Attenuation of 3,4-Methylenedioxymethamphetamine (MDMA) Induced Neurotoxicity With the Serotonin Precursors Tryptophan and 5-Hydroxytryptophan

8/3/2019 Attenuation of 3,4-Methylenedioxymethamphetamine (MDMA) Induced Neurotoxicity With the Serotonin Precursors

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Pergamon0024-3205(94) 00252.5

Life Sc iences , V ol. 55, No . 15, pp. 1193-1198, 1994Copy r igh t 1994 E ls e v ier Sc ie nc e L td

P r i n t e d i n t h e U S A . A l l r i g h t s r e s e r v e d0024-3205194 $6.00 + .00

A T T E N U A T I O N O F 3 , 4 - M E T H Y L E N E D I O X Y M E T H A M P H E T A M I N E ( M D M A )I N D U C E D N E U R O T O X I C I T Y W I T H T H E S E R O T O N I N P R E C U R S O R S T R Y P T O P H A NAND 5 - H Y D R O X Y T R Y P T O P H A N

J o n E . S p r a g u e 1, X u e m e i H u a n g I , A r th i K a n t h a s a m y 2 , an d D av id E . N ich o l s l , 21 D e p a r t m e n t o f P h a r m a c o l o g y a n d T o x i c o l o g y & 2 D e p a r tm e n t o f M e d i c in a l C h e m i s t r y a n d

P h a r m a c o g n o s y , S c h o o l o f P h a r m a c y a n d P h a r m a c a l S c i e n c e s , P u rd u e U n i v e r s i ty ,W es t La fay e t t e , IN. 4 7 9 0 7

(Rec eived in final form July 29, 1994)Summary

T r e a t m e n t o f r a t s w i th s e r o to n i n ( 5 - H T ) p r e c u r s o r s t r y p t o p h a n ( T R P , 4 0 0m g / k g ) a n d 5 - h y d r o x y t r y p t o p h a n ( 5 - H T P , 5 0 m g / k g ) w a s s h o w n t o a t t e n u a t eM D M A ( 2 0 m g / k g ) i n d u c e d s e r o t o n e r g i c n e u r o t o x i c i t y a s m e a s u r e d b y [ 3 H ] -p a ro x e t i n e b in d in g i n t h e s t r i a tu m, h ip p o cam p u s , an d f ro n t a l co r t ex o f t h e ra t b ra in .H i p p o c a m p a l 5 - H T a n d 5 - H I A A l e v e ls w e r e a l s o i n d ic a t iv e o f t h e p r o t e c ti v e e f f e c tso f T R P a n d 5 - H T P . T h e s e r e s u lt s s ug g e s t t h a t d e p l et io n o f 5 - H T s t or e s isi m p o r t a n t f o r M D M A - i n d u c e d n e u r o to x i c it y . T h e p o s s i b le s i g n if i c a n ce o f th i s 5 -H T d e p l e t i o n i n M D M A - i n d u c e d s e r o n t o n e r g i c t e r m i n a l d e g e n e r a t i o n i s a l s od i scu ssed .

Key Words: 5-hydroxytryptamine,neurotoxicity, MD MA , tryptophan

3 , 4 - m e t h y l e n e d i o x y m e t h a m p h e t a m i n e ( M D M A o r " E c s t a sy " ) w a s f ir s t s y n th e s i z e d in t h eea r ly 1 9 0 0 's a s an an o rex i c ag en t . Ho we v er , M D M A h ad n o t rece iv ed mu ch a t t en t i o n un t i l t h ep as t d ecad e . In 1 9 85 , th e Dru g En fo rcem en t Ad m in i s t ra t io n i n v o k ed th e i r em erg en cy p o w ers top l a c e M D M A i n to S c h e d u l e 1, b e c a u s e o f it 's w i d e - s p re a d u s e a m o n g y o u n g p r o f e s s i o n a ls a n dco l l eg e s t u d en t s (1 ) . Th i s p o p u l a r i t y is p ro b ab ly d u e t o b o th i ts eu p h o r i c ac t i o n an d i t s u n iq u ee f fec t s o n emo t io n , wi th o u t t h e sen so ry d i s t o r t i o n seen wi th s t ro n g p sy ch ed e l i c s su ch a s l y se rg i ca c i d d i e t h y l a m i d e ( L S D ) . M D M A h a s b e e n r e p o r te d to in c r e a se i n te r p e r s o n al c o m m u n i c a t i o n a n de m p a t h y , l e ad i n g s e v e r a l t h er a p is t s t o p r o p o s e th e u s e o f M D M A a s a n a d j u n ct t o p s y c h o t h e r a p y( 2 ). H o w e v e r , n o d o u b le - b l in d e x p e r i m e n t s h a v e b e e n u n d e rt a k e n t o e x a m i n e M D M A ' sth e rap eu t i c e f f i cacy i n t h i s se t ti n g .

M D M A h a s a l s o b e e n s h o w n t o d e p le t e s e le c t iv e l y t h e c o n c en t r a ti o n s o f s e r o t o n in ( 5 - H T )an d i t s ma jo r me tab o l i t e , 5 -h y d ro x y in d o l eace t i c ac id (5 -HIAA) , an d t o i n d u ce se ro to n e rg i c n eu ro nt e rmin a l d eg en e ra t i o n i n th e b ra in o f ro d en ts an d n o n -h u ma n p r ima te s fo l l o win g ad min i s t ra t i o n o fa s in g l e h ig h d o se (3 ,4 ) . Th i s l a t t e r e f fec t h as fu r t h e r h in d e red c l in i ca l ev a lu a t i o n o f M D M A .S t u d ie s o f th e m e c h a n i s m s b y w h i c h M D M A d e p l et e s th e s e m a r k e r s s u g g e s t a r o le f o r d o p a m i n e( D A ) i n th e n e u r o t o x ic i t y o f M D M A ( 5 ,6 ) . In vitro (6) and in vivo (7 ) s t u d i e s h av e sh o wn th a tM D M A in d u ces a d ram a t i c i n c rease i n t h e ex t race l l u la r co n cen t ra t i o n o f D A in t h e s tr i a t u m. Na shet al. ( 5 ) a l s o d e m o n s t r a t e d t h a t M D M A s t i m u l a t e s D A b i o s y n t h e s i s in vivo, a s m e a s u r e d b yd i h y d r o x y p h e n y l a l a n in e ( D O P A ) a c c u m u l a t io n i n th e s tr i at u m . T h i s e f f e c t w a s b l o c k e d b y t h e 5 -


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1194 5-H T and MD M A-induced Neurotoxicity Voi. 55, No. 15, 1994

H T 2A / 2C a n t a g o n i s t , k e t a n s e r i n , w h i c h h a s b e e n s h o w n t o a t t e n u a t e t h e M D M A - i n d u c e d i n c r e a s ein s t r ia ta l DA i n v i v o a n d a l s o t o a tt e n u a te t h e n e u r o t o x i c it y i n d u c e d b y M D M A ( 7) . T h e s e l e c t i v e5 - H T 2 A r e c e p t o r a n t a g o n is t , M D L 2 8 , 1 3 3 A h a s a l s o re c e n tl y b e e n s h o w n t o b l o c k M D M Ai n d u c e d D A s y n th e s is a n d re l ea s e a n d t o p r e v e n t M D M A - i n d u c e d n e u r o t o x ic i t y (8 ). T h u s ,p r o p o s e d c o m p o n e n t s o f t h e m e c h a n i s m o f M D M A - i n d u c e d n e u r o t o x i c i ty m a y b e a s f o l lo w s : 1 )M D M A i n d u c e s a r e le a s e o f 5 - H T ; 2 ) T h i s 5 - H T a c t iv a t e s t h e 5 -H T 2 A / 2C r e c e p t o r c o m p l e x ; 3 )T h i s a c t i v a t i o n , i n c o n j u n c t i o n w i t h M D M A - i n d u c e d D A r e l e a s e , l e a d s t o a p r o f o u n d i n c r e a s e i nD A s y n t h e s i s a n d r e l e a s e ; 4 ) T h i s i n c r e a s e d D A t h e n s o m e h o w p l a y s a m a j o r r o l e i n t h en e u r o t o x i c i t y p r o c e s s . T h e e x a c t m e c h a n i s m s o f a ll th e s t ep s a r e c u r r e n t l y u n d e r i n v e s t i g a ti o n .

I n a r e c e n t r e p o rt , H u a n g a n d N i c h o l s ( 9 ) p r o p o s e d t h a t 5 - H T d e p l e t i o n f r o m n e u r o nt e r m i n a l s m a y r e n d e r t h e m v u l n e r a b l e t o D A t o x i c i ty o r th e a c t i o n o f s o m e o t h e r t o x in . I n o r d e r t oi n v e s t i g a te t h is h y p o t h e s i s , w e p r e tr e a t ed a n i m a l s w i t h 5 - H T p r e c u r s o r s , n a m e l y , t r y p t o p h a n ( T R P )a n d 5 - h y d r o x y t r y p t o p h a n ( 5 - H T P ) .

M a t e r i a l s a n d M e t h o d s

T h i r t y - t w o a d u l t , m a l e S p r a g u e - D a w l e y r a t s ( 1 7 5 - 1 9 9 g ; H a r l a n , I n d i a n a p o l i s , I N ) w e r eu s e d i n th i s s t u d y . U p o n a r r i v a l a n im a l s w e r e t ra n s f e r re d t o i n d i v id u a l c a g e s w i t h a d l i b a c c e s s t of o o d a n d w a t e r .

T h e a n i m a l s w e r e th e n r a n d o m l y a s s i g n e d to fo u r t r e a t m e n t g r o u p s ( n = 8 ). T r e a t m e n t sw e r e g i v e n t w i c e d a i l y f o r f o u r d a y s a t 7 A M a n d 7 P M . T h i s t r e a tm e n t r e g i m e n w a s s e l e c te ds i n c e i t i s k n o w n t o p r o d u c e t h e m o s t e x t e n s i v e s e rt o n e r g i c d e f ic i ts . T h e e x p e r i m e n t a l g r o u p sw e r e a s f o l l o w s :

G r o u p I = s a l i n eG r o u p I I = M D M A ( 2 0 m g / k g )G r o u p I I I = M D M A + T R P ( 4 00 m g / kg )G r o u p I V = M D M A + R O 4 - 4 6 0 2 ( 5 0 m g / k g ) + 5 - H T P ( 5 0 m g / k g )

A l l d o s e s o f M D M A w e r e g i v e n s u b c u t a n e o u s ly ; a ll o t h e r d r u g s w e r e g i v e ni n t r ap e r i to n e a l l y . R O 4 - 4 6 0 2 w a s u s e d i n c o n j u n c t i o n w i t h 5 - H T P t o i n h i b i t t h e p e r i p h e ra ld e c a r b o x y l a s e . T h e d r u g p re t re a t m e n ts w e r e g i v e n 3 0 m i n u te s b e fo r e M D M A . O n e w e e k a f te r t h el a st t r e a tm e n t , t h e a n i m a l s w e r e s a c r i f i c e d b y d e c a p it a t io n ; t h e b ra i n r e g i o n s w e r e d i s s e c t e d o u ta n d s t o r e d a t - 7 0 C u n t i l a s sa y . A n e s t i m a t e o f s e r o t o n e r g i c n e u r o t o x i c i t y i n t h e s t r ia t u m ,h i p p o c a m p u s , a n d f ro n t a l co r t e x w a s m a d e u s i n g a [ 3 H ] p a r o x e ti n e b i n d in g m e t h o d t o l a b e l 5 - H Tu p t a k e s i te s ( 1 1 ,1 2 ) . 5 - H T a n d 5 - H I A A l e v e ls in th e h i p p o c a m p u s w e r e a l so e s ti m a t e d a t t h ist im e . T h e h i p p o c a m p u s w a s s e le c te d s i n c e it w a s o n e o f th e r eg i o n s s h o w n t o h a v e l o n g t e r mr e d u c t io n s i n 5 - H T a n d 5 - H I A A l e ve l s af te r M D M A t r ea t m e n t ( 3 ,4 )

M D M A h y d r o c h l o r i d e w a s s y n t h e s iz e d i n o u r l a b o ra t o ry (1 0 ). [ 3 H ] P a ro x e t in e w a sp u r c h a s e d f r o m N e w E n g l a n d N u c l e a r ( B o s t o n , M A ) a t a s p e c if ic a c t i vi ty o f 2 0 .5 C i / m m o l .F l u o x e t i n e w a s g e n e r o u s l y p r o v i d e d b y E l i L i l l y a n d C o . (I n d i a n a p o l is , I N ) . R O 4 - 4 6 0 2 w a sk i n d l y d o n a t e d b y H o f f m a n - L A R o c h e ( N u tl e y , N J ). A l l o t h e r r e ag e n ts w e r e p u r c h a s e d f r o mS i g m a C h e m i c a l C o . ( S t. L o u i s , M O ) .

A m o d i f i e d p r o c e d u r e o f M a r c u s s o n e t a l . ( 1 1 ) w a s e m p l o y e d t o m e a s u r e [ 3 H ] p a r o x e t i n eb i n d i n g s i te s . S i n c e i t h a s b e e n p r e v i o u s l y r e p o r t e d t h a t o n l y t h e B m a x a n d n o t t h e K D v a l u e s a r e


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Vol. 55, No. 15, 1994 5-HT and MDM A-induced Neurotoxicity 1195

altered after MDMA treatment (12), it is possible to estimate the number of 5-HT uptake sites witha single saturating (1 nM) concentration of [3H]paroxetine. Nonspecific binding was determinedwith 1 p.M fluoxetine.

Hippocampal 5-HT and 5-HIAA levels were also determined. Tissue samples weresonicated for 15 seconds in 0.1N HCIO4 containing 0.05% Na2EDTA, and 0.1% Na2S205. Thesamples were then centrifuged at 14,000 x g for 4 minutes with a tabletop centrifuge. Thesupematant was collected and 50 ~tl injected onto the HPLC column (Brownlee C18, Anspec; AnnArbor, MI.), A model 400 EG & G Princeton electrochemical detector (Princeton, NJ) with seriesdual electrodes was utilized. The mobile phase consisted of 50 mM NaH2PO4, 30 mM citric acid,0.1 mM Na2EDTA, 0.034 % sodium octyl sulfate and 25% methanol. Peaks were integrated withthe Dynamax Methods Manger software (Rainin; Woburn, MA.).

Statistical treatment of groups was performed with an ANOVA. Differences betweentreatment groups were determined with a Student Neuman-Keuls multiple range test withsignificance set at p


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1196 5-HT and MD M A-induced Neurotoxicity Vol. 55 , No . 15 , 1994

1 4 0

SALINEM D M AMDMA + T R PM D M A + 5 - H T P

1 2 0

100,-.1"" 80-Z

60

40

20

IaY

STRIATUM FRONTALCORTEX

H I P P O C A M P U S

F i g . 1[ 3 H ]p a r o xe t in e b i n d i n g o n e w e e k a ft er m u l t ip l e d o s e s o f M D M A a n d M D M A d r ug

c o m b i n a t i o n s. S A L = s a li n e t re a tm e n t g r ou p ; M D M A + T R P = M D M A + T R P p r et re at m en t;M D M A + 5 - H T P = M D M A + 5 - H T P p r et re a tm e n t . B a s a l [ 3H ] p a r o x e t in e b i n d i n g si te d e n s i t ie s i nt h e s tr i a tu m , h i p p o c a m p u s , a n d fr o n t a l c o r t e x w e r e 2 7 . 9 + 3 . 4 , 1 6 . 6 + 2 . 0 , a n d 1 8 . 4 + 1 . 0 f m o l / gw e t w e i g h t , r e s p e c t i v e l y , a = s i g n i f i c a n t l y d i ff e r e n t f r o m s a l i n e c o n t r o l , b = s i g n i f i c a n t l y d i f f e r e n tf r o m a l l o t h e r tr e a t m e n t g r o u p s .

D i s c u s s i o nT h e r e s u l ts o f th e p r e s e n t s t u d y s u g g e s t t h a t i n o r d e r f o r M D M A t o in d u c e s e r o t o n e r g i c

n e u r o t o x i c i t y , t h e n e u r o n te r m i n a l s m u s t b e d e p l e t e d o f s e r o t o n i n . S t u d i e s in m a n y l a b o r a to r i e sh a v e s h o w n t ha t M D M A a n d s tr uc tu ra l a n a l o g u e s ( e .g . N - m e t h y l - l - ( 1 , 3 - b e n z o d i o x o l - 5 - y l ) - 2 -b u t a n a m i n e (M B D B ) , a n d N - e t h y l -3 , 4 -m e t h y l e n e n e d i o x y a m p h e t a m i n e ( M D E ) ) w h i c h a c u t el yd e p l e t e 5 - H T , l e a d t o l o n g t e r m d e c r e a s e s i n c o n c e n t r a t i o n s o f 5 - H T a n d 5 - H I A A i n t h e b r a i n o fr o d e n t s an d n o n - h u m a n p r i m a t es ( 1 3 , 1 4 , 1 5 , 1 6 ,) . T h e s e d e c r e a s e s i n s e r o t on e r g i c p a r am e t e r s h a v ea l s o b e e n l i n k e d t o 5 - H T a x o n t e r m i n a l d e g e n e r a t i o n ( 1 7 ) . F u r t h er su p p o r t f o r a r o l e o f 5 - H Td e p l e t io n r e c e n tl y c a m e f r o m B r a d b er r y e t a l . ( 1 8 ), w h o s h o w e d t h a t n e u r o t o x i c it y i n d u c e d b yp a r a - c h l o r o a m p h e t a m i n e ( P C A ) c o u l d b e b l o c k e d b y T R P p r e tr e at m e n t.

A c u t e M D M A t r ea t m e n t h a s a l so b e e n s h o w n t o d e c r e as e t r y p to p h a n h y d r o x y l a s e a c t iv i ty( 1 9 ) . T h i s o r i g in a l r e p o r t s u g g e s t e d t h a t t h e d e c r e a s e i n tr y p t o p h a n h y d r o x y l a s e a c t i v i t y m i g h t


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play an important role in MDMA-induced neurotoxicity. However, the results of the present studyshowing that 5-HTP was no more effective that TRP suggest that the decrease in tryptophanhydroxylase activity may not be important.

Berger e t a l . (20) have shown that if monoamine stores are depleted with reserpine prior toPCA treatment, serotonergic neurotoxicity can be blocked. These authors speculated that 5-HTitself or a toxic metabolite of 5-HT (i.e. 5,6- or 5,7-dihydroxytryptamine) might therefore beresponsible for the neurotoxicity. The present results suggest that such toxins are probably notresponsible for serotonergic neurotoxicity. If in fact this were the case, pretreatment with TRP or5-HTP would be expected to enhance the concentrations of these substances and potentiate theneurotoxicity.

In summary, the results of this study suggest that depletion of 5-HT stores increases thevulnerability of serotonergic neurons to the toxic effects of MDMA. This is a necessary but notsufficient condition to produce neurotoxicity (21). This depletion may then allow the carrier-mediated uptake of the ultimate toxicant (DA?) into the 5-HT terminal. Such a scenario wouldexplain why MDMA or PCA-induced neurotoxicity can still be inhibited by the 5-HT uptakeblocker fluoxetine up to six hours after drug administration (22,23).

AcknowledgementsThis work was supported by USPHS Grant DA04758 from the National Institute on Drug

Abuse. The authors thank Stewart Frescas for the synthesis of MDMA.References

1. D.M. BARNES, Science. 240 24 (1988).2. G. GREER, Advances. 2 57-59 (1985).3. C.J. SCHMIDT, J.A. LEVIN and W. LOVENBERG, Biochem. Pharmacol. 36 747-755(1987).4. D.L. COMMINS, G. VOSMER, R.M. VIRUS, W.L. WOOLVERTON, C.R. SCHUSTERand L.S. SEIDEN, J. Pharmacol. Exp. Ther. 241 338-345 (1987).5. J. NASH, H. MELTZER and G. GUDELSKY, J. Neurochem. 54 1062-1067 (1990).6 M.P. JOHNSON, A.J. HOFFMAN and D.E. NICHOLS, Eur. J. Pharmacol. 132 269-276(1986).7. J.F. NASH, Life Sci. 47 2401-2408 (1990).8. C.J. SCHMIDT, C.K. BLACK, V.L. TAYLOR, G.M. FADAYEL, T.M. HUMPHREYS,

T.R. NEIDUZAK and S.M. SORENSEN, Eur. J. Pharmacol. 220 151-159 (1992).9. X. HUANG and D.E. NICHOLS, Eur. J. Pharmacol. 238 291-296 (1993).10. D.E. NICHOLS, A.J. HOFFMAN, R. OBERLENDER, D. JACOB III and A.T. SHULGIN,J. Med. Chem. 29 2009-2015(1986).11. J.O. MARCUSSON, M. BERGSTROM, ERIKSSON and S.B. ROSS, J. Neurochem. 501783-1790 (1988).12. G. BATTAGLIA, S.Y. O'HEARN, E. MOLLIVER, M.E. KUHAR and E.B. DESOUZA, J.Pharmacol. Exp. Ther. 242 911-916 (1987).13. D.M. STONE, D.C. STAHL, G.R. HANSON and J.W. GIBB, Eur. J. Pharmacol. 128 41-48(1986).14. G.A. RICAURTE, L.S. FORNO, L.E. WILSON, L.E. DE LANNEY, I. IRWIN, M.E.MOLLIVER and J.W. LANGSTON, J. Am. Med. Assoc. 260 51-55(1988).15. M.P. JOHNSON, and D.E. NICHOLS, Pharmacol. Biochem. Behav. 33 105-108(1989).16. J.W. GIBB, D. STONE, M. JOHNSON and G.R. HANSON, The ClincalPharmacological and Neurotoxicological Effec ts of the Drug MDMA, Peroutka (ed) 133Kluwer Publishers, Norwell, MA. (1990).


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17. E. O'HEARN, G. BATTAGLIA, E.B. DESOUZA, M.J. KUHAR and M.E. MOLLIVER, J.Neurosci. 8 2788-2803(1988).18. C.W. BRADBERRY, R.N. IYER, J.S. SPROUSE, G.K. AGHAJANIAN and R.H. ROTH,23th Neurosci. Meeting Abst. 19(2) 1483 (1993).19. C.J. SCHMIDT and V.L. TAYLOR, Biochem. Pharmacol. 36 4095-4102 (1987).20. U.B. BERGER, R. GRZANNA and M.E. MOLLIVER, Brain Res. 578 177-185 (1992).21. M.P. JOHNSON, X. HUANG, and D.E. NICHOLS, Pharmacol. Biochem. Behav. 40, 915-922 (1991).22. R.W. FULLER, H.D. SNODDY, K.W. PERRY, F.P. BYMASTER, and D.T. WONG,Biochem. Pharmacol. 27 193-198 (1978).23. C.J. SCHMIDT, J. Pharmacol . Exp. Ther. 240 1-7 (1987).

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Attenuation of 3,4-Methylenedioxymethamphetamine (MDMA) Induced Neurotoxicity With the Serotonin Precursors Tryptophan and 5-Hydroxytryptophan