423
Chem. Res. Toxicol. 1990, 3, 423-427
Studies on the Cytochrome P-450 Catalyzed Ring a-Carbon Oxidation of the Nigrostriatal Toxin I-Methyl-4-phenyl-I ,2,3,6-tetrahydropyridine (MPTP) Susanne Ottoboni,? Timothy J. Carlson,s William F. Trager,s Kay Castagnoli,' and Neal Castagnoli, Jr.**+ Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, and Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195 Received March 5, 1990 In vitro metabolic studies have established that rat liver cytochromes P-450IIB1 and P-45OIA1 but not rabbit liver cytochrome P-450IIB4 catalyze the oxidation of the Parkinsonian inducing (MPTP) to the corresponding dineurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine hydropyridinium (MPDP+) and pyridinium (MPP+)species. Kinetic experiments with the most effective isozyme, cytochrome P-45OIA1, indicate that the reaction proceeds at a moderate velocity [ V, = 20.1 nmol/(min.nmol of P-450IAl)l and high K (0.87 mM). Futhermore, kinetic deuterium isotope effect measurements provided D V and b( V / K )values of 2.99 and 1.04, respectively. A comparison with the corresponding values for the monoamine oxidase B (MAO-B) catalyzed reaction (4.37 and 9.35, respectively) suggests that either these enzymes catalyze the ring a-carbon oxidation of MPTP by different pathways or that the initial one-electron transfer to generate an aminium radical intermediate previously proposed for both enzyme systems is reversible in the case of MAO-B and irreversible in the case of cytochrome P-450IA1.
Introduction The in vivo oxidation of l-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP,l 1; see Chart I) to the corresponding 2,3-dihydropyridiniumintermediate MPDP' (2) and subsequently to the pyridinium species MPP+ (3)is an obligatory process for the expression of the nigrostriatal toxicity of this Parkinsonian inducing agent (I). A variety of studies has established that the conversion of MPTP to MPDP+ is efficiently catalyzed by MAO-B (2), an enzyme that generally is associated with the oxidation of primary and secondary amines (3). Although the MAO-B pathway appears to be responsible for the in vivo bioactivation of MPTP, several reports in the literature suggest that oxidases other than MA0 may catalyze the formation of MPDP+. For example, the conversion of MPTP to MPP+ by cultured astrocytes (4) and freshly prepared rat hepatocytes (5) was only partially blocked by the MAOA/B inhibitor pargyline. Furthermore, the in vivo formation of MPP+has been observed in MPTP-treated mice which had been pretreated with deprenyl, a selective MAO-B inhibitor (6). The structural features of MPTP, a cyclic tertiary allylamine, suggest that it should be a substrate for the cytochrome P-450 monooxygenases (7). Early attempts to demonstrate the cytochrome P-450 catalyzed formation of MPDP+ employing 100 pM MPTP and rat liver microsomal preparations led to the characterization of the NADPH-dependent formation of 4-phenyl-1,2,3,6-tetrahydropyridine (4)and MPTP N-oxide (5) but not MPDP+ (8). Subsequent studies, however, established that mouse liver microsomal preparations do catalyze the NADPHdependent formation of MPDP+ but only at high (1 mM) MPTP concentrations, suggesting that the reaction has a
* Address correspondence to this author. 'Virginia Polytechnic Institute and State University. 1 University of Washington.
Chart I
Q Q QQQQ /
/
high K , (9). Our interests in the mechanismb) by which enzymes catalyze the a-carbon oxidation of cyclic tertiary amines (10) and the potential toxicological importance of pyridinium metabolite formation (11) have led us to examine the interactions of MPTP with purified cytochrome P-450 isozymes.
Experimental Section Chemicals. All chemicals and solvents were reagent or HPLC grade. MPDP+C10; (12),MPTP N-oxide (8), and MPTP-d,.HCl (13)were synthesized as previously described. MPTP-HC1 and MPP+Cl- were purchased from Research Biochemicals, Inc., Natick, MA. HPLC analyses were performed as described previously (14) on a Beckman 114M chromatograph employing an H P Model 1040A diode array detector. Separations were achieved on an Altex Ultrasil SCX cation-exchange column (10 wm, 25 cm X 4.6 mm i.d.) a t a flow rate of 1.8 mL/min using the following mobile phase: 8% acetonitrile and 92% 0.1 M acetic acid containing 0.075 M triethylamine hydrochloride and adequate formic acid to adjust the pH to 2.3. The purifications of cytochrome P-450IIB4 (I@, cytochrome P-450IIB1 (16), and NADPH-dependent cytochrome P-450 reductase (17) were accomplished as previously described. Protein
Abbreviations: MAO, monoamine oxidase; MPTP, l-methyl-4phenyl-1,2,3,6-tetrahydropyridine;MPDP+, l-methyl-4-phenyl-2,3-dihydropyridinium; MPP+, 1-methyl-4-phenylpyridinium; NADPH, reduced nicotinamide adenine dinucleotide phosphate; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol.
0893-228x/90/2103-0423$02.50/0 0 1990 American Chemical Society
Ottoboni et al.
424 Chem. Res. Toxicol., Vol. 3, No. 5, 1990 Table I. Characteristics of the Oxidation of 1 mM MPTP by Various Cytochromes P-4500 Ti' of total MPDP+ time MPTP PTP N-oxide isozyme 60 92.4 trace 3.6 3.9 P-450IIB4 100 NDb P-450IIB1 0 ND ND 15 95.7 2.3 2.0 ND 30 90.0 6.1 ND 3.6 4.4 45 88.5 6.6 ND ND 5.5 60 84.4 9.5 ND 100 ND ND P-450IA1 0 9.0 89.1 1.3 15 trace 16.5 70.9 3.2 7.6 30 22.0 11.0 61.3 2.9 45 23.2 15.8 51.6 4.9 60 ND 100 ND ND 60 P-450IA1 - NADPH 12.5 22.1 59.6 3.5 60 P-450IA1 + pargyline a
Incubations were carried out at 37
O C
in the presence of 1 nmol of purified enzyme.
content was determined by the method of Lowry (18) and the specific P-450 content by the method of Omura and Sat0 (19). Specific contenb of purified P-450IIB4 and P-450IIB1 were found to be 14.2 and 12.8 nmol of P-450/mg of protein, respectively. The preparation of cytochrome P-450IA1 was achieved by a modification of the general method reported by Wolf et al. (20) as follows: Long Evans rats (80 g) were treated with 3-methylcholanthrene (40 mg/kg) via intraperitoneal injection once a day for 3 days. On the fourth day the rata were killed by decapitation and microsomes were prepared and solubilized with 3 g of sodium cholate/g of protein. After dialysis (2 X 12 h with lox volume) against 100 mM potassium phosphate (pH 7.4) containing 20% glycerol, 1 mM EDTA, 1 mM DTT, 0.4% cholate, and 0.1 mM phenylmethanesulfonyl fluoride, the solubilized microsomes were loaded onto an octyl-Sepharose column (200 mL) which had been equilibrated with dialysis buffer. The column was washed with equilibration buffer to yield the red-colored cytochrome b5 band (800 mL) and by equilibration buffer containing 0.1% Emulgen to yield the amber-colored cytochrome P-450 band (500 mL). The 448 nm, reduced difmajor P-450IAl-containing fractions (A, ference spectra) were combined, dialyzed against 10 mM potassium phosphate (pH 7.71, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5% cholate, and 0.2% Emulgen, and loaded onto a DE-53 cellulose column that had been equilibrated with dialysis buffer. After washing with equilibration buffer, a buffer gradient of 0-40 mM KCl (400 mL) was run followed by a 40-80 mM KCl (200 mL) gradient. The large band collected from the higher salt gradient was P-450IA1 [as determined by the reduced difference spectrum and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] with one minor contaminant of lower molecular weight. The P-450IAl-containing fraction was concentrated by using an Amicon membrane filter, to about 20 mL, and dialyzed against 2 X 500 mL of 10 mM potassium phosphate (pH 6.5) containing 20% glycerol, 0.1 mM EDTA, 0.1 mM Dl'T, and 0.2% Lubrol PX. The resulting isolate was loaded onto a CM-Sepharose CL-6B column. Bound P-450 was washed extensively (20 column volumes) with the equilibration buffer minus Lubrol PX and was eluted in wash buffer which had the phosphate concentration increased to 200 mM. The P-450IA1 that eluted was found to be pure by SDS-PAGE and had a specific content of 11.8 nmol of P-450/mg of protein. Kinetic Studies. Typical incubations with purified enzymes (final volume 1 mL) included 1 nmol of cytochrome P-450, 1.2 nmol of cytochrome P-450 reductase, 30 of phosphatidylcholine, 10 pmol of glucose 6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 5 pmol of magnesium chloride, and 0.5 pmol of NADPH in 100 mM potassium phosphate buffer, pH 7.4. Incubations were initiated by addition of MPTP, MPTP-d,, or MPDP+ at the concentrations indicated in the tables. A t the appropriate time points (Tables I and 11),the incubations were stopped by addition of 1mL of 5% trichloroacetic acid solution containing 1.25 pg of 4-phenylpyridine/mL as internal standard. The mixture was centrifuged to precipitate the protein, and the soluble portion was stored a t -70 "C until analyzed. The rates of oxidation of MPTP to MPDP+/MPP+ and to MPTP N-oxide were found to be linear over the first 20 min at substrate concentrations between 100 pM and 1mM, and therefore all reaction
MPP+ trace ND ND 0.3 0.5 0.6 ND 0.6 1.9 2.8 4.4
ND 3.3
* ND = not detected.
rates were estimated after a 15-min incubation period. Lineweaver-Burk analyses of the data established the K, and V,, values.
Results and Discussion Incubations with 1 mM MPTP were carried out in a reconstituted system with purified rabbit liver cytochrome P-450IIB4 and r a t liver cytochromes P-450IIB1 and P450IAl. HPLC analyses of these mixtures revealed significant differences in enzyme activity and in product composition (Table I). Cytochrome P-450IIB4, which was the least active of the three enzyme preparations, produced approximately equal amounts of the demethylated metabolite PTP (4) and the N-oxide 5. Only trace amounts of MPDP+ a n d MPP+ were detected. Cytochrome P450IIB1, which was intermediate in activity, catalyzed the formation of MPDP+/MPP+ and PTP but n o t t h e Noxide. HPLC analyses of timed aliquots indicated that ring a-carbon oxidation proceeded at a rate that was approximately 50% that of the N-demethylation reaction (Table I). T h e most active isozyme was cytochrome P-450IA1, which metabolized almost 50% of the 1 mM substrate in 60 min. In this case ring a-carbon oxidation was t h e main pathway (60% of total metabolism) followed by N-oxidation (30%) and N-demethylation (10%). Plots of the kinetic data summarized in Table I establish t h a t the catalytic activities for all three pathways are h e a r for the first 30 min, after which activity falls off. Pargyline (30 pM), which completely inhibits t h e MAO-B-catalyzed oxidation of MPTP t o MPDP+ (2), was without effect on t h e corresponding cytochrome P-450IA1 catalyzed oxidation which, however, was dependent on NADPH. These results document t h e isozyme selectivity of MPTP metabolism by cytochromes P-450 a n d clearly demonstrate t h a t members of this important family of enzymes are capable of catalyzing t h e conversion of MPTP (and presumably other tetrahydropyridines and structurally related compounds of potential toxicological interest) t o t h e corresponding dihydropyridinium a n d pyridinium species. T h e formation of MPTP N-oxide by the IIB4 a n d IA1 forms of t h e enzyme also is of interest since tertiary amine N-oxide formation generally is thought to proceed via flavin monooxygenase catalysis (21). I n addition to M P D P + , we also detected significant amounts of M P P + in t h e cytochromes P-450IIB1 a n d P-450IA1 incubation mixtures. Previous studies have established that MPDP+ is converted t o MPP' via autoxidation and a disproportionation reaction in which the free base of M P D P + acts as a hydride donor and MPDP+ as hydride acceptor (22). MAO-B also catalyzes this oxidation although a t only 3% t h e rate at which i t catalyzes
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 425
P-450-Catalyzed Oxidation of MPTP Table 11. Catalytic Activity of Cytochrome P-450IA1 on the Oxidation of 100 pM MPDP+a % of total conditions time,min MPDP+ MPP' buffer 10 93.4 6.6 20 92.8 7.2 30 91.9 8.1 P-450IA1 (-NADPH) 10 93.4 6.6 20 91.9 8.1 30 90.4 9.6 P-450IA1 (+NADPH) 10 90.0 10.0 20 84.7 15.3 30 79.0 21.0 aIncubations were carried out at 37 O C in the presence of 1 nmol of purified enzyme.
O
-0 5
05
15
2 5
m1 Figure 1. Plots of 1/ V versus 1/[S] for the cytochrome P-45OI.41 catalyzed N-and C-oxidation of MPTP.
the oxidation of MPTP (23). Results of studies designed to assess the catalytic activity of cytochrome P-450IA1 on the oxidation of 100 pM MPDP+ (a concentration at which autoxidation is the only reaction that occurs in the absence of enzyme) to MPP+ are summarized in Table 11. The NADPH-dependent enhancement of the rate of this conversion in the presence of cytochrome P-450IA1 supports a catalytic role for the enzyme in this reaction. Rough estimates suggest that at this concentration the corresponding rate for the cytochrome P-450IA1 catalyzed oxidation of MPTP to MPDP+ is about 10 times greater than the rate of enzymatic conversion of MPDP+ to MPP'. Studies on the substrate concentration dependence of the cytochrome P-450IA1 catalyzed oxidation of MPTP to MPDP+/MPP+ and to MPTP N-oxide provided estimates of the K, and V,, values for these transformations. Lineweaver-Burk analyses of the data established the K , for the ring a-carbon oxidation pathway to be 0.87 mM and for the N-oxidation pathway to be 0.78 mM (Figure 1). The corresponding V,, values were 20.1 nmol/ (minanmol of P-450IA1) and 15.6 nmol/(min.nmol of P450IA1), respectively. While the V,, values indicate that MPTP is a reasonable substrate for P-450IA1 [O-deethylation of 7-ethoxycoumarin, an excellent substrate for P450IA1, has a V,, of 144 nmol/(min.nmol of enzyme) and a K , of 20 pM ( 2 4 ) ] ,the K , values indicate that MPTP has little affinity for the enzyme. Thus, the inherent ability of the enzyme to turn over this substrate as assessed by the V,,J K, ratio is relatively poor. The fact that both cytochrome P-450IA1 and MAO-B catalyze the oxidation of MPTP to MPDP+ provides an opportunity to assess possible similarities and differences in the mechanismb) by which this conversion is achieved.
The MAO-B-catalyzed oxidations of amines are thought to proceed via initial one-electron transfer from the nitrogen lone pair of the substrate (6, eq l) to the oxidized RCH,N2'R2
RCH2N'+R2 k-i
7
k2
RC'HN2+R2 8
-
RCH=N+R~ 9 (1) form of FAD. In the case of N-cyclopropyl- and Ncyclobutyl-containing substrates, the resulting aminium radicals (7) undergo rapid ring opening to form carboncentered radicals that inactivate the enzyme (25). Similarly, cytochrome P-450 catalyzed N-dealkylationreactions of tertiary amines are believed to proceed via the corresponding aminium radical intermediates since the majority of these reactions display low intermolecular and intramolecular deuterium isotope effects (26). Subsequent proton loss from 7 then yields the carbon-centered radical 8 which undergoes a second one-electron oxidation to give the iminium product 9. The low magnitudes of the observed isotope effects2 associated with the cytochrome P-450 reactions are consistent with the generally held notion that proton loss from aminium radicals is only partially rate limiting, that is, the rate of proton loss is comparableto but faster than the rate of initial electron transfer (27). The ring a-carbon oxidation of MPTP formally is equivalent to the initiating event of an N-dealkylation reaction, and therefore one might expect the MAO-B-catalyzed oxidation of MPTP to proceed with a modest isotope effect. Recent studies, however, have demonstrated that the MAO-B-catalyzed ring a-carbon oxidation of MPTP vs MPTP-d, proceeds with an intermolecular deuterium isotope effect of 9.35 f 0.54 on V / K and 4.37 f 0.23 on V,, (13). In the present investigation we have determined the V,, and K, values for the cytochrome P-45OIA1 catalyzed ring a-carbon oxidation of MPTP and MPTP-d7 and have calculated D ( V / K )and DV to be 1.04 and 2.99, respectively. These contrasting results, particularly the virtual lack of an isotope effect on V / K for the P-450IAl-catalyzedreaction, would suggest either that these two enzymes are operating by different mechanisms or that other factors differentially alter the balance between kl and k2 in eq 1 in a way that leads to a large disparity in the observed isotope effects. One possible approach to a better understanding of how the balance between kl and k, might be altered in these systems could come from the direct measurement of the isotope effect associated with the deprotonation of an aminium radical species. Fortunately, recent model chemical studies by Dinnocenzo have established an isotope effect of 7.68 for the deprotonation of p-anisylmethylaminium hexafluoroarsenate in acetonitrile by quinuclidine (28). This result argues that enzyme-catalyzed reactions proceeding according to eq 1may display a large isotope effect provided that the initially formed radical cation has sufficient lifetime to allow its expression, i.e., kl > k,. The MAO-B-catalyzed reaction might be expected to display such a kinetic relationship since initial electron transfer is believed to be a reversible process (25). If electron transfer is reversible and rapid relative to deprotonation, isotopic discrimination can occur and the observed isotope effect should approach the intrinsic isoThe term "observed isotope effect" is used to indicate the actual value of the isotope effect obtained experimentally and to distinguish it from the 'intrinsic isotope effect" for the reaction, Le., the isotope effect associated solely with the bond breaking step. The intrinsic isotope effect, the magnitude of which must be known before mechanistic conclusions can be reached, generally can only be approximated by direct experimental measurement.
426 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
tope effect for the reaction. The large isotope effect on V / K is consistent with a low commitment to catalysis, i.e., k, >> k2. The lower value of the isotope effect on V,, (4.37), however, suggests that a moderate amount of suppression is still operative (29). Since D Vfor the cytochrome P-450IA1 catalyzed reaction is 2.99 and D(V/K) is only 1.04, the expression of the isotope effect is highly suppressed relative to the MAOB-catalyzed reaction. Complete suppression of the D( V/K) isotope effect, as measured by the rate of disappearance of substrate or the rate of total product formation, is expected if an irreversible step precedes the isotopically sensitive step of hydrogen atom removal (30). Evidence to suggest that this may be the case with the cytochrome P-450's has been provided by Harada and co-workers (31), who found not only that the initial formation of the reactive oxene form of the enzyme is irreversiblebut also that once formed, it is highly committed to substrate oxidation. Consequently, the back-reaction of electron transfer from the one-electron-reduced oxygen-cytochrome P-450 complex to the aminium radical would be highly unlikely relative to the forward deprotonation reaction. On the basis of these considerations, the only isotope effect that might be observed in the cytochrome P-450 catalyzed reaction would be the secondary isotope effect associated with initial electron transfer from the d, substrate ion. Also consistent with this view is the low ( k H / k D = 1.35) isotope effect observed in the electrochemical oxidation of MPTP/MPTP-2,2,6,6-d, to MPDP' (13). Although the above arguments may be consistent with the formation of a common aminium radical intermediate in both the MAO-B and cytochrome P-450IA1 catalyzed oxidations of MPTP to MPDP+, the subsequent fate of this intermediate does not appear to be the same. The sole product resulting from the MAO-B-catalyzed reaction is MPDP+, which is consistent with the thermodynamically preferred product produced exclusively in the electrochemical oxidation of MPTP (13). On the other hand, the cytochrome P-450 isozymes IIBl and IA1 yield the ring a-carbon oxidation product MPDP', and all three isozymes (IIB1, IA1, and IIB4) yield the demethylated product PTP. Furthermore, the ratios of the cytochrome P-450 catalyzed a-carbon oxidation products are isozyme dependent-the ratio of (MPDP+ + MPP+)/(PTP)at the 30-min time point is 0.64 for the cytochrome P-450IIB1 catalyzed reaction, 5.40 for the cytochrome P-450IA1 catalyzed reaction, and 0 for the cytochrome P-450IIB4 catalyzed reaction. These data argue that, at least in the case of the cytochrome P-450's, the enzyme must be mechanistically involved in the deprotonation step. Upon further consideration, it must be noted that these results do not rule out the possibility that these two enzymes catalyze the ring a-carbon oxidation of MPTP by different pathways. For example, a hydrogen abstraction pathway cannot be ruled out for the cytochrome P-450IA1 catalyzed reaction. This statement is based on the cited evidence (31)that oxene formation in cytochrome P-450 catalyzed reactions is an irreversible process and that the reactive intermediate is highly committed to substrate oxidation. Theoretical considerations have established that if an irreversible step (oxene formation) occurs prior to any isotopically sensitive step (carbon-hydrogen bond cleavage), the reaction will have a D(V/K)of 1 (29, 30), irrespective of the specific pathway followed, provided that no branching occurs. The formation of the three observed products in the cytochrome P-450IA1 catalyzed oxidation of MPTP-d,, however, implies the possibility of metabolic switching, i.e., the ratio of N-oxidation/C-D oxidation will
Ottoboni et al.
be greater than N-oxidation/C-H oxidation. Because metabolic switching generally leads to unmasking of intrinsic isotope effects, a relatively large isotope effect should be observed if hydrogen atom abstraction were the first step in substrate oxidation. The low observed D( V / K ) of 1.04 for MPDP' formation, therefore, would seem to imply that hydrogen atom abstraction cannot be the first catalytic event. On the other hand, the degree of unmasking of the intrinsic isotope effect for a-carbon oxidation that can occur because of branching to the N-oxide pathway is directly dependent upon the quantitative importance of the N-oxide pathway-the more favored the N-oxide pathway, the greater the degree of unmasking. Since the N-oxide pathway is less favored than the MPDP' pathway, significant unmasking would not be expected. In addition, the flanking of the nitrogen atom by seven deuterium atoms should result in a significant secondary isotope effect that would disfavor N-oxide formation. As a consequence, the ability of the N-oxide pathway to unmask the isotope effect associated with the other two sites would be reduced further. Thus, when all the factors are considered, even the possibility of branching, there is no reason to expect a significant isotope effect in this cytochrome P-450 catalyzed ring a-carbon oxidation of MPTP. On the basis of these considerations, the D( V / K ) of 1.04 observed for the cytochrome P-450IA1 catalyzed oxidation of MPTP is consistent with C-H bond cleavage resulting either from initial electron transfer followed by proton loss or from direct hydrogen atom abstraction. In conclusion, these kinetic results demonstrate that rat liver cytochrome P-450IA1 catalyzes the ring a-carbon oxidation of MPTP. The isotope effect is similar to those observed for cytochrome P-450 catalyzed oxidative N-dealkylation reactions but considerably smaller than that reported for the corresponding MAO-B-catalyzed reaction. Product analysis argues that all three cytochrome P-450 isozymes participate in the deprotonation step. If both catalytic pathways proceed via an initial one-electron transfer step, this step is likely to be irreversible in the cytochrome P-450IA1 catalyzed reaction and reversible in the MAO-B-catalyzed reaction. Alternatively, the possibility that the MAO-B and cytochrome P-45OIA1 catalyzed reactions do not share a common intermediate or that they both proceed via a hydrogen atom abstraction pathway cannot be discounted unequivocally. The demonstration that cytochrome P-450 can catalyze the conversion of MPTP to MPDP' has toxicological implications for this family of isozymes with respect to the possible bioactivation of partially oxidized piperidinecontaining xenobioticswhich are not MA0 substrates. For example, the neuroleptic drug haloperidol [a l-substituted-4-(p-chlorophenyl)-4-piperidinol derivative] is transformed in the rat to the corresponding pyridinium metabolite (32). Since haloperidol is not a substrate for MAO-B, it seems reasonable to speculate that this in vivo transformation is catalyzed by cytochrome P-450. The recent report (33) that cytochrome P-450IID1 (debrisoquine hydroxylase) can be solubilized from neuronal membranes isolated from canine striata provides further evidence for the potential role of the cytochrome P-450 mediated bioactivation of neurotoxic xenobiotics. Acknowledgment. Supported by USPHS Grants NS 23066 and GM 36922 and the Harvey W. Peters Research Center for Parkinson's Disease and Disorders of the Central Nervous System. Registry No. 1, 28289-54-5; 2, 94613-45-3; 3, 48134-75-4; 4, 10338-69-9; 5, 95969-40-7; cytochrome P-450, 9035-51-2.
P-450-Catalyzed Oxidation of MPTP
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