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Comparative effects of dehydroepiandrosterone and related steroids on peroxisome proliferation in rat liver
Life Sciences, Vol. 52, pp. 1709-1716 Printed in the USA
Pergamon Press
C O M P A R A T I V E EFFECTS OF D E H Y D R O E P I A N D R O S T E R O N E AND R E L A T E D STEROIDS ON PEROXISOME P R O L I F E R A T I O N IN RAT LIVER M. S. Rao, H. Ide, K. Alvares, V. Subbarao, J.K. Reddy, O. Hechter* and A.V. Yeldandi D e p a r t m e n t of P a t h o l o g y and *Department of Physiology, N o r t h w e s t e r n U n i v e r s i t y Medical School, Chicago, IL 60611 (Received in final form March 15, 1993) Summary D e h y d r o e p i a n d r o s t e r o n e (DHEA) is known to induce p e r o x i s o m e p r o l i f e r a t i o n and peroxisomal e n o y l - C o A h y d r a t a s e / 3 - h y d r o x y a c y l - C o A d e h y d r o g e n a s e (PBE) m R N A in the rat liver. We have compared the effects of 6 i n t e r m e d i a t e m e t a b o l i t e s of DHEA on the induction of p e r o x i s o m e p r o l i f e r a t i o n and PBE mRNA. Administration of epiandrosterone, etiocholanolone, androstenedione, e s t r o n e or estradiol for 2 weeks in the diet at 0.45% c o n c e n t r a t i o n to adult male F-344 rats failed to induce s i g n i f i c a n t increases in p e r o x i s o m e p r o l i f e r a t i o n and PBE m R N A when c o m p a r e d to the parent c o m p o u n d DHEA. Dietary a d m i n i s t r a t i o n of 5-androstene-3~,17~-diol (ADIOL) for 2 weeks at 0.45% c o n c e n t r a t i o n caused an increase in PBE m R N A and p e r o x i s o m e p r o l i f e r a t i o n but to a lesser extent than DHEA. Following a single i n t r a g a s t r i c dose of DHEA an increase in PBE mRNA level was o b s e r v e d in the liver at 1 hr and c o n t i n u e d to 16 hrs., but not with its metabolites. These results strongly suggest that DHEA or p o s s i b l y a n o t h e r yet to be identified m e t a b o l i t e might be r e s p o n s i b l e for p e r o x i s o m e proliferation. Dehydroepiandrosterone (DHEA) is a steroid secreted by adrenal c o r t e x in certain m a m m a l s p a r t i c u l a r l y humans (i). The functions of DHEA in humans are not fully understood. However, in e x p e r i m e n t a l animals which make little or no DHEA l o n g - t e r m administration of this steroid inhibits many pathological p r o c e s s e s i n c l u d i n g the d e v e l o p m e n t of tumors, diabetes, obesity and a t h e r o s c l e r o s i s (2-4). Recently, DHEA was shown to induce peroxisome proliferation, and to increase the activity of certain peroxisome-associated enzymes in the liver (5-8). Although it exerts a n t i c a r c i n o g e n i c effects in a v a r i e t y of tissues, l o n g - t e r m a d m i n i s t r a t i o n of this agent in the diet at a c o n c e n t r a t i o n of 0.45% for 84 weeks resulted in the d e v e l o p m e n t of h e p a t o c e l l u l a r c a r c i n o m a s in the rat (3,9-11). Accordingly, peroxisome proliferative property appears to make DHEA a hepatocarcinogen in rats, a function exhibited by other M.S. Rao, Dept. of Pathology, N o r t h w e s t e r n Univ. 303 E. C h i c a g o Ave., Chicago, IL 60611
Medical
0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
School,
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p e r o x i s o m e p r o l i f e r a t o r s (12,13). The question arises w h e t h e r DHEA itself is the active agent for the induction of p e r o x i s o m e proliferation, or serves as the source of an active m e t a b o l i t e formed ~ vivo which induces p e r o x i s o m e proliferation. The failure of DHEA to induce peroxisome proliferation in isolated h e p a t o c y t e s (14), and its inability to activate PPAR, a peroxisome proliferator activated receptor under ~ vitro conditions (15,16), suggests that DHEA in itself is not r e s p o n s i b l e for the p e r o x i s o m e p r o l i f e r a t i v e effects observed vivo. In this study, we examined the p e r o x i s o m e p r o l i f e r a t i v e effect of 6 recognized metabolites of DHEA. Except 5androstene-3~,17~-diol (ADIOL), none of the m e t a b o l i t e s tested showed a significant increase in peroxisome proliferation. A D I O L induced 50% less p e r o x i s o m e p r o l i f e r a t i o n than DHEA at the same dose level. These results suggest that DHEA in itself, DHEA with a c o f a c t o r or p o s s i b l y a DHEA m e t a b o l i t e other than those studied is r e s p o n s i b l e for the hepatic p e r o x i s o m e p r o l i f e r a t i o n o b s e r v e d ~ vivo. M a t e r i a l s and M e t h o d s Male F-344 rats, weighing 90-100g, were p u r c h a s e d from Charles River L a b o r a t o r i e s (Wilmington, Mass., USA). After a brief p e r i o d of a c c l i m a t i z a t i o n d e h y d r o e p i a n d r o s t e r o n e acetate, epiandrosterone, 4-androstenedione, estrone, 17~-estradiol, 3~e t i o c h o l a n o l o n e or 5-androstene-3~,17~-diol acetate (Sigma Chem. Co., St. Louis, MO) was fed to 3 to 5 rats at a c o n c e n t r a t i o n of 0.45% for a 2 week c o m p a r a t i v e study. Control animals were m a i n t a i n e d on normal rat chow. All rats were killed under ether anesthesia. For morphological evaluation and quantitative m o r p h o m e t r i c analysis of p e r o x i s o m e s liver was p r o c e s s e d for light and e l e c t r o n m i c r o s c o p y as p r e v i o u s l y d e s c r i b e d (17). To v i s u a l i z e the change in p e r o x i s o m e p r o l i f e r a t i o n a s s o c i a t e d Mr 80,000 p o l y p e p t i d e (PPA-80), 30 ~g of s o l u b i l i z e d p o s t n u c l e a r fractions were analyzed by SDS-PAGE (17,18). For immunoblotting, c y t o s o l i c p r o t e i n (20 ~g protein), from 1400xg s u p e r n a t a n t of 10% (w/v) h o m o g e n a t e s of liver in i0 mM p h o s p h a t e buffer (pH 7), was resolved by SDS-PAGE, transferred to nitrocellulose and immunostained with anti-PBE (19). For a s s e s s i n g the level of PBE mRNA total liver RNA was p r e p a r e d and a n a l y z e d by N o r t h e r n blot h y b r i d i z a t i o n using 3 2 p - l a b e l e d PBE cDNA (7,19). The levels of mRNA were m e a s u r e d by d e n s i t o m e t r i c scanning of autoradiographs. Further, to evaluate the rapidity with w h i c h the m R N A coding for peroxisomal PBE is induced rats were given a single dose of DHEA by gavage (450 m g / 1 0 0 g body wt in 2 ml corn oil) and s a c r i f i c e d in groups of 2 at i, 8 and 16 hours. Three control rats were given corn oil only and killed at 16 hours. A d d i t i o n a l groups of 2 rats were given a single intragastric dose (450 m g / 1 0 0 g body wt) of epiandrosterone, androstenedione, estrone, 17~-estradiol or A D I O L and killed after 8 hours. Results and D i s c u s s i o n Body and liver weights and the volume density of p e r o x i s o m e s in h e p a t o c y t e s of controls, and rats fed DHEA and its i n t e r m e d i a t e m e t a b o l i t e s in diet for 2 weeks are p r e s e n t e d in Table I. Liver w e i g h t for 100g body w e i g h t in DHEA and A D I O L treated groups increased s i g n i f i c a n t l y (P<0.001) w h e n c o m p a r e d
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to o t h e r g r o u p s . Ultrastructural e x a m i n a t i o n of l i v e r c e l l s of r a t s t r e a t e d w i t h D H E A a n d A D I O L for 2 w e e k s r e v e a l e d a l a r g e n u m b e r of p e r o x i s o m e s w h e r e a s no s u c h i n c r e a s e w a s n o t e d in r a t s treated with other steroids tested. Morphometric analysis showed a 6 and 2.6 fold increase in t h e v o l u m e density of peroxisomes in D H E A a n d A D I O L t r e a t e d rats, r e s p e c t i v e l y (Table I). Figures 1 and 2 show SDS-PAGE profiles of p o s t n u c l e a r f r a c t i o n s p r e p a r e d f r o m l i v e r s of r a t s a f t e r 2 w e e k s of e x p o s u r e to D H E A a n d o t h e r s t e r o i d s . In D H E A a n d A D I O L t r e a t e d rats, a marked increase in the amount of peroxisome proliferationassociated PPA-80 was observed.
Fiq.
2
I
1 2 3 4 5 6 7 8 9
Fiq.
IA
3
4
IB
SDS-polyacrylamide s l a b gel e l e c t r o p h o r e s i s of p o s t n u c l e a r f r a c t i o n s of l i v e r f r o m c o n t r o l (1-3), D H E A (4-6) a n d e t i o c h o l a n o l o n e (7-9) (Fig. IA) a n d A D I O L (Fig. IB) t r e a t e d rats. The arrows indicate the p o s i t i o n of p e r o x i s o m e p r o l i f e r a t o r - a s s o c i a t e d 80,000 mol wt polypeptide. 1
2
3
4
5
Fiq.
6
7
8
9
2
S D S - P A G E of p o s t - n u c l e a r f r a c t i o n of l i v e r f r o m e s t r a d i o l (1-3), e s t r o n e (4-6) a n d a n d r o s t e n e d i o n e (7-9) t r e a t e d rats. N o i n c r e a s e in t h e a m o u n t of 8 0 , 0 0 0 m o l w t p o l y p e p t i d e is n o t e d (arrows). In c o n t r o l r a t s a n d r a t s t r e a t e d w i t h D H E A m e t a b o l i t e s no increase in the amount of this protein was evident. Immunoblotting using polyclonal antibodies to PBE further confirmed the increase in PBE in D H E A a n d A D I O L t r e a t e d rats. N o r t h e r n b l o t a n a l y s i s of t o t a l l i v e r R N A f r o m r a t s t r e a t e d w i t h DHEA and ADIOL for 2 weeks showed a marked increase in t h e l e v e l s of PBE m R N A (Fig. 3). N o a p p r e c i a b l e c h a n g e in PBE m R N A
178-estradiol
Estrone
3 ~Etiocholanolone
Control
5
6
7
8
3
3
using
"t" t e s t
6.9 ~ 0.4
7.5 ~ 0.2
6.4 ~ 0.3
6.7 ± 0.i
6.7 ! 0.2
6.5 ± 0.i
i0.0 ~ 0.4
11.5 ± 0.5
5.1 ~ 0.3
5.2 ~ 0.i
6.3 ~ 0.i
6.0 ± 0.I
4.9 ~ 0.2
5.4 ± 0.i
7.3 ~ 0.i
8.4 ± 0.2
NS
P<0.01
P<0.OI
NS
NS
P<0.001
P<0.001
and Peroxisome
Liver Weight/ 100g b.wt
Liver Weight
Liver Weight
student's
136 ~ 3
143 ~ 3
i01 ~
113 ± 2
137 ~ 3
2
136 ~ 3 121 ~
I
on B o d y Weight,
Weight
136 ~
Body
of D H E A
obtained
4-androstenedione
4
are
Epiandrosterone
3
P values
5-androstene-3B,178-diol
its
2
and
Dehydroepiandrosterone
Treatment
of M e t a b o l i t e s
1
Group
Effects
TABLE
Density
1.0
0.i
1.8 ! 0.2
1.3 ~ 0.4
i.i ~
1.6 ± 0.3
i.i ~ 0.i
1.7 ! 0.2
4.6 ~ 0.8
ii.0 !
Peroxisome volume density
Volume
O
tu
Z O
O
m
o
a;
o M
m o~
m
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in other g r o u p s was evident (Fig. 3). Epiandrosterone not induce PBE m R N A (not shown in the picture).
1
2
34
5
6 7 8
also did
9
1
2
3
4
5
6
t g g
Fiq.
3A
Fiq.
3B
N o r t h e r n blot analysis for PBE m R N A in liver of rats t r e a t e d with DHEA (1-3), e t i o c h o l a n o l o n e (4,5), andros t e n e d i o n e (6,7), or estradiol (8-9) (Fig. 3A), and A D I O L (1-4) and control (5,6) (Fig. 3B) for 2 weeks in diet at 0.45% c o n c e n t r a t i o n : Total ~ A (20~g/lane was a n a l y z e d by N o r t h e r n b l o t t i n g with P-labeled PBE cDNA. The size of PBE mRNA is 3.0 kilobases.
Changes in the levels of PBE mRNA in the liver of rats at d i f f e r e n t time points after a large single i n t r a g a s t r i c dose of DHEA are shown in Figure 4. Levels of this mRNA increased A
A
A
B
B
C
C
D
I
............
Fiq.
D
e
4
N o r t h e r n blot analysis for PBE mRNA in livers of control (A) and rats killed at 1 (B), 8 (C), and 16 (D) hours after a single i n t r a g a s t r i c dose of DHEA (450 m g / 1 0 0 g body wt). Blot a n a l y s i s was carried out as d e s c r i b e d in Fig. 3. The r e l a t i v e amounts of 28s and 18s RNA used for N o r t h e r n blot a n a l y s i s is similar. at 1 hour and c o n t i n u e d to rise up to 16 hours. Quantitative d e n s i t o m e t r i c analysis showed 4.8, 6.8 and 9.2 fold increase at i, 8 and 16 hours respectively. There was no increase in PBE m R N A in rats t r e a t e d with DHEA metabolites. Peroxisome proliferative effects of DHEA are well c h a r a c t e r i z e d in rats and mice (5-7). The results of this study further c o r r o b o r a t e and extend these o b s e r v a t i o n s c o n c e r n i n g the effect of DHEA after a d m i n i s t r a t i o n of a single dose or feeding c o n t i n u o u s l y for 2 weeks. A d m i n i s t r a t i o n of DHEA for 2 weeks
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caused s i g n i f i c a n t hepatomegaly, p e r o x i s o m e p r o l i f e r a t i o n and increased levels of PBE p r o t e i n and PBE mRNA. A f t e r a large single dose of DHEA (450 m g / 1 0 0 g body wt) a rapid increase in the rate of PBE m R N A a c c u m u l a t i o n was noted. This increase was o b s e r v e d 1 hour after DHEA dosing and c o n t i n u e d up to 16 hours. However, at a lower dose level (50 m g / 1 0 0 g body wt), increase in PBE m R N A was evident only at 24 hours (7). The m e c h a n i s m by w h i c h DHEA causes p e r o x i s o m e p r o l i f e r a t i o n is not clear. Since DHEA appears to be ineffective in a c t i v a t i n g PPAR or inducing p e r o x i s o m e p r o l i f e r a t i o n under ~ vitro conditions, the p o s s i b i l i t y arises that one of the i n t e r m e d i a t e metabolites of DHEA may play an important role in inducing p e r o x i s o m e proliferation. DHEA is known to be c o n v e r t e d to androgens and estrogens, as well as to other intermediate metabolites. We selected epiandrosterone, androstenedione, ~estradiol, estrone, etiocholanolone and A D I O L in this study because they are e s t a b l i s h e d m e t a b o l i t e s of DHEA. Several of these metabolites appear to be more potent than DHEA in p r e v e n t i n g and obesity (e.g., etiocholanolone) and inhibiting m i t o c h o n d r i a l r e s p i r a t i o n (e.g., epiandrosterone) (21,22). The present study shows that, except ADIOL, none of the compounds tested had any a p p r e c i a b l e effect on p e r o x i s o m e v o l u m e density, PPA-80 or PBE m R N A in the liver after dietary a d m i n i s t r a t i o n for 2 weeks at 0.45% concentration. ADIOL, a m e t a b o l i t e of DHEA produced in a reaction catalyzed by 17~-hydroxysteroid oxidoreductase also causes qualitatively similar pleiotropic effects in the liver as the parent compound. However, the p e r o x i s o m e p r o l i f e r a t i v e effect of A D I O L was 50% less than DHEA. S i m i l a r type of effects were also observed by Prough and Wu (23). A single large dose of DHEA m e t a b o l i t e s including A D I O L had no effect on PBE m R N A levels at 8 hrs. These results indicate that either DHEA itself, DHEA plus a c o f a c t o r or some DHEA m e t a b o l i t e other than those used in this study may be r e s p o n s i b l e for inducing p e r o x i s o m e proliferation. S t e r o i d h o r m o n e s exert their p h y s i o l o g i c a l effects through a r e c e p t o r m e d i a t e d mechanism. It is not clear w h e t h e r the p e r o x i s o m e p r o l i f e r a t i v e effect of DHEA is m e d i a t e d through a r e c e p t o r mechanism. Studies by Mohan and Cleary (24) indicate that rat h e p a t o c y t e s do not possess a DHEA b i n d i n g protein. However, studies by Kalimi and R e g e l s o n (25) and Meikle et al. (26) have identified specific DHEA binding p r o t e i n in rat liver and m u r i n e T cells which may be DHEA receptors. R e s o l v i n g this question of r e c e p t o r m e d i a t e d m e c h a n i s m of action of DHEA is important not only to u n d e r s t a n d the biological basis for the general effects of DHEA but also to gain an u n d e r s t a n d i n g of the m o l e c u l a r m e c h a n i s m s r e s p o n s i b l e for p e r o x i s o m e proliferation. Peroxisome proliferators, such as clofibric acid, exert peroxisome proliferative effect through a receptor mediated m e c h a n i s m (15,16,27) involving PPAR, the receptor a c t i v a t e d by p e r o x i s o m e proliferators, which belongs to steroid r e c e p t o r gene s u p e r f a m i l y (15). All p e r o x i s o m e p r o l i f e r a t o r s except DHEA so far tested have been shown to activate PPAR (15,16). Recent evidence suggests a coupling of the p e r o x i s o m e p r o l i f e r a t o r and 9 - c i s - r e t i n o i c acid signalling pathways (28). The failure of DHEA to p r o d u c e p e r o x i s o m e p r o l i f e r a t i o n under in WtFO conditions might be a t t r i b u t e d to short periods of incubation. However, the results of single dose DHEA e x p e r i m e n t s w h i c h d e m o n s t r a t e
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marked increase in PBE m R N A levels w i t h i n 1 h o u r show that failure to o b s e r v e DHEA effects in isolated h e p a t o c y t e s cannot be a t t r i b u t e d to short p e r i o d s of incubation. With clofibric acid and its analogs, the rapid increase in the levels of ~o x i d a t i o n e n z y m e mRNAs is due to increase in the t r a n s c r i p t i o n of these genes (29). M a r k e d increase in PBE m R N A levels w i t h i n one hour after DHEA a d m i n i s t r a t i o n suggest a s i m i l a r mechanism. Acknowledqements This research was R e v i e w Program, M a y b e r r y
supported Foundation
by V e t e r a n s Affairs and NIH Grant GM23750.
Merit
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6. 7. 8.
9. I0. ii. 12. 13. 14. 15. 16. 17.
18. 19.
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D.L. C O L E M A N , E n d o c r i n o l , 1172279-2283 (1985). P.F. M O H A N a n d M.P. CLEARY, Int. J. Biochem, 2_! 1 1 0 3 - 1 1 0 7 (1989). R..A. PROUGH,H-Q. W U a n d L. M I L E W I C H . The Biologic Role of Dehydmep~ndros~mne (DHEA). M. Kalimi and W. Ragelson (Eds.). 253-279, W a l t e r de G r u y t e r & Co., Berlin, N e w Y o r k (1990). P.F. M O H A N a n d M.P. CLEARY, S t e r o i d s 5_/7 2 4 4 - 2 4 7 (1992). M. K A L I M I a n d W. R E G E L S O N , Biochem. B i o p h y s Res. Commu. 156 22-29 (1988). A.W. M E I K L E , R.W. DORCHUCK, B.A. ARANEO, J.D. STRINGHAM, T.G. EVANS, S.L. SPRUANCE and R.A. DAYNES, J. Steroid B i o c h e m . Molec. Biol. 4 2 2 9 3 - 3 0 4 (1992). N.D. LALWANI, K. A L V A R E S , M.K. REDDY, M.N. REDDY, I. P A R I K H a n d J.K. REDDY, Proc. Natl. Acad. Sci. U S A 8_44 5 2 4 2 - 5 2 4 6 (1987). S.A. K L I E W E R , K. U M E S O N O , D.J. NOONAN, R.A. H E Y M A N a n d R.M. EVANS, N a t u r e 358 7 7 1 - 7 7 4 (1992). J.K. REDDY, S.K. GOEL, M.R. NEMALI, J.J. CAPRINO, T.G. L A F F L E R , M.K. REDDY, S.J. S P E R B E C K , T. OSUMI, T. H A S H I M O T O , N.D. L A L W A N I a n d M.S. RAO, Proc. Natl. Acad. sci. U S A 8_/3 1 7 4 7 - 1 7 5 1 (1986).
Pergamon Press
C O M P A R A T I V E EFFECTS OF D E H Y D R O E P I A N D R O S T E R O N E AND R E L A T E D STEROIDS ON PEROXISOME P R O L I F E R A T I O N IN RAT LIVER M. S. Rao, H. Ide, K. Alvares, V. Subbarao, J.K. Reddy, O. Hechter* and A.V. Yeldandi D e p a r t m e n t of P a t h o l o g y and *Department of Physiology, N o r t h w e s t e r n U n i v e r s i t y Medical School, Chicago, IL 60611 (Received in final form March 15, 1993) Summary D e h y d r o e p i a n d r o s t e r o n e (DHEA) is known to induce p e r o x i s o m e p r o l i f e r a t i o n and peroxisomal e n o y l - C o A h y d r a t a s e / 3 - h y d r o x y a c y l - C o A d e h y d r o g e n a s e (PBE) m R N A in the rat liver. We have compared the effects of 6 i n t e r m e d i a t e m e t a b o l i t e s of DHEA on the induction of p e r o x i s o m e p r o l i f e r a t i o n and PBE mRNA. Administration of epiandrosterone, etiocholanolone, androstenedione, e s t r o n e or estradiol for 2 weeks in the diet at 0.45% c o n c e n t r a t i o n to adult male F-344 rats failed to induce s i g n i f i c a n t increases in p e r o x i s o m e p r o l i f e r a t i o n and PBE m R N A when c o m p a r e d to the parent c o m p o u n d DHEA. Dietary a d m i n i s t r a t i o n of 5-androstene-3~,17~-diol (ADIOL) for 2 weeks at 0.45% c o n c e n t r a t i o n caused an increase in PBE m R N A and p e r o x i s o m e p r o l i f e r a t i o n but to a lesser extent than DHEA. Following a single i n t r a g a s t r i c dose of DHEA an increase in PBE mRNA level was o b s e r v e d in the liver at 1 hr and c o n t i n u e d to 16 hrs., but not with its metabolites. These results strongly suggest that DHEA or p o s s i b l y a n o t h e r yet to be identified m e t a b o l i t e might be r e s p o n s i b l e for p e r o x i s o m e proliferation. Dehydroepiandrosterone (DHEA) is a steroid secreted by adrenal c o r t e x in certain m a m m a l s p a r t i c u l a r l y humans (i). The functions of DHEA in humans are not fully understood. However, in e x p e r i m e n t a l animals which make little or no DHEA l o n g - t e r m administration of this steroid inhibits many pathological p r o c e s s e s i n c l u d i n g the d e v e l o p m e n t of tumors, diabetes, obesity and a t h e r o s c l e r o s i s (2-4). Recently, DHEA was shown to induce peroxisome proliferation, and to increase the activity of certain peroxisome-associated enzymes in the liver (5-8). Although it exerts a n t i c a r c i n o g e n i c effects in a v a r i e t y of tissues, l o n g - t e r m a d m i n i s t r a t i o n of this agent in the diet at a c o n c e n t r a t i o n of 0.45% for 84 weeks resulted in the d e v e l o p m e n t of h e p a t o c e l l u l a r c a r c i n o m a s in the rat (3,9-11). Accordingly, peroxisome proliferative property appears to make DHEA a hepatocarcinogen in rats, a function exhibited by other M.S. Rao, Dept. of Pathology, N o r t h w e s t e r n Univ. 303 E. C h i c a g o Ave., Chicago, IL 60611
Medical
0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
School,
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p e r o x i s o m e p r o l i f e r a t o r s (12,13). The question arises w h e t h e r DHEA itself is the active agent for the induction of p e r o x i s o m e proliferation, or serves as the source of an active m e t a b o l i t e formed ~ vivo which induces p e r o x i s o m e proliferation. The failure of DHEA to induce peroxisome proliferation in isolated h e p a t o c y t e s (14), and its inability to activate PPAR, a peroxisome proliferator activated receptor under ~ vitro conditions (15,16), suggests that DHEA in itself is not r e s p o n s i b l e for the p e r o x i s o m e p r o l i f e r a t i v e effects observed vivo. In this study, we examined the p e r o x i s o m e p r o l i f e r a t i v e effect of 6 recognized metabolites of DHEA. Except 5androstene-3~,17~-diol (ADIOL), none of the m e t a b o l i t e s tested showed a significant increase in peroxisome proliferation. A D I O L induced 50% less p e r o x i s o m e p r o l i f e r a t i o n than DHEA at the same dose level. These results suggest that DHEA in itself, DHEA with a c o f a c t o r or p o s s i b l y a DHEA m e t a b o l i t e other than those studied is r e s p o n s i b l e for the hepatic p e r o x i s o m e p r o l i f e r a t i o n o b s e r v e d ~ vivo. M a t e r i a l s and M e t h o d s Male F-344 rats, weighing 90-100g, were p u r c h a s e d from Charles River L a b o r a t o r i e s (Wilmington, Mass., USA). After a brief p e r i o d of a c c l i m a t i z a t i o n d e h y d r o e p i a n d r o s t e r o n e acetate, epiandrosterone, 4-androstenedione, estrone, 17~-estradiol, 3~e t i o c h o l a n o l o n e or 5-androstene-3~,17~-diol acetate (Sigma Chem. Co., St. Louis, MO) was fed to 3 to 5 rats at a c o n c e n t r a t i o n of 0.45% for a 2 week c o m p a r a t i v e study. Control animals were m a i n t a i n e d on normal rat chow. All rats were killed under ether anesthesia. For morphological evaluation and quantitative m o r p h o m e t r i c analysis of p e r o x i s o m e s liver was p r o c e s s e d for light and e l e c t r o n m i c r o s c o p y as p r e v i o u s l y d e s c r i b e d (17). To v i s u a l i z e the change in p e r o x i s o m e p r o l i f e r a t i o n a s s o c i a t e d Mr 80,000 p o l y p e p t i d e (PPA-80), 30 ~g of s o l u b i l i z e d p o s t n u c l e a r fractions were analyzed by SDS-PAGE (17,18). For immunoblotting, c y t o s o l i c p r o t e i n (20 ~g protein), from 1400xg s u p e r n a t a n t of 10% (w/v) h o m o g e n a t e s of liver in i0 mM p h o s p h a t e buffer (pH 7), was resolved by SDS-PAGE, transferred to nitrocellulose and immunostained with anti-PBE (19). For a s s e s s i n g the level of PBE mRNA total liver RNA was p r e p a r e d and a n a l y z e d by N o r t h e r n blot h y b r i d i z a t i o n using 3 2 p - l a b e l e d PBE cDNA (7,19). The levels of mRNA were m e a s u r e d by d e n s i t o m e t r i c scanning of autoradiographs. Further, to evaluate the rapidity with w h i c h the m R N A coding for peroxisomal PBE is induced rats were given a single dose of DHEA by gavage (450 m g / 1 0 0 g body wt in 2 ml corn oil) and s a c r i f i c e d in groups of 2 at i, 8 and 16 hours. Three control rats were given corn oil only and killed at 16 hours. A d d i t i o n a l groups of 2 rats were given a single intragastric dose (450 m g / 1 0 0 g body wt) of epiandrosterone, androstenedione, estrone, 17~-estradiol or A D I O L and killed after 8 hours. Results and D i s c u s s i o n Body and liver weights and the volume density of p e r o x i s o m e s in h e p a t o c y t e s of controls, and rats fed DHEA and its i n t e r m e d i a t e m e t a b o l i t e s in diet for 2 weeks are p r e s e n t e d in Table I. Liver w e i g h t for 100g body w e i g h t in DHEA and A D I O L treated groups increased s i g n i f i c a n t l y (P<0.001) w h e n c o m p a r e d
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to o t h e r g r o u p s . Ultrastructural e x a m i n a t i o n of l i v e r c e l l s of r a t s t r e a t e d w i t h D H E A a n d A D I O L for 2 w e e k s r e v e a l e d a l a r g e n u m b e r of p e r o x i s o m e s w h e r e a s no s u c h i n c r e a s e w a s n o t e d in r a t s treated with other steroids tested. Morphometric analysis showed a 6 and 2.6 fold increase in t h e v o l u m e density of peroxisomes in D H E A a n d A D I O L t r e a t e d rats, r e s p e c t i v e l y (Table I). Figures 1 and 2 show SDS-PAGE profiles of p o s t n u c l e a r f r a c t i o n s p r e p a r e d f r o m l i v e r s of r a t s a f t e r 2 w e e k s of e x p o s u r e to D H E A a n d o t h e r s t e r o i d s . In D H E A a n d A D I O L t r e a t e d rats, a marked increase in the amount of peroxisome proliferationassociated PPA-80 was observed.
Fiq.
2
I
1 2 3 4 5 6 7 8 9
Fiq.
IA
3
4
IB
SDS-polyacrylamide s l a b gel e l e c t r o p h o r e s i s of p o s t n u c l e a r f r a c t i o n s of l i v e r f r o m c o n t r o l (1-3), D H E A (4-6) a n d e t i o c h o l a n o l o n e (7-9) (Fig. IA) a n d A D I O L (Fig. IB) t r e a t e d rats. The arrows indicate the p o s i t i o n of p e r o x i s o m e p r o l i f e r a t o r - a s s o c i a t e d 80,000 mol wt polypeptide. 1
2
3
4
5
Fiq.
6
7
8
9
2
S D S - P A G E of p o s t - n u c l e a r f r a c t i o n of l i v e r f r o m e s t r a d i o l (1-3), e s t r o n e (4-6) a n d a n d r o s t e n e d i o n e (7-9) t r e a t e d rats. N o i n c r e a s e in t h e a m o u n t of 8 0 , 0 0 0 m o l w t p o l y p e p t i d e is n o t e d (arrows). In c o n t r o l r a t s a n d r a t s t r e a t e d w i t h D H E A m e t a b o l i t e s no increase in the amount of this protein was evident. Immunoblotting using polyclonal antibodies to PBE further confirmed the increase in PBE in D H E A a n d A D I O L t r e a t e d rats. N o r t h e r n b l o t a n a l y s i s of t o t a l l i v e r R N A f r o m r a t s t r e a t e d w i t h DHEA and ADIOL for 2 weeks showed a marked increase in t h e l e v e l s of PBE m R N A (Fig. 3). N o a p p r e c i a b l e c h a n g e in PBE m R N A
178-estradiol
Estrone
3 ~Etiocholanolone
Control
5
6
7
8
3
3
using
"t" t e s t
6.9 ~ 0.4
7.5 ~ 0.2
6.4 ~ 0.3
6.7 ± 0.i
6.7 ! 0.2
6.5 ± 0.i
i0.0 ~ 0.4
11.5 ± 0.5
5.1 ~ 0.3
5.2 ~ 0.i
6.3 ~ 0.i
6.0 ± 0.I
4.9 ~ 0.2
5.4 ± 0.i
7.3 ~ 0.i
8.4 ± 0.2
NS
P<0.01
P<0.OI
NS
NS
P<0.001
P<0.001
and Peroxisome
Liver Weight/ 100g b.wt
Liver Weight
Liver Weight
student's
136 ~ 3
143 ~ 3
i01 ~
113 ± 2
137 ~ 3
2
136 ~ 3 121 ~
I
on B o d y Weight,
Weight
136 ~
Body
of D H E A
obtained
4-androstenedione
4
are
Epiandrosterone
3
P values
5-androstene-3B,178-diol
its
2
and
Dehydroepiandrosterone
Treatment
of M e t a b o l i t e s
1
Group
Effects
TABLE
Density
1.0
0.i
1.8 ! 0.2
1.3 ~ 0.4
i.i ~
1.6 ± 0.3
i.i ~ 0.i
1.7 ! 0.2
4.6 ~ 0.8
ii.0 !
Peroxisome volume density
Volume
O
tu
Z O
O
m
o
a;
o M
m o~
m
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Steroids and Peroxisomes
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in other g r o u p s was evident (Fig. 3). Epiandrosterone not induce PBE m R N A (not shown in the picture).
1
2
34
5
6 7 8
also did
9
1
2
3
4
5
6
t g g
Fiq.
3A
Fiq.
3B
N o r t h e r n blot analysis for PBE m R N A in liver of rats t r e a t e d with DHEA (1-3), e t i o c h o l a n o l o n e (4,5), andros t e n e d i o n e (6,7), or estradiol (8-9) (Fig. 3A), and A D I O L (1-4) and control (5,6) (Fig. 3B) for 2 weeks in diet at 0.45% c o n c e n t r a t i o n : Total ~ A (20~g/lane was a n a l y z e d by N o r t h e r n b l o t t i n g with P-labeled PBE cDNA. The size of PBE mRNA is 3.0 kilobases.
Changes in the levels of PBE mRNA in the liver of rats at d i f f e r e n t time points after a large single i n t r a g a s t r i c dose of DHEA are shown in Figure 4. Levels of this mRNA increased A
A
A
B
B
C
C
D
I
............
Fiq.
D
e
4
N o r t h e r n blot analysis for PBE mRNA in livers of control (A) and rats killed at 1 (B), 8 (C), and 16 (D) hours after a single i n t r a g a s t r i c dose of DHEA (450 m g / 1 0 0 g body wt). Blot a n a l y s i s was carried out as d e s c r i b e d in Fig. 3. The r e l a t i v e amounts of 28s and 18s RNA used for N o r t h e r n blot a n a l y s i s is similar. at 1 hour and c o n t i n u e d to rise up to 16 hours. Quantitative d e n s i t o m e t r i c analysis showed 4.8, 6.8 and 9.2 fold increase at i, 8 and 16 hours respectively. There was no increase in PBE m R N A in rats t r e a t e d with DHEA metabolites. Peroxisome proliferative effects of DHEA are well c h a r a c t e r i z e d in rats and mice (5-7). The results of this study further c o r r o b o r a t e and extend these o b s e r v a t i o n s c o n c e r n i n g the effect of DHEA after a d m i n i s t r a t i o n of a single dose or feeding c o n t i n u o u s l y for 2 weeks. A d m i n i s t r a t i o n of DHEA for 2 weeks
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caused s i g n i f i c a n t hepatomegaly, p e r o x i s o m e p r o l i f e r a t i o n and increased levels of PBE p r o t e i n and PBE mRNA. A f t e r a large single dose of DHEA (450 m g / 1 0 0 g body wt) a rapid increase in the rate of PBE m R N A a c c u m u l a t i o n was noted. This increase was o b s e r v e d 1 hour after DHEA dosing and c o n t i n u e d up to 16 hours. However, at a lower dose level (50 m g / 1 0 0 g body wt), increase in PBE m R N A was evident only at 24 hours (7). The m e c h a n i s m by w h i c h DHEA causes p e r o x i s o m e p r o l i f e r a t i o n is not clear. Since DHEA appears to be ineffective in a c t i v a t i n g PPAR or inducing p e r o x i s o m e p r o l i f e r a t i o n under ~ vitro conditions, the p o s s i b i l i t y arises that one of the i n t e r m e d i a t e metabolites of DHEA may play an important role in inducing p e r o x i s o m e proliferation. DHEA is known to be c o n v e r t e d to androgens and estrogens, as well as to other intermediate metabolites. We selected epiandrosterone, androstenedione, ~estradiol, estrone, etiocholanolone and A D I O L in this study because they are e s t a b l i s h e d m e t a b o l i t e s of DHEA. Several of these metabolites appear to be more potent than DHEA in p r e v e n t i n g and obesity (e.g., etiocholanolone) and inhibiting m i t o c h o n d r i a l r e s p i r a t i o n (e.g., epiandrosterone) (21,22). The present study shows that, except ADIOL, none of the compounds tested had any a p p r e c i a b l e effect on p e r o x i s o m e v o l u m e density, PPA-80 or PBE m R N A in the liver after dietary a d m i n i s t r a t i o n for 2 weeks at 0.45% concentration. ADIOL, a m e t a b o l i t e of DHEA produced in a reaction catalyzed by 17~-hydroxysteroid oxidoreductase also causes qualitatively similar pleiotropic effects in the liver as the parent compound. However, the p e r o x i s o m e p r o l i f e r a t i v e effect of A D I O L was 50% less than DHEA. S i m i l a r type of effects were also observed by Prough and Wu (23). A single large dose of DHEA m e t a b o l i t e s including A D I O L had no effect on PBE m R N A levels at 8 hrs. These results indicate that either DHEA itself, DHEA plus a c o f a c t o r or some DHEA m e t a b o l i t e other than those used in this study may be r e s p o n s i b l e for inducing p e r o x i s o m e proliferation. S t e r o i d h o r m o n e s exert their p h y s i o l o g i c a l effects through a r e c e p t o r m e d i a t e d mechanism. It is not clear w h e t h e r the p e r o x i s o m e p r o l i f e r a t i v e effect of DHEA is m e d i a t e d through a r e c e p t o r mechanism. Studies by Mohan and Cleary (24) indicate that rat h e p a t o c y t e s do not possess a DHEA b i n d i n g protein. However, studies by Kalimi and R e g e l s o n (25) and Meikle et al. (26) have identified specific DHEA binding p r o t e i n in rat liver and m u r i n e T cells which may be DHEA receptors. R e s o l v i n g this question of r e c e p t o r m e d i a t e d m e c h a n i s m of action of DHEA is important not only to u n d e r s t a n d the biological basis for the general effects of DHEA but also to gain an u n d e r s t a n d i n g of the m o l e c u l a r m e c h a n i s m s r e s p o n s i b l e for p e r o x i s o m e proliferation. Peroxisome proliferators, such as clofibric acid, exert peroxisome proliferative effect through a receptor mediated m e c h a n i s m (15,16,27) involving PPAR, the receptor a c t i v a t e d by p e r o x i s o m e proliferators, which belongs to steroid r e c e p t o r gene s u p e r f a m i l y (15). All p e r o x i s o m e p r o l i f e r a t o r s except DHEA so far tested have been shown to activate PPAR (15,16). Recent evidence suggests a coupling of the p e r o x i s o m e p r o l i f e r a t o r and 9 - c i s - r e t i n o i c acid signalling pathways (28). The failure of DHEA to p r o d u c e p e r o x i s o m e p r o l i f e r a t i o n under in WtFO conditions might be a t t r i b u t e d to short periods of incubation. However, the results of single dose DHEA e x p e r i m e n t s w h i c h d e m o n s t r a t e
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Steroids and Peroxisomes
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marked increase in PBE m R N A levels w i t h i n 1 h o u r show that failure to o b s e r v e DHEA effects in isolated h e p a t o c y t e s cannot be a t t r i b u t e d to short p e r i o d s of incubation. With clofibric acid and its analogs, the rapid increase in the levels of ~o x i d a t i o n e n z y m e mRNAs is due to increase in the t r a n s c r i p t i o n of these genes (29). M a r k e d increase in PBE m R N A levels w i t h i n one hour after DHEA a d m i n i s t r a t i o n suggest a s i m i l a r mechanism. Acknowledqements This research was R e v i e w Program, M a y b e r r y
supported Foundation
by V e t e r a n s Affairs and NIH Grant GM23750.
Merit
References i. 2. 3. 4. 5.
6. 7. 8.
9. I0. ii. 12. 13. 14. 15. 16. 17.
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