The biodisposition of MPP+ in mouse brain

Neurosclence Letters, 101 (1989) 83-88

83

Elsevier ScientificPubhshers Ireland Ltd. NSL 06110

The biodisposition o f M P P + in m o u s e brain Ian Irwin, Louis E. DeLanney, Donato Di Monte and J. William Langston The Instaute for Medwal Research and Cahforma Parkinson "s Foundatton, San Jose, CA 95128 (U S A )

(Recewed 15 December 1988, Accepted 16 January 1989) Key words

l-Methyl-4-phenyl-1,2,3,6-tetrahydropyndme, 1-Methyl-4-phenylpyndlnmm1on, Neurotoxlcology, BloactlvaUon,Blotransformatlon

These stu&es assessed the role of biotransformatlon m the rapid ehmmation of MPP + from the central nervous system (CNS) compartment of mice Micewere given either MPP + via the mtracerebroventncular 0 c v ) route, or MPTP mtraperitoneally The ehmmatmn of MPP ÷ from the brain over 10 h was slmdar m both groups and followed exponentml kinetics Using hqmd sontfllation counting, high-pressure hqmd chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis, no compounds other than MPP ÷ were detected m brain 2-10 h after the i c v administration of radlolabelled [14C/3H]MPP+ MPP + was also unchanged after mcubatmn with brain homogenates These data re&care that MPP + is not removed from the CNS compartment wa biotransformat~on Because of ~ts pos~twe charge and kinetics of ehmmatlon, the possibd~tyof an acuve transport system is suggested

The n e u r o t o x i c a n t M P T P

( l - m e t h y l - 4 - p h e n y l - l , 2 , 3 , 6 - t e t r a h y d r o p y r i d i n e ) pro-

duces m a n y o f the n e u r o p a t h o l o g i c a l , n e u r o c h e m l c a l a n d behavioral features o f idiopathic P a r k l n s o n ' s disease in primates (for review, see ref. 17). Interestingly, rodents are m u c h less sensitive to the toxic effects of this c o m p o u n d [3, 4, 8, 22], a l t h o u g h relatively large doses o f M P T P p r o d u c e long-lasting depletions o f s t n a t a l d o p a m m e and d e g e n e r a t i o n o f d o p a m i n e r g i c nigrostriatal n e u r o n s in mice [9, 22] Several e x p l a n a t i o n s have been p u t forward to a c c o u n t for this difference in species susceptlbdlty, i n c l u d i n g the absence of n e u r o m e l a n m from r o d e n t b r a i n [5, 11] a n d the presence of higher levels o f m o n o a m i n e oxtdase B m cerebral capillary e n d o t h e l i a (CCE) of rodents [21]. The earhest experimental o b s e r v a t i o n whxch m a y have a b e a r i n g o n this question relates to the b l o d i s p o s l t i o n o f M P T P . It has been k n o w n for some time that M P P ÷ ( l - m e t h y l - 4 - p h e n y l p y n d i n i u m ion), the putative toxic m e t a b o l i t e o f M P T P , is m o r e rapidly r e m o v e d from the central n e r v o u s system (CNS) c o m p a r t m e n t in rodents t h a n in p r i m a t e s [11, 14, 19, 20]. W h e n toxic doses of M P T P are gwen to mice a n d m o n k e y s , similar c o n c e n t r a t i o n s o f M P P ÷ a p p e a r within the C N S in the first h o u r [11, 12]; however, M P P ÷ disappears f r o m the b r a i n o f the m o u s e within 24 h [18, Correspondence J W Langston, Cahfornla Parkmson's Foundation, 2444 Moorpark Avenue, State 316, San Jose, CA 95128, U S A

0304-3940/89/$ 03 50 © 1989 Elsevier ScientificPubhshers Ireland Ltd

84 20], while detectable levels of MPP + persist in the brain of the monkey for at least 10 days [! 1, 20]. In spite of this potentially important observation, little is known regarding the mechamsm(s) responsible for the rapid elimination of MPP + from the mouse CNS For this reason we undertook the present studies, which were designed to characterize the kinetics of elimination of M P P + from the CNS c o m p a r t m e n t in mice, and to investigate the mechanism(s) underlying its disappearance_ Male C57BL/6 mice, 6--8 weeks old, were used for all studies. Animals were housed 5 to a cage under constant temperature (22°C) and humidity in a room illuminated 12 h/day with food and water available ad libitum. M P T P was obtained from Aldrich Chemical Company, and converted to its hydrochlonde salt as previously described [10]. [14C]MPTP (phenyl ring-labelled, 7.23 mCi/mmol) was obtained from New England Nuclear, and converted to [14C]MPP+ by incubation with homogenates of mouse brain_ Brains from 4 mice were homogenized in 36 ml of 50 m M KH2PO4 (pH 7 4). T e n / t m o l of [14C]MPTP (dissolved in 2.0 ml of the buffer) were added to the homogenate, which was then incubated in a mechanical shaker bath at 37°C for 24 h Assay of M P P + by high-pressure liquid c h r o m a t o g r a p h y (HPLC) as previously described [19] revealed that 77.5% of the M P T P was converted to M P P + The [14C]MPP + formed was purified by extraction of the iodide ion pair into CHC13 [16]. Chromatographic analysis showed that this material consisted solely of M P P +. M P P + , 3H-labelled in the methyl group (85 Ci/mmol), was obtained from N E N , Solvents and chemicals used for chromatography were nanograde (Malllnkrodt, St. Louis, MO) All other chemicals were reagent grade. Our first experiments were designed to (1) assess if lntracerebroventricular[y (1.c v )-admlmstered MPP + provides an accurate model to study the elimination of M P P + from the brain after the systemic administration of MPTP; and (2) determine the kinetics of elimination of M P P + from the CNS c o m p a r t m e n t in the mouse. Mice were given either M P T P (30 mg/kg) lntraperitoneally (i.p.) or MPP + (20/~g) administered l.C.V. [13]. Five animals from each treatment group were sacrificed 2, 4, 8 and 10 h after mjectlon. These time points were chosen based on previous work showing that concentrations of MPP + m the mouse brain decline to undetectable concentrations during this time period [18] The brains were removed, homogemzed in methanol (5.0 ml) and MPP ÷ was assayed by gas chromatography/mass spectrometry (GC/ MS) as previously descnbed [12]. Both i c.v -administered M P P + and M P P ÷ derived from the systemic administration of M P T P were rapidly eliminated. Regression analysis of these data using linear, exponential, logarithmic and power transformations (least squares method) gave the best correlation for exponential kinetics (Fig. I). Comparison of the slopes, using a Student's t-test, showed that although the concentration of MPP ÷ was much higher after the l.C.V, regimen, the slope of the curves were not significantly different. Thus, the elimination of i.c.v-admimstered M P P ÷ appears to be similar to that which occurs when MPP + is generated in the CNS after systemic administration of MPTP_ The next experiments were performed using the i c.v. model to determine if the blotransformation o f M P P ÷ accounts for Its rapid disappearance from the brain. M P P +

85

~f"l 6 L L I--I

5 Z

,J

T IME

(HRS)

Fig 1 E h m m a U o n o f M P P + from the CNS c o m p a r t m e n t after mtracerebroventricular (i.c v ) adrmnistratlon o f M P P + (squares) and mtrapentoneal (i.p) admlmstratlon of M P T P (diamonds) Inmal concentrations were determined by G C / M S and expressed in total ng/ml of extract Symbols represent mean and S.E.M values for 5 ammals at each ttme point. Kinetic constants for the ehnunation o f M P P + were similar after both i c v M P P + ( Y = - 0 2195 X + 7 182, R2=0.989359) and M P P + after i.p M P T P ( Y = - 0 . 2 3 X + 5 461, R 2 = 0 989369).

(20 gg, containing both [3H]MPP+ and [14C]MPP+ was administered i.c.v, to mice. Groups of mice (n = 5/group) were sacrificed 2, 4, 8 and 10 h after injection and methanohc extracts of the brains were prepared as described above. To assess in vivo metabohsm of MPP + and possibly identify any products of this metabolism, 3 different assays were employed. (1) Liquid Scintillation Counting (LSC): counts per minute (cpm) for each isotope were determined by LSC of duplicate 1.0 ml ahquots of the methanolic extracts. Disintegrations per minute (dpm) were calculated by adding known amounts of each isotope to each sample (standard additions method), recounting and correcting for efficiency. MPP + values were calculated based on the specific activity of MPP + and the dpm of each sample. N o significant difference between the elimination o f either isotope was detected; the ratio of 3H/14C remained constant at all time points studied (2 h = 2.87 + 0.07, 4 h = 3.03 + 0.15, 8 h = 2.90 + 0 11, 10 h = 2 . 6 6 + 0 . 0 9 ) and was the same as the ratio in the injection solution (2.88 +0.10). (2) HPLC analysis the following procedure was repeated for samples collected at each o f the 4 time points studied. Aliquots of the extracts of the brains (n = 5/time point) were pooled, concentrated by a stream of nitrogen and assayed by HPLC. Fractions of the eluate were collected at 0.50 min intervals and counted by LSC. H P L C recovery of radmactivity from the extracts was the same as that noted for the [3Hfl4C]MPP+ standard solution (88%). Only a single peak, which co-chromatographed with an MPP + standard, was detected in the pooled sample from each time point (Fig. 2). (3) GC/MS assay, an aliquot of each methanolic extract was assayed for MPP + using GC/MS. No significant difference between MPP + values was observed regardless of whether this was measured by GC/MS, or calculated from 3 H

86

800 j~

:/ \

600 q a. fJ

400-

200

! FRACTION

NUMBER

Fig 2 H P L C analysis of brain extracts from ammals given 1 c.v_-admlmstered rachoactlve M P P ÷ Counts/ m m (CPM) were determined m fractions collected every 0 5 min. Each scan represents data olatmm~d from pooled, concentrated brain extracts of a m m a l s kdled at indicated ttme points (see text). For d a n t y , scans are offset by 50 clam The major peak (fractions 12-18) co-chromatographed with an authentic standard o f M P P ÷ Data shown are for 14C; s]milar results were obtained for 3H

or 14C dpm (Table I), indicating that all the radloactivtty could be accounted for by this single chemical species. While these results are highly compatible with the conclusion that MPP + is not blotransformed in mouse brain, they do not completely rule out the posslbdity that a metabolite of MPP + ts formed and ehminated so rapidly that it escaped detection in this in vivo model. To exclude this possibility, the following in vitro experiment was performed. MPP + (100 #M) was mcubated with bram homogenates prepared from mice as described above. Tnplicate incubations were carried out at 37°C for 1, 8, and 24 h and stopped by the addition of 3 voltmaes of ice-cold acetonitrile. After centrifugatton, an aliquot of the supernatant was analyzed for MPP ÷ by HPLC. No change in MPP + concentration was detected at any time point m this closed system, making tt unlikely that a rapidly eliminated metabolite ts formed by the brain.

TABLE I M P P ÷ C O N C E N T R A T I O N IN W H O L E B R A I N A T V A R I O U S T I M E POINTS A F T E R T H E I N T R A C E R E B R O V E N T R I C U L A R A D M I N I S T R A T I O N O F 3H/~'42 M P P ÷ Values for JH and ~4C were calculated from dpm, and G C / M S values were determined directly as described m text Values are expressed as gg total in extract + S E.M Time

3H

~4C

GC/MS

2 4 8 10

4 7 ±0_5 3 0 ±0_3 15 ± 0 . 2 071±03

4.3 ± 0 5 2 7 ±0_2 15 ±0_3 072±02

47 ±05 2.8 ± 0 3 1 6 ±0_3 082±01

h h h h

87

Taken together, our results address questions raised by prewous studies on the fate of M P P ÷ in the CNS compartment. For example, metabolites other than M P P ÷ have been detected in the brains of rodents following the systemic admimstration of M P T P [1, 15, 20]. Because of the multicompartment distribution and peripheral metabolism of systemically admimstered MPTP, the source of these metabohtes could not be determined [1], raising the posslbihty th.at they may have been generated from M P P ÷ within the CNS compartment. Our results indicate that these other compounds are unlikely to be products of further biotransformation of M P P ÷ w~thin the rodent CNS The fact that M P P ÷ ~s not further metabohzed also effectively rules out biotransformat~on as an explanaaon for Its rapid ehmmatton from the CNS I f blotransformation is not revolved in the removal of M P P + from the brain, the question remains as to what alternative mechanism(s) might be responsible for its rapid disappearance. One possibility is that, in the mouse, M P P + simply diffuses out of the CNS c o m p a r t m e n t into the periphery This seems unlikely in view of the fact that MPP + is a highly polar, charged compound which does not easily cross membranes [6] and does not enter the CNS when admimstered systemically (Irwin et a l , unpublished observation) If M P P + does not diffuse into the brain, then its removal by passive diffusion would not be expected either. Further, if this mechanism explains the rap~d removal of M P P + from the brain of the mouse, one would have to postulate a sequestration mechanism in the monkey that limits the availabdity of M P P + for diffusion Binding of M P P + to neuromelantn, which is present in primate but not rodent brain, m a y represent one such mechanism [5, 1 l]. However, binding to neuromelanin cannot provide a complete explanation because MPP + persists m the nonpigmented areas of monkey brain as well as regions where neuromelanin-containlng neurons are present [1 l] Another, and perhaps more intriguing, possibility is that, m the mouse, MPP + is eliminated from the CNS c o m p a r t m e n t by an active transport system that is either absent or less efficient in the primate. The exponential kinetics of elimination of MPP + reported here provide the first experimental support for this hypothesis. That such a transport system might exist for M P P + should not be too surprising since a number of selective carriers m the cerebral capillary epithehum (CCE) have been identified [7]. Whde some of these are bi-directional, others have been decrlbed which allow for the passage of substrates across capillary membranes in only one direction [2]. Unidirectional systems are possible by wrtue of the fact that the luminal and antiluminal membranes of the CCE appear to be functionally distract [2]. Further studies, both in vitro and in vivo, are needed to determine whether or not a similar process ts involved m the elimination of M P P + from the CNS compartment in mice. The authors wish to thank John Skratt and David Remmler for technical assistance, and to gratefully acknowledge David Rosner and Pamela Schmidt for their help in the preparation of this manuscript. D. DI M. is a recipient of the Lillian Schorr Research Fellowship from the Parkinson's Disease Foundation, New York This work was supported m part by the California Parklnson's Foundation, the United Parkinson Foundation, the Parkinson's Disease Foundation, and the National Institute of Aging (R01 AG07348-01). 1 Arora, P K , Rmchl, N J , Harlk, S 1_ and Sayre, L M , Chemical oxidation of 1-methyl-4-phenyl1,2,3,6-tetrahydropyndme (MPTP) and its m wvo metabohsm m rat brain and hver, Blochem BIO--L-

-

n _ _

~

. . . . . . . .

I

g,,,ql

[II~OOX

i-l,')l

~1

1,.)A,-

I

88 2 Bet.z, A L. and Goldstem, G . W , Polarity of the blood-brain barrier neutral amino acid transport into isolated brain capillanes, Scaence, 202 (1978) 225-227 3 Boyce, S., Kelly, E , Reavill, C., Jenner, P. and Marsden, C.D, Repeated adm,nistratlon of n-methyl4-phenyl-1,2,5,6-tetrahydropyndine to rats is not toxic to striatal dopamine neurones, Biochem Pharmacol., 33 (1984) 1747-1752 4 Chiueh, C C , Markey, S.P., Burns, R S, Johannessen, J.N., Pert, A. and Kopin, I_J, Neurochemlcal and behavioral effects of systemic and mtramgral admimstratlon of N-methyl-4-phenyl-l,2,3,6-tetrahydropyndme m the rat, Eur J_ Pharmacol, 100 (1984) 189-194. 5 D'Amato, R.J_, Alexander, G M , Schwartzmann, R.J., Kltt, C A , Pnce, D L. and Snyder, S.H., Evidence of neuromelanin involvement m MPTP-mduced neurotoxlclty, Nature (Lond_), 327 (1987) 324-326 6 DI Monte, D , Ekstrom, G , Shmka, T., Smith, M T , Trevor, A J. and Castagnoh, Jr_, N , Role of l-methyl-4-phenylpyndmmm ton formation and accumulation m 1-methyl-4-phenyl-l,2,3,6-tetrahydropyndme toxacaty to asolated hepatocytes, Chem Baol. Interact_, 62 (1987) 105-116 7 Goldstem, G W_ and Betz, A L , Blood vessels and the blood-brain barner In A.K. Asbury, G_M McKhann and W I McDonald (Eds.), Daseases of the Nervous System - Clinical Neuroblology, Saunders, Phdadelphm, 1986, pp. 172-184 8 Hallman, H , Lange, J , OIson, L , Str6mberg, I and Jonsson, G , Neurochemlcal and histochemlcal characterization of neurotoxac effects of l-methyl-4-phenyl-l,2,3,6-tetrahydropyndineon brain catecholamme neurones m the mouse, J. Neurochem., 44 0985) 117-127 9 Heakkda, R E , Hess, A and Duvmsm, R C , Dopammergic neurotoxlclty of 1-methyl-4-phenyl1,2,5,6-tetrahydropyndme m mice, Science, 224 (I 984) 1451-453 10 Irwin, I and Langston, J W , Safety and handling of MPTP, Neurology, 35 0985) 619. I l Irwin, I and Langston, J W , Selectwe accumulatmn o f M P P + in the substantm nigra a key to neurotoxlcaty?, Life Scl, 36 0985) 207-212 12 Irwin, I., Langston, J W_ and DeLanney, L E., 4-Phenylpyndme (4-PP) and M P T P the relataonshap between stnatal MPP + concentratmns and neurotoxlclty, Life Sci, 40 (1987) 731-740 13 Irwin, I , Rlcaurte, G A_, DeLanney, L E and Langston, J W , The sensitivity of nigrostnatal dopamine neurons to MPP + does not increase with age, Neuroscl. Lett, 87 (1988) 51-56_ 14 Johannessen, J_N, Chmeh, C C , Burns, R.S and Markey, S_P_, Differences m metabolism of MPTP in the rodent and pnmate parallel differences m sensmvaty to its neurntoxlc effects, Lafe Sea, 36 0985) 219 224. 15 Johannessen, J N , Chmeh, C C , Herkenham, M A , Markey, S P., Burns, R S, Adams, J D and Schuller, H M , Relatmnshlp of the in VlVOmetabolism of MPTP to toxicaty_ In S P Markey, N Castagnoli Jr, A J Trevor and I J Kopm (Eds), MPTP A Neurotoxm Producing a Parkinsoman Syndrome, Academic Press, New York, 1986, pp_ 173-189 16 Langston, J.W, Irwin, I , Langston, E B. and Forno, L S_, l-Methyl-4-phenylpyndmmmion (MPP+): Identlficatmn of a metabolate of MPTP, a toxin selectwe to the substantm nigra, Neuroscl Lett, 48 (1984) 87-92 17 Langston, J W and Irwin, I., MPTP current concepts and controversies, Clin Neuropharmacol, 9 (1986) 485-507 18 Langston, J W , Irwin, I , Langston, E_B_, DeLanney, L E. and Ricaurte, G A , MPTP-mduced parkmsomsm m humans a review of the syndrome and observations relatang to the phenomenon of tardwe toxicity In S P Markey, N. Castagnoll, J r , A J Trevor and I J. Kopan (Eds.), MPTP A Neurotoxm Producing a Parkmsonlan Syndrome, Academic Press, New York, 1986, .pp. 9-21. 19 Langston, J W , Irwin, I. and DeLanney, L.E_, The bmtransformatlon of MPTP and dmposation of MPP ÷ the effects of aging, Life SCl., 40 (1987) 749-754 20 Markey, S P , Johannessen, J N , Chmeh, C C , Burns, R.S and Herkenham, M . A , lntraneuronal generatmn of a pyrldlnlum metabohte may cause drug-induced parkmsomsm, Nature (Lond), 311 (1984) 464-467 21 Rtaehl, N J , Hank, S I , Kalarm, R N_ and Sayre, L M , On the mechanisms underlying I-methyl-4phenyl-l,2,3,6-tetrahydropyndlne neurotoxlcaty. II. Susceptlbihty among mammalian species correlates wath the toxm's metabohc patterns an brain macrovessels and laver, J Pharmacol., Exp Ther, 244 (1988) 443-448 22 Rlcaurte, G A , Langston, J W , DeLanney, L_E, Irwin, I , Peroutka, S J and Forno, L S_, Fate of nlgrostnatal neurons in young mature mice gwen 1-methyl-4-phenyl-l,2,3,6-tetrahydropyndlne