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The effect of diethyldithiocarbamate on the biodisposition of MPTP: an explanation for enhanced neurotoxicity
European Journal of Pharmacology, 141 (1987) 209-217
209
Elsevier EJP 00905
The effect of diethyldithiocarbamate on the biodisposition of MPTP: an explanation for enhanced neurotoxicity I a n Irwin 1, Ellen Y. W u 2, Louis E. D e L a n n e y 1, A n t h o n y T r e v o r 2 a n d J. William L a n g s t o n 1,. t The Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128, and 2 Department of Pharmacology, University of California, San Francisco, CA 94143, U.S.A.
Received 12 May 1987, accepted 16 June 1987
Diethyldithiocarbamate (DDC) has been reported to exacerbate the neurotoxic effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in mice. In this study, the effects of DDC on the biotransformation and distribution of MPTP and 1-methyl-4-phenylpyridinium ion (MPP ÷, the putative toxic metabolite of MPTP) were investigated. When DDC was administered prior to a standardized dosage of MPTP, the initial concentrations of MPTP in striatum, ventral mesencephalon and frontal cortex were markedly increased when compared to animals given MPTP alone. The pre-administration of DDC also produced increased concentrations of MPP ÷ in these regions at all time points studied. Further, the rate of disappearance of MPP ÷ from brain was significantly less in DDC pretreated animals, when compared to animals given MPTP alone. In vitro studies, using either brain homogenates or partially purified MAO-B, showed that DDC enhanced the biotransformation of MPTP. These results suggest that DDC enhances MPTP-induced neurotoxicity by increasing brain concentrations of MPP ÷. Factors contributing to this increase appear to include greater delivery of MPTP to the central nervous system (CNS), increased biotransformation of MPTP to MPP ÷ via MAO, and possibly reduced clearance of MPP ÷ from the brain. Parkinson's disease; Neurotoxicity; MPTP; MPP÷; Dopamine; Biotransformation; Diethyldithiocarbamate (DDC)
I. Introduction During the last three years a great deal has been learned about the mechanism of action of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Langston and Irwin, 1986; Lewin, 1986), a compound which is toxic to the dopaminergic neurons of the substantia nigra (Burns et al., 1983; Langston et al., 1983), and could hold clues to the cause of nigral cell degeneration in Parkinson's disease (Lewin, 1985; Langston and Irwin, 1986). After systemic administration, MPTP gains access to the central nervous system (CNS) and is oxidized to 1-methyl-4-phenylpyridinium ion (MPP ÷) (Langston et al., 1984a; Markey et al.,
* To whom all correspondenceshould be addressed.
1984) by monoamine oxidase B (MAO-B) (Chiba et al., 1984). Once formed, MPP + probably enters catecholaminergic (CA) neurons via the CA uptake system (Chiba et al., 1985; Javitch and Snyder, 1985), where it induces cell death by a mechanism which is as yet unknown (Irwin, 1986; Langston and Irwin, 1986). These observations have led to the discovery of several ways of preventing MPTP toxicity in the mouse, including the irreversible (Markey et al., 1984; Heikkila et al., 1984) and competitive (Melamed and Youdim, 1985) inhibition of MAOB, as well as blocking the CA uptake system (Sundstrom and Jonsson, 1985; Javitch and Snyder, 1985; Ricaurte et al., 1985). Although MAO inhibitors also prevent neurotoxicity in the primate (Langston et al., 1984b; Cohen et al., 1985), it has not yet proved possible to prevent
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
210 MPTP-induced degeneration of the substantia nigra by blocking the CA uptake system in this species (Langston and Irwin, 1986; Schultz et al., 1986). Diethyldithiocarbamate (DDC) has been reported to increase the neurotoxic effects of MPTP (Corsini et al., 1985). This observation is of particular interest because it could open up an entirely new avenue of investigation into the mechanism of action of MPTP. Because D D C inhibits superoxide dismutase (SOD), it was suggested that this finding supported a free radical-mediated mechanism of MPTP toxicity (Corsini et al., 1985) via the redox cycling of MPP + in a manner similar to paraquat (Bus et al., 1974). However, current evidence indicates that MPP + is very stable and unlikely to undergo redox cycling (Sayre et al., 1986). Further, a direct comparison of the effects of MPP + with those of paraquat in isolated hepatocytes (DiMonte et al., 1986) has established that MPP + does not produce the lipid peroxidation or free radical damage which is characteristic of paraquat. We now provide evidence pointing to an alternative explanation for the effect of DDC on MPTP neurotoxicity which involves changes in the metabolism and biodisposition of MPTP and its putative toxic metabolite MPP +.
All dosages are expressed as 'free' drug on a m g / k g basis, and were administered as single i.p. injections. When DDC was given in combination with MPTP, it was administered as a single injection 0.5 h prior to MPTP.
2. Materials and methods
2.5. DDC and MPTP-induced striatal dopamine depletion
2.1. Animals
Male C57B1/6J IMR males, age six to eight weeks, were used for all in vivo studies. Animals were housed five to a cage, under constant temperature and humidity conditions in a room illuminated for 12 h / d a y . All animals were killed by cervical dislocation and their brains rapidly removed. Appropriate regions were dissected, frozen and stored in liquid nitrogen, and analyzed as previously described (Ricaurte et al., 1986). 2.2. Drugs and chemicals
MPTP (Aldrich Chemical Co., Milwaukee, WI) was prepared as previously described (Ricaurte et al., 1986). A sterile solution of the disodium salt of
DDC (Sigma, St. Louis, MO) was prepared immediately before use. Benzylamine was purchased from Aldrich Chemical Company (Milwaukee, WI). Solvents for HPLC were purchased from Burdick and Jackson (Muskegon, MI). All other reagents were analytical grade. 2.3. MA O-B purification
MAO-B was extracted and purified from bovine liver mitochondria by methods previously described (Salach, 1979; Weyler and Salach, 1981). Enzyme activities were determined spectrophotometrically at 3 0 ° C by initial rate measurements with benzylamine as substrate (Salach, 1979). The final enzyme preparation was estimated to be > 50% homogeneous, based on its specific activity of 1 /~M of benzylamine oxidized/min per mg protein, or 1 U n i t / m g (Weyler and Salach, 1981). 2.4. Drug administration
Experimental mice, along with saline controls, were given either D D C alone, MPTP alone, or MPTP in combination with D D C (table 1), and killed one week later. Striatal dopamine determinations were carried out utilizing High Pressure Liquid Chromatography (HPLC) coupled to electrochemical detection (ECD) as previously described (Ricaurte et al., 1986). 2.6. The effect of D D C on M P T P and M P P + concentrations after M P T P treatment
Mice were given either D D C alone, MPTP alone, or D D C in combination with MPTP (fig. 1). Animals were killed at 0.5, 1.0 and 2.0 h, and striata, ventral mesencephalon and frontal cortex were assayed for MPTP and MPP +, using a gas
211 c h r o m a t o g r a p h y / m a s s spectrometry method previously described (Irwin et al., 1987).
2.7. The effects of DDC on the biotransformation of M P T P by homogenates of mouse brain In three separate experiments, the effect of various concentrations of D D C on biotransformation of MPTP in brain homogenates of mice was determined. Whole brains were rapidly homogenized in 22 volumes of 50 mM phosphate buffer, pH 7.4. Aliquots (0.45 ml) of this homogenate were preincubated at 37 ° C after the addition of 0.025 ml buffer containing D D C (0 to 20 mM). Preincubation was carried out for five min, then 0.025 ml MPTP (2 mM) was added. The final concentration of MPTP in the incubation mixture was 100/~M. Finally concentrations of D D C were 0, 12.5, 25, 50, 100, 200, 500 and 1000 #M. Incubations were also carried out using homogenates which had been inactivated by boiling for 10 min and in homogenates which had been preincubated w i t h 10 - 6 M deprenyl for 5 rain before the addition of MPTP or DDC. After 1 h of incubation at 37 ° C, four volumes of cold acetonitrile were added and the MPP + which had been formed was assayed by HPLC as previously described (Langston et al., 1984a; Irwin et al., 1987). Protein was assayed by the method of Lowry et al. (1951).
2.8. The effects of DDC on M P T P oxidation by semi-purified MA O-B
added to 0.99 ml of the assay mixture containing 3.3 mM benzylamine and initial rates of benzaldehyde formation were measured spectrophotometrically.
3. Results
3.1. The effect of DDC on MPTP-induced striatal dopamine depletion MPTP, given as a single intraperitoneal injection, did not produce statistically significant dopamine depletions at doses less than 30 m g / k g (table 1). Although D D C alone had no statistically significant effect on striatal dopamine, the dopamine-depleting effect of MPTP was greatly enhanced when D D C was given prior to MPTP (table 1). For example, the 30 m g / k g dose of MPTP produced only a 50% depletion of dopamine, whereas this same dose of MPTP, when given after DDC, produced a 97% depletion of dopamine.
3.2. The effect of DDC on M P T P and M P P + concentrations in striata, oentral mesencephalon and frontal cortex after M P T P administration MPTP was detected in all three brain regions only in animals killed at the 30 min time point. However, at 30 min MPTP concentrations were TABLE 1
Samples of partially purified MAO-B (0.006 U) in 50 mM sodium phosphate buffer containing 0.2% ( w / v ) Triton X-100, p H 7.2, were pre-incubated with D D C (0.10 to 1.0 mM) at 3 0 ° C for 5 rain. MPTP (3.3 mM) was added and the formation of M P D P + was monitored spectrophotometrically at 343 nm at time points up to 90 min. To assess the effects of D D C on the inactivation of MAO-B by MPTP, 0.6 U of enzyme was incubated at 3 0 ° C in the standard assay mixture with 3.3 mM MPTP and either 0.05, 0.25 or 0.5 mM DDC. Control samples were similarly incubated with 3.3 mM MPTP alone, D D C alone, and in the absence of both MPTP and DDC. Portions (10 /~1) were periodically removed and
Striatal dopamine concentrations in mice receiving increasing dosages of MPTP alone, and in combination with DDC. MPTP was given as a single i.p. injection. DDC (400 mg/kg) was administered 0.5 h prior to MPTP injection. Animals were killed by cervical dislocation, 1 week followingdrug treatment. Striata were rapidly removed and assayed for dopamine as previously described (Ricaurte et al., 1986). Dopamine concentrations are in ng/mgztzS.E.M. Values for saline control were 13.0-+0.32 and for DDC alone (400 mg/kg) were 12.8-+ 0.32. These were not significantlydifferent. MPTP dose
MPTPalone
MPTP+ DDC
10 mg/kg 20 mg/kg 30 mg/kg
12.6_+0.3 (n = 5) 12.1 + 0.5 (n = 5) 6.1 _+0.2 (n = 10) a
9.5 + 0.5 (n = 5) a.b 3.1 _+0.2(n = 5) a.b 0.4 + 0.1 (n = 10) a.b
a Significantly different than control (P < 0.001). b Significantly different from MPTP alone (P < 0.001).
212 m u c h higher in the a n i m a l s receiving the p r i o r injection of D D C (fig. 1). W h e n D D C was given p r i o r to M P T P , the c o n c e n t r a t i o n s of M P P + were higher when c o m p a r e d to a n i m a l s receiving M P T P a l o n e (fig. 2). Differences were a p p a r e n t in the frontal cortex at 0.5 h, a n d at 1.0 a n d 2.0 h M P P + levels were significantly higher in all three regions. T r e a t m e n t with D D C a l o n e p r o d u c e d no d e t e c t a ble M P T P o r M P P + u n d e r these c o n d i t i o n s .
3.3. Effect of DDC on the biotransformation of MPTP by brain homogenates H o m o g e n a t e s of m o u s e whole b r a i n p r o d u c e d M P P + at a rate o f 0.06 _+ 0.002 n m o l / m i n p e r m g protein. T h e a d d i t i o n of D D C resulted in a signific a n t increase in the rate of M P P + p r o d u c t i o n (fig. 3), which was d e p e n d e n t on the c o n c e n t r a t i o n of D D C . Boiling h o m o g e n a t e s p r i o r to i n c u b a t i o n with M P T P e h m i n a t e d the p r o d u c t i o n of M P P +. T h e a d d i t i o n of D D C to b o i l e d h o m o g e n a t e s d i d n o t result in the c o n v e r s i o n of M P T P to M P P +. W h e n i n c u b a t i o n s c o n t a i n e d o n l y M P T P , the add i t i o n of d e p r e n y l (10 -6 M) r e d u c e d M P P + p r o d u c t i o n b y 90% (0.006 n m o l / m i n p e r m g protein).
10.0
[]~] MPTP alone m DDC +MPTP
g
g
5.0
FC
15.0 /
E c2~
g 7 13g..
A
10,0
%
\
\ I
oo.'54
i
oo'. 1
Time (hours)
Fig. 2. Concentrations of MPP + at 0.5, 1.0 and 2.0 h in striata (STR), ventral mesencephalon (VME) and frontal cortex (FC) of mice given 30 mg/kg of MPTP alone (triangles) or 0.5 h after a 400 mg/kg dose of DDC (circles). Values are means + S.E.M. for each group (n = 5group). Concentrations of MPP ÷ in DDC-pretreated animals differed significantly from those in animals given MPTP alone as follows: 0.5 h, FC only (P < 0.005); 1.0 h, STR (P < 0.001), VME (P < 0.005), FC (P < 0.001); 2 h, STR (P < 0.001), VME (P < 0.005), FC (P < 0.001).
A d d i n g 100 btM D D C to these d e p r e n y l - c o n t a i n ing h o m o g e n a t e s d i d not result in the p r o d u c t i o n of a n y a d d i t i o n a l M P P +.
3.4. The effect of DDC on the oxidation of MPTP by partially purified MA O-B
1-13..
0 ~
VME
STR
STR
VME
FC
Fig. 1. Effect of DDC on [MPTP] in various regions of mouse brain following MPTP treatment. Concentration of MPTP in various brain regions 30 min following the administration of a single 30 mg/kg dose of MPTP alone (striped bars), or 0.5 h after a 400 mg/kg dose of DDC (solid bars). Values are means_+ S.E.M. for each group (n = 5/group). Concentrations of MPTP in DDC-pretreated animals differed significantly from those in animals given MPTP alone as follows: striata (STR, P < 0.001); ventral mesencephalon (VME, P < 0.005); frontal cortex (FC P < 0.001).
Purified M A O - B c a t a l y z e d the a l p h a c a r b o n o x i d a t i o n o f M P T P (3.3 m M ) , f o r m i n g its 2-elect r o n o x i d a t i o n p r o d u c t 2 , 3 - M P D P + at an initial rate of 300 n m o l / m i n p e r mg protein. A s shown in fig. 4, the f o r m a t i o n of M P D P + was n o t linear with time, reaching a p l a t e a u at a p p r o x i m a t e l y 60 m i n due to m e c h a n i s m - b a s e d i n a c t i v a t i o n of the e n z y m e (Singer et al., 1985). D D C i n c r e a s e d the initial rate of M P T P o x i d a t i o n b y M A O - B a n d increased the f o r m a t i o n of M P D P + in a concentration-dependent manner. When incubated with 100 /~M M P D P + in buffer, b u t w i t h o u t en-
213
60
g ~= 50 "o
,.° I..,, o (3.
12.5
I
I
I
I
I
50
100
200
300
500
[ DDC] pM
Fig. 3. In vitro enhancement of MPP + production by DDC. The effect of increasing concentrations of DDC on the production of MPP ÷ in whole brain homogenates prepared from mice and incubated with MPTP. Values are means+ S.E.M. of three separate experiments.
zyme, D D C (0.5 m M ) h a d n o effect o n the o x i d a tion of this m e t a b o l i t e . I n e x p e r i m e n t s o n M A O - B i n a c t i v a t i o n b y 3.3 m M M P T P , the half-life of e n z y m e i n a c t i v a t i o n was 29.6 + 9.3 rain. T h e add i t i o n of D D C (0.05, 0.25 or 0.5 m M ) to i n c u b a tions of M A O - B with M P T P d i d n o t significantly alter the half-life for the i n a c t i v a t i o n of the enz y m e b y the n e u r o t o x i n (the half-lives for these c o n c e n t r a t i o n s of D D C were 23.2 + 3.7, 20.8 + 3.9, 28.9 + 11.6 respectively (P > 0.1).
120
100
~60 I ~40
4. D i s c u s s i o n ~" 20
6'0 r,Mt (Mmrtsl
a'0
Fig. 4. Effect of DDC on oxidation of MPTP by partially purified MAO-B. The oxidation of MPTP by partially purified MAO-B in the absence of (solid triangles) and presence of 100 #M (open triangles), 500 #M (solid circles) and 1000 #M (open circles) DDC. Results represent the means of six determinations at different time points.
A direct r e l a t i o n s h i p b e t w e e n striatal M P P + c o n c e n t r a t i o n s a n d the degree of M P T P - i n d u c e d d e p l e t i o n of striatal d o p a m i n e has r e c e n t l y b e e n shown ( I r w i n et at., 1987). F u r t h e r , c o m p o u n d s which i n h i b i t the b i o t r a n s f o r m a t i o n of M P T P a n d thus reduce the c o n c e n t r a t i o n of M P P + in the C N S ( L a n g s t o n et al., 1984b; M a r k e y et al., 1984; H e i k k i l a et al., 1984; Irwin et al., 1987) also r e d u c e the n e u r o t o x i c i t y of M P T P in the r o d e n t
214
and primate. The results reported here suggest that the converse may be true as well. In these studies, DDC-induced enhancement of toxicity is accompanied by dramatic increases in brain concentrations of the putative toxic metabolite of MPTP, MPP +. These differences are detectable thirty min after drug treatment and, by 2 h, MPP + concentrations are three to eight times higher in animals given D D C prior to MPTP administration. Hence, our results suggest that higher levels of MPP + in the CNS explain the effects of D D C on MPTP neurotoxicity. At least three factors may combine to increase the CNS concentrations of MPP + in animals receiving DDC prior to MPTP administration: increased delivery of MPTP to the CNS, reduced elimination of MPP + from the CNS and increased biotransformation of MPTP to MPP +. That the first of these plays a role is suggested by the finding that 30 min after administration, the concentration of MPTP in the CNS was two to three times higher in DDC-treated animals (fig. 1). The mechanism accounting for this increase is at present unclear. One possible explanation is that D D C may reduce the peripheral biotransformation or elimination of MPTP, thereby shifting the burden from the systemic organs to the brain. For example, DDC is an inhibitor of plasma amine oxidase (PAO). This copper-containing enzyme, which is found in the plasma of mammals (Pettersson, 1985), oxidizes monoamines, including benzylamine and phenylethylamine (Pettersson, 1985). It is possible that PAO could account for some fraction of the peripheral metabolism of MPTP and, therefore, its inhibition could result in increased delivery of MPTP to the CNS. Our experiments also suggest that D D C may alter the elimination of MPP +. When animals are given MPTP alone, the concentration of MPP + decreases in a time-dependent manner in all three brain regions studied (fig. 2). The rate of decrease in MPP + concentration is greatest in the FC and the VME, but the concentration of MPP + also decreases with time in the striatum. D D C appears to drastically alter this pattern, significantly decreasing the rate of disappearance of MPP + from the CNS. The persistence of higher MPP + levels over time is difficult to attribute to continued
production of MPP + because MPTP was no longer present at the 1 or 2 h time points. These results suggest that DDC, either directly or indirectly, alters the elimination of MPP + as well. The third and perhaps most interesting factor contributing to DDC-induced enhancement of neurotoxicity is suggested by the in vitro effects of D D C on MPTP biotransformation. Our data provide evidence that D D C increases the rate of biotransformation of MPTP in brain homogenates. This effect was concentration-dependent; the highest concentration of D D C (500/~M) caused nearly a 60% increase in the rate of MPP + production (fig. 3). To date, no other compounds have been described which have this effect. The fact that the MAO-B inhibitor deprenyl abolished this effect suggested that D D C could be interacting with MAO-B to enhance the enzyme activity present in the brain homogenates. To explore this possibility further, in vitro experiments with partially purified MAO-B were carried out. These confirmed that D D C increased the rate of oxidation of MPTP as measured by the formation of its metabolite M P D P + (fig. 4). As shown in this final set of experiments (Section 3.4.), the effect of DDC on the MAO-B mediated oxidation of MPTP does not appear to involve protection against the mechanism-based inactivation of the enzyme caused by MPTP, or the prevention of non-enzymic oxidation of MPDP +. The mechanisms underlying the effect of D D C on the MAO-B-catalyzed oxidation of MPTP remains to be elucidated. MAO-B is a complex enzyme, and at least two different mechanisms have been identified which vary with different substrates (Husain et al., 1982). Many questions remain regarding multiple active sites, the role of metal ions, the significance of membrane and protein environments, and the biological regulation of this enzyme (Murphy, 1978; Von Korff, 1979; Youdim and Holzbauer, 1976). Physiological increases in brain MAO activity, associated with changes in endogenous steroids, adrenalectomy or hypophysectomy have been noted (for review see Sourkes, 1979; Youdim and Holzbauer, 1976) and an 'activating factor' for platelet and rat liver MAO has been reported to be present in human plasma (Yu and Bolton, 1979). However, to the
215 best of our knowledge D D C is the first specific compound observed which stimulates increased enzyme activity. For this reason, D D C may prove a useful tool in understanding the function and regulation of this important enzyme. For example, determining whether or not the ability to increase M A O activity is shared by other chelating agents may help clarify the role of metal ions in MAO activity and regulation. The removal of metals via chelation could reduce the formation of free radicals resulting in increased levels of peroxide. This action could be important in view of the recent demonstration that H 2 0 2 in the absence of catalase inhibition induces M A O activity (Konradi et al., 1986). It will also be of particular interest to determine whether or not D D C enhances the biotransformation of other MAO substrates, particularly other neurotoxic tetrahydropyridines (Youngster et al., 1986; Wilkening et al., 1986; Fuller and Hemrick-Luecke, 1986; Johannessen et al., 1987; Zimmerman et al., 1986). It is interesting to note that changes in the biodisposition and neurotoxicity of MPTP after D D C administration parallel those which are seen with age (Ricaurte et al., 1987; Langston et al., 1987). Compared to young animals, the administration of equivalent doses of MPTP to older animals results in greater DA depletions, with higher concentrations of MPP ÷ in the CNS. Further, homogenates of brain tissue from older animals exhibit enhanced biotransformation of MPTP and this can account, at least in part, for the increased susceptibility of older animals to MPTP (Ricaurte et al., 1986; Langston et al., 1987). It is possible, therefore, that D D C may also provide a useful tool for studying the age-related effects of MPTP. That D D C exacerbates the neurotoxicity of MPTP by enhancing or amplifying factors which produce, deliver and maintain increased concentrations of toxin in the CNS may have relevance to the usefulness of MPTP in the study of Parkinson's disease. Endogenous (Glover et al., 1983), and naturally occurring (Irwin et al., 1987) inhibitors of MPTP biotransformation have previously been described, and factors present in human plasma which increase M A O activity have also been reported (Yu and Bolton, 1979). Our
observation regarding the activation of an MAOB-dependent pathway raises the possibility that the activity of this important enzyme may be enhanced by endogenous, dietary or other factors. If this enzyme serves as a major xenobiotic metabolizing system for the CNS, then such factors could play a role in the vulnerability of the brain to toxic and perhaps neurodegenerative processes.
Acknowledgements The authors wish to thank John Skratt and Lorrene DavisRitchie for technical assistance. This work was supported in part by the United Parkinson Foundation, the Retirement Research Foundation, the Institute for Medical Research, Santa Clara Valley Medical Center, and NIEHS Grant 1 R01 ES03697-03.
References Bums, R.S., C. Chieuh, S.P. Markey, M.H. Ebert, D.M. Jacobowitz and I.J. Kopin, 1983, A primate model of parkinsonism: Selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by MPTP, Proc. Natl. Acad. Sci. 80, 4546. Bus, J.S., S.D. Aust and J.E. Gibson, 1974, Superoxide- and singlet oxygen-catalyzedlipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity, Biochem. Biophys. Res. Commun. 58, 749. Chiba, K., A. Trevor and N. Castagnoli, Jr., 1984, Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase, Biochem. Biophys. Res. Commun. 120, 574. Chiba, K., A.J. Trevor and N. Castagnoli, Jr., 1985, Active uptake of MPP÷, a metabolite of MPTP, by brain synaptosomes, Biochem. Biophys. Res. Commun. 128, 1229. Cohen, G., P. Pasik, B. Cohen, A. Leist, C. Mytilineou and M.D. Yahr, 1985, Pargyline and deprenyl prevent the neurotoxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) in monkeys, European J. Pharmacol. 106, 209. Corsini, G.U., S. Pintus, C.C. Chiueh, J.F. Weiss and I.J. Kopin, 1985, 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) neurotoxicityin ntice is enhanced by pretreatment with diethyldithiocarbamate, European J. Pharmacol. 119, 127. DiMonte, D., M.S. Sandy, G. Ekstrom and M.T. Smith, 1986, Comparative studies on the mechanisms of paraquat and 1-methyl-4-phenylpyridine (MPP÷ ) cytotoxicity,Biochem. Biophys. Res. Commun. 137, 303. Fuller, R.W. and S.K. Hemrick-Luecke, 1986, Persistent depletion of striatal dopamine in mice by m-hydroxy-MPTP, Res. Comm. Chem. Pathol. Pharmacol. 53 (2), 167.
216 Glover, V., I. Armando, A. Clow and M. Sandier, 1983, Endogenous urinary monoamine oxidase inhibitor: The benzodiazepine connection, Mod. Probl. Pharmacopsychiat. 19, 118. Heikkila, R.E., L. Manzino, F.S. Cabbat and R.C. Duvoisin, 1984, Protection against the dopaminergic neurotoxicity of 1-methyl-4-phenyl-l,2,5,6-tetrahydropyridine by monoamine oxidase inhibitors, Nature 311,467. Husain, M., D.E. Edmundson and T.P. Singer, 1982, Kinetic studies in the catalytic mechanism of liver monoamine oxidase, Biochemistry 21, 595. Irwin, I., 1986, The neurotoxin 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP): A key to Parkinson's disease?, Pharmacol. Res. 3, 7. Irwin, I., J.W. Langston and L.E. DeLanney, 1987, 4-Phenylpyridine (4PP) and MPTP: The relationship between striatal MPP + concentrations and neurotoxicity, Life Sci. 40, 731. Javitch, J.A. and S.H. Snyder, 1985, Uptake of MPP(+) by dopamine neurons explains selectivity of parkinsonism-inducing neurotoxin, MPTP, European J. Pharmacol. 106, 455. Johannessen, J.N., J.M. Savitt, C.J. Markey, J.P. Bacon, A. Weisz, D.S. Hanselman and S.P. Markey, 1987, I. The development of amine substituted analogues of MPTP as unique tools for the study of MPTP toxicity and Parkinson's disease, Life Sci. 40, 697. Konradi, C., P. Riederer and M.B.H. Youdim, 1986, Hydrogen peroxide enhances the activity of monoamine oxidase type-B but not of type-A: A pilot study, J. Neural. Transm. (Suppl.) 22, 61. Langston, J.W., P.A. Ballard, J.W. Tetrud and I. Irwin, 1983, Chronic parkinsonism in humans due to a product of meperidine-analog synthesis, Science 219, 979. Langston, J.W. and I. Irwin, 1986, MPTP: Current concepts and controversies, Clin. Neuropharmacol. 9, 485. Langston, J.W., I. Irwin and L.E. DeLanney, 1987, The biotransformation of MPTP and disposition of MPP+: The effects of aging, Life Sci. 40, 749. Langston, J.W., I. Irwin, E.B. Langston and L.S. Forno, 1984a, 1-Methyl-4-phenylpyridinium ion (MPP ÷ ): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra, Neurosci. Lett. 48, 87. Langston, J.W., I. Irwin, E.B. Langston and L.S. Fomo, 1984b, Pargyline prevents MPTP-induced parkinsonism in primates, Science 225, 1480. Lewin, R., 1985, Parkinson's disease: An environmental cause? Science 229, 257. Lewin, R., 1986, Age factors loom in parkinsonian research, Science 234, 1200. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Markey, S.P., J.N. Johannessen, C.C. Chiueh, R.S. Bums and M.A. Herkenham, 1984, Intraneuronal generation of a pyridinium metabolite may cause drag-induced parkinsonism, Nature 311,464. Melamed, E. and M.B. Youdim, 1985, Prevention of dopaminergic toxicity of MPTP in mice by phenylethyl-
amine, a specific substrate of type B monoamine oxidase, Br. J. Pharmacol. 86, 529. Murphy, D.L., 1978, Substrate-selective monoamine oxidases inhibitor, tissue, species and functional differences, Biochem. Pharmacol. 27, 1889. Pettersson, G., 1985, Plasma amine oxidase, in: Structure and Functions of Amine Oxidases, ed. B. Mondovi (CRC Press, Inc., Boca Raton, Florida) p. 105. Ricaurte, G.A., I. Irwin, L.S. Forno, L.E. DeLanney, E.B. Langston and J.W. Langston, 1987, Aging and MPTP-induced degeneration of dopaminergic neurons in the substantia nigra, Brain Res. 403, 43. Ricaurte, G.A., J.W. Langston, L.E. DeLanney, I. Irwin and J.D. Brooks, 1985, Dopamine uptake blockers protect against the dopamine depleting effect of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) in the mouse striatum, Neurosci. Lett. 59, 259. Ricaurte, G.A., J.W. Langston, L.E. DeLanney, I. Irwin, S.J. Peroutka and L.S. Forno, 1986, Fate of nigrostriatal neurons in young mature mice given 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP): A neurochemical and morphological reassessment, Brain Res. 376 (1), 117. Salach, J.l., 1979, Monoamine oxidase from beef liver mitochondria: Simplified isolation procedure, properties, and determination of its cysteinyl flavin content, Arch. Biochem. Biophys. 192 (1), 128. Sayre, L.M., P.K. Arora, S.C. Feke and F.L. Urbach, 1986, Mechanism of induction of Parkinson's disease by 1methyl-4-phenyl-l,2,3,6-tetrahydropyridine(MPTP). Chemical and electrochemical characterization of a geminaldimethyl-blocked analog of a postulated toxic metabolite, J. Amer. Chem. Soc, 108, 2464. Schultz, W., E. Scarnati, E. Sundstrom, T. Tsutsumi and G: Jonsson, 1986, The catecholamine uptake blocker nomifensine protects against MPTP-induced parkinsonism in monkeys, Exp. Brain Res. 63, 216. Singer, T.P., J.I. Salach and D. Crabtree, 1985, Reversible inhibition and mechanism-based irreversible inactivation of monoamine oxidases by l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), Biochem. Biophys. Res. Commun. 127, 707. Sourkes, T.L, 1979, Influence of hormones, vitamins and metals on monoamine oxidase activity, in: Monoamine Oxidase: Structure, Function and Altered Functions, eds. T.P. Singer, R.W. Von Korff and D.L. Murphy (Academic Press, New York) p. 291. Sundstrom, E. and G. Jonsson, 1985, Pharmacological interference with the neurotoxic action of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) on central catecholamine neurons in the mouse, European J. Pharmacol. 110, 293. Von Korff, R.W., 1979, Monoamine oxidase: Unanswered questions, in: Monoamine Oxidase: Structure, Function and Altered Functions, eds. T.P. Singer, R.W. Von Korff and D.L. Murphy (Academic Press, New York) p. 1. Weyler, W. and J.I. Salach, 1981, Iron content and spectral properties of highly purified bovine fiver monoamine oxidase, Arch. Biochem. Biophys. 212 (1), 147.
217 Wilkening, D., V.G. Vernier, L.E. Arthaud, G. Treacy, J.P. Kenney, V.J. Nickolson, R. Clark, D.H. Smith, C. Smith and G. Boswell, 1986, A parkinson-like neurologic deficit in primates is caused by a novel 4-substituted piperidine, Brain ires. 368, 239. Youdim, M.B.H. and M. Holzbauer, 1976, Physiological aspects of the oxidative deamination of monoamines, in: Monoamine Oxidase and its Inhibition, CIBA Foundation Symposium 39 (Elsevier, North-Holland) p. 105. Youngster, S.K., R.C. Duvoisin, A. Hess, P.K. Sonsalla, M.V. Kindt and R.E. Heikkila, 1986, 1-Methyl-4-(2'-methyl-
phenyl)-l,2,3,6-tetrahydropyridine (2'-CH3-MPTP) is a. more potent dopaminergic neurotoxin than MPTP in mice, European J. Pharmacol. 122, 283. Yu, P.H. and A.A. Boulton, 1979, Activation of platelet monoamine oxidase by plasma in the human, Life Sci. 25, 31. Zimmerman, D.M., B.E. Cantrell, J.K. Reel, S.K. HemrickLuecke and R.W. Fuller, 1986, Characterization of the neurotoxic potential of m-methoxy-MPTP and the use of its n-ethyl analogue as a means of avoiding exposure to a possible parkinsonism-causing agent, J. Med. Chem. 29, 1517.
209
Elsevier EJP 00905
The effect of diethyldithiocarbamate on the biodisposition of MPTP: an explanation for enhanced neurotoxicity I a n Irwin 1, Ellen Y. W u 2, Louis E. D e L a n n e y 1, A n t h o n y T r e v o r 2 a n d J. William L a n g s t o n 1,. t The Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128, and 2 Department of Pharmacology, University of California, San Francisco, CA 94143, U.S.A.
Received 12 May 1987, accepted 16 June 1987
Diethyldithiocarbamate (DDC) has been reported to exacerbate the neurotoxic effects of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in mice. In this study, the effects of DDC on the biotransformation and distribution of MPTP and 1-methyl-4-phenylpyridinium ion (MPP ÷, the putative toxic metabolite of MPTP) were investigated. When DDC was administered prior to a standardized dosage of MPTP, the initial concentrations of MPTP in striatum, ventral mesencephalon and frontal cortex were markedly increased when compared to animals given MPTP alone. The pre-administration of DDC also produced increased concentrations of MPP ÷ in these regions at all time points studied. Further, the rate of disappearance of MPP ÷ from brain was significantly less in DDC pretreated animals, when compared to animals given MPTP alone. In vitro studies, using either brain homogenates or partially purified MAO-B, showed that DDC enhanced the biotransformation of MPTP. These results suggest that DDC enhances MPTP-induced neurotoxicity by increasing brain concentrations of MPP ÷. Factors contributing to this increase appear to include greater delivery of MPTP to the central nervous system (CNS), increased biotransformation of MPTP to MPP ÷ via MAO, and possibly reduced clearance of MPP ÷ from the brain. Parkinson's disease; Neurotoxicity; MPTP; MPP÷; Dopamine; Biotransformation; Diethyldithiocarbamate (DDC)
I. Introduction During the last three years a great deal has been learned about the mechanism of action of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (Langston and Irwin, 1986; Lewin, 1986), a compound which is toxic to the dopaminergic neurons of the substantia nigra (Burns et al., 1983; Langston et al., 1983), and could hold clues to the cause of nigral cell degeneration in Parkinson's disease (Lewin, 1985; Langston and Irwin, 1986). After systemic administration, MPTP gains access to the central nervous system (CNS) and is oxidized to 1-methyl-4-phenylpyridinium ion (MPP ÷) (Langston et al., 1984a; Markey et al.,
* To whom all correspondenceshould be addressed.
1984) by monoamine oxidase B (MAO-B) (Chiba et al., 1984). Once formed, MPP + probably enters catecholaminergic (CA) neurons via the CA uptake system (Chiba et al., 1985; Javitch and Snyder, 1985), where it induces cell death by a mechanism which is as yet unknown (Irwin, 1986; Langston and Irwin, 1986). These observations have led to the discovery of several ways of preventing MPTP toxicity in the mouse, including the irreversible (Markey et al., 1984; Heikkila et al., 1984) and competitive (Melamed and Youdim, 1985) inhibition of MAOB, as well as blocking the CA uptake system (Sundstrom and Jonsson, 1985; Javitch and Snyder, 1985; Ricaurte et al., 1985). Although MAO inhibitors also prevent neurotoxicity in the primate (Langston et al., 1984b; Cohen et al., 1985), it has not yet proved possible to prevent
0014-2999/87/$03.50 © 1987 ElsevierSciencePublishers B.V. (BiomedicalDivision)
210 MPTP-induced degeneration of the substantia nigra by blocking the CA uptake system in this species (Langston and Irwin, 1986; Schultz et al., 1986). Diethyldithiocarbamate (DDC) has been reported to increase the neurotoxic effects of MPTP (Corsini et al., 1985). This observation is of particular interest because it could open up an entirely new avenue of investigation into the mechanism of action of MPTP. Because D D C inhibits superoxide dismutase (SOD), it was suggested that this finding supported a free radical-mediated mechanism of MPTP toxicity (Corsini et al., 1985) via the redox cycling of MPP + in a manner similar to paraquat (Bus et al., 1974). However, current evidence indicates that MPP + is very stable and unlikely to undergo redox cycling (Sayre et al., 1986). Further, a direct comparison of the effects of MPP + with those of paraquat in isolated hepatocytes (DiMonte et al., 1986) has established that MPP + does not produce the lipid peroxidation or free radical damage which is characteristic of paraquat. We now provide evidence pointing to an alternative explanation for the effect of DDC on MPTP neurotoxicity which involves changes in the metabolism and biodisposition of MPTP and its putative toxic metabolite MPP +.
All dosages are expressed as 'free' drug on a m g / k g basis, and were administered as single i.p. injections. When DDC was given in combination with MPTP, it was administered as a single injection 0.5 h prior to MPTP.
2. Materials and methods
2.5. DDC and MPTP-induced striatal dopamine depletion
2.1. Animals
Male C57B1/6J IMR males, age six to eight weeks, were used for all in vivo studies. Animals were housed five to a cage, under constant temperature and humidity conditions in a room illuminated for 12 h / d a y . All animals were killed by cervical dislocation and their brains rapidly removed. Appropriate regions were dissected, frozen and stored in liquid nitrogen, and analyzed as previously described (Ricaurte et al., 1986). 2.2. Drugs and chemicals
MPTP (Aldrich Chemical Co., Milwaukee, WI) was prepared as previously described (Ricaurte et al., 1986). A sterile solution of the disodium salt of
DDC (Sigma, St. Louis, MO) was prepared immediately before use. Benzylamine was purchased from Aldrich Chemical Company (Milwaukee, WI). Solvents for HPLC were purchased from Burdick and Jackson (Muskegon, MI). All other reagents were analytical grade. 2.3. MA O-B purification
MAO-B was extracted and purified from bovine liver mitochondria by methods previously described (Salach, 1979; Weyler and Salach, 1981). Enzyme activities were determined spectrophotometrically at 3 0 ° C by initial rate measurements with benzylamine as substrate (Salach, 1979). The final enzyme preparation was estimated to be > 50% homogeneous, based on its specific activity of 1 /~M of benzylamine oxidized/min per mg protein, or 1 U n i t / m g (Weyler and Salach, 1981). 2.4. Drug administration
Experimental mice, along with saline controls, were given either D D C alone, MPTP alone, or MPTP in combination with D D C (table 1), and killed one week later. Striatal dopamine determinations were carried out utilizing High Pressure Liquid Chromatography (HPLC) coupled to electrochemical detection (ECD) as previously described (Ricaurte et al., 1986). 2.6. The effect of D D C on M P T P and M P P + concentrations after M P T P treatment
Mice were given either D D C alone, MPTP alone, or D D C in combination with MPTP (fig. 1). Animals were killed at 0.5, 1.0 and 2.0 h, and striata, ventral mesencephalon and frontal cortex were assayed for MPTP and MPP +, using a gas
211 c h r o m a t o g r a p h y / m a s s spectrometry method previously described (Irwin et al., 1987).
2.7. The effects of DDC on the biotransformation of M P T P by homogenates of mouse brain In three separate experiments, the effect of various concentrations of D D C on biotransformation of MPTP in brain homogenates of mice was determined. Whole brains were rapidly homogenized in 22 volumes of 50 mM phosphate buffer, pH 7.4. Aliquots (0.45 ml) of this homogenate were preincubated at 37 ° C after the addition of 0.025 ml buffer containing D D C (0 to 20 mM). Preincubation was carried out for five min, then 0.025 ml MPTP (2 mM) was added. The final concentration of MPTP in the incubation mixture was 100/~M. Finally concentrations of D D C were 0, 12.5, 25, 50, 100, 200, 500 and 1000 #M. Incubations were also carried out using homogenates which had been inactivated by boiling for 10 min and in homogenates which had been preincubated w i t h 10 - 6 M deprenyl for 5 rain before the addition of MPTP or DDC. After 1 h of incubation at 37 ° C, four volumes of cold acetonitrile were added and the MPP + which had been formed was assayed by HPLC as previously described (Langston et al., 1984a; Irwin et al., 1987). Protein was assayed by the method of Lowry et al. (1951).
2.8. The effects of DDC on M P T P oxidation by semi-purified MA O-B
added to 0.99 ml of the assay mixture containing 3.3 mM benzylamine and initial rates of benzaldehyde formation were measured spectrophotometrically.
3. Results
3.1. The effect of DDC on MPTP-induced striatal dopamine depletion MPTP, given as a single intraperitoneal injection, did not produce statistically significant dopamine depletions at doses less than 30 m g / k g (table 1). Although D D C alone had no statistically significant effect on striatal dopamine, the dopamine-depleting effect of MPTP was greatly enhanced when D D C was given prior to MPTP (table 1). For example, the 30 m g / k g dose of MPTP produced only a 50% depletion of dopamine, whereas this same dose of MPTP, when given after DDC, produced a 97% depletion of dopamine.
3.2. The effect of DDC on M P T P and M P P + concentrations in striata, oentral mesencephalon and frontal cortex after M P T P administration MPTP was detected in all three brain regions only in animals killed at the 30 min time point. However, at 30 min MPTP concentrations were TABLE 1
Samples of partially purified MAO-B (0.006 U) in 50 mM sodium phosphate buffer containing 0.2% ( w / v ) Triton X-100, p H 7.2, were pre-incubated with D D C (0.10 to 1.0 mM) at 3 0 ° C for 5 rain. MPTP (3.3 mM) was added and the formation of M P D P + was monitored spectrophotometrically at 343 nm at time points up to 90 min. To assess the effects of D D C on the inactivation of MAO-B by MPTP, 0.6 U of enzyme was incubated at 3 0 ° C in the standard assay mixture with 3.3 mM MPTP and either 0.05, 0.25 or 0.5 mM DDC. Control samples were similarly incubated with 3.3 mM MPTP alone, D D C alone, and in the absence of both MPTP and DDC. Portions (10 /~1) were periodically removed and
Striatal dopamine concentrations in mice receiving increasing dosages of MPTP alone, and in combination with DDC. MPTP was given as a single i.p. injection. DDC (400 mg/kg) was administered 0.5 h prior to MPTP injection. Animals were killed by cervical dislocation, 1 week followingdrug treatment. Striata were rapidly removed and assayed for dopamine as previously described (Ricaurte et al., 1986). Dopamine concentrations are in ng/mgztzS.E.M. Values for saline control were 13.0-+0.32 and for DDC alone (400 mg/kg) were 12.8-+ 0.32. These were not significantlydifferent. MPTP dose
MPTPalone
MPTP+ DDC
10 mg/kg 20 mg/kg 30 mg/kg
12.6_+0.3 (n = 5) 12.1 + 0.5 (n = 5) 6.1 _+0.2 (n = 10) a
9.5 + 0.5 (n = 5) a.b 3.1 _+0.2(n = 5) a.b 0.4 + 0.1 (n = 10) a.b
a Significantly different than control (P < 0.001). b Significantly different from MPTP alone (P < 0.001).
212 m u c h higher in the a n i m a l s receiving the p r i o r injection of D D C (fig. 1). W h e n D D C was given p r i o r to M P T P , the c o n c e n t r a t i o n s of M P P + were higher when c o m p a r e d to a n i m a l s receiving M P T P a l o n e (fig. 2). Differences were a p p a r e n t in the frontal cortex at 0.5 h, a n d at 1.0 a n d 2.0 h M P P + levels were significantly higher in all three regions. T r e a t m e n t with D D C a l o n e p r o d u c e d no d e t e c t a ble M P T P o r M P P + u n d e r these c o n d i t i o n s .
3.3. Effect of DDC on the biotransformation of MPTP by brain homogenates H o m o g e n a t e s of m o u s e whole b r a i n p r o d u c e d M P P + at a rate o f 0.06 _+ 0.002 n m o l / m i n p e r m g protein. T h e a d d i t i o n of D D C resulted in a signific a n t increase in the rate of M P P + p r o d u c t i o n (fig. 3), which was d e p e n d e n t on the c o n c e n t r a t i o n of D D C . Boiling h o m o g e n a t e s p r i o r to i n c u b a t i o n with M P T P e h m i n a t e d the p r o d u c t i o n of M P P +. T h e a d d i t i o n of D D C to b o i l e d h o m o g e n a t e s d i d n o t result in the c o n v e r s i o n of M P T P to M P P +. W h e n i n c u b a t i o n s c o n t a i n e d o n l y M P T P , the add i t i o n of d e p r e n y l (10 -6 M) r e d u c e d M P P + p r o d u c t i o n b y 90% (0.006 n m o l / m i n p e r m g protein).
10.0
[]~] MPTP alone m DDC +MPTP
g
g
5.0
FC
15.0 /
E c2~
g 7 13g..
A
10,0
%
\
\ I
oo.'54
i
oo'. 1
Time (hours)
Fig. 2. Concentrations of MPP + at 0.5, 1.0 and 2.0 h in striata (STR), ventral mesencephalon (VME) and frontal cortex (FC) of mice given 30 mg/kg of MPTP alone (triangles) or 0.5 h after a 400 mg/kg dose of DDC (circles). Values are means + S.E.M. for each group (n = 5group). Concentrations of MPP ÷ in DDC-pretreated animals differed significantly from those in animals given MPTP alone as follows: 0.5 h, FC only (P < 0.005); 1.0 h, STR (P < 0.001), VME (P < 0.005), FC (P < 0.001); 2 h, STR (P < 0.001), VME (P < 0.005), FC (P < 0.001).
A d d i n g 100 btM D D C to these d e p r e n y l - c o n t a i n ing h o m o g e n a t e s d i d not result in the p r o d u c t i o n of a n y a d d i t i o n a l M P P +.
3.4. The effect of DDC on the oxidation of MPTP by partially purified MA O-B
1-13..
0 ~
VME
STR
STR
VME
FC
Fig. 1. Effect of DDC on [MPTP] in various regions of mouse brain following MPTP treatment. Concentration of MPTP in various brain regions 30 min following the administration of a single 30 mg/kg dose of MPTP alone (striped bars), or 0.5 h after a 400 mg/kg dose of DDC (solid bars). Values are means_+ S.E.M. for each group (n = 5/group). Concentrations of MPTP in DDC-pretreated animals differed significantly from those in animals given MPTP alone as follows: striata (STR, P < 0.001); ventral mesencephalon (VME, P < 0.005); frontal cortex (FC P < 0.001).
Purified M A O - B c a t a l y z e d the a l p h a c a r b o n o x i d a t i o n o f M P T P (3.3 m M ) , f o r m i n g its 2-elect r o n o x i d a t i o n p r o d u c t 2 , 3 - M P D P + at an initial rate of 300 n m o l / m i n p e r mg protein. A s shown in fig. 4, the f o r m a t i o n of M P D P + was n o t linear with time, reaching a p l a t e a u at a p p r o x i m a t e l y 60 m i n due to m e c h a n i s m - b a s e d i n a c t i v a t i o n of the e n z y m e (Singer et al., 1985). D D C i n c r e a s e d the initial rate of M P T P o x i d a t i o n b y M A O - B a n d increased the f o r m a t i o n of M P D P + in a concentration-dependent manner. When incubated with 100 /~M M P D P + in buffer, b u t w i t h o u t en-
213
60
g ~= 50 "o
,.° I..,, o (3.
12.5
I
I
I
I
I
50
100
200
300
500
[ DDC] pM
Fig. 3. In vitro enhancement of MPP + production by DDC. The effect of increasing concentrations of DDC on the production of MPP ÷ in whole brain homogenates prepared from mice and incubated with MPTP. Values are means+ S.E.M. of three separate experiments.
zyme, D D C (0.5 m M ) h a d n o effect o n the o x i d a tion of this m e t a b o l i t e . I n e x p e r i m e n t s o n M A O - B i n a c t i v a t i o n b y 3.3 m M M P T P , the half-life of e n z y m e i n a c t i v a t i o n was 29.6 + 9.3 rain. T h e add i t i o n of D D C (0.05, 0.25 or 0.5 m M ) to i n c u b a tions of M A O - B with M P T P d i d n o t significantly alter the half-life for the i n a c t i v a t i o n of the enz y m e b y the n e u r o t o x i n (the half-lives for these c o n c e n t r a t i o n s of D D C were 23.2 + 3.7, 20.8 + 3.9, 28.9 + 11.6 respectively (P > 0.1).
120
100
~60 I ~40
4. D i s c u s s i o n ~" 20
6'0 r,Mt (Mmrtsl
a'0
Fig. 4. Effect of DDC on oxidation of MPTP by partially purified MAO-B. The oxidation of MPTP by partially purified MAO-B in the absence of (solid triangles) and presence of 100 #M (open triangles), 500 #M (solid circles) and 1000 #M (open circles) DDC. Results represent the means of six determinations at different time points.
A direct r e l a t i o n s h i p b e t w e e n striatal M P P + c o n c e n t r a t i o n s a n d the degree of M P T P - i n d u c e d d e p l e t i o n of striatal d o p a m i n e has r e c e n t l y b e e n shown ( I r w i n et at., 1987). F u r t h e r , c o m p o u n d s which i n h i b i t the b i o t r a n s f o r m a t i o n of M P T P a n d thus reduce the c o n c e n t r a t i o n of M P P + in the C N S ( L a n g s t o n et al., 1984b; M a r k e y et al., 1984; H e i k k i l a et al., 1984; Irwin et al., 1987) also r e d u c e the n e u r o t o x i c i t y of M P T P in the r o d e n t
214
and primate. The results reported here suggest that the converse may be true as well. In these studies, DDC-induced enhancement of toxicity is accompanied by dramatic increases in brain concentrations of the putative toxic metabolite of MPTP, MPP +. These differences are detectable thirty min after drug treatment and, by 2 h, MPP + concentrations are three to eight times higher in animals given D D C prior to MPTP administration. Hence, our results suggest that higher levels of MPP + in the CNS explain the effects of D D C on MPTP neurotoxicity. At least three factors may combine to increase the CNS concentrations of MPP + in animals receiving DDC prior to MPTP administration: increased delivery of MPTP to the CNS, reduced elimination of MPP + from the CNS and increased biotransformation of MPTP to MPP +. That the first of these plays a role is suggested by the finding that 30 min after administration, the concentration of MPTP in the CNS was two to three times higher in DDC-treated animals (fig. 1). The mechanism accounting for this increase is at present unclear. One possible explanation is that D D C may reduce the peripheral biotransformation or elimination of MPTP, thereby shifting the burden from the systemic organs to the brain. For example, DDC is an inhibitor of plasma amine oxidase (PAO). This copper-containing enzyme, which is found in the plasma of mammals (Pettersson, 1985), oxidizes monoamines, including benzylamine and phenylethylamine (Pettersson, 1985). It is possible that PAO could account for some fraction of the peripheral metabolism of MPTP and, therefore, its inhibition could result in increased delivery of MPTP to the CNS. Our experiments also suggest that D D C may alter the elimination of MPP +. When animals are given MPTP alone, the concentration of MPP + decreases in a time-dependent manner in all three brain regions studied (fig. 2). The rate of decrease in MPP + concentration is greatest in the FC and the VME, but the concentration of MPP + also decreases with time in the striatum. D D C appears to drastically alter this pattern, significantly decreasing the rate of disappearance of MPP + from the CNS. The persistence of higher MPP + levels over time is difficult to attribute to continued
production of MPP + because MPTP was no longer present at the 1 or 2 h time points. These results suggest that DDC, either directly or indirectly, alters the elimination of MPP + as well. The third and perhaps most interesting factor contributing to DDC-induced enhancement of neurotoxicity is suggested by the in vitro effects of D D C on MPTP biotransformation. Our data provide evidence that D D C increases the rate of biotransformation of MPTP in brain homogenates. This effect was concentration-dependent; the highest concentration of D D C (500/~M) caused nearly a 60% increase in the rate of MPP + production (fig. 3). To date, no other compounds have been described which have this effect. The fact that the MAO-B inhibitor deprenyl abolished this effect suggested that D D C could be interacting with MAO-B to enhance the enzyme activity present in the brain homogenates. To explore this possibility further, in vitro experiments with partially purified MAO-B were carried out. These confirmed that D D C increased the rate of oxidation of MPTP as measured by the formation of its metabolite M P D P + (fig. 4). As shown in this final set of experiments (Section 3.4.), the effect of DDC on the MAO-B mediated oxidation of MPTP does not appear to involve protection against the mechanism-based inactivation of the enzyme caused by MPTP, or the prevention of non-enzymic oxidation of MPDP +. The mechanisms underlying the effect of D D C on the MAO-B-catalyzed oxidation of MPTP remains to be elucidated. MAO-B is a complex enzyme, and at least two different mechanisms have been identified which vary with different substrates (Husain et al., 1982). Many questions remain regarding multiple active sites, the role of metal ions, the significance of membrane and protein environments, and the biological regulation of this enzyme (Murphy, 1978; Von Korff, 1979; Youdim and Holzbauer, 1976). Physiological increases in brain MAO activity, associated with changes in endogenous steroids, adrenalectomy or hypophysectomy have been noted (for review see Sourkes, 1979; Youdim and Holzbauer, 1976) and an 'activating factor' for platelet and rat liver MAO has been reported to be present in human plasma (Yu and Bolton, 1979). However, to the
215 best of our knowledge D D C is the first specific compound observed which stimulates increased enzyme activity. For this reason, D D C may prove a useful tool in understanding the function and regulation of this important enzyme. For example, determining whether or not the ability to increase M A O activity is shared by other chelating agents may help clarify the role of metal ions in MAO activity and regulation. The removal of metals via chelation could reduce the formation of free radicals resulting in increased levels of peroxide. This action could be important in view of the recent demonstration that H 2 0 2 in the absence of catalase inhibition induces M A O activity (Konradi et al., 1986). It will also be of particular interest to determine whether or not D D C enhances the biotransformation of other MAO substrates, particularly other neurotoxic tetrahydropyridines (Youngster et al., 1986; Wilkening et al., 1986; Fuller and Hemrick-Luecke, 1986; Johannessen et al., 1987; Zimmerman et al., 1986). It is interesting to note that changes in the biodisposition and neurotoxicity of MPTP after D D C administration parallel those which are seen with age (Ricaurte et al., 1987; Langston et al., 1987). Compared to young animals, the administration of equivalent doses of MPTP to older animals results in greater DA depletions, with higher concentrations of MPP ÷ in the CNS. Further, homogenates of brain tissue from older animals exhibit enhanced biotransformation of MPTP and this can account, at least in part, for the increased susceptibility of older animals to MPTP (Ricaurte et al., 1986; Langston et al., 1987). It is possible, therefore, that D D C may also provide a useful tool for studying the age-related effects of MPTP. That D D C exacerbates the neurotoxicity of MPTP by enhancing or amplifying factors which produce, deliver and maintain increased concentrations of toxin in the CNS may have relevance to the usefulness of MPTP in the study of Parkinson's disease. Endogenous (Glover et al., 1983), and naturally occurring (Irwin et al., 1987) inhibitors of MPTP biotransformation have previously been described, and factors present in human plasma which increase M A O activity have also been reported (Yu and Bolton, 1979). Our
observation regarding the activation of an MAOB-dependent pathway raises the possibility that the activity of this important enzyme may be enhanced by endogenous, dietary or other factors. If this enzyme serves as a major xenobiotic metabolizing system for the CNS, then such factors could play a role in the vulnerability of the brain to toxic and perhaps neurodegenerative processes.
Acknowledgements The authors wish to thank John Skratt and Lorrene DavisRitchie for technical assistance. This work was supported in part by the United Parkinson Foundation, the Retirement Research Foundation, the Institute for Medical Research, Santa Clara Valley Medical Center, and NIEHS Grant 1 R01 ES03697-03.
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