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1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig
Life Sciences, Vol. 36, pp. 1233-1238 Printed in the U.S.A.
Pergamon Press
1-METHYL-4-PHENYL-I,2,3,6-TETRAHYDROPYRIDINE (MPTP) DOES NOT DESTROY NIGROSTRIATAL NEURONS IN THE SCORBUTIC GUINEA PIG Thomas L. Perry 1, Voon Wee Yong, Masatoshi Ito, Karen Jenes, Richard A. Wall, James G. Foulks, James M. Wright, and Stephen J. Kish* Department of Pharmacology Therapeutics, University of B r i t i s h Columbia, Vancouver, Canada, V6T IW5; and Human Brain Laboratory*, Clarke I n s t i t u t e of Psychiatry, Toronto, Canada, M5T 1R8 (Received in final form January 22, 1984)
Summary Guinea pigs were injected subcutaneously with 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTPtreated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The r e l a t i v e lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.
The parenteral i n j e c t i o n or inhalation of 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP) has resulted in the development of severe and l a s t ing symptoms of Parkinson's disease in human subjects (1-3). Monkeys of 3 d i f f e r e n t species injected repeatedly with low doses of MPTP develop neurological signs that closely resemble human Parkinson's disease, and t h e i r brains l a t e r show a s t r i a t a l depletion of dopamine and a loss of neurons in the zona compacta of the substantia nigra (4-7). In the squirrel monkey, MPTP has been shown to be r a p i d l y metabolized to 1-methyl-4-phenylpyridinium ion (MPP+), only the l a t t e r compound being detectable in brain one hour after systemic i n j e c t i o n of MPTP (7). Until the recent demonstration that MPTP damages n i g r o s t r i a t a l dopaminergic neurons in mice (8,9), e f f o r t s to produce a model of Parkinson's disease in small laboratory rodents generally had been unsuccessful. A possible exception was the reported 50 reduction of s t r i a t a l dopamine in guinea pigs chronically treated with MPTP (10). IAddressee for r e p r i n t s .
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Neurotoxic Effects of MPTP in Guinea Pig
Vol. 36, No. ]3, 1985
We wished to develop a small mammal model of Parkinson's disease, using MPTP, in order to explore the a b i l i t y of certain drugs to protect neurons in the substantia nigra from the neurotoxic effects of MPTP. We had e a r l i e r observed that the content of glutathione was lower in the substantia nigra than in other regions in autopsied human brain, and that glutathione was esp e c i a l l y depleted in the substantia nigra in patients dying with Parkinson's disease (11). I t seemed possible that MPTP might cause dopamine to accumulate in n i g r o s t r i a t a l neuronal c e l l bodies with subsequent production of o x i dizing species such as H202, 02 , HO , and dopamine semiquinone r a d i cals. These oxidizing species in turn might cause l i p i d peroxidation of cell membranes and eventual neuronal death. A l t e r n a t i v e l y , MPTP i t s e l f might be metabolized to MPP+ (7), with some of the presently u n i d e n t i f i e d intermediate oxidation products causing damage to n i g r o s t r i a t a l dopaminergic neurons. Since the guinea pig i s , l i k e primates, unable to synthesize ascorbic acid, and since ascorbic acid might exert an antioxidant protective e f f e c t in the substantia nigra, we have compared the neurochemical and behavioral effects of chronic MPTP administration in normal and scorbutic guinea pigs. Materials and Methods Animals Young male Hartley guinea pigs (Charles River, Canada) weighing approximately 450 g were used in our experiments. They were allowed free access to food and water, and were maintained on a 12 h l i g h t - d a r k cycle. Animals were fed e i t h e r normal guinea pig chow (Ralston Purina Co.), or ascorbic acidd e f i c i e n t guinea pig p e l l e t s ( N u t r i t i o n a l Biochemicals). Scorbutic guinea pigs were fed the ascorbate-deficient d i e t for 10 days before MPTP injections were started, and t h i s d i e t was continued f o r 15 days f u r t h e r during MPTP i n j e c t i o n s . Control animals maintained on a scorbutic diet began to lose weight a f t e r 12 days, and at the end of 25 days on this d i e t , i t was necessary not only to return a l l animals to normal d i e t but also to give them a single subcutaneous rescue i n j e c t i o n of 20 mg ascorbic acid in order to prevent numerous deaths from scurvy. Dru 9 Administration C r y s t a l l i n e MPTP (Aldrich, 97 ) was converted to i t s hydrochloride s a l t and dissolved in 0.9 NaCI s o l u t i o n , adjusted to ph 6-7, in a concentration equivalent to 5 mg of free base per ml. Animals were given d a i l y subcutaneous injections of MPTP, 8 mg/kg. This dose caused animals to become immobile for up to 4h. A s l i g h t l y higher dose, I0 mg/kg, was found to k i l l some animals a f t e r a single i n j e c t i o n , and the dosage of 8 mg/kg appeared to be the highest that animals could survive on a d a i l y basis. Control animals received d a i l y subcutaneous injections of 0.9 NaCI. Biochemical Methods During the periods of MPTP i n j e c t i o n s , 24-h urine specimens were collected from both control and MPTP-injected animals. Guinea pigs were then s a c r i f i c e d by cervical d i s l o c a t i o n , and f r o n t a l cortex and striatum were dissected out and stored at -80°C u n t i l analyzed. All determinations of enzyme a c t i v i t i e s and measurements of neurotransmitters and t h e i r metabolites in brain were done blind to avoid possible bias. Tyrosine hydroxylase (TH) a c t i v i t y was measured in one h a l f of each striatum, using a s l i g h t modification of the method of Waymire et a l . (12), in which d i t h i o t h r e i t o l replaced the 2-mercaptoethanol. Choline a c e t y l t r a n s -
Vol. 36, No. 13, 1985
Neurotoxic
Effects of MPTP in Guinea Pig
ferase (CAT) a c t i v i t y was measured in f r o n t a l cortex, using 0.5 brain homogenates and the method of Fonnum (13).
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Triton-X-lO0
Contents of dopamine (DA), noradrenaline (NA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxy-4hydroxyphenylethyleneglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were measured simultaneously in striatum by high performance l i q u i d chromatography (HPLC) and electrochemical detection, using a modification of the method of Wagner et al. (14). B r i e f l y , half s t r i a t a were homogenized in 1.0 ml of 0.1 M ice-cold HCIO4 containing 0.4 mM NaHSO3, Homogenates were centrifuged at 10,000 x g for I0 min at 4°C, and a f t e r supernatants were f i l t e r e d through a M i l l i p o r e SJHV f i l t e r , 4 mm in diameter with 0.45 um pores, 25 ul of each sample were injected into the HPLC system. This included a reverse-phase chromatographic column (60 x 4.6 mm ODS Hypersil, dp 3~m) and an LC-4A amperometric detector (Bioanal~tical Systems) with a glassy carbon electrode. The potential was set at 0.7 V with respect to an Ag/AgCI reference electrode. A 2-channel recorder set at 2 and 20 nA f u l l scale deflections was used, since the tissue contents of compounds of i n t e r est often d i f f e r e d by more than an order of magnitude. The mobile phase was an aqueous solution of NaH2PO4 (0.1 M, containing 60 mg Na2EDTA and 100 mg sodium octyl sulfate per l i t e r ) . The pH was adjusted to 3.6 m 0.01 with 3 M H3PO4, and the flow rate was 1.0 ml/min. External standards were injected a f t e r every second to fourth sample of striatum to compensate for s l i g h t decreases in s e n s i t i v i t y due to electrode and column changes. Brain contents of compounds were calculated from the amplitude of the peak d e f l e c tions which they produced on chromatograms. MPTP and i t s metabolites in guinea pig urine were separated on an HPLC column (125 mm x 4.6 mm I . D . , ) packed with ODS Hypersil (dp = 5 ~m) and eluted (at a flow rate of 1.0 ml/min) with an aqueous solution (pH 4.00) containing NaH2PO4 (50 mM), sodium dodecyl sulfate (2 mM), N,N-dimethylhexylamine (4 mM), and a c e t o n i t r i l e (30 , v / v ) . Measurement of the UV spect r a of MPTP and of 4-phenylpyridine in the eluent indicated that 245 nm was an appropriate setting f o r the photometric detector. Direct i n j e c t i o n of f i l t e r e d urine into the HPLC column allowed detection of MPTP (retention time 24 min) in concentrations as low as 2 ~M with this system. Urine specimens were tested both before and after digestion with a mixture of sulfatase and glucuronidase derived from Helix pomatia (Sigma). After digestion, urine specimens were extracted with ethyl acetate, and the evaporated extracts were dissolved in water p r i o r to i n j e c t i o n into the HPLC column. Results Non-Scorbutic Guinea Pigs Guinea pigs fed a normal d i e t and given 10 d a i l y injections of MPTP (8 mg/kg) f a i l e d to show any neurological signs other than sedation and hypotonia f o r about 4 h a f t e r each i n j e c t i o n . Mean values for s t r i a t a l tyrosine hydroxylase a c t i v i t y , dopamine and i t s 2 metabolites, noradrenaline, and 5-HIAA are shown in Table I f o r control animals and for treated animals k i l l e d at 2 h and at 30 days a f t e r the last MPTP i n j e c t i o n . Shortly a f t e r the f i n a l MPTP i n j e c t i o n , s t r i a t a l dopamine content was s l i g h t l y elevated, and DOPAC and HVA contents were decreased, possibly r e f l e c t i n g the weak inh i b i t i o n of monoamine oxidase by MPTP which has been reported by some invest i g a t o r s (15,16). Animals k i l l e d one month l a t e r showed no changes from cont r o l s in s t r i a t a l tyrosine hydroxylase a c t i v i t y or in contents of dopamine and its metabolites.
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TABLE I 7yrosine Hydroxylase A c t i v i t y , Dopamine, and Other Compounds in Striatum of Non-Scorbutic Guinea Pigs Injected with MPTP (8 mg/kg) f o r 10 Days Parameter
Control
(8) TH DA DOPAC HVA NA 5-HIAA
7.5*0.3 31.4"4.1 2 0 . 4 * 1.9 16.5 * 1.0 0 . 5 * 0.1 I.i* 0.i
MPTP (killed 2 h after last injection)
(4)
6.7*0.3 39.9"4.1 8.7 * 1.5" 1 3 . 2 " 2.6 0.4* 0 1.9"0.5
MPTP ( k i l l e d 30 d a f t e r last injection)
(9)
6.6*0.3 34.3*3.8 ] 7 . 3 * 1.7 1 6 . 7 " 1.4 0 . 4 * 0.1 1.4"0.2
Tyrosine hydroxylase a c t i v i t y expressed in nmol/h/mg p r o t e i n , and neurotransmitters and metabolites in nmol/g wet weight. Values are means * SEM. Number of animals shown in parentheses. *Signific a n t l y d i f f e r e n t from c o n t r o l s ; P < 0.01. Scorbutic Guinea Pigs Table I I l i s t s mean values f o r these and a d d i t i o n a l parameters f o r guinea pigs made severely scorbutic with an a s c o r b a t e - d e f i c i e n t d i e t and given 15 d a i l y i n j e c t i o n s of MPTP (8 mg/kg) or s a l i n e . The MPTP-treated and control animals l o s t weight at the same rate while on the a s c o r b a t e - d e f i c i e n t d i e t . MPTP produced sedation and hypotonia f o r about 4h a f t e r i n j e c t i o n s , but no abnormal neurological signs were evident e i t h e r at the end of 15 d a i l y MPTP i n j e c t i o n s , or a f t e r 2 months recovery on a normal d i e t . There was a s l i g h t ( 2 2 ) reduction in s t r i a t a l t y r o s i n e hydroxylase a c t i v i t y in MPTP-treated animals immediately as well as 2 months a f t e r the end of i n j e c t i o n s . Reduct i o n s in the s t r i a t a l contents of dopamine and i t s metabolites were small and of no or marginal s t a t i s t i c a l significance. Choline a c e t y l t r a n s f e r a s e a c t i v i t y in f r o n t a l cortex was not a l t e r e d by chronic treatment with MPTP. Urinary Metabolites of MPTP A l i q u o t s of 24-hour urine specimens from guinea pigs on both normal and a s c o r b a t e - d e f i c i e n t d i e t s were analysed by HPLC f o r the presence of MPTP and i t s m e t a b o l i t e s . MPTP i t s e l f was not detectable in any urine specimens c o l lected during MPTP treatment, but a compound having r e t e n t i o n and photometric c h a r a c t e r i s t i c s very s i m i l a r to those of the 4-phenylpyridinium ion was observed in a l l such urines. This compound was well separated from the MPTP locus on chromatograms, and i t s r e t e n t i o n was such that i t s i d e n t i t y with MPP+, a metabolite of MPIP which has been i s o l a t e d from s q u i r r e l monkey brain ( 7 ) , is probable. In a d d i t i o n , several compounds not present in the urine of control animals were found a f t e r s u l f a t a s e / g l u c u r o n i d a s e digestion of urine from MPTP-treated animals. These u n i d e n t i f i e d compounds (presumably transformation products of MPTP) were not detectable without enzymatic hydrolysis. Their l i p o p h i l i c i t y (less than MPTP or MPP+) suggests t h a t they may be hydroxylated d e r i v a t i v e s of MPTP.
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Effects of MPTP in Guinea Pig
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TABLE I I Effects of 15 Daily Injections of MPTP (8 mg/kg) on Enzymes, Neurotransmitters, and Metabolites in Scorbutic Guinea Pigs Parameter
Animals k i l l e d 24 h a f t e r MPTP injections Control (8)
CAT TH DA DOPAC HVA NA MHPG 5-HT 5-H IAA
6.3*0.2 5 8 . 7 * 2.2 8 . 4 * 0.4 10.5"0.9 1.7-0.2 0 . 7 * 0.1 0 . 9 * 0.1 0 . 6 * 0.1
MPTP (8)
4.9*0.2* 4 9 . 3 , 3.7t 7.6*0.7 1 1 . 4 " 1.5 1.1- O.lt 0.5, 0.i f 0.8 • 0.2 0 . 7 * 0.1
Animals k i l l e d 60 d a f t e r MPTP injections Control (15) 11.2"0.5 5.5-0.1 3 9 . 7 * 4.5 2 0 . 1 - 0.7 2 2 . 9 * 1.4 0.8*0.2 0.3* 0 0.7"0.1 0 . 9 * 0.1
MPTP (15) 11.0"0.5 4 . 3 - 0.~ 3 2 . 7 * 2.0 1 5 . 4 " 0.~ 1 5 . 9 " 1.~ 1.0" 0.i 0 . 3 * 0.] 0 . 7 * 0.1 0.7- 0.i
Choline acetyltransferase a c t i v i t y (nmol/lO min/mg protein) measured in f r o n t a l cortex. A l l other measurements are in striatum. Tyrosine hydroxylase a c t i v i t y expressed in nmol/h/mg protein. Neurotransmitters and metabolites expressed in nmol/g wet weight. Values are means * SEM. Number of animals shown in parentheses. *Significantly d i f f e r e n t from controls k i l l e d at same time; P < 0.001. +Sig_ n i f i c a n t l y d i f f e r e n t from controls k i l l e d at same time; P < 0.05. Discussion The neurochemical findings of t h i s study indicate that in non-scorbutic guinea pigs chronic treatment with sub-lethal doses of MPTP does not cause permanent damage to n i g r o s t r i a t a l dopaminergic neurons. The reduction in s t r i a t a l DOPAC content and the small but n o n - s i g n i f i c a n t elevation in dopamine content at the end of the treatment period resemble the more marked neurochemical abnormalities which acute MPTP treatment produces in rhesus monkeys (4), but in our experiments with guinea pigs, these changes were transient. The 50 reduction in s t r i a t a l dopamine content of guinea pigs reported by Chiueh et a l . (10) conceivably may have resulted from a higher dosage or a longer administration of MPTP by these i n v e s t i g a t o r s , although they did not provide d e t a i l s . I t is also obvious that very l i t t l e lasting damage is done to n i g r o s t r i a t a l dopaminergic neurons in guinea pigs rendered severely scorbutic and treated with MPTP in the highest dosage and f o r the longest period compatible with t h e i r s u r v i v a l . C l e a r l y , ascorbic acid def i c i e n c y did not make neurons in the substantia nigra s i g n i f i c a n t l y more susceptible to the neurotoxic effects of MPTP. The f a i l u r e of MPTP to damage n i g r o s t r i a t a l dopaminergic neurons in the guinea pig and r a t , while such damage occurs r e a d i l y in primates and the mouse, remains unexplained. Our HPLC analyses of urine suggest that MPTP is metabolized to MPP÷ in the guinea pig, at least in peripheral tissues, and indicate the presence of other metabolites as w e l l . The r e l a t i v e protection of dopaminergic n i g r o s t r i a t a l neurons in the guinea pig might be due to metabolism of MPTP in the brain by pathways which d i f f e r from those in primates
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Neurotoxic Effects of MPTP in Guinea Pig
Vol. 36, No. 13, 1985
and the mouse, or could r e s u l t from more e f f e c t i v e protective mechanisms against the toxic effects of MPTP or i t s metabolites within the substantia nigra of the guinea pig. Acknowledgements This research was supported by of Canada, the Parkinson Foundation cal Services Foundation. V.W.Y. is ship from the Huntington Society of of the Ontario Ministry of Health.
grants from the Medical Research Council of Canada, and the B r i t i s h Columbia Medithe r e c i p i e n t of a predoctoral scholarCanada, and S.J.K. is a Career Scientist
References 1. G. C. DAVIS, A. C. WILLIAMS, S, P. MARKEY, M. H. EBERT, E. D. CAINE, C. M. REICHERT, and I. J. KOPIN, Psychiatry Res. 1:249-254 (1979). 2. J. W. LANGSTON, Po BALLARD, J. W. TETRUI~, and I. IRWIN, Science 219:979-980 (1983). 3. J. M. WRIGHT, R. A. WALL, T. L. PERRY, and D. W. PATY, New Engl. J. Med. 310:325 (1984). 4. R. S. BURNS, C. C. CHIUEH, S. P. MARKEY, M. H. EBERT, D. M. JACOBOWITZ, and I. J. KOPIN, Proc. Natl. Acad. Sci. (USA) 80:4546-4550 (1983). 5. R. S. BURNS, S. P. MARKEY, J. M. PHILLIPS, and C. C. CHIUEH, Canad. J. Neurol. Sci. i i , No. 1 (Supplement):166-168 (1984). 6. J. W. LANGSTON, L. S. FORNO, C. S. REBERT, and I. IRWIN, Brain Res. 292:390-394 (1984). 7. J. W. LANGSTON, I. IRWIN, E. B. LANGSTON, and L. S. FORNO, Neurosci. Lett. 48:87-92 (1984). 8. R. E. H~IKKILA, A. HESS, and R. C. DUVOISIN, Science 224:1451-1453 (1984). 9. R. A. WALLACE, R. BOLDRY, T. SCHMITTGEN, D. MILLER, and N. URETSKY, Life Sci. 35:285-291 (1984). I0. C. C. CHIUEH, S. P. MARKEY, R. S. BURNS, J. JOHANNESSEN, D. M. JACOBOWITZ, and I. J. KOPIN, The Pharmacologist 2__55:(3):131 (Abst. 109) (1983). i i . T. L. PERRY, D. V. GODIN, and S. HANSEN, Neurosci. Lett. 33:305-310 (1982). 12. J. C. WAYMIRE, R. BJUR, and N. WEINER, Anal. Biochem. 43:588-600 (1971). 13. F. FONNUM, J. Neurochem. 24:407-409 (1975). 14. J. WAGNER, P. VITALI, M. G. PALFREYMAN, M. ZRAIKA, and S. HUOT, J. Neurochem. 38:1241-1254 (1982). 15. J. A. JAVlTCH, G. R. UHL, and S. H. SNYDER, Proc. Natl. Acad. Sci (USA) 81:4591-4595 (1984). 16. B. PARSONS, and T. C. RAINBOW, Eur. J. Pharmacol. 102:375-377 (1984).
Pergamon Press
1-METHYL-4-PHENYL-I,2,3,6-TETRAHYDROPYRIDINE (MPTP) DOES NOT DESTROY NIGROSTRIATAL NEURONS IN THE SCORBUTIC GUINEA PIG Thomas L. Perry 1, Voon Wee Yong, Masatoshi Ito, Karen Jenes, Richard A. Wall, James G. Foulks, James M. Wright, and Stephen J. Kish* Department of Pharmacology Therapeutics, University of B r i t i s h Columbia, Vancouver, Canada, V6T IW5; and Human Brain Laboratory*, Clarke I n s t i t u t e of Psychiatry, Toronto, Canada, M5T 1R8 (Received in final form January 22, 1984)
Summary Guinea pigs were injected subcutaneously with 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTPtreated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The r e l a t i v e lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.
The parenteral i n j e c t i o n or inhalation of 1-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP) has resulted in the development of severe and l a s t ing symptoms of Parkinson's disease in human subjects (1-3). Monkeys of 3 d i f f e r e n t species injected repeatedly with low doses of MPTP develop neurological signs that closely resemble human Parkinson's disease, and t h e i r brains l a t e r show a s t r i a t a l depletion of dopamine and a loss of neurons in the zona compacta of the substantia nigra (4-7). In the squirrel monkey, MPTP has been shown to be r a p i d l y metabolized to 1-methyl-4-phenylpyridinium ion (MPP+), only the l a t t e r compound being detectable in brain one hour after systemic i n j e c t i o n of MPTP (7). Until the recent demonstration that MPTP damages n i g r o s t r i a t a l dopaminergic neurons in mice (8,9), e f f o r t s to produce a model of Parkinson's disease in small laboratory rodents generally had been unsuccessful. A possible exception was the reported 50 reduction of s t r i a t a l dopamine in guinea pigs chronically treated with MPTP (10). IAddressee for r e p r i n t s .
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Neurotoxic Effects of MPTP in Guinea Pig
Vol. 36, No. ]3, 1985
We wished to develop a small mammal model of Parkinson's disease, using MPTP, in order to explore the a b i l i t y of certain drugs to protect neurons in the substantia nigra from the neurotoxic effects of MPTP. We had e a r l i e r observed that the content of glutathione was lower in the substantia nigra than in other regions in autopsied human brain, and that glutathione was esp e c i a l l y depleted in the substantia nigra in patients dying with Parkinson's disease (11). I t seemed possible that MPTP might cause dopamine to accumulate in n i g r o s t r i a t a l neuronal c e l l bodies with subsequent production of o x i dizing species such as H202, 02 , HO , and dopamine semiquinone r a d i cals. These oxidizing species in turn might cause l i p i d peroxidation of cell membranes and eventual neuronal death. A l t e r n a t i v e l y , MPTP i t s e l f might be metabolized to MPP+ (7), with some of the presently u n i d e n t i f i e d intermediate oxidation products causing damage to n i g r o s t r i a t a l dopaminergic neurons. Since the guinea pig i s , l i k e primates, unable to synthesize ascorbic acid, and since ascorbic acid might exert an antioxidant protective e f f e c t in the substantia nigra, we have compared the neurochemical and behavioral effects of chronic MPTP administration in normal and scorbutic guinea pigs. Materials and Methods Animals Young male Hartley guinea pigs (Charles River, Canada) weighing approximately 450 g were used in our experiments. They were allowed free access to food and water, and were maintained on a 12 h l i g h t - d a r k cycle. Animals were fed e i t h e r normal guinea pig chow (Ralston Purina Co.), or ascorbic acidd e f i c i e n t guinea pig p e l l e t s ( N u t r i t i o n a l Biochemicals). Scorbutic guinea pigs were fed the ascorbate-deficient d i e t for 10 days before MPTP injections were started, and t h i s d i e t was continued f o r 15 days f u r t h e r during MPTP i n j e c t i o n s . Control animals maintained on a scorbutic diet began to lose weight a f t e r 12 days, and at the end of 25 days on this d i e t , i t was necessary not only to return a l l animals to normal d i e t but also to give them a single subcutaneous rescue i n j e c t i o n of 20 mg ascorbic acid in order to prevent numerous deaths from scurvy. Dru 9 Administration C r y s t a l l i n e MPTP (Aldrich, 97 ) was converted to i t s hydrochloride s a l t and dissolved in 0.9 NaCI s o l u t i o n , adjusted to ph 6-7, in a concentration equivalent to 5 mg of free base per ml. Animals were given d a i l y subcutaneous injections of MPTP, 8 mg/kg. This dose caused animals to become immobile for up to 4h. A s l i g h t l y higher dose, I0 mg/kg, was found to k i l l some animals a f t e r a single i n j e c t i o n , and the dosage of 8 mg/kg appeared to be the highest that animals could survive on a d a i l y basis. Control animals received d a i l y subcutaneous injections of 0.9 NaCI. Biochemical Methods During the periods of MPTP i n j e c t i o n s , 24-h urine specimens were collected from both control and MPTP-injected animals. Guinea pigs were then s a c r i f i c e d by cervical d i s l o c a t i o n , and f r o n t a l cortex and striatum were dissected out and stored at -80°C u n t i l analyzed. All determinations of enzyme a c t i v i t i e s and measurements of neurotransmitters and t h e i r metabolites in brain were done blind to avoid possible bias. Tyrosine hydroxylase (TH) a c t i v i t y was measured in one h a l f of each striatum, using a s l i g h t modification of the method of Waymire et a l . (12), in which d i t h i o t h r e i t o l replaced the 2-mercaptoethanol. Choline a c e t y l t r a n s -
Vol. 36, No. 13, 1985
Neurotoxic
Effects of MPTP in Guinea Pig
ferase (CAT) a c t i v i t y was measured in f r o n t a l cortex, using 0.5 brain homogenates and the method of Fonnum (13).
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Triton-X-lO0
Contents of dopamine (DA), noradrenaline (NA), serotonin (5-HT), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxy-4hydroxyphenylethyleneglycol (MHPG), and 5-hydroxyindoleacetic acid (5-HIAA) were measured simultaneously in striatum by high performance l i q u i d chromatography (HPLC) and electrochemical detection, using a modification of the method of Wagner et al. (14). B r i e f l y , half s t r i a t a were homogenized in 1.0 ml of 0.1 M ice-cold HCIO4 containing 0.4 mM NaHSO3, Homogenates were centrifuged at 10,000 x g for I0 min at 4°C, and a f t e r supernatants were f i l t e r e d through a M i l l i p o r e SJHV f i l t e r , 4 mm in diameter with 0.45 um pores, 25 ul of each sample were injected into the HPLC system. This included a reverse-phase chromatographic column (60 x 4.6 mm ODS Hypersil, dp 3~m) and an LC-4A amperometric detector (Bioanal~tical Systems) with a glassy carbon electrode. The potential was set at 0.7 V with respect to an Ag/AgCI reference electrode. A 2-channel recorder set at 2 and 20 nA f u l l scale deflections was used, since the tissue contents of compounds of i n t e r est often d i f f e r e d by more than an order of magnitude. The mobile phase was an aqueous solution of NaH2PO4 (0.1 M, containing 60 mg Na2EDTA and 100 mg sodium octyl sulfate per l i t e r ) . The pH was adjusted to 3.6 m 0.01 with 3 M H3PO4, and the flow rate was 1.0 ml/min. External standards were injected a f t e r every second to fourth sample of striatum to compensate for s l i g h t decreases in s e n s i t i v i t y due to electrode and column changes. Brain contents of compounds were calculated from the amplitude of the peak d e f l e c tions which they produced on chromatograms. MPTP and i t s metabolites in guinea pig urine were separated on an HPLC column (125 mm x 4.6 mm I . D . , ) packed with ODS Hypersil (dp = 5 ~m) and eluted (at a flow rate of 1.0 ml/min) with an aqueous solution (pH 4.00) containing NaH2PO4 (50 mM), sodium dodecyl sulfate (2 mM), N,N-dimethylhexylamine (4 mM), and a c e t o n i t r i l e (30 , v / v ) . Measurement of the UV spect r a of MPTP and of 4-phenylpyridine in the eluent indicated that 245 nm was an appropriate setting f o r the photometric detector. Direct i n j e c t i o n of f i l t e r e d urine into the HPLC column allowed detection of MPTP (retention time 24 min) in concentrations as low as 2 ~M with this system. Urine specimens were tested both before and after digestion with a mixture of sulfatase and glucuronidase derived from Helix pomatia (Sigma). After digestion, urine specimens were extracted with ethyl acetate, and the evaporated extracts were dissolved in water p r i o r to i n j e c t i o n into the HPLC column. Results Non-Scorbutic Guinea Pigs Guinea pigs fed a normal d i e t and given 10 d a i l y injections of MPTP (8 mg/kg) f a i l e d to show any neurological signs other than sedation and hypotonia f o r about 4 h a f t e r each i n j e c t i o n . Mean values for s t r i a t a l tyrosine hydroxylase a c t i v i t y , dopamine and i t s 2 metabolites, noradrenaline, and 5-HIAA are shown in Table I f o r control animals and for treated animals k i l l e d at 2 h and at 30 days a f t e r the last MPTP i n j e c t i o n . Shortly a f t e r the f i n a l MPTP i n j e c t i o n , s t r i a t a l dopamine content was s l i g h t l y elevated, and DOPAC and HVA contents were decreased, possibly r e f l e c t i n g the weak inh i b i t i o n of monoamine oxidase by MPTP which has been reported by some invest i g a t o r s (15,16). Animals k i l l e d one month l a t e r showed no changes from cont r o l s in s t r i a t a l tyrosine hydroxylase a c t i v i t y or in contents of dopamine and its metabolites.
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Neurotoxic Effects of MPTP in Guinea Pig
Vol. 36, No. 13, 1985
TABLE I 7yrosine Hydroxylase A c t i v i t y , Dopamine, and Other Compounds in Striatum of Non-Scorbutic Guinea Pigs Injected with MPTP (8 mg/kg) f o r 10 Days Parameter
Control
(8) TH DA DOPAC HVA NA 5-HIAA
7.5*0.3 31.4"4.1 2 0 . 4 * 1.9 16.5 * 1.0 0 . 5 * 0.1 I.i* 0.i
MPTP (killed 2 h after last injection)
(4)
6.7*0.3 39.9"4.1 8.7 * 1.5" 1 3 . 2 " 2.6 0.4* 0 1.9"0.5
MPTP ( k i l l e d 30 d a f t e r last injection)
(9)
6.6*0.3 34.3*3.8 ] 7 . 3 * 1.7 1 6 . 7 " 1.4 0 . 4 * 0.1 1.4"0.2
Tyrosine hydroxylase a c t i v i t y expressed in nmol/h/mg p r o t e i n , and neurotransmitters and metabolites in nmol/g wet weight. Values are means * SEM. Number of animals shown in parentheses. *Signific a n t l y d i f f e r e n t from c o n t r o l s ; P < 0.01. Scorbutic Guinea Pigs Table I I l i s t s mean values f o r these and a d d i t i o n a l parameters f o r guinea pigs made severely scorbutic with an a s c o r b a t e - d e f i c i e n t d i e t and given 15 d a i l y i n j e c t i o n s of MPTP (8 mg/kg) or s a l i n e . The MPTP-treated and control animals l o s t weight at the same rate while on the a s c o r b a t e - d e f i c i e n t d i e t . MPTP produced sedation and hypotonia f o r about 4h a f t e r i n j e c t i o n s , but no abnormal neurological signs were evident e i t h e r at the end of 15 d a i l y MPTP i n j e c t i o n s , or a f t e r 2 months recovery on a normal d i e t . There was a s l i g h t ( 2 2 ) reduction in s t r i a t a l t y r o s i n e hydroxylase a c t i v i t y in MPTP-treated animals immediately as well as 2 months a f t e r the end of i n j e c t i o n s . Reduct i o n s in the s t r i a t a l contents of dopamine and i t s metabolites were small and of no or marginal s t a t i s t i c a l significance. Choline a c e t y l t r a n s f e r a s e a c t i v i t y in f r o n t a l cortex was not a l t e r e d by chronic treatment with MPTP. Urinary Metabolites of MPTP A l i q u o t s of 24-hour urine specimens from guinea pigs on both normal and a s c o r b a t e - d e f i c i e n t d i e t s were analysed by HPLC f o r the presence of MPTP and i t s m e t a b o l i t e s . MPTP i t s e l f was not detectable in any urine specimens c o l lected during MPTP treatment, but a compound having r e t e n t i o n and photometric c h a r a c t e r i s t i c s very s i m i l a r to those of the 4-phenylpyridinium ion was observed in a l l such urines. This compound was well separated from the MPTP locus on chromatograms, and i t s r e t e n t i o n was such that i t s i d e n t i t y with MPP+, a metabolite of MPIP which has been i s o l a t e d from s q u i r r e l monkey brain ( 7 ) , is probable. In a d d i t i o n , several compounds not present in the urine of control animals were found a f t e r s u l f a t a s e / g l u c u r o n i d a s e digestion of urine from MPTP-treated animals. These u n i d e n t i f i e d compounds (presumably transformation products of MPTP) were not detectable without enzymatic hydrolysis. Their l i p o p h i l i c i t y (less than MPTP or MPP+) suggests t h a t they may be hydroxylated d e r i v a t i v e s of MPTP.
Vol. 36, No. 13, 1985
Neurotoxic
Effects of MPTP in Guinea Pig
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TABLE I I Effects of 15 Daily Injections of MPTP (8 mg/kg) on Enzymes, Neurotransmitters, and Metabolites in Scorbutic Guinea Pigs Parameter
Animals k i l l e d 24 h a f t e r MPTP injections Control (8)
CAT TH DA DOPAC HVA NA MHPG 5-HT 5-H IAA
6.3*0.2 5 8 . 7 * 2.2 8 . 4 * 0.4 10.5"0.9 1.7-0.2 0 . 7 * 0.1 0 . 9 * 0.1 0 . 6 * 0.1
MPTP (8)
4.9*0.2* 4 9 . 3 , 3.7t 7.6*0.7 1 1 . 4 " 1.5 1.1- O.lt 0.5, 0.i f 0.8 • 0.2 0 . 7 * 0.1
Animals k i l l e d 60 d a f t e r MPTP injections Control (15) 11.2"0.5 5.5-0.1 3 9 . 7 * 4.5 2 0 . 1 - 0.7 2 2 . 9 * 1.4 0.8*0.2 0.3* 0 0.7"0.1 0 . 9 * 0.1
MPTP (15) 11.0"0.5 4 . 3 - 0.~ 3 2 . 7 * 2.0 1 5 . 4 " 0.~ 1 5 . 9 " 1.~ 1.0" 0.i 0 . 3 * 0.] 0 . 7 * 0.1 0.7- 0.i
Choline acetyltransferase a c t i v i t y (nmol/lO min/mg protein) measured in f r o n t a l cortex. A l l other measurements are in striatum. Tyrosine hydroxylase a c t i v i t y expressed in nmol/h/mg protein. Neurotransmitters and metabolites expressed in nmol/g wet weight. Values are means * SEM. Number of animals shown in parentheses. *Significantly d i f f e r e n t from controls k i l l e d at same time; P < 0.001. +Sig_ n i f i c a n t l y d i f f e r e n t from controls k i l l e d at same time; P < 0.05. Discussion The neurochemical findings of t h i s study indicate that in non-scorbutic guinea pigs chronic treatment with sub-lethal doses of MPTP does not cause permanent damage to n i g r o s t r i a t a l dopaminergic neurons. The reduction in s t r i a t a l DOPAC content and the small but n o n - s i g n i f i c a n t elevation in dopamine content at the end of the treatment period resemble the more marked neurochemical abnormalities which acute MPTP treatment produces in rhesus monkeys (4), but in our experiments with guinea pigs, these changes were transient. The 50 reduction in s t r i a t a l dopamine content of guinea pigs reported by Chiueh et a l . (10) conceivably may have resulted from a higher dosage or a longer administration of MPTP by these i n v e s t i g a t o r s , although they did not provide d e t a i l s . I t is also obvious that very l i t t l e lasting damage is done to n i g r o s t r i a t a l dopaminergic neurons in guinea pigs rendered severely scorbutic and treated with MPTP in the highest dosage and f o r the longest period compatible with t h e i r s u r v i v a l . C l e a r l y , ascorbic acid def i c i e n c y did not make neurons in the substantia nigra s i g n i f i c a n t l y more susceptible to the neurotoxic effects of MPTP. The f a i l u r e of MPTP to damage n i g r o s t r i a t a l dopaminergic neurons in the guinea pig and r a t , while such damage occurs r e a d i l y in primates and the mouse, remains unexplained. Our HPLC analyses of urine suggest that MPTP is metabolized to MPP÷ in the guinea pig, at least in peripheral tissues, and indicate the presence of other metabolites as w e l l . The r e l a t i v e protection of dopaminergic n i g r o s t r i a t a l neurons in the guinea pig might be due to metabolism of MPTP in the brain by pathways which d i f f e r from those in primates
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Neurotoxic Effects of MPTP in Guinea Pig
Vol. 36, No. 13, 1985
and the mouse, or could r e s u l t from more e f f e c t i v e protective mechanisms against the toxic effects of MPTP or i t s metabolites within the substantia nigra of the guinea pig. Acknowledgements This research was supported by of Canada, the Parkinson Foundation cal Services Foundation. V.W.Y. is ship from the Huntington Society of of the Ontario Ministry of Health.
grants from the Medical Research Council of Canada, and the B r i t i s h Columbia Medithe r e c i p i e n t of a predoctoral scholarCanada, and S.J.K. is a Career Scientist
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