Document MM0eXnQ0o2kjyyYwwa6E2BkJV

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 255, No. 1, May 15, pp. 14-18, 1987 Comparative Toxicity and Antioxidant Activity of 1-Methyl-4-phenyl1,2,3,6-tetrahydropyridine and Its Monoamine Oxidase B-Generated Metabolites in Isolated Hepatocytes and Liver Microsomes' GUNILLA EKSTRijM,' DONATO DI MONTE, MARTHA S. SANDY, AND MARTYN T. SMITH3 Department of Biom.edical and Environmental Health Sciences, School of Public Health, University of Califwnia, Berkeley, California 94720 Received October 16, 1986 MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is converted by monoamine ox- idase B to its putative toxic metabolite MPP+ (1-methyl-4-phenylpyridinium ion) via MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium ion). Both the parent compound and these two major metabolites were toxic to isolated rat hepatocytes with MPDP+ being the most toxic and MPPf the least effective. MPP+ produced a slight increase in lipid peroxidation above control levels in hepatocytes, while both MPTP and MPDP+ showed antioxidant effects. The latter two compounds also protected against chemically and nonchemically induced lipid peroxidation in rat liver microsomes. MPDP+ was effective at much lower concentrations than MPTP. MPDP+ was also markedly more efficient when NADPH was used to induce microsomal lipid peroxidation. Lipid peroxidation as a consequence of oxygen radical generation is therefore unlikely to be involved in MPTP toxicity in vitro and the rationale of using chain-breaking antioxidants as protective agents in vivo needs a more careful evaluation. 0 1987 Academic Press, Inc. The neurotoxic effects of MPTP4 (lmethyl-4-phenyl-1,2,3,6-tetrahydropyridine), which ultimately lead to a clinical picture of parkinsonism in humans and other primates (l), require its metabolic 1This work was supported by the Odd Fellows Lodge, Stockholm, Sweden (G.E.), the Health Effects Component of University of California Toxic Sub- stances Program (D.D.), the Northern California Oc- cupational Health Center (M.S.S.), and the National Foundation for Cancer Research (M.T.S.). ' Present address: Department of Physiological Chemistry, Sweden. Karolinska Institute, 104 01 Stockholm, ' To whom all correspondence should be addressed. * Abbreviations used: MPTP, l-methyl-l-phenyl- 1,2,3,6-tetrahydropyridine; MAO-B, monoamine oxi- dase B; MPP+, l-methyl-4-phenylpyridinium ion; MPDP+, 1-methyl-4-phenyl-2,3-dihydrupyridinium ion; TCA, trichloroacetic acid; TBA, thiobarbituric acid; 1.2-MPDP, dine. 1-methyl-4-phenyl-1.2-dihydropyri- activation by monoamine oxidase B (MAO- B) (2). This oxidative pathway produces the putative toxic metabolite MPP+ (l-methyl- 4-phenylpyridinium ion) via a dihydropyr- idinium intermediate MPDP+ (1-methyl-4- phenyl-2,3-dihydropyridinium ion) (3). The toxic effects of MPP+ have been attributed to the generation of active oxygen species and the subsequent oxidative destruction of cell membrane lipids (4,5). Attempts to prevent the toxic consequences of MPTP exposure in vivo via the use of antioxidants have led, however, to controversial results (6, 7). Furthermore, in recent work with isolated rat hepatocytes, we have disso- ciated the cytotoxic effects of MPP+ and MPTP from the production of oxygen rad- icals (8, 9). In this study we extend our investiga- tions to MPDP+. The role played by this unstable metabolite in MPTP toxicity is still relatively unknown despite the fact 0003-9861/87 $3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 14 TOXICITY AND ANTIOXIDANT ACTIVITY OF MPTP 15 that it has been shown to produce damage to dopaminergic neurons in the substantia nigra (10). Furthermore, we relate the cy- totoxicity of MPTP and its MAO-B-gen- erated metabolites to their effects on lipid peroxidation, in order to test the rationale of using antioxidants as protective agents. MATERIALS AND METHODS Hepatocytes were isolated from male Sprague- Dawley rats (180-250 g) and incubated at 37C in Krebs-Henseleit buffer, pH 7.4 (ll), under a 95% O,/ 5% COP atmosphere. Cell viability was assessed by trypan blue exclusion (11). Liver microsomes were prepared from similar male Sprague-Dawley rats as described in Ref. (12). Protein was determined as de- scribed by Lowry et al. (13). Reaction mixtures con- tained microsomes corresponding to 0.5 mg of protein in 50 mM potassium phosphate buffer, pH 7.4, in a final volume of 1 ml. The incubations were started by the addition of 100 ~1 of 500 FM cumene hydroperoxide or 50 ~1 of 4.8 mM NADPH and terminated by the addition of 0.25 ml 40% trichloroacetic acid (TCA) and 0.125 ml 5 M HCL Control incubations were carried out by adding cumene hydroperoxide or NADPH after the addition of TCA. The uv irradiation was performed at 254 nm at a distance of 4 cm for 10 min. The effi- ciency of the lamp was 580 W/cm2 at 15 cm. Micro- somes were diluted to 0.5 mg/ml with 50 mM potas- sium phosphate buffer, pH 7.4, in small petri dishes. MPTP and MPDP+ were added just prior to irradia- tion. Lipid peroxidation products were measured using the thiobarbituric acid (TBA) assay (14). Cytochrome c reduction was measured spectrophotometrically at 550 nm. MPTP, MPDP+, and MPP+ were kindly sup- plied by Professor Neal Castagnoli, Jr., Division of Toxicology, Department of Pharmacy, University of California, San Francisco. RESULTS AND DISCUSSION When isolated rat hepatocytes were exposed to equimolar concentrations of the parent compound MPTP or either of the two major metabolites (MPDP+, MPP+), results as in Fig. IA were obtained. The loss of cell viability ultimately occurred in 100% of hepatocytes after addition of any of the three compounds, but was most rapid after MPDP+ exposure, and occurred only after a relatively long lag period in the presence of MPP+. Due to their charged structures, both MPDP+ and MPP+ could be expected to have limited accessibility to hepatocytes. MPDP+, however, was the most toxic of the three compounds studied and is likely to be readily taken up by hepatocytes, since rapid intracellular biochemical changes followed its addition to the incubation mixtures (data not shown). The passive diffusion across cell membranes of the lipophilic free base 1,2-MPDP (1-methyl-4-phenyl-1,2-dihydropyridine) (Fig. 2) might account for the ready accessibility of the corresponding charged acid (MPDP+) to the cell (15). It has been suggested that a free radicalmediated mechanism could be involved in MPTP toxicity (4,5) and antioxidants have been proposed as protective agents against oxygen radical-induced lipid peroxidation after MPTP exposure (7). We therefore measured lipid peroxidation products in hepatocyte incubations after the addition of 1.5 mM MPTP, MPDP+, or MPP+ (Fig. 1B). A slightly increased formation of these B. 3 I I MPP+ Time (mid 01 II L 0 60 120 1.30 Time (mid FIG. 1. Cell viability (A) and lipid peroxidation (B) after exposure of isolated hepatocytes mM MPTP (0), 1.5 mM MPDP+ (O), 1.5 mM MPP+ (A), or no addition (0). to 1.5 16 EKSTRijM ET AL. MPTP MPDP+ 1,2-MPDP MPP+ FIG. 2. Structures of MPTP and its major metabo- lites: MPTP, l-methyl-4-phenyl-1,2,3,6-tetrahydro- pyridine; MPDP+, l-methyl-4-phenyl-2,3-dihydro- pyridinium ion; 1,2-MPDP, I-methyl-4-phenyl-1,2- dihydropyridine; MPP+, 1-methyl-4-phenylpyridinium ion. products, as compared to control cells, was observed only in the presence of MPP+, the least toxic of the three pyridine compounds. On the other hand, exposure to both MPTP and MPDP+ produced rates of lipid per- oxidation even lower than those measured in untreated cells. We decided to further investigate these antioxidant properties of MPTP and MPDP+ in a noncellular system, namely rat liver microsomes. Organic hydroperoxides can initiate microsomal lipid peroxidation by the cytochrome P-450-dependent formation of peroxy and alkoxy radicals (16). The generation of TBA-reactive products following a 5-min incubation of microsomes with 50 PM cumene hydroperoxide was inhibited by 50% in the presence of 1 mM MPTP and by 75% in the presence of 0.5 MM MPDP+ (Fig. 3). No effect was observed after the addition of 1 IBM MPP+ (not shown). Lipid per- oxidation can also be induced nonchemi- tally by uv irradiation. The presence of ei- ther MPTP or MPDP+ during irradiation protected microsomes against peroxidative damage (Fig. 4), which shows that these compounds are direct chemical antioxi- dants. MPDP+ was more effective in pre- venting this lipid peroxidation than MPTP, producing an 80% inhibition at concentra- tions 10 times lower than those of MPTP. When NADPH was added to microsomal incubations to induce lipid peroxidation via NADPH-cytochrome P-450 (c) reductase (l?), MPDP+ was even more efficient at in- hibiting the formation of TBA-reactive products. It was able to protect signifi- cantly at concentrations as low as 0.05 mM, 100 times lower than with MPTP (Fig. 5). MPDP+ also decreased the rate of NADPH- dependent cytochrome c reduction in liver microsomes (Fig. 6). This effect, however, was significant only at concentrations much higher than 0.05 mM, indicating that the protective effect of MPDP+ against NADPH-induced lipid peroxidation cannot simply be a consequence of inhibition of the activity of NADPH-cytochrome P-450 (c) reductase. A more probable antioxidant mechanism is that MPDP+, and to a lesser extent MPTP, reacts directly with the ini- tiating species, a proposed superoxide rad- ical/iron complex (18). In conclusion, both MPTP and MPDP+ do not induce, but indeed inhibit lipid per- oxidation in rat hepatocytes and liver mi- Time (mid FIG. 3. Effect of MPTP (A) and MPDP+ (B) on liver microsomal lipid peroxidation initiated by 50 pM eumene hydroperoxide. Reaction mixtures contained microsomes corresponding to 0.5 mg of protein alone (0) and with 1 mM MPTP (V), 5 mM MPTP (+), 0.1 mM MPDP+ (Cl), or 0.5 mM MPDP+ (W). Incubations were performed at 30C. Results shown are the means of four separate incubations. TOXICITY AND ANTIOXIDANT ACTIVITY OF MPTP 17 OoL d lRRAOlAT!ON III I 5 10 15 0" 0 Time (mid 1I 5 10 15 FIG. 4. Protective effect of MPTP (A) and MPDP+ (B) against formation of TBA-reactive products induced by uv irradiation. Samples were taken at indicated time points (following 10 min of irradiation) from petri dishes containing microsomes (0.5 mg of protein/ml) alone (0), with 5 mM MPTP (+), or with 0.5 mM MPDP' (m). The amount of TBA-reactive products in the samples prior to irradiation has been subtracted. Results shown are the means of four separate experiments. crosomes. This effect in the presence of MPTP does not seem to be related to its metabolism. Pargyline, an inhibitor of lOr-----l MAO-B, did not modify the antioxidant ef- fects of MPTP in isolated hepatocytes (data not shown). Furthermore, MPTP was able to protect against lipid peroxidation in microsomes even in the absence of NADPH, when no evidence of metabolite formation has been reported (19). The results reported here lead to the conclusion that lipid peroxidation is unlikely to be involved in the general cytotoxic effects of MPTP. Whether or not lipid peroxidation plays any role in the selective toxicity of this compound to 4 IO- B. IaI Time (mid FIG. 5. Inhibitory effects of MPTP (A) and MPDP+ (B) on NADPH-dependent microsomal lipid peroxidation. Reaction mixtures (1 ml) consisted of microsomes corresponding to 0.5 mg of protein and 0.2 mg NADPH (0) with 1 mM MPTP (0), 5 mM MPTP (+), 0.05 mM MPDP+ (O), or 0.075 mM MPDP+ (m). Incubations were performed at 3O'C. Results shown are the means of three separate incubations. MPOP+ (mYI FIG. 6. Effect of MPDP+ on NADPH-dependent cytochrome c reduction in liver microsomes. Rat liver microsomes (0.5 mg/ml) were incubated at 30C in 50 mM potassium phosphate buffer (pH 7.6) in the presence of cytochrome c (0.6 mg/ml) and NADPH (20 /rg/ ml). Cytochrome c reduction was monitored at 550 nm following the addition of increasing concentrations of MPDP*. Data represent the means of duplicate experiments. 18 EKSTRiiM ET AL. the dopaminergic neurons of the substantia nigra remains an open question, however. An alternative mechanism of cytotoxicity involving accumulation of MPP+ in mitochondria (20), the subsequent inhibition of mitochondrial respiration (9,21), and disruption of calcium homeostasis (22) seems more likely. The latter has been shown to be important in the cytotoxicity of numerous compounds to hepatocytes (23) and may well be important in the neuronal toxicity of MPTP. Why nigrostriatal neurons should be so sensitive to the toxic effects of MPTP and/or its metabolites is a subject for further study. The fact that both MPTP and MPDP+ are quite potent antioxidants in their own right cannot be ignored, however, and the rationale of using antioxi- dants as protective agents against parkinsonism induced by MPTP or "MPTP-like" substances needs careful evaluation. ACKNOWLEDGMENTS We thank Drs. Neal Castagnoli, Jr., and Anthony Trevor for much helpful discussion. REFERENCES 1. LANGSTON, J. W., BALLARD, P., TETRUD, J. W., AND IRWIN, I. (1983) Science 219,979-980. 2. HEIKKILA, R. E., MANZINO, L., CABBAT, F. S., AND DUVOISIN, R. C. (1984) Nature @n&m) 311,467- 469. 3. CHIBA, K., TREVOR, A. J., AND CASTAGNOLI, N., JR. (1984) Biochem Biophys. Res. Commun 120, 54'7-578. 4. MARKEY, S. P., JOHANESSEN, J. N., CHIUEH, C. C., BURNS, R. S., AND HERKENHAM, M. A. (1984) Nature (London) 311,464-467. 5. SINHA, B. K., SINGH, Y., AND KRISHNA, G. (1986) B&hem Biophys. Res. 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