banned. However, they are still entering the environment in several ways [1, 2]. T h e results concerning toxic effects of PCBs on mammalian reproduction are enlarged [3-9]. Studies in rats have shown that a single in vivo dose: of the coplanar PCB isomer, 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl produced a severe reduction in the plasma testosterone concentration [3]. Also, the postnatal exposure of male rats to PCBs caused impaired reproductive function in adults [4]. However, lack of the effect of PCBs on plasma testosterone levels, as well as on in vitro androgen production was demonstrated in the mouse [5]. T h e r e is ample evidence that PCBs could influence reproductive function in females [6-9]. Since the effects on reproduction may be attributed to alterations of steroid-metabolizing enzymes, the aim of the present study was to investigate the effects of PCBs on hCG-stimulated androgen production by suspension of Leydig cells from adult rat testis. In order to determine the possible site of PCBs action on the steroidogenic enzymes, the effect on testosterone production supported by progesterone or androstenedine as a substrate was also investigated. *Correspondence to R. Kova&:vi~. Received 19 Sep. 1994; accepted 18 Jan. 1995. 595
Chemicals PCB mixture (stock solution; PCBs dissolved in toluol) contained tetra (2, 3, 4, 5; 0.01 mg/ml), penta (2, 2', 4, 5, 5'; 0.01mg/ml), hexa (2, 2', 3, 3', 6, 6'; 0.007mg/ml) and octa (2, 2 ~, 3, 3', 4, 4', 5, 5'; 0.022 mg/ml) congeners and was kindly donated by D r B. Webster (University of Manitoba, Canada) and antitestosterone serum No. 250 by D r G. D. Niswender (Colorado State University, U . S . A ) . M e d i u m 199 (M199), bovine serum albumine (BSA, Fraction V), collagenase ( T y p e I), testosterone, progesterone, androstenedione and antiprogesterone serum (raised against progesterone-llct-BSA) were purchased from Sigma; h C G (Pregnyl, 3000 IU/mg) was obtained from Organon Inc.; and [1, 2, 6, 7-3H(N)]progesterone and [1, 2, 6, 7-3H(N)]testosterone were obtained from New England Nuclear. All other reagents were of analytical grade.
Animals and cell dispersion Male Wistar rats, random bred in our laboratory, were raised under controlled environmental conditions, with food and water ad libitum. Adult rats (around 3 months old) were killed by decapitation, testes quickly removed, decapsulated and enzymatically dispersed with collagenase as previously described [10]. A trypan blue exclusion test was used to determine total cell counts and viable cell number.
R. Kova~evi6 et al.
596
Testosterone production in vitro A l i q u o t s (10 6 cells/0.2 ml) o f c r u d e cell s u s p e n s i o n o f L e y d i g cells w e r e a d d e d to 12 × 7 5 m l p l a s t i c t u b e s c o n t a i n i n g 0 . 1 m l o f h C G ( 1 0 n g ) or s t e r o i d substrates (2#M) with or without 0.2ml of PCB m i x t u r e so t h a t final v o l u m e was 0.5 ml. T h e d e s i r e d c o n c e n t r a t i o n o f P C B s was p r e p a r e d b y e v a p o r a t i n g the n e c e s s a r y a m o u n t o f stock s o l u t i o n a n d d i s s o l v i n g it in t h e m e d i u m 199 e n r i c h e d b y B S A ( 0 . 1 % ) , ( M 1 9 9 B S A ) . All t u b e s w e r e i n c u b a t e d for 2 h in a s h a k i n g w a t e r b a t h o s c i l l a t i n g at 100 c y c l e s / m i n u n d e r an a t m o s p h e r e o f 9 5 % O ~ - 5 % CO2. T h e t u b e s w e r e c e n t r i f u g e d for 5 m i n at 400 g at 4°C a n d t h e s u p e r n a t a n t was s t o r e d at - 2 0 ° C p r i o r to m e a s u r e m e n t o f testosterone and progesterone by RIA. Since the antit e s t o s t e r o n e - l l - B S A s e r u m N o . 250 ( f r o m G . D . N i s w e n d e r ) shows h i g h c r o s s - r e a c t i v i t y w i t h d i h y d r o t e s t o s t e r o n e (and n o t w i t h o t h e r steroids), assay values for a n d r o g e n p r o d u c t i o n are r e f e r r e d to as " T + D H T " c o n c e n t r a t i o n s . T h e p e l l e t e d cells in t h e t u b e w e r e r e s u s p e n d e d in M 1 9 9 - B S A ( 0 . 4 m l / t u b e ) , a n d the cell v i a b i l i t y was assessed b y t r y p a n b l u e e x c l u s i o n test.
RESULTS T h e results s h o w n in T a b l e 1, i n d i c a t e t h a t P C B mixture induced a dose-dependent decrease of Leydig cell v i a b i l i t y after 2 h i n c u b a t i o n . Since in t h e p r e s e n c e o f t h e lowest dose o f P C B s (5 x 10 -6 M ) , t h e n u m b e r o f v i a b l e cells after 2 h i n c u b a t i o n was n o t a l t e r e d t h a t dose o f P C B s was u s e d in t h e later e x p e r i m e n t s . P C B s i n h i b i t e d a n d r o g e n p r o d u c t i o n in t h e p r e s e n c e o f s a t u r a t e d dose o f h C G , w h i l e p r o g e s t e r o n e levels w e r e u n a f f e c t e d ( T a b l e 2). C o n v e r s i o n o f t h e i n d i v i d u a l metabolites of the microsomal part of the steroidogenic p a t h w a y s ( p r o g e s t e r o n e , a n d r o s t e n e d i o n e ) to t e s t o s t e r one was t e s t e d in t h e p r e s e n c e o f 5 x 10 -6 M o f P C B s ( T a b l e 3). A n i n h i b i t o r y effect on t e s t o s t e r o n e p r o d u c t i o n was o b s e r v e d if p r o g e s t e r o n e was u s e d as a
Table 1. Effect of different doses of PCBs on viability of adult rat Leydig cells after 2 h incubation
Group Controls PCBs
PCBs (M) -5.20 x 1.04 x 1.56 x 2.08 x
10 -6 10 5 10 -5 10 5
Percent of viable cells after 2 h incubation 100 100 71.03 42.48 2.20
Leydig cells (1 x 106) were incubated for 2 h in the presence of different concentrations of PCBs (5 replicates for each group). At the end of incubation the tubes were centrifuged at 400 g, pelleted cells were resuspended in 0.4 ml of M199-BSA (combined together for each group) and the cell viability was assessed by Trypan blue exclusion test.
Table 2. Effect of PCBs on hCG-stimulated production of progesterone and androgen (T ÷ D H T ) by suspension of adult rat Leydig cells
Group Controls PCBs
Production of progesterone (pg/106 L.c.)
Production of T + DHT (ng/106 L.c.)
131.75 +6.11 (5) 137.97 _+20.20 (4)
16.39+ 1.03 (5) 11.94 ___1.25 (4)*
Leydig cells (1 x 106) were incubated for 2 h in the presence of a saturated dose of hCG (10 ng/106 L . c . ) with or without PCBs (5.2 X 10 -6 M). Numbers represent mean ___SEM. Numbers in brackets represent number of replicates. Significance: *P < 0.05 vs control. Results are representative of 3 experiments. L.c., Leydig cells.
substrate. However, was n o t affected.
conversion of androstenedione
DISCUSSION I n t h e p r e s e n t s t u d y , t h e i n h i b i t o r y effect o f P C B s on hCG-stimulated androgen production by suspension of rat L e y d i g cells has b e e n r e c o r d e d . S i n c e t h e p r o g e s t e r one level in t h e s e i n c u b a t i o n s was u n c h a n g e d , the i n h i b i t o r y effect at t h e level o f m i c r o s o m a l e n z y m e s w o u l d b e e x p e c t e d . S u c h an effect was c o n f i r m e d b y decrease of progesterone supported testosterone prod u c t i o n w h e n L e y d i g cells w e r e e x p o s e d to P C B s . O n the o t h e r h a n d , the c o n v e r s i o n o f a n d r o s t e n e d i o n e to t e s t o s t e r o n e was n o t c h a n g e d b y P C B s t r e a t m e n t . A c c o r d i n g to o u r r e s u l t s , it c o u l d b e s u p p o s e d t h a t P C B s d e c r e a s e t h e a c t i v i t y o f C21 s i d e - c h a i n cleavage P 4 5 0 , w h i c h is k n o w n to catalyze 1 7 c t - h y d r o x y l a t i o n a n d C17, 20 lyase a c t i v i t y [11]. H o w e v e r , the results o f G o l d m a n a n d Y a w e t z [12] have s h o w n t h a t A r o c l o r 1254 i n d u c e d no c h a n g e s in t h e a c t i v i t y o f 1 7 e h y d r o x y l a s e / C 1 7 lyase in g u i n e a p i g testis m i c r o s o m e s , e i t h e r o f p r e t r e a t e d a n i m a l s o r w h e n A r o c l o r 1254 (13.3 # M ) was p r e s e n t in t h e r e a c t i o n p r e p a r a t i o n s . A t the p r e s e n t m o m e n t it is n o t p o s s i b l e to give a clear e x p l a n a t i o n for such a d i s c r e p a n c y . H o w e v e r , the differences in P C B c o n g e n e r s c o n t a i n e d in A r o c l o r
Table 3. Effect of PCBs (5.2;< l O - 6 M ) on androgen (T + D H T ) production by suspension of adult rat Leydig cells in the presence of progesterone ( 2 p M ) and androstenedione (2 # M ) as substrate Levels of T + DHT (ng/106 L.c.) Substrate Progesterone Androstenedione
Controls
PCBs
32.59___ 1.72 (5) 80.70 + 10.25 (4)
25.85_+1.91 (5)* 68.83 + 4.83 (5)
Leydig cells (1 x 106) were incubated for 2h in the presence of progesterone or androstenedione, with or without PCBs (5.2 x 10-6M). Numbers represent mean _+SEM. Numbers in brackets represent number of replicates. Significance: *P < 0.001 vs. control. Results are representative of 2 experiments. L.c., Leydig cells.
Effect of PCB on Leydig Cells 1254 ( p o l y c h l o r o b i p h e n y l c o n t a i n i n g 5 4 % c h l o r i n e ; tetra, 1 1 % ; p e n t a , 4 9 % ; hexa, 34% ; a n d h e p t a c o n g e n e r s , 6 % ) a n d in o u r m i x t u r e ( h i g h a m o u n t o f o c t a - c o n g e n e r s , 4 9 % ) c o u l d a c c o u n t f o r such results. Also, the differences in th e test m o d e l u s e d ( m i c r o somal p r e p a r a t i o n s or w h o l e cell s y s te m ) w o u l d n o t be neglected. A c c o r d i n g to the p r e s e n t e d data t h e m e c h a n i s m by w h i c h P C B s d e c r e a s e d 1:he a c t i v i t y o f C21 side c h a i n c l e v e a g e P 4 5 0 c o u l d not be p r e c i s e l y defined. F u r t h e r e x p e r i m e n t s are n e c e s s a r y to clarify if the m i x t u r e o f P C B s act d i r e c t l y on t h e e n z y m e a n d / o r t h e r e is an i n d i r e c t effect t h r o u g h a c h a n g e in the h y d r o p h o b i c e n v i r o n m e n t in w h i c h th e e n z y m e is e m b e d d e d . S e v eral r e p o r t s h a v e s h o w n that t h e c h a n g e s in m e m b r a n e p h o s p h o l i p i d c o m p o s i t i o n c o u l d alter t h e a c t i v i t y o f s o m e rat t e s t i c u l a r enzy~rnes [13-15]. O u r data also s h o w e d that h i g h e r doses o f P C B s (>/ 1 0 -5 M ) d e c r e a s e cell v i a b i l i t y after 2 h i n c u b a t i o n , w h i c h i n d i c a t e d t h e t o x ic effect o f P C B s o n p l a s m a m e m b r a n e i n t e g r i t y . H o w e v e r , the dose o f 5 x 10 -6 M w h i c h a l t e r e d a n d r o g e n p r o d u c t i o n o f L e y d i g cells, h a d no effect on cell v i a b i l i t y T h a t dose c o r r e s p o n d s to t h e r a n g e o f 1-2 p p m . C o n s i d e r i n g the resuRs f r o m o u r study, t o g e t h e r w i t h the data f r o m the l i t e r a t u r e [3-9, 14, 16] it c o u l d be s u p p o s e d t h a t P C B s are e n v i r o n m e n t a l p o l l u t a n t s w i t h c e r t a i n n e g a t i v e i n f l u e n c e on r e p r o d u c t i v e f u n c tion. Acknowledgements--We are grateful to Dr B. Webster for the gift of PCBs and to Dr G. D. Niswender for the supply of antiserum. This research was supported by a joint grant from the Serbia Research Association.
REFERENCES 1. Ahlborg U. G., Brouwer A., Fingerhut M. A., Jacobson J. L., Jacobson S. W., Kennedy S. W., Kettrup A. A. F., Koeman J. H., Poiger H., Rappe C., Safe S. H., Seegal R. F., Tuomisto J. and van den Berg M.: Impact of polychlorinated dibenzo-pdioxins, dibenzofurans and biphenyls on human and environmental health, with special emphasis on application of the toxic equivalency factor concept. Fur. J. Pharmac. Eur. Toxic. Pharm. Sec. 228 (1992) 179-199.
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2. Vojinovi6-Milorado~"M., Marjanovi6 P., Buzarov D., Pavkov S., Dimitrijevi6 Lj. and Miloradov M.: Bioaccumulation of polychlorinated biphenyls and organochlorine pesticides in selected fish species as an indicator of the pollution of aquatic resources in Voivodina, Yugoslavia. W. Sci. Tech. 26 (1992) 2361-2364. 3. Yeowell H. N., Waxman D. J., Wadhera A. and Goldstein J. A.: Suppression of the constutive, male specific rat hepatic cytochrome P-450 2c and its mRNA by 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl and 3-methylcholanthrene. Molec. Pharmac. 32 (1987) 340-347. 4. Sager D. B.: Effect of postnatal exposure to polychlorinated biphenyls on adult male reproductive function. Environ. Res. 31 (1983) 76-94. 5. Johansson B.: Lack of effects of polychlorinated biphenyls on testosterone synthesis in mice. Pharmac. Toxic. 61 (1987) 220-223. 6. Brezner E., Terkel J. and Perry A. S.: The effect of Aroclor 1254 (PCB) on the physiology of reproduction in the female rat. Comp. Biochem. Physiol. 77C (1984) 65-70. 7. Smitsvabprooije A. E., Lammers J. H. C. M., Waalkemsberendsen D. H., Kulig B. M. and Snoij N. J.: Effects of the PCB 3, 4, 5, Y, 4', 5'-hexachlorobiphenyl on the reproduction capacity of Wistar rats. Chemosphere 27 (1993) 395-400. 8. Jonsson H. T., Keil J. E., Gaddy R. G., Loadholt C. B., Henniger G. R. and Walker E. M.: Prolonged ingestion of commercial DDT and PCB: effects on progesterone levels and reproduction in the mature female rat. Arch. Environ. Contain. Toxic. 3 (1976) 279-290. 9. Backlin B. M. and Bergman A.: Morphological aspects on the reproductive organs in female mink (Mustela Vison) exposed to polychlorinated biphenyls and fractions thereof. Ambio 21 (1992) 596-601. 10. Ko~acevi~ R. and Sara6 M.: Bromocriptine-induced inhibition of hydroxylase/lyase activity of adult rat Leydig cells. J. Steroid Biochem. Molec. Biol. 46 (1993) 841-845. 11. Nakajin S., Shikita M. and Hall P. F.: Microsomal cytochrome P450 from neonatal pig testis: purification and properties of a C21 side-chain cleavage system (17-hydroxylase-C17-20 lyase). J. Biol. Chem. 256 (1981) 3871-3876. 12. Goldman D. and Yawetz A.: The interference of Aroclor 1254 with progesterone metabolism in guinea pig adrenal and testes microsomes. J. Biochem. Toxic. 5 (1990) 99-107. 13. Bogovich K. and Payne A. H.: Purification of rat testicular microsomal 17-ketosteroid reductase. Evidence that 17-ketosteroid reductase and 17fl-hydroxysteroid dehydrogenase are distinct enzymes, ft. Biol. Chem. 260 (1980) 5552-5559. 14. Cooke G. M. and Robaire B.: Modulation of epididymal A4steroid 5a-reductase activity in vitro by the phospholipid environment, ft. Biol. Chem. 260 (1985) 7489-7495. 15. Cooke G. M. and Robaire B.: Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro. Biol. Reprod. 39 (1988) 329-339. 16. Miranda C. L., Henderson M. C., Wang J. L., Chang H. S., Hendricks J. D. and Buhler D. R.: Differential effects of 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl (HCB) on interrenal steroidogenesis in male and female rainbow trout Oncorhynchus Mykiss. Comp. Biochem. Physiol. 103C (1992) 153-157.
Pergamon
Effect of P C B s on A n d r o g e n P r o d u c t i o n by S u s p e n s i o n of A d u l t Rat Leydig Cells in v i t r o R. K o v a ~ e v i d , 1. M . V o j i n o v i ~ - M i l o r a d o v ,
2 I. T e o d o r o v i C
a n d S. A n d r i C
;Institute of Biology and 2Institute of Chemistry, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia The effects of P C B s ( m i x t u r e of 2, 3, 4, 5-tetra; 2, 2', 4, 5, 5'-penta; 2, 2', 3, 3', 6, 6"-hexa and 2, 2', 3, 3', 4, 4', 5, 5'-octa congeners) on a n d r o g e n p r o d u c t i o n were investigated by suspension of Leydig cells f r o m adult rat testis, h C G - s t i m u l a t e d a n d r o g e n p r o d u c t i o n was significantly inhibited by P C B s while p r o g e s t e r o n e level was not affected. P r o g e s t e r o n e s u p p o r t e d testosterone p r o d u c t i o n was also decreased by P C B s , while c o n v e r s i o n o f a n d r o s t e n e d i o n e to testosterone was unchanged. These results suggest that the activity of m i c r o s o m a l e n z y m e C21 side-chain cleveage P450 was decreased by P C B t r e a t m e n t of Leydig cells in vitro.
37. Steroid Biochem. Moh~c. Biol., Vol. 52, No. 6, pp. 595-597, 1995
EXPERIMENTAL
INTRODUCTION T h e polychlorinated biphenyls (PCBs) have been widely identified as environmental pollutants. T h e i r industrial use lasted from the early '30s until the 1980s when they were banned. However, they are still entering the environment in several ways [1, 2]. T h e results concerning toxic effects of PCBs on mammalian reproduction are enlarged [3-9]. Studies in rats have shown that a single in vivo dose: of the coplanar PCB isomer, 3, 4, 5, 3', 4', 5'-hexachlorobiphenyl produced a severe reduction in the plasma testosterone concentration [3]. Also, the postnatal exposure of male rats to PCBs caused impaired reproductive function in adults [4]. However, lack of the effect of PCBs on plasma testosterone levels, as well as on in vitro androgen production was demonstrated in the mouse [5]. T h e r e is ample evidence that PCBs could influence reproductive function in females [6-9]. Since the effects on reproduction may be attributed to alterations of steroid-metabolizing enzymes, the aim of the present study was to investigate the effects of PCBs on hCG-stimulated androgen production by suspension of Leydig cells from adult rat testis. In order to determine the possible site of PCBs action on the steroidogenic enzymes, the effect on testosterone production supported by progesterone or androstenedine as a substrate was also investigated. *Correspondence to R. Kova&:vi~. Received 19 Sep. 1994; accepted 18 Jan. 1995. 595
Chemicals PCB mixture (stock solution; PCBs dissolved in toluol) contained tetra (2, 3, 4, 5; 0.01 mg/ml), penta (2, 2', 4, 5, 5'; 0.01mg/ml), hexa (2, 2', 3, 3', 6, 6'; 0.007mg/ml) and octa (2, 2 ~, 3, 3', 4, 4', 5, 5'; 0.022 mg/ml) congeners and was kindly donated by D r B. Webster (University of Manitoba, Canada) and antitestosterone serum No. 250 by D r G. D. Niswender (Colorado State University, U . S . A ) . M e d i u m 199 (M199), bovine serum albumine (BSA, Fraction V), collagenase ( T y p e I), testosterone, progesterone, androstenedione and antiprogesterone serum (raised against progesterone-llct-BSA) were purchased from Sigma; h C G (Pregnyl, 3000 IU/mg) was obtained from Organon Inc.; and [1, 2, 6, 7-3H(N)]progesterone and [1, 2, 6, 7-3H(N)]testosterone were obtained from New England Nuclear. All other reagents were of analytical grade.
Animals and cell dispersion Male Wistar rats, random bred in our laboratory, were raised under controlled environmental conditions, with food and water ad libitum. Adult rats (around 3 months old) were killed by decapitation, testes quickly removed, decapsulated and enzymatically dispersed with collagenase as previously described [10]. A trypan blue exclusion test was used to determine total cell counts and viable cell number.
R. Kova~evi6 et al.
596
Testosterone production in vitro A l i q u o t s (10 6 cells/0.2 ml) o f c r u d e cell s u s p e n s i o n o f L e y d i g cells w e r e a d d e d to 12 × 7 5 m l p l a s t i c t u b e s c o n t a i n i n g 0 . 1 m l o f h C G ( 1 0 n g ) or s t e r o i d substrates (2#M) with or without 0.2ml of PCB m i x t u r e so t h a t final v o l u m e was 0.5 ml. T h e d e s i r e d c o n c e n t r a t i o n o f P C B s was p r e p a r e d b y e v a p o r a t i n g the n e c e s s a r y a m o u n t o f stock s o l u t i o n a n d d i s s o l v i n g it in t h e m e d i u m 199 e n r i c h e d b y B S A ( 0 . 1 % ) , ( M 1 9 9 B S A ) . All t u b e s w e r e i n c u b a t e d for 2 h in a s h a k i n g w a t e r b a t h o s c i l l a t i n g at 100 c y c l e s / m i n u n d e r an a t m o s p h e r e o f 9 5 % O ~ - 5 % CO2. T h e t u b e s w e r e c e n t r i f u g e d for 5 m i n at 400 g at 4°C a n d t h e s u p e r n a t a n t was s t o r e d at - 2 0 ° C p r i o r to m e a s u r e m e n t o f testosterone and progesterone by RIA. Since the antit e s t o s t e r o n e - l l - B S A s e r u m N o . 250 ( f r o m G . D . N i s w e n d e r ) shows h i g h c r o s s - r e a c t i v i t y w i t h d i h y d r o t e s t o s t e r o n e (and n o t w i t h o t h e r steroids), assay values for a n d r o g e n p r o d u c t i o n are r e f e r r e d to as " T + D H T " c o n c e n t r a t i o n s . T h e p e l l e t e d cells in t h e t u b e w e r e r e s u s p e n d e d in M 1 9 9 - B S A ( 0 . 4 m l / t u b e ) , a n d the cell v i a b i l i t y was assessed b y t r y p a n b l u e e x c l u s i o n test.
RESULTS T h e results s h o w n in T a b l e 1, i n d i c a t e t h a t P C B mixture induced a dose-dependent decrease of Leydig cell v i a b i l i t y after 2 h i n c u b a t i o n . Since in t h e p r e s e n c e o f t h e lowest dose o f P C B s (5 x 10 -6 M ) , t h e n u m b e r o f v i a b l e cells after 2 h i n c u b a t i o n was n o t a l t e r e d t h a t dose o f P C B s was u s e d in t h e later e x p e r i m e n t s . P C B s i n h i b i t e d a n d r o g e n p r o d u c t i o n in t h e p r e s e n c e o f s a t u r a t e d dose o f h C G , w h i l e p r o g e s t e r o n e levels w e r e u n a f f e c t e d ( T a b l e 2). C o n v e r s i o n o f t h e i n d i v i d u a l metabolites of the microsomal part of the steroidogenic p a t h w a y s ( p r o g e s t e r o n e , a n d r o s t e n e d i o n e ) to t e s t o s t e r one was t e s t e d in t h e p r e s e n c e o f 5 x 10 -6 M o f P C B s ( T a b l e 3). A n i n h i b i t o r y effect on t e s t o s t e r o n e p r o d u c t i o n was o b s e r v e d if p r o g e s t e r o n e was u s e d as a
Table 1. Effect of different doses of PCBs on viability of adult rat Leydig cells after 2 h incubation
Group Controls PCBs
PCBs (M) -5.20 x 1.04 x 1.56 x 2.08 x
10 -6 10 5 10 -5 10 5
Percent of viable cells after 2 h incubation 100 100 71.03 42.48 2.20
Leydig cells (1 x 106) were incubated for 2 h in the presence of different concentrations of PCBs (5 replicates for each group). At the end of incubation the tubes were centrifuged at 400 g, pelleted cells were resuspended in 0.4 ml of M199-BSA (combined together for each group) and the cell viability was assessed by Trypan blue exclusion test.
Table 2. Effect of PCBs on hCG-stimulated production of progesterone and androgen (T ÷ D H T ) by suspension of adult rat Leydig cells
Group Controls PCBs
Production of progesterone (pg/106 L.c.)
Production of T + DHT (ng/106 L.c.)
131.75 +6.11 (5) 137.97 _+20.20 (4)
16.39+ 1.03 (5) 11.94 ___1.25 (4)*
Leydig cells (1 x 106) were incubated for 2 h in the presence of a saturated dose of hCG (10 ng/106 L . c . ) with or without PCBs (5.2 X 10 -6 M). Numbers represent mean ___SEM. Numbers in brackets represent number of replicates. Significance: *P < 0.05 vs control. Results are representative of 3 experiments. L.c., Leydig cells.
substrate. However, was n o t affected.
conversion of androstenedione
DISCUSSION I n t h e p r e s e n t s t u d y , t h e i n h i b i t o r y effect o f P C B s on hCG-stimulated androgen production by suspension of rat L e y d i g cells has b e e n r e c o r d e d . S i n c e t h e p r o g e s t e r one level in t h e s e i n c u b a t i o n s was u n c h a n g e d , the i n h i b i t o r y effect at t h e level o f m i c r o s o m a l e n z y m e s w o u l d b e e x p e c t e d . S u c h an effect was c o n f i r m e d b y decrease of progesterone supported testosterone prod u c t i o n w h e n L e y d i g cells w e r e e x p o s e d to P C B s . O n the o t h e r h a n d , the c o n v e r s i o n o f a n d r o s t e n e d i o n e to t e s t o s t e r o n e was n o t c h a n g e d b y P C B s t r e a t m e n t . A c c o r d i n g to o u r r e s u l t s , it c o u l d b e s u p p o s e d t h a t P C B s d e c r e a s e t h e a c t i v i t y o f C21 s i d e - c h a i n cleavage P 4 5 0 , w h i c h is k n o w n to catalyze 1 7 c t - h y d r o x y l a t i o n a n d C17, 20 lyase a c t i v i t y [11]. H o w e v e r , the results o f G o l d m a n a n d Y a w e t z [12] have s h o w n t h a t A r o c l o r 1254 i n d u c e d no c h a n g e s in t h e a c t i v i t y o f 1 7 e h y d r o x y l a s e / C 1 7 lyase in g u i n e a p i g testis m i c r o s o m e s , e i t h e r o f p r e t r e a t e d a n i m a l s o r w h e n A r o c l o r 1254 (13.3 # M ) was p r e s e n t in t h e r e a c t i o n p r e p a r a t i o n s . A t the p r e s e n t m o m e n t it is n o t p o s s i b l e to give a clear e x p l a n a t i o n for such a d i s c r e p a n c y . H o w e v e r , the differences in P C B c o n g e n e r s c o n t a i n e d in A r o c l o r
Table 3. Effect of PCBs (5.2;< l O - 6 M ) on androgen (T + D H T ) production by suspension of adult rat Leydig cells in the presence of progesterone ( 2 p M ) and androstenedione (2 # M ) as substrate Levels of T + DHT (ng/106 L.c.) Substrate Progesterone Androstenedione
Controls
PCBs
32.59___ 1.72 (5) 80.70 + 10.25 (4)
25.85_+1.91 (5)* 68.83 + 4.83 (5)
Leydig cells (1 x 106) were incubated for 2h in the presence of progesterone or androstenedione, with or without PCBs (5.2 x 10-6M). Numbers represent mean _+SEM. Numbers in brackets represent number of replicates. Significance: *P < 0.001 vs. control. Results are representative of 2 experiments. L.c., Leydig cells.
Effect of PCB on Leydig Cells 1254 ( p o l y c h l o r o b i p h e n y l c o n t a i n i n g 5 4 % c h l o r i n e ; tetra, 1 1 % ; p e n t a , 4 9 % ; hexa, 34% ; a n d h e p t a c o n g e n e r s , 6 % ) a n d in o u r m i x t u r e ( h i g h a m o u n t o f o c t a - c o n g e n e r s , 4 9 % ) c o u l d a c c o u n t f o r such results. Also, the differences in th e test m o d e l u s e d ( m i c r o somal p r e p a r a t i o n s or w h o l e cell s y s te m ) w o u l d n o t be neglected. A c c o r d i n g to the p r e s e n t e d data t h e m e c h a n i s m by w h i c h P C B s d e c r e a s e d 1:he a c t i v i t y o f C21 side c h a i n c l e v e a g e P 4 5 0 c o u l d not be p r e c i s e l y defined. F u r t h e r e x p e r i m e n t s are n e c e s s a r y to clarify if the m i x t u r e o f P C B s act d i r e c t l y on t h e e n z y m e a n d / o r t h e r e is an i n d i r e c t effect t h r o u g h a c h a n g e in the h y d r o p h o b i c e n v i r o n m e n t in w h i c h th e e n z y m e is e m b e d d e d . S e v eral r e p o r t s h a v e s h o w n that t h e c h a n g e s in m e m b r a n e p h o s p h o l i p i d c o m p o s i t i o n c o u l d alter t h e a c t i v i t y o f s o m e rat t e s t i c u l a r enzy~rnes [13-15]. O u r data also s h o w e d that h i g h e r doses o f P C B s (>/ 1 0 -5 M ) d e c r e a s e cell v i a b i l i t y after 2 h i n c u b a t i o n , w h i c h i n d i c a t e d t h e t o x ic effect o f P C B s o n p l a s m a m e m b r a n e i n t e g r i t y . H o w e v e r , the dose o f 5 x 10 -6 M w h i c h a l t e r e d a n d r o g e n p r o d u c t i o n o f L e y d i g cells, h a d no effect on cell v i a b i l i t y T h a t dose c o r r e s p o n d s to t h e r a n g e o f 1-2 p p m . C o n s i d e r i n g the resuRs f r o m o u r study, t o g e t h e r w i t h the data f r o m the l i t e r a t u r e [3-9, 14, 16] it c o u l d be s u p p o s e d t h a t P C B s are e n v i r o n m e n t a l p o l l u t a n t s w i t h c e r t a i n n e g a t i v e i n f l u e n c e on r e p r o d u c t i v e f u n c tion. Acknowledgements--We are grateful to Dr B. Webster for the gift of PCBs and to Dr G. D. Niswender for the supply of antiserum. This research was supported by a joint grant from the Serbia Research Association.
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