Metabolic Brain Disease, Vol. 7, No. 3, 1992
Differential Effects of Metal Ions on Type A and Type B M o n o a m i n e Oxidase Activities in Rat Brain and Liver Mitochondria Thomas K.C. Leung, 1' 2 Louis Lim, 1 and James C.K. Lai 3.4 Received June 8, 1992 ; accepted July 30, 1992 To investigate the hypothesis that neurotoxic metals can exert their toxicity through the direct inhibition of monoamine oxidases (MAOs), the effects of several neurotoxic metal ions on type A (MAO-A) and type B (MAO-B) monoamine oxidase activities in rat forebrain nonsynaptic mitochondria and rat liver mitochondria were studied. At pathophysiological levels (10-100 p.M), Cu 2+ and Cd 2+ are good inhibitors of brain mitochondrial MAO-A and, to a lesser extent, liver mitochondrial MAO-A. The inhibition of MAO-B activities in brain and liver mitochondria by Cu 2+ and Cd 2+ is only detected at the higher end of the concentration range (i.e., 50-100 p.M). At the pathophysiological level of 0.5 mM, A13+ only inhibits brain mitochondrial MAO-A but at the higher level of 2.5 mM, it inhibits both forms of MAO in brain as well as liver mitochondria. Even at toxic levels (e.g., 5 mM), neither Mn 2§ nor Li § inhibits the activities of MAO-A and MAO-B in brain and liver mitochondria. Our results are consistent with the hypothesis that some neurotoxic metals can exert their toxicity through the direct inhibition of the isoforms of MAO. Our data also suggest that the selective inhibition of brain MAO-A by Cu 2+ and Cd 2+ may assume pathophysiological importance in the neurotoxicity of copper and cadmium.
KEY WORDS: metal toxicity; monoamine oxidases; metal inhibition of monoamine oxidases.
1 Department of Neurochemistry, Institute of Neurology, University of London, Queen Square, London WC1N 3BG, U.K. 2 Present address is: Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511. 3 Department of Pharmaceutical Sciences and Center for Toxicology Research, College of Pharmacy, Idaho Slate University, Pocatello, ID 83209, U.S.A. 4 To whom correspondence should be addressed at the Department of Pharmaceutical Sciences, College of Pharrnacy, Idaho State University, Campus Box 8334, Pocatello, ID 83209-0009, U.S.A.
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INTRODUCTION There is good evidence that monoamine oxidase (MAO; monoamine: 0 2 oxidoreductase, EC 1.4.3.4) in mammalian tissues (including brain) exist in at least two forms (known, respectively, as type A (MAO-A) and type B (MAO-B) MAO) (Tipton e t a / . , 1976; Holzbauer and Youdim, 1977; Bach et al., 1988; Shih et al., 1990). The initial classification of MAO into types A and B is based upon substrate-and inhibitor- specificities (Johnston, 1968; Knoll and Magyar, 1972; Tipton et al., 1977; Holzbauer and Youdim, 1977). For example, MAO-A selectively deaminates neurotransmitter amines such as serotonin (5-HT) and norepinephrine (NE) and is preferentially inhibited by clorgyline whereas MAO-B selectively deaminates benzylamine and phenylethylamine and is preferentially inhibited by deprenyl (see Tipton et al. (1977), Owen et al. (1977), and Lai et al. (1982) and Refs. cited therein). Results from immunochemical studies with anti-MAO monoclonal antibodies reveal that there are unique epitopes on human MAO-A not shared by human MAO-B (Kochersperger et al., 1985). Thus, the immunochemical data (Kochersperger et al., 1985) provide some support for the hypothesis that MAO-A and MAO-B are distinct proteins or isozymes. More definitive support for this hypothesis comes from recent structural studies on MAO: the peptide and cDNA sequences indicate that MAO-A and MAO-B are encoded in different genes and have some homology at the amino acid level (Bach et al., 1988; Hsu et al., 1988; Powell et al., 1989). Tissue-specific and age-related differential expressions of MAO-A and MAO-B genes have been noted (Shih et al., 1990; Grimsby et al., 1990). Metal ions play many critical roles in the "neurotransmitter metabolic cycle" (see Lai et al., 1985 for discussion and Refs.). Since MAOs (together with catechol-O-methyl transferase) are the key enzymes that regulate the metabolism of biogenic amines, especially neurotransmitter amines such as serotonin, dopamine, and norepinephrine (see Lai et at. (1983, 1985) for discussion and Refs.), it has been speculated that, in the neurotoxicity of several metals (including manganese), one underlying mechanism may be the inhibition by the metal ions of the MAOs and thereby altering amine metabolism (see Lai et al. (1983, 1992) for a detailed discussion and Refs.). The present study was initiated to address the hypothesis that neurotoxic metals can exert their toxicity through the direct inhibition of MAOs. The effects of several metal ions (i.e., Li§ Mn2§ Cd2§ Cu2+, and A13§ known to be neurotoxic, on MAO-A and MAO-B activities in rat brain and liver mitochondria were investigated since the distributions of MAO isoforms in brain and liver are different. M A T E R I A L S AND METHODS
Analytical grade chemicals were used and purchased either from BDH Chemicals (Enfield, Middlesex, U.K.) or from Sigma Chemicals (St. Louis, MO, U.S.A.). BBS3 was obtained from Beckman Instruments Inc. (Fullerton, CA, U.S.A.) and butyl-PBD (5-(4-biphenyl)-2-(4-t-butylphenyl)-l-oxa-3,4-diazole) was from CIBA Ltd. (Horsham, Sussex, U.K.). 5-Hydroxy-side chain 2-[14C]tryptamine creatinine sulfate (serotonin) (specific activity 58 mCi/mmol) and [7-I4C]benzylamine hydrochloride (specific activity 60 mCi/mmol) were purchased from the Radiochemical Centre, Amersham (Buckinghamshire, U.K.). All solutions were prepared using deionized glass-distilled water.
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Male Wistar (M.R.C. Porton strain) rats (150-170 g; 50-65 days old) were employed. For the isolation of forebrain non-synaptic mitochondria, four rats were used in each experiment; the procedure was that of Lai and Clark (Lai et al., 1977; Lai and Clark, 1979) except that none of the media contained EDTA and the mitochondria were finally washed twice without the inclusion of albumin in the isolation medium. For the preparation of liver mitochondria, two rats were used in each experiment. Liver mitochondria were isolated by differential centrifugation (Lai and Barrow, 1984) and then further purified using the discontinuous Ficoll-sucrose gradient of Lai and Clark (1979, 1989); none of the media contained EDTA. The forebrain non-synaptic mitochondria and liver mitochondria so isolated were metabolically active and relatively free from non-mitochondrial material (Lai and Clark, 1979, 1989 and J.C.K. Lai, unpublished observations). MAO-A (serotonin-oxidizing) and MAO-B (benzylamine-oxidizing) activities were assayed as described previously (Owen et al., 1977; Leung et al., 1980) but with minor modifications (Leung et al., 1981, 1982; Lai et al., 1982). Potassium phosphate (40 mM) buffer, pH 7.4 was used instead of the Tris-HC1 buffer. Final concentrations of all substrates were 1 mM and 0.1 mg of mitochondrial protein was employed per assay tube. The temperature was 37 ~ C. and pre-incubation time was 5 minutes. (Where appropriate, the metal ions (at the specified concentration) were added at the beginning of the pre-incubation.) When serotonin was the substrate, the assay incubation time was 30 minutes, whereas in the case of benzylamine, it was 60 minutes. A 10-minute-boiled tissue sample was used as the blank and this was processed as for the reaction mixture. The reaction was stopped by the addition of 0.1 ml of 6 M HC1 to the reaction mixture. The reaction products of serotonin and benzylamine were extracted with 2 ml of benzene:ethyl acetate mixture (1:1, v/v) and 2 ml of toluene, respectively. After centrifugation at 1000 g for 5 minutes, 1 ml of the organic phase was transferred to a vial containing 10 ml of scintillation fluid, which contained 0.4% (w/v) of butyl-PBD in a mixture of toluene and Bio-Solv BBS3 (9:1, v/v). The radioactivity therein was determined by liquid scintillation spectrometry. The difference in the extracted radioactivity from the reaction mixture and that obtained using the boiled tissue blank was used to calculate the enzymatic activity (in mU/mg protein). (1 mU was defined as 1 nmole of product formed per minute.) Protein determinations were carded out by the method of Lowry et al. (1951). Under the assay conditions employed in the present study, MAO-A and MAO-B activities were linear with respect to the reaction time and tissue concentrations. Statistical analyses were carried out using a BASIC program (written by J.C.K. Lai) for analysis of variance (ANOVA) with the post-hoe Tukey test for multiple comparisons. Only P values < 0.05 were regarded as being significant.
RESULTS Effects of LiCI and MnCI2 on MAO-A and MAO-B activities in Brain and Liver Mitochondria
The effects ofLi + were investigated at 5 mM because this concentration is slightly above the therapeutic range although toxic symptoms have been noted (see Underwood (1977) and
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Atterwill and Tordoff (1982) for discussion). However, at this level, Li § did not affect M A O - A and M A O - B activities in brain and liver mitochondria (Tables I and II). At pathophysiological levels (0.1-1 mM) (see Lai et al. (1992) for discussion and Refs.), Mn 2+ did not significantly alter the activities of M A O - A and M A O - B in brain (Table I) and liver (Table II) mitochondria. Even at toxic levels (5-10 mM), Mn 2§ was still ineffective (Tables I and H).
Effects of AICi3 on MAO-A and MAO-B activities in Brain and Liver Mitochondria At the pathophysiological level of 0.5 m M (Lai and Blass, 1984a), A13§ did not alter the activities of M A O - B in brain mitochondria (Table I). Nor did A13+ (at 0.5 mM) markedly influence the activities o f M A O - A and M A O - B in liver mitochondria although the M A O - A
Table I. Effects of Metal Ions on Type A (Serotonin-Oxidizing) and Type B (BenzylamineOxidizing) Monoamine Oxidase Activities in Rat Brain Mitochondia a
Monamine Oxidase Activity (% of control) Metal Ions
Concentration SertoninOxidizing
LiC1
BenzylamineOxidizing
5mM
96 + 8
96 + 10
MnC12
0.1mM 1 mM 5mM 10mM
90 + 5 88 + 6 96+3 97+1
A1C13
0.5 mM 2.5 mM
78 + 4 b 44+3 c
93 + 5 46+5 c
CdC12
10 glVl 100gM
53 + 9 c 3+1c
97 + 8 5+1c
CuC12
10 gM 50gM
70 + 10 b 18+8c
91 + 4 3+2c
108 + 104 + 111+ 111+
5 17 1 1
a Values are Means + SD of 3-4 separate experiments, expressed as % of corresponding control activities (MAO-A, 4.22 + 0.19 mU/mg protein; MAO-B, 2.22 + 0.18 mU/mg protein). Other details are as described in MATERIALS AND METHODS. t~ p < 0.05 versus corresponding control activity. c p < 0.01 versus corresponding control activity.
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143
activity showed a trend of being decreased (Table II). However, 0.5 m M A13§ did mildly but significantly inhibit M A O - A in brain mitochondria (by 20%; Table I). At the higher level of 2.5 mM, AI 3§ definitely inhibited (by approximately 50%) both M A O - A and MAO-B activities in brain (Table I) and liver (Table II) mitochondria. Effects of CdCI2 on MAO-A and MAO-B activities in Brain and Liver Mitochondria At 10 gM, Cd 2§ significantly inhibited M A O - A activities in brain (by 45%; Table I) and liver (by 25%; Table II) mitochondria. However, at this level, Cd 2§ was without effect on MAO-B activities in brain and liver mitochondria (Tables I and II). At the higher and more toxic level of 100 gM, Cd 2§ strongly inhibited (by >80%) both types of M A O activities in brain and liver mitochondria (Tables I and II).
Table II. Effects of Metal Ions on Type A (Serotonin-Oxidizing) and Type B (BenzylamineOxidizing) Monoamine Oxidase Activities in Rat Liver Mitochondria a
Monoamine Oxidase Activity (% of Control) Metal Ions
Concentration S erotoninOxidizing
BenzylamineOxidizing
5 mM
105 + 7
101 + 6
MnC12
0.1 mM lmM 5raM 10 mM
98 + 6 93+6 100+2 96+ 1
93 + 6 108+3 102+3 94+ 1
A1C13
0.5 mM 2.5 mM
85 + 5 52+2 c
91 + 2 49+2 c
CdC12
10 ~Vl 100 p.M
74 + 4 b 2+1 c
90 + 8 15+1 c
CuC12
10 gM 500M
88 + 4 3+1 c
101 + 5 2+1 c
LiC1
a Values are Means + SD of 3-4 separate experiments, expressed as % of corresponding control activities (MAO-A, 7.04 _+ 0.78 mU/mg protein; MAO-B, 5.21 + 0.17 mU/mg protein). Other details are as described in MATERIALS AND METHODS. b p < 0.05 versus corresponding control activity. c p< 0.01 versus corresponding control activity.
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Effects of CuCI2 on MAO-A and MAO.B activities in Brain and Liver Mitochondria
At the pathophysiological level of 10 I~M (see Lai and Blass (1984b) for discussion and Refs.), Cu 2§ only significantly inhibited (by 20-30%) MAO-A (but not MAO-B) in brain mitochondria (Table I). Neither MAO-A nor MAO-B in liver mitochondria was significantly affected by Cu 2+ at this concentration although the MAO-A activity showed a trend of being decreased (Table I). At the higher (but still pathophysiological) level of 50 I.tM, Cu 2§ almost abolished MAO-A and MAO-B activities in brain (Table I) and liver (Table II) mitochondria.
DISCUSSION The results of this study demonstrate that, at pathophysiological levels, several neurotoxic metal ions, including A13+, Cd 2+, Cu 2+ (but not Mn 2+ or Li+), are good inhibitors of both MAO-A and MAO-B activities in brain and liver mitochondria (Tables I and II). Moreover, MAO-A in brain mitochondria is more susceptible to the inhibitory effects of A13§ Cd 2§ and Cu 2+ than MAO-B in brain and liver mitochondria (compare the data in Tables I and II). (However, brain MAO-A is only marginally more susceptible than liver MAO-A to these metal ions.) In general, whenever inhibitory effects of A13+, Cd 2§ Cu 2§ are detected, the inhibition of MAO-A is consistently more pronounced than that of MAO-B (Tables I and H). Thus, taken together, our results provide some support for the hypothesis that some neurotoxic metals can exert direct inhibitory effects on the multiple forms of MAO. The inhibition of brain mitochondrial MAO-A by Cu 2+ and Cd 2§ may assume some pathophysiologicai importance in the neurotoxicity of copper and cadmium (see Lai et al. (1980) and Lai and Blass (1984b) for additional discussion and Refs.). Because at pathophysiological levels of 10-50 IxM, Cu 2§ and Cd 2§ are good inhibitors of brain mitochondrial MAO-A and, to a lesser extent, liver mitochondrial MAO-A (Tables I and II), the metal inhibition of MAO-A (and hence the metal-induced inhibition of the metabolism of neurotransmitter amines such as serotonin, norepinephrine, and dopamine) is a quantitatively more important mechanism than the metal inhibition of MAO-B. Since the inhibitory effects of A13§ on MAO-A and MAO-B are only detected at high metal ion concentrations (Tables I and II), this inhibition by A13§ is unlikely to assume pathophysiological importance in aluminum toxicity. However, the concentrations (0.5-2.5 mM) of A13§ at which the inhibition of brain mitochondrial MAO-A was detected, do fall within the range of brain levels of aluminum reportedly found in patients who died of diaiysis encephalopathy (see Lai and Blass (1984a) for additional discussion and Refs.). Even at high and toxic levels (see Lai et al. (1983, 1985, 1992), Underwood (1977), and Atterwill and Tordoff (1982) for discussions and Refs.), neither Mn 2+ nor Li § inhibits the activities of MAO-A and MAO-B in brain (Table I) and liver (Table II) mitochondria. Thus, one could reasonably argue against the possibility of the inhibition of the multiple forms of MAO by these metal ions playing a role in the toxicity of manganese and lithium. The selective inhibition of brain MAO-A by Cu 2§ and Cd 2§ led us to speculate that the decreases in neurotransmitter metabolism (e.g., metabolism of serotonin, norepinephrine, and dopamine) induced by these metal ions may be one of the mechanisms underlying the
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neurotoxic effects of copper and cadmium. Clearly, this is an interesting area for further investigation.
ACKNOWLEDGMENTS We thank Professors A.N. Davison and J.B. Clark for their interests and continued support. J.C.K. Lai wishes to thank Dean Arthur A. Nelson, Jr., and Dr. Dana L. Diedrich for their continued support and encouragement. The study was supported, in part, by the Worshipful Company of Pewterers, U.K. (J.C.K. Lai and T.K.C. Leung), the Brain Research Trust (L. Lim), the N.I.H., U.S.A. (J.C.K. Lai), and the Idaho State University College of Pharmacy Research Funds (J.C.K. Lai).
REFERENCES
Atterwill, C.K., and Tordoff, A.F.C. (1982). Effects of repeated lithium administration on the subcellular distribution of 5-hydroxytryptamine in rat brain. Brit. J. Pharmacol. 76:413-421. Bach, A.W., Lan, N.C., Johnson, D.L., Abell, C.W., Bembenek, M.E., Kwan, S.-W., Seeburg, P.H., and Shih, J.C. (1988). eDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties. Proc. Natl. Acad. Sci. USA 85:4934-4938. Grimsby, J., Lan, N.C., Neve, R., Chen, K., and Shih, J.C. (1990). Tissue distribution of human monoamine oxidase A and B mRNA. J. Neurochem. 55:1166-1169. Holzbauer, M., and Youdim, M.B.H. (1977). Physiological control of monoamine oxidases. In Usdin, E., Weiner, N., and Youdim, M.B.H. (eds.), Structure and Function of Monoamine Enzymes, Marcel Dekker, New York, pp. 601-627. Hsu, Y.-P.P., Weyler, W., Chen, S., Sims, K.B., Rinehart, W.B., Utterback, M.C., Powell, J.F., and Breakefield, X.O. (1988). Structural features of human monoamine oxidase A elucidated from eDNA and peptide sequence. J. Neurochem. 51:1321-1324. Johnston, J.P. (1968). Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol. 17: 1285-1297. Knoll, J., and Magyar, K. (1972). Some puzzling pharmacological effects of monoamine oxidase inhibitors. Adv. Biochem. Psychopharmacol. 5:393-408. Kochersperger, L.M., Waguespack, A., Patterson, J.C., Hsieh, C.C., Weyler, W., Salach, J.I., and Denney, R.M. (1985). Immunological uniqueness of human monoamine oxidases A and B: new evidence from studies with monoclonal antibodies to human monoamine oxidase A. J. Neurosci. 5:2874-2881. Lai, J.C.K., and Barrow, H.N. (1984). Comparison of the inhibitory effects of mercuric chloride on cytosolic and mitochondrial hexokinase activities in rat brain, kidney and spleen. Comp. Biochem. Physiol. 78C:81-87. Lai, J.C.K., and Blass, J.P. (1984a). Inhibition of brain glycolysis by aluminum. J. Neurochem. 42:438-446. Lai, J.C.K., and Blass, J.P. (1984b). Neurotoxic effects of copper: inhibition of glycolysis and glycolytic enzymes. N eurochem. Res. 9:1699-1710. Lai, J.C.K., Chan, A.W.K., Leung, T.K.C., Minski, M.J., and Lim, L. (1992). Neurochemical changes in rats chronically treated with a high concentration of manganese chloride. Neurochem. Res. (in press).
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Lai, J.C.K., and Clark, J.B. (1979). Preparation of synaptie and non-synaptic mitochondria from mammalian brain. In Fleischcr, S., and Packer, L. (eds.),Methods in Enzymology, Vol. 55, Part F, Academic Press, New York, pp. 51-60. Lai, J.C.K., and Clark, J.B. (1989). Isolation and characterization of synaptic and non-synaptic mitocbondria from mammalian brain. In Boulton, A.A., Baker, G.B., and Butterworth, R.F. (eds.), NeuroMethods, Vol. 11, Humana Press, Clifton,NJ, pp. 43-98. Lai, J.C.K., Guest, J.F.,Leung, T.K.C., Lira,L., and Davison, A.N. (1980). The effectsof cadmium, manganese and aluminum on sodium-potassium-activated and magnesium-activated adenosine Iriphosphatase activity and choline uptake in rat brain synaptosomes. Biochem. Pharmacol. 29:141-146. Lai, J.C.K., Leung, T.K.C., and Lira, L. (1982). Monoamine oxidase activities in liver, heart, spleen and kidney of the rat: organ-specific changes in aging and after chronic manganese chloride administration. Exp. Gerontol. 17:219-225. Lai, J.C.K., Leung, T.K.C., and Lim, L. (1985). Effects of metal ions on neurotransmitter function and metabolism. In Gabay, S., Harris, J., and Ho, B.T. (eds.), Metal Ions in Neurology and Psychiatry (Neurology andNeurobiology), Vol. 15, Alan IAss, New York, pp. 177-197. Lai, J.C.K., Walsh, J.M., Dennis, S.C., and Clark, J.B. (1977). Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J. Neurochem. 28:625-631, Lai, J.C.K., Wong, P.C.L., and Lim, L. (1983). Structure and function of synaptosomal and mitochondrial membranes: elucidation using neurotoxic metals and neuromodulatory agents. In Sun, G.Y., Bazan, N., Wu, J.-Y., Porcellati, G., and Sun, A.Y. (eds.), NeuralMembranes, Humana Press, Clifton, NJ, pp. 355-374. Leung, T.K.C., Lai, J.C.K., Mart, W., and Lim, L. (1980). The activities of the A and B forms of monoamine oxidase in liver, hypothalamus and cerebral cortex of the female rat: effects of administxation of ethinyl oestradiol and the progestogens norethisterone acetate and D-norgestrel. Biochem. Soc. Trans. 8:615-616. Leung, T.K.C., Lai, J.C.K., and Lira, L. (1981). The regional distribution of monoamine oxidase activities towards different substrates: effects in rat brain of chronic administration of manganese chloride and of ageing. J. Neurochem. 36:2037-2043. Leung, T.K.C., Lai, J.C.K., and Lira, L. (1982). The effects of chronic manganese feeding on the activity of monoamine oxidase in various organs of the developing rats. Comp. Biochem. Physiol. 71C:223-228. Owen, F., Bourne, R.C., Lai, J.C.K., and Williams, R. (1977). The heterogeneity of monoamine oxidase in distinct populations of rat brain mitochondria. Biochem. Pharmacol. 26:289-292. Powell, J.F., Hsu Y.-P.P., Weyler, W., Chen, S., Salach, J.I., Andrikopoulos, K., Mallet, J., and Breakefield, X.O. (1989). The primary structure of bovine monoamine oxidase type A: comparison with peptide sequences of bovine monoamine oxidase type B and other flavoenzymes. Biochem. J. 259, 407-413. Shih, J.C., Grimsby, J., and Chert, K. (1990). The expression of human MAO-A and B genes. J. Neural Transm. Suppl. 32, 41-47. Tipton, K.F., Houslay, M.D., and Mantle, TJ. (1976). The nature and locations of the multiple forms of monoamine oxidase. In Monoamine Oxidase and Its Inhibition (Ciba Foundation Symposium 39, new series), Elsevier, Amsterdam, pp. 5-31. Underwood, EJ. (1977). Trace Elements in Human and Animal Nutrition, 4th Ed., Academic Press, New York, pp. 440-442.
Differential Effects of Metal Ions on Type A and Type B M o n o a m i n e Oxidase Activities in Rat Brain and Liver Mitochondria Thomas K.C. Leung, 1' 2 Louis Lim, 1 and James C.K. Lai 3.4 Received June 8, 1992 ; accepted July 30, 1992 To investigate the hypothesis that neurotoxic metals can exert their toxicity through the direct inhibition of monoamine oxidases (MAOs), the effects of several neurotoxic metal ions on type A (MAO-A) and type B (MAO-B) monoamine oxidase activities in rat forebrain nonsynaptic mitochondria and rat liver mitochondria were studied. At pathophysiological levels (10-100 p.M), Cu 2+ and Cd 2+ are good inhibitors of brain mitochondrial MAO-A and, to a lesser extent, liver mitochondrial MAO-A. The inhibition of MAO-B activities in brain and liver mitochondria by Cu 2+ and Cd 2+ is only detected at the higher end of the concentration range (i.e., 50-100 p.M). At the pathophysiological level of 0.5 mM, A13+ only inhibits brain mitochondrial MAO-A but at the higher level of 2.5 mM, it inhibits both forms of MAO in brain as well as liver mitochondria. Even at toxic levels (e.g., 5 mM), neither Mn 2§ nor Li § inhibits the activities of MAO-A and MAO-B in brain and liver mitochondria. Our results are consistent with the hypothesis that some neurotoxic metals can exert their toxicity through the direct inhibition of the isoforms of MAO. Our data also suggest that the selective inhibition of brain MAO-A by Cu 2+ and Cd 2+ may assume pathophysiological importance in the neurotoxicity of copper and cadmium.
KEY WORDS: metal toxicity; monoamine oxidases; metal inhibition of monoamine oxidases.
1 Department of Neurochemistry, Institute of Neurology, University of London, Queen Square, London WC1N 3BG, U.K. 2 Present address is: Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511. 3 Department of Pharmaceutical Sciences and Center for Toxicology Research, College of Pharmacy, Idaho Slate University, Pocatello, ID 83209, U.S.A. 4 To whom correspondence should be addressed at the Department of Pharmaceutical Sciences, College of Pharrnacy, Idaho State University, Campus Box 8334, Pocatello, ID 83209-0009, U.S.A.
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INTRODUCTION There is good evidence that monoamine oxidase (MAO; monoamine: 0 2 oxidoreductase, EC 1.4.3.4) in mammalian tissues (including brain) exist in at least two forms (known, respectively, as type A (MAO-A) and type B (MAO-B) MAO) (Tipton e t a / . , 1976; Holzbauer and Youdim, 1977; Bach et al., 1988; Shih et al., 1990). The initial classification of MAO into types A and B is based upon substrate-and inhibitor- specificities (Johnston, 1968; Knoll and Magyar, 1972; Tipton et al., 1977; Holzbauer and Youdim, 1977). For example, MAO-A selectively deaminates neurotransmitter amines such as serotonin (5-HT) and norepinephrine (NE) and is preferentially inhibited by clorgyline whereas MAO-B selectively deaminates benzylamine and phenylethylamine and is preferentially inhibited by deprenyl (see Tipton et al. (1977), Owen et al. (1977), and Lai et al. (1982) and Refs. cited therein). Results from immunochemical studies with anti-MAO monoclonal antibodies reveal that there are unique epitopes on human MAO-A not shared by human MAO-B (Kochersperger et al., 1985). Thus, the immunochemical data (Kochersperger et al., 1985) provide some support for the hypothesis that MAO-A and MAO-B are distinct proteins or isozymes. More definitive support for this hypothesis comes from recent structural studies on MAO: the peptide and cDNA sequences indicate that MAO-A and MAO-B are encoded in different genes and have some homology at the amino acid level (Bach et al., 1988; Hsu et al., 1988; Powell et al., 1989). Tissue-specific and age-related differential expressions of MAO-A and MAO-B genes have been noted (Shih et al., 1990; Grimsby et al., 1990). Metal ions play many critical roles in the "neurotransmitter metabolic cycle" (see Lai et al., 1985 for discussion and Refs.). Since MAOs (together with catechol-O-methyl transferase) are the key enzymes that regulate the metabolism of biogenic amines, especially neurotransmitter amines such as serotonin, dopamine, and norepinephrine (see Lai et at. (1983, 1985) for discussion and Refs.), it has been speculated that, in the neurotoxicity of several metals (including manganese), one underlying mechanism may be the inhibition by the metal ions of the MAOs and thereby altering amine metabolism (see Lai et al. (1983, 1992) for a detailed discussion and Refs.). The present study was initiated to address the hypothesis that neurotoxic metals can exert their toxicity through the direct inhibition of MAOs. The effects of several metal ions (i.e., Li§ Mn2§ Cd2§ Cu2+, and A13§ known to be neurotoxic, on MAO-A and MAO-B activities in rat brain and liver mitochondria were investigated since the distributions of MAO isoforms in brain and liver are different. M A T E R I A L S AND METHODS
Analytical grade chemicals were used and purchased either from BDH Chemicals (Enfield, Middlesex, U.K.) or from Sigma Chemicals (St. Louis, MO, U.S.A.). BBS3 was obtained from Beckman Instruments Inc. (Fullerton, CA, U.S.A.) and butyl-PBD (5-(4-biphenyl)-2-(4-t-butylphenyl)-l-oxa-3,4-diazole) was from CIBA Ltd. (Horsham, Sussex, U.K.). 5-Hydroxy-side chain 2-[14C]tryptamine creatinine sulfate (serotonin) (specific activity 58 mCi/mmol) and [7-I4C]benzylamine hydrochloride (specific activity 60 mCi/mmol) were purchased from the Radiochemical Centre, Amersham (Buckinghamshire, U.K.). All solutions were prepared using deionized glass-distilled water.
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Male Wistar (M.R.C. Porton strain) rats (150-170 g; 50-65 days old) were employed. For the isolation of forebrain non-synaptic mitochondria, four rats were used in each experiment; the procedure was that of Lai and Clark (Lai et al., 1977; Lai and Clark, 1979) except that none of the media contained EDTA and the mitochondria were finally washed twice without the inclusion of albumin in the isolation medium. For the preparation of liver mitochondria, two rats were used in each experiment. Liver mitochondria were isolated by differential centrifugation (Lai and Barrow, 1984) and then further purified using the discontinuous Ficoll-sucrose gradient of Lai and Clark (1979, 1989); none of the media contained EDTA. The forebrain non-synaptic mitochondria and liver mitochondria so isolated were metabolically active and relatively free from non-mitochondrial material (Lai and Clark, 1979, 1989 and J.C.K. Lai, unpublished observations). MAO-A (serotonin-oxidizing) and MAO-B (benzylamine-oxidizing) activities were assayed as described previously (Owen et al., 1977; Leung et al., 1980) but with minor modifications (Leung et al., 1981, 1982; Lai et al., 1982). Potassium phosphate (40 mM) buffer, pH 7.4 was used instead of the Tris-HC1 buffer. Final concentrations of all substrates were 1 mM and 0.1 mg of mitochondrial protein was employed per assay tube. The temperature was 37 ~ C. and pre-incubation time was 5 minutes. (Where appropriate, the metal ions (at the specified concentration) were added at the beginning of the pre-incubation.) When serotonin was the substrate, the assay incubation time was 30 minutes, whereas in the case of benzylamine, it was 60 minutes. A 10-minute-boiled tissue sample was used as the blank and this was processed as for the reaction mixture. The reaction was stopped by the addition of 0.1 ml of 6 M HC1 to the reaction mixture. The reaction products of serotonin and benzylamine were extracted with 2 ml of benzene:ethyl acetate mixture (1:1, v/v) and 2 ml of toluene, respectively. After centrifugation at 1000 g for 5 minutes, 1 ml of the organic phase was transferred to a vial containing 10 ml of scintillation fluid, which contained 0.4% (w/v) of butyl-PBD in a mixture of toluene and Bio-Solv BBS3 (9:1, v/v). The radioactivity therein was determined by liquid scintillation spectrometry. The difference in the extracted radioactivity from the reaction mixture and that obtained using the boiled tissue blank was used to calculate the enzymatic activity (in mU/mg protein). (1 mU was defined as 1 nmole of product formed per minute.) Protein determinations were carded out by the method of Lowry et al. (1951). Under the assay conditions employed in the present study, MAO-A and MAO-B activities were linear with respect to the reaction time and tissue concentrations. Statistical analyses were carried out using a BASIC program (written by J.C.K. Lai) for analysis of variance (ANOVA) with the post-hoe Tukey test for multiple comparisons. Only P values < 0.05 were regarded as being significant.
RESULTS Effects of LiCI and MnCI2 on MAO-A and MAO-B activities in Brain and Liver Mitochondria
The effects ofLi + were investigated at 5 mM because this concentration is slightly above the therapeutic range although toxic symptoms have been noted (see Underwood (1977) and
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Atterwill and Tordoff (1982) for discussion). However, at this level, Li § did not affect M A O - A and M A O - B activities in brain and liver mitochondria (Tables I and II). At pathophysiological levels (0.1-1 mM) (see Lai et al. (1992) for discussion and Refs.), Mn 2+ did not significantly alter the activities of M A O - A and M A O - B in brain (Table I) and liver (Table II) mitochondria. Even at toxic levels (5-10 mM), Mn 2§ was still ineffective (Tables I and H).
Effects of AICi3 on MAO-A and MAO-B activities in Brain and Liver Mitochondria At the pathophysiological level of 0.5 m M (Lai and Blass, 1984a), A13§ did not alter the activities of M A O - B in brain mitochondria (Table I). Nor did A13+ (at 0.5 mM) markedly influence the activities o f M A O - A and M A O - B in liver mitochondria although the M A O - A
Table I. Effects of Metal Ions on Type A (Serotonin-Oxidizing) and Type B (BenzylamineOxidizing) Monoamine Oxidase Activities in Rat Brain Mitochondia a
Monamine Oxidase Activity (% of control) Metal Ions
Concentration SertoninOxidizing
LiC1
BenzylamineOxidizing
5mM
96 + 8
96 + 10
MnC12
0.1mM 1 mM 5mM 10mM
90 + 5 88 + 6 96+3 97+1
A1C13
0.5 mM 2.5 mM
78 + 4 b 44+3 c
93 + 5 46+5 c
CdC12
10 glVl 100gM
53 + 9 c 3+1c
97 + 8 5+1c
CuC12
10 gM 50gM
70 + 10 b 18+8c
91 + 4 3+2c
108 + 104 + 111+ 111+
5 17 1 1
a Values are Means + SD of 3-4 separate experiments, expressed as % of corresponding control activities (MAO-A, 4.22 + 0.19 mU/mg protein; MAO-B, 2.22 + 0.18 mU/mg protein). Other details are as described in MATERIALS AND METHODS. t~ p < 0.05 versus corresponding control activity. c p < 0.01 versus corresponding control activity.
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143
activity showed a trend of being decreased (Table II). However, 0.5 m M A13§ did mildly but significantly inhibit M A O - A in brain mitochondria (by 20%; Table I). At the higher level of 2.5 mM, AI 3§ definitely inhibited (by approximately 50%) both M A O - A and MAO-B activities in brain (Table I) and liver (Table II) mitochondria. Effects of CdCI2 on MAO-A and MAO-B activities in Brain and Liver Mitochondria At 10 gM, Cd 2§ significantly inhibited M A O - A activities in brain (by 45%; Table I) and liver (by 25%; Table II) mitochondria. However, at this level, Cd 2§ was without effect on MAO-B activities in brain and liver mitochondria (Tables I and II). At the higher and more toxic level of 100 gM, Cd 2§ strongly inhibited (by >80%) both types of M A O activities in brain and liver mitochondria (Tables I and II).
Table II. Effects of Metal Ions on Type A (Serotonin-Oxidizing) and Type B (BenzylamineOxidizing) Monoamine Oxidase Activities in Rat Liver Mitochondria a
Monoamine Oxidase Activity (% of Control) Metal Ions
Concentration S erotoninOxidizing
BenzylamineOxidizing
5 mM
105 + 7
101 + 6
MnC12
0.1 mM lmM 5raM 10 mM
98 + 6 93+6 100+2 96+ 1
93 + 6 108+3 102+3 94+ 1
A1C13
0.5 mM 2.5 mM
85 + 5 52+2 c
91 + 2 49+2 c
CdC12
10 ~Vl 100 p.M
74 + 4 b 2+1 c
90 + 8 15+1 c
CuC12
10 gM 500M
88 + 4 3+1 c
101 + 5 2+1 c
LiC1
a Values are Means + SD of 3-4 separate experiments, expressed as % of corresponding control activities (MAO-A, 7.04 _+ 0.78 mU/mg protein; MAO-B, 5.21 + 0.17 mU/mg protein). Other details are as described in MATERIALS AND METHODS. b p < 0.05 versus corresponding control activity. c p< 0.01 versus corresponding control activity.
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Effects of CuCI2 on MAO-A and MAO.B activities in Brain and Liver Mitochondria
At the pathophysiological level of 10 I~M (see Lai and Blass (1984b) for discussion and Refs.), Cu 2§ only significantly inhibited (by 20-30%) MAO-A (but not MAO-B) in brain mitochondria (Table I). Neither MAO-A nor MAO-B in liver mitochondria was significantly affected by Cu 2+ at this concentration although the MAO-A activity showed a trend of being decreased (Table I). At the higher (but still pathophysiological) level of 50 I.tM, Cu 2§ almost abolished MAO-A and MAO-B activities in brain (Table I) and liver (Table II) mitochondria.
DISCUSSION The results of this study demonstrate that, at pathophysiological levels, several neurotoxic metal ions, including A13+, Cd 2+, Cu 2+ (but not Mn 2+ or Li+), are good inhibitors of both MAO-A and MAO-B activities in brain and liver mitochondria (Tables I and II). Moreover, MAO-A in brain mitochondria is more susceptible to the inhibitory effects of A13§ Cd 2§ and Cu 2+ than MAO-B in brain and liver mitochondria (compare the data in Tables I and II). (However, brain MAO-A is only marginally more susceptible than liver MAO-A to these metal ions.) In general, whenever inhibitory effects of A13+, Cd 2§ Cu 2§ are detected, the inhibition of MAO-A is consistently more pronounced than that of MAO-B (Tables I and H). Thus, taken together, our results provide some support for the hypothesis that some neurotoxic metals can exert direct inhibitory effects on the multiple forms of MAO. The inhibition of brain mitochondrial MAO-A by Cu 2+ and Cd 2§ may assume some pathophysiologicai importance in the neurotoxicity of copper and cadmium (see Lai et al. (1980) and Lai and Blass (1984b) for additional discussion and Refs.). Because at pathophysiological levels of 10-50 IxM, Cu 2§ and Cd 2§ are good inhibitors of brain mitochondrial MAO-A and, to a lesser extent, liver mitochondrial MAO-A (Tables I and II), the metal inhibition of MAO-A (and hence the metal-induced inhibition of the metabolism of neurotransmitter amines such as serotonin, norepinephrine, and dopamine) is a quantitatively more important mechanism than the metal inhibition of MAO-B. Since the inhibitory effects of A13§ on MAO-A and MAO-B are only detected at high metal ion concentrations (Tables I and II), this inhibition by A13§ is unlikely to assume pathophysiological importance in aluminum toxicity. However, the concentrations (0.5-2.5 mM) of A13§ at which the inhibition of brain mitochondrial MAO-A was detected, do fall within the range of brain levels of aluminum reportedly found in patients who died of diaiysis encephalopathy (see Lai and Blass (1984a) for additional discussion and Refs.). Even at high and toxic levels (see Lai et al. (1983, 1985, 1992), Underwood (1977), and Atterwill and Tordoff (1982) for discussions and Refs.), neither Mn 2+ nor Li § inhibits the activities of MAO-A and MAO-B in brain (Table I) and liver (Table II) mitochondria. Thus, one could reasonably argue against the possibility of the inhibition of the multiple forms of MAO by these metal ions playing a role in the toxicity of manganese and lithium. The selective inhibition of brain MAO-A by Cu 2§ and Cd 2§ led us to speculate that the decreases in neurotransmitter metabolism (e.g., metabolism of serotonin, norepinephrine, and dopamine) induced by these metal ions may be one of the mechanisms underlying the
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145
neurotoxic effects of copper and cadmium. Clearly, this is an interesting area for further investigation.
ACKNOWLEDGMENTS We thank Professors A.N. Davison and J.B. Clark for their interests and continued support. J.C.K. Lai wishes to thank Dean Arthur A. Nelson, Jr., and Dr. Dana L. Diedrich for their continued support and encouragement. The study was supported, in part, by the Worshipful Company of Pewterers, U.K. (J.C.K. Lai and T.K.C. Leung), the Brain Research Trust (L. Lim), the N.I.H., U.S.A. (J.C.K. Lai), and the Idaho State University College of Pharmacy Research Funds (J.C.K. Lai).
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