Manganese poisoning and the attack of trivalent manganese upon catecholamines

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 256, No. 2, August 1, pp. 638-650, 1987

Manganese Poisoning and the Attack of Trivalent Manganese upon Catecholamines’ FREDERICK Department of Microbiology Received

S. ARCHIBALD’

AND

CURTIS

TYREE

and Immunology, McGill University, Montreal, January

12,1987,

and in revised

form

March

Quebec,

Canada

20, 1987

Human manganese poisoning or manganism results in damage to the substantia nigra of the brain stem, a drop in the level of the inhibitory neurotransmitter dopamine, and symptoms resembling those of Parkinson’s disease. Manganic (Mr?) manganese ions were shown to be readily produced by 0; in vitro and spontaneously under conditions obtainable in the human brain. Mn3+ as its pyrophosphate complex was shown to rapidly and efficiently carry out four-electron oxidations of dopamine, its precursor dopa (3,4dihydroxyphenylalanine), and its biosynthetic products epinephrine and norepinephrine. Mn3+-pyrophosphate was shown to specifically attack dihydroxybenzene derivatives, but only those with adjacent hydroxyl groups. Further, the addition of Mn’+-pyrophosphate to a system containing a flux of 02 and dopamine greatly accelerated the oxidation of dopamine. The oxidation of dopamine by Mn3+ neither produced nor required 02, and Mn3+ was far more efficient than Mn2+, Mn4+ (MnOz), OS, or H202 in oxidizing the catecholamines. A higher oxidation state, Mn(OH)3, formed spontaneously in an aqueous Mn(OH)2 precipitate and slowly darkened, presumably being oxidized to Mn02. Like reagent Mn02, it weakly catalyzed dopamine oxidation. However, both Mn02 preparations showed dramatically increased abilities to oxidize dopamine in the presence of pyrophosphate due to enhancement of the spontaneous formation of the Mn3+ complex. These results strongly suggest that the pathology of manganese neurotoxicity is dependent on the ease with which simple Mn3+ complexes are formed under physiological conditions and the efficiency with which they destroy catecholamines. 0 1987 Academic Press, Inc.

Human manganese poisoning or manganism was first described by Couper and Glasgow in 1837 in workers refining manganese ores (1). Recently, manganism has received considerable attention, both because of its high incidence among miners and factory workers exposed to manganese dusts, and because its symptoms and pathology bear a striking resemblance to those of Parkinson’s disease (2-4). Symp-

toms frequently observed in manganism are disorientation, memory loss, anxiety, compulsive laughing and crying, mask-like facies, akinesia, rigidity, and tremor. Parkinson’s disease is believed to be caused in large measure by a reduction in the level of the catecholamine neurotransmitter dopamine in the caudate nucleus due to death of more than 80% of the dopaminergic cells of the substantia nigra. In both manganism and Parkinson’s, the amount of neuromelanin, an intracellular polymer of dopamine-derived quinones, is greatly reduced in the substantia nigra due to the loss of dopaminergic cells (2-5). At present, the principal therapy for Parkinson’s is administration of the dopamine precursor L-

i This work was supported by a research grant from the Medical Research Council of Canada. a To whom correspondence should be addressed at Pulp and Paper Research Institute of Canada, 570 St. John’s Boulevard, Pointe Claire, Quebec, Canada H9R

359. 0003-9861187 Copyright All rights

$3.00

0 198’7 by Academic Press, Inc. of repmduction in any form reserved.

638

MANGANESE

AND

CATECHOLAMINE

dopa (3,4-dihydroxy-L-phenylalanine) which can cross the endothelial bloodbrain barrier (unlike dopamine) and restore dopamine levels. Likewise, treatment of drug-abusing patients whose dopaminergic cells have been killed by MPTP (lmethyl-4-phenyltetrahydropyridine) and miners suffering from the hypokinetic form of chronic manganese poisoning with Ldopa results in a reduction or elimination of rigidity and hypokinesia (3, 6-8). However, L-dopa is frequently ineffective and the resultant controversy has generated the hypothesis that there are two types of manganese intoxication, one having symptoms of increased muscle tone and rigidity which responds to L-dopa treatment, and a second type showing symptoms of bradykinesis which doesn’t respond to L-dopa therapy (9). In his recent review, Barbeau emphasized that manganese neurotoxicity often exhibits additional symptoms not seen in Parkinsonism, including hyperkinetic dyskinesis, pallidal degeneration, and more diffuse brain pathology, leading him to conclude that manganism is best described as a “low dopamine syndrome” due to an early increase in dopamine turnover (3). The mechanisms whereby the inhalation of manganese dusts results in this severe disease are at present completely unknown. Hypotheses put forth to explain manganese neurotoxicity include (a) direct toxicity of Mnzf or Mn in a higher oxidation state for the dopaminergic cells (2); (b) Mn2+ exacerbation of the production of superoxide (O;), hydrogen peroxide, or hydroxyl (OH’) free radicals which may, in turn, attack dopamine, dopaminergic cells, or dopamine receptors (2, 11-13); (c) toxicity resulting from the diminution of the peroxidase and catalase content of the substantia nigra by manganese; (d) Mn2+catalyzed production of 6-hydroxydopamine or other toxic catecholamines and decrease in protective thiols (2,10-15); and a Abbreviations used: L-dopa, L-3,4-dihydroxyphenylalanine; MPTP, 1-methyl-4-phenyltetrahydropyridine; 6-OHDA, 6-hydroxydopamine; ODU, optical density units; SOD, bovine erythrocyte Cu-Zn cofactored superoxide dismutase.

OXIDATION

639

(e) Mn may oxidize or enhance the autooxidation of dopamine and thus both eliminate this neurotransmitter and produce from it cytotoxic quinones (2-5, 9-13, 16-23). Cohen and Heikkila (12) have shown that several oxidized catecholamines including 6-hydroxydopamine and 6-aminodopamine could form the cytotoxic species OH’, O,, and H202, and this was later confirmed by Graham et al. (11, 18). Graham et al. also showed that the quinone autooxidation products of L-dopa and dopamine exerted toxicity on Cl300 neuroblastoma cells in vitro via their nucleophilic reactivity (18). It has been observed that the substantia nigra has detectable levels of H202 and high levels of monoamine oxidase, able to evolve H202 (14). The polymerization of the catecholamine-derived quinones to form the neuromelanin for which the substantia nigra is named is a 0, evolving autooxidative process (18). Ambani et al. have reported lowered levels of peroxidase and catalase in the substantia nigra of Parkinson’s patients (14). Proponents of free radical mechanisms are often quick to point out the very low reported levels of catalase (23) and glutathione (23) reported in the human brain, but any such explanation must also take into account the relatively high reported levels of SOD (24), nigral peroxidase and catalase (14), a-tocopherol (23), and carotenoids found there (23). Jonsson (17) and Rotman et al. (21) showed that MnC12 treatment produced a loss of dopamine from the corpus striatum of rats. Finally, Donaldson et al. have presented evidence that Mn2+ in vitro increases dopamine autooxidation and propose that Mn in a higher oxidation state may efficiently oxidatively attack dopamine (26). Donaldson et al. also have data for an apparently contradictory effect, i.e., that Mn2+-pyrophosphate, Mn2+-tartarate, and some other Mn2+ complexes retard lipid peroxidation in brain homogenates (10). There are a number of other references to the efficient blocking of 0;) microsomal, or ionizing radiation-mediated lipid peroxidation by simple Mn2+ complexes (15,27-29,35). The difficulties in determining the true mechanism of Mn toxicity stem from the for-

640

ARCHIBALD

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midable technical problems of experimentally observing the neurochemistry of the brain, and from a lack of understanding of the probable in viva chemical reactions among various oxy radicals, manganese ions and complexes, and catecholamines. Recently the manganese biochemistry of Lactobacillus plantarum was investigated after this bacterium was found to use millimolar levels of nonenzymatic manganese as a scavenger of 0; in place of the micromolar superoxide dismutase (SOD) carrying out this function in most other organisms (30). Some of the findings of this and subsequent work on manganese (31-35) appear to be relevant in explaining human manganism. They include (i) Mn2+ scavenges 0, and is efficiently converted to Mn3+ in a number of simple complexes and chelates (Eq. [l]); (ii) many simple Mn3+ complexes are relatively stable and can selectively oxidize a variety of specific biological molecules; (iii) some Mn chelates can act as a true SOD, dismuting 2 0; to O2 and Hz02 (Eqs. [l], [2a]). Under no conditions have Mn2+ or Mn3+ been seen to evolve 0, and unlike the related 02-reducing transition metals Cu and Fe, Mn2+ and Mn3+ do not produce OH’ via Fenton’s or Haber-Weiss reactions (28, 29, 34-36). However, while Mn2+ and Mn3+ complexes appear to have little or no ability to scavenge OH’ directly, by scavenging 0; they can prevent the Fe3+-EDTA catalyzed production of OH’ in y irradiation and xanthine oxidase 0; generating systems (Cheton and Archibald, unpublished). Mn2+ complex + 0; + 2 H+ + Mn3+ complex + H202

[l]

Mn3+ complex + 02 + Mn2+ + O2

Dal

2 Mn3+ complex + AH2 + 2 Mn2+ + A

[2b]

2 Mn3+complex

+ H202 --f 2 Mn2+ + O2 [2c]

Therefore, a stable and physiologically relevant Mn3+ complex, Mn3+-pyrophosphate, was reacted with a variety of catecholamines derived from tyrosine, ineluding dopa, dopamine, norepinephrine (arterenol), epinephrine (adrenalin), and 6hydroxydopamine (B-OHDA), as well as

TYREE

with related dihydroxy and quinone compounds. Mn4+, the other high oxidation state found in viva, was also tested as hydrous or reagent Mn02 for its ability to oxidize dopamine. The results eliminate a number of the proposed toxic mechanisms and suggest that Mn3+ is the proximal toxic species in manganism. METHODS

AND

MATERIALS

Assays. Superoxide (0;) was generated by using xanthine oxidase to oxidize acetaldehyde to acetic acid in a system derived from the classical cytochrome c superoxide dismutase assay (37). EDTA was omitted from all assay mixtures unless otherwise stated, and all assays employing 0; were performed at pH 7.8 and 25’C, using sufficient xanthine oxidase to produce an initial rate of reduction of 0.025 ODU/min at 550 nm for 10 pM cytochrome c. All difference and wavelength scans employed a Pye Unicam SP8-400 uv/vis spectrophotometer, recorder, and 3-ml silica cuvettes. Fixed wavelength measurements also employed a Gilford 2000 instrument. Both instruments were calibrated with a holmium oxide filter and were accurate to f0.7 nm near the measured uv and visible peaks. A neutral density standard ensured that OD values were accurate to within 2%. Oxygen consumption measurements used a stirred 3-ml polarographic oxygen measuring cell and a chart recorder (Rank Bros., Bottisham) at 25°C. Reagents. To avoid complex formation and precipitation, MnCl, was dissolved in distilled water. All other reagents were dissolved in 10 mM potassium phosphate buffer, pH 7.2, unless otherwise stated. The Mn3+-pyrophosphate complex was prepared by combining 100 nM Na-pyrophosphate, pH 7.0, and 10 mM MnCla with excess (-20 mM) MnOa dust. This mixture was gently stirred for 24 h at 25°C. Upon centrifugation (25OOg, 5 min) to remove the residual insoluble MnOz, the deep red color of Mn3+-pyrophosphate was observed. The mangani pyrophosphate millimolar extinction coefficient at 259 nm reported by Kenten and Mann (6.20) (38) was used to calculate the final Mna+ concentration (18.2 mM). The visible peak of M& pyrophosphate at X = 478 nm (c mM = 0.104) was also observed. To ensure a good yield of stable Mn3+-pyrophosphate the pyrophosphate buffer was extracted for Fe using the 8-hydroxyquinoline-chloroform method of Waring and Werkman (39) and then autoclaved to remove the traces of chloroform. This preparation is stable for years. All solutions of dopamine, DL-dopa, norepinephrine, epinephrine, and tyrosine were made fresh daily and kept on ice and in the dark until used. The 6-OHDA solutions had to be made under low light and pOs and inserted into the spectrophotometer as quickly as possible. The xanthine oxidase (from bovine cream)

MANGANESE

AND

CATECHOLAMINE

was daily diluted from stock, and its activity, including the ability of superoxide dismutase to inhibit its reduction of cytochrome c, was assessed using the cytochrome c SOD assay. Acetaldehyde was stored at -20°C and distilled at 23°C on the day of use. An increase in the boiling point of stock acetaldehyde to 25°C or greater indicated it was unusable. Catalase (bovine liver) and copper-zinc cofactored bovine erythrocyte superoxide dismutase were dialyzed and filter-sterilized (0.22 pm pore size) before use. Horse heart cytochrome c (Sigma type III) was also made up fresh daily. Granular MnOz was washed three times in 10 mM K+ phosphate buffer, pH 7.2, by agitation and decanting of the supernatant to remove the small amount of MnOz dust present. The hydrous Mn4+ complex was produced by adding 10% (w/v) reagent MnClz * 4HzO to 10 mM Tris base at its natural pH of 9.2. The pH of the resulting solution (7.1) was raised with NaOH to 8.3 and the resulting heavy tan precipitate of Mn(OH)3 was collected and washed three times (5 min, 25OOg) with excess water. The resulting thick slurry (pH 6.9) progressed to a dark brown over the next 24 h, indicating the formation of MnOz. All assays were performed using 10 mM K+ phosphate, pH 7.2, as buffer, except those using xanthine oxidase, which were run at pH 7.8. SOD, catalase, all organic reagents, and the manganese salts were obtained from the Sigma Chemical Co., except the MnOz dust which was from Fisher Scientific. RESULTS

When DL-DOPA in a physiological buffer was mixed with Mn3+-pyrophosphate there was an immediate (~5 s) appearance of visible color, easily discernible from the Mn3+-pyrophosphate color by its much greater intensity and different visible maximum (476 nm) (Table I). Parallel trials using Mn2+ -pyrophosphate, or a lOOO-fold molar excess of MnC12 or M&O4 produced no reaction with DL-dopa. Increasing the Mn3+:DL-dopa ratio (Table I) and observing 600-200 nm spectral scans (not shown) demonstrated the successive production of two oxidized species from DLdopa by Mn3+ and the disappearance of Mn3+, as evidenced by the disappearance of its strong 259 nm absorbance (t mM = 6.20). The first oxidized species formed from DLdopa showed a X,,, at 282 nm and maximal concentration at a Mn3+:DL-dopa ratio of 2:l and is probably the ortho-quinone (18). This absorbance disappeared completely at a Mn3+:DL-dopa ratio of 4:l. The second and final product had absorbance maxima at

641

OXIDATION TABLE

I

OXIDATIONOFDL-DOPABYMX?+-PYROPHOSPHATE

Absorbance maxima (e mM) appearing upon Mn3+ addition DL-dopa:Mn3’ molar ratio 1:0.5 1:l 1:2 1:3 1:4 1:5 1:6 1:8

476 nm

302 nm

280 nm

0.48 0.92 1.80 2.64 3.64 3.92 3.88 4.05

ND 2.72 5.20 7.84 10.16 10.92 10.44 11.80

3.64 4.40 5.12 ND ND ND ND ND

Note. Reaction mixtures contained 0.1 InM DL-dopa, 10 mM K+-phosphate, pH 7.2, to which was added Mn3+ from a stock solution containing 18.2 ItIM Mn3+ and 6.2 nM Mn2+ in 50 mM Na+-pyrophosphate, pH 7.0. All reactions were carried out at 25°C and were complete in <5 s, i.e. by the time the added Mn3+ was thoroughly mixed into the 3-ml cuvette reaction mixture. ND, not detectable.

476 and 302 nm and was stable for at least several hours. These maxima are in reasonable agreement with those for dopachrome, the dehydro-indole-0-quinone derivative of dopa (18). Further evidence that the observed reaction was an oxidation of DL-dopa and a reduction of Mn3+-pyrophosphate was the appearance of a small amount of white precipitate ([Mn(OH)2k). If reduction of the catecholamine by Mn3+ were occurring, then a black precipitate of highly insoluble Mn02 would be expected. Oxidation of dopamine was also expected because Mn3+ is a powerful oxidant in most complexes and most catecholamines are readily oxidized. The results show that Mn3+ can efficiently degrade the immediate biochemical precursor of dopamine. When Mn3+-pyrophosphate was added to buffered dopamine, the results were identical to those with DL-dopa except for slightly different absorbance maxima (Table II). The fact that both DL-dopa and dopamine go to their two-electron oxidized intermediates (stable over at least 30 min) when the Mn3+:catecholamine ratio is 2:l indicates that the susceptibility of the original catecholamines to Mn3’-pyro-

642

ARCHIBALD TABLE

OXIDATION

0F DOPAMINE

II

BY Mn’+-PYROPHOSPHATE

Absorbance Dopamine:Mn3+ ratio 1:0.5 1:l 1:2 1:3 1:4 1:5 1:6 1:8

maxima appearing

(6 mM)

472 nm

298 nm

280 nm

0.50 0.84 1.65 2.50 3.30 3.74 3.67 3.95

ND ND 6.02 8.90 11.60 13.00 14.90 14.50

3.40 4.15 6.01 ND ND ND ND ND

Note. Assays were performed I). ND, not detectable.

as for DL-dopa

(Table

phosphate oxidation is greater than that of the oxidized intermediates. Figure 1 shows the relationship between the quantity of the final products of Mn3+:DL-dopa and dopamine oxidations and the Mn3+: catecholamine ratio, confirming that the oxidations are apparently four-electron. However, if reduced oxygen species were being produced in our system, this apparent four-electron oxidation of DL-dopa and dopamine by Mn3+ could be incorrect. Earlier work in a nonbiological system (0.52.0 M HClO& also suggested that Mn3+ initially displaces two protons from dihydroxy compounds including catecholamines and that this is followed by rapid reduction of Mn3+ to Mn2+ with concomitant quinone formation (40). When the mixtures used in the assays shown in Tables I and II were reacted in a closed 3-ml polarographic 02measuring cell (p02 -100 mm Hg), there was no net O2 consumption or evolution over a lo-min period, even though a net production or consumption of 1 O2 per 100 catecholamine molecules would have been detectable (data not shown). Addition of 100 U ml-’ of bovine liver catalase to the oxygen cell yielded no burst of 02, thus indicating no H202 production as well. It should be noted that if the Mn3+-oxidized DL-dopa and dopamine were left in aqueous solution, the solution blackened over several hours, suggesting a slow melanin-like

AND

TYREE

polymerization of the quinones. It was not determined if this process consumed or evolved 02. The mammalian biosynthesis of catecholamines starts from phenylalanine and successively produces tyrosine, L-dopa, dopamine, norepinephrine, and epinephrine. Table III demonstrates that Mn3+-pyrophosphate (but not Mn2+-pyrophosphate) rapidly oxidizes all these compounds, except tyrosine, which was completely unaffected. As in the case of DL-dopa, the colored products of the Mn3+ attack on dopamine, norepinephrine, and epinephrine have absorbance maxima corresponding (reasonably well) to those of the dehydroindole-0-quinones dopachrome, aminochrome, noradrenochrome, and adrenochrome, respectively (18). In the absence of Mn3+, oxidation of the neurotoxic 6OHDA to a stable colored compound in the 02-containing reaction mixture took several minutes, while Mn3+ accelerated this to completion in <5 s. With both Mn3+ and O2 per se, 6-OHDA absorbance maxima

Catecholamine:

Mnt3

ratio

FIG. 1. Stoichiometry of Mn3+-pyrophosphate-mediated DL-dopa and dopamine oxidation. Assays were run at 23”C, pH 7.2, in 10 mM K+-pyrophosphate buffer containing 100 PM of DL-dopa or dopamine. After addition of Mn’+-pyrophosphate from an 18.2 mEd stock, the absorbance was determined at 472 nm for dopamine (0) or 476 nm for DL-dopa (Cl).

MANGANESE

AND

CATECHOLAMINE

TABLE WAVELENGTH

PEAKS

SUBSTANCES

-.

-

Substance

AND

ALONE

643

OXIDATION

III

EXTINCTION COEFFICIENTSFOR DOPAMINE AND RELATED AND AFTER ADDITION OF Mn2+ OR Mn3+ (1:4 RATIO) A,, (nm)

fmM

Lax (nm)

emM

Tyrosine Tyrosine + Mna+ Tyrosine + Mngf

274 274 274

0.70 0.70 0.69

-

DL-dopa DL-dopa + Mna+ DL-dopa + Mn3+

280 280

3.14 3.14

-

-

476

3.64

472

3.30

302

10.2

Dopamine Dopamine + Mn’+ Dopamine + Mn3+

280 280

Norepinephrine Norepinephrine Norepinephrine

280

2.96

280

2.96

483

Epinephrine Epinephrine Epinephrine

+ Mn’+ + Mn3+ + Mn2+ + Mn3+

298

2.20 2.20

11.6

-

291

8.76

278

3.20

289

3.20

-

302

6.92

485

3.04 3.88

Note. The reactions were performed under the conditions described in Table I, using 0.1 mM of the appropriate substrate combined with 0.4 mM of Mna+-pyrophosphate or Mn3+-pyrophosphate.

appeared at 268 and 487 nm with c mM values of 23.1 and 2.5, respectively; i.e., the spontaneous and Mn3+-accelerated oxidation products appeared to be identical, but not identical to the dopamine oxidation product. It is likely that the Mn3+-produced 6-OHDA product is the para-quinone (18). The Mn3+-oxidized products of the catecholamines were not identified, but were generally similar in absorbance maxima to the products of tyrosinase (polyphenol oxidase)-treated L-dopa, dopamine, and norepinephrine reported by Graham (18). Whether the up to 5 nm discrepancies can be accounted for by instrument error, the breadth of the peaks, or reaction mixture differences, or whether Mn3+ produces different oxidized products from mushroom tyrosinase (polyphenol oxidase) is not known. The observation that phenylalanine and tyrosine were not oxidized by Mn3+, although their dihydroxy derivatives were, suggested the site of Mn3+ attack on the catecholamines. Therefore, a variety of non-, mono-, and dihydroxylated benzene ring compounds were tested for their sus-

ceptibility to Mn3+-pyrophosphate-mediated oxidation (Table IV). Only one of the three monohydroxylated compounds reacted and that one, guaiacol, had adjacent hydroxy and methoxy groups. Of the dihydroxy compounds, all those and only those with adjacent hydroxy groups were oxidized; i.e., the placement of the hydroxyl groups was all-important in the reactivity of these compounds with Mn3+. Since Mn3+ has a similar ionic radius and chelating behavior to Fe3+, and Fe3+ chelates very strongly to catechols (41), it is likely that chelation of Mn3+ by the dihydroxy moiety is the first step in the oxidative attack of Mn3+ upon catecholamines. Hydroquinone appeared exceptional in being oxidized yet having nonadjacent hydroxyl groups. It is particularly susceptible to oxidation and rereduction under a variety of conditions and may be reacting with Mn3+ via a different mechanism than the other hydroxylated compounds tested. Interestingly, the dihydroxy nature of the catecholamines may also in part determine their reactivity with O;, as norepinephrine reportedly reacts to some extent with 02, while oc-

644

ARCHIBALD

AND TABLE

SUSCEPTIBILITYOF

TYREE

IV

SELECTED CATECHOLAMINES,QUINONES,ANDBENZENEDERIVATIVES TOMn3+-PYROPHOSPHATE-MEDIATED OXIDATION Spectral changes

Substrate Benzoate Phenol Catechol

None None <20 s

pHydroxybenzoate 2,3-Dihydroxybenzoate

None
2,4-Dihydroxybenzoate 2,5-Dihydroxybenzoate 2,6-Dihydroxybenzoate 3,4-Dihydroxybenzoate 3,5-Dihydroxybenzoate 1,2-Dimethoxybenzene 1-Hydroxy-2-methoxybenzene

(/3-resorcylic acid) (gentisic acid) (protocatechuic

None None None 110 s

acid)

(veratrole) (guaiacol)

Benzoquinone Hydroquinone

None None
Note. Reactions were carried out in 10 mM K+-phosphate and 0.4 mM Mn3+ as the Mn3+-pyrophosphate complex. a These absorbances were shoulders, making the extinction

topamine, the identical molecule less the 3-hydroxy group, is much less reactive (42). The observation that Mnzf-pyrophosphate was apparently unable to oxidize dopamine (Table III) appeared to contradict some findings of Donaldson et al. (26), who reported that 10 PM of Znzf, Ni’+, Cu’+, and especially Mn2+ enhanced the autooxidation of 0.5 mM dopamine in 50 mM Tris buffer (pH ‘I.@, as measured by aminochrome formation at 480 nm. Table V is a repeat of their experiment with Mn2+, with some additional parameters added. We also found that the presence of Mn2+ enhances dopamine autooxidation, but that the pH and buffer used varied the rate by more than ninefold. Under the most “physiological” conditions (10 mM K+-phosphate, pH 7.2), the autooxidation rate in the presence of Mn2+ was barely higher than in its absence, while in Tris buffer at a more alkaline pH (7.8), as Donaldson et al. employed, Mn2+ exerted a significant effect. The tendency of Mn2+ to consume O2 and

Product 1 (nm)

buffer,

Absorbances emM

-

-

386 270 -

2.40 3.05’

403 256

1.22” 4.36’=

-

-

390 287 -

1.28 0.824” -

460 419 -

5.97 6.21 -

426 246

0.016 19.0

pH 7.2, at 25°C

coefficients

difficult

using

to calculate

0.1

mM

substrate

accurately.

precipitate at even slightly alkaline pH and of the precipitate to spontaneously form the +3 and +4 valence states is the most obvious explanation for this phenomenon (43). It is noteworthy that this tendency to spontaneously form Mn3+ is also the basis of the classic Winkler O2 assay in which O2 is consumed quantitatively by the addition of base to Mnzf forming Mn(OH)3 (44,45). It should be noted that even the most rapid Mn-mediated oxidation observed in the Mn2+-Tris system was thousands of times slower than the observed reaction of Mn3+pyrophosphate with dopamine. At this point one of the more attractive hypotheses was that an elevated level of brain stem Mn2+ in a form able to scavenge 02 might be efficiently converted to Mn3+ by the ambient flux of catecholamine-derived 0;; Mn thereby amplifying Oi-mediated damage. First the ability of 02 to efficiently oxidize Mn2+- to Mn3+-pyrophosphate, demonstrated previously (33), was confirmed using acetaldehyde and

MANGANESE

AND

CATECHOLAMINE TABLE

COMPARISON

OF THE RATE OF AUTOOXIDATION BUFFERS

645

OXIDATION

V

OF 0.5 XIIM DOPAMINE ALONE AT DIFFERENT pH VALUES

AND WITH Mn2+

Incubation Buffer

-

10 10 50 50 10 10 50 50 Note.

mM K+-phosphate mM K+-phosphate mM Tris-HCl mM Tris-HCI mM Kc-phosphate + Mn2+ mM K+-phosphate + Mn2+ mM Tris-HCl + Mn” mM Tris-HCl

Mn*+

+ MnzC

concentration

IN DIFFERENT

time

PH

20 min

40 min

60 min

7.2 7.8 7.2 7.8 7.2 7.8 7.2 7.8

0.008 0.020 0.012 0.030 0.012 0.042 0.021 0.143

0.013 0.036 0.021 0.051 0.020 0.077 0.037 0.219

0.020 0.051 0.030 0.072 0.031 0.108 0.053 0.282

was 10 pM (as MnCla)

and assays

xanthine oxidase. The production of Mn3+ from 0.4 mM Mnzf in 5 mM pyrophosphate observed at 259 nm was totally blocked by SOD (data not shown). In addition, the Mn3+-pyrophosphate was stabilized by the presence of bovine liver catalase. Direct reactivity of 0; with DL-dopa, dopamine, norepinephrine, and epinephrine was assessed in the acetaldehyde-xanthine oxidase assay. Epinephrine was quite reactive (comparable to cytochrome coX), but the other three catecholamines were very weakly reactive with 0, and Hz02 generated in the assay (Tables VI, VII). These results indicated that Mn2+, which reacts rapidly with 0; to form Mn3+ which, in turn, reacts rapidly with the catecholamines, greatly increases the ability of a given flux of 0; to oxidize catecholamines. Table VI bears this out. Maximal dopamine oxidation occurred only when a complete 0; generating system was present with Mn2+. In the absence of Mn2+, dopamine oxidation was negligible except in the presence of Fe3+-EDTA. Thus, hydroxyl radicals appeared to attack dopamine unaided (and unscavenged) by Mn, as indicated by the ability of Fe3+-EDTA to enhance dopamine oxidation and the ability of the OH. scavenger mannitol to reduce it. (Table VI; (36)). Clearly, 0; and H202 per se do not. The large inhibition of Mn2+dependent dopamine oxidation caused by SOD demonstrates the role of 02 in producing Mn3+.

were

performed

at 23°C.

The results in Table VII indicate that the oxidative destruction of DL-dopa, norepinephrine, and epinephrine were likewise greatly enhanced by the presence of Mn2+, and dependent upon the presence of 0, but not H202. As a number of established assays for 0, and SOD use the epinephrine to adrenochrome oxidation as their colorimetric detector (46), the high Mn-free oxidation rate was not surprising; but even here the presence of Mn2+ increased the rate of O;-dependent oxidation. It is worth noting that Mn3+ -pyrophosphate will readily react with millimolar H202 to produce water and O2 (Eq. [2c]) (33) but, when confronted with micromolar levels of H202 in the xanthine oxidase system and any of the four catecholamines tried, it preferentially oxidized the catecholamine (Tables VI, VII). In living tissue, Mn has been found as Mn2+, Mn3+, and Mn4+. We have shown that Mn2+ does not react with the catecholamines while Mn3+ does so vigorously. What about Mn4+? Mn4+ is known in biological systems exclusively as the highly insoluble dark oxide (Mn02), a good true catalyst for the dismutation of H202 to 02 and H20. When dopamine in orthophosphate buffer was exposed to a 4-fold and 200-fold molar excess of Mn02 in granular and dust forms, there was an immediate appearance of oxidized dopamine (Fig. 2). However, only a small proportion of the dopamine was oxidized and the rate of ox-

ARCHIBALD TABLE

AND

idation diminished greatly after the first 3 min. The MnOz dust with its much greater surface area gave substantially more dopamine oxidation per mole than the granular form, suggesting that the reaction stopped far short of completion due to coating of the MnOz particles with unreactive Mn2+ precipitates. The post-3-min plateaus and the poor reactivity of granular MnOz previously exposed to dopamine (Fig. 2) further supported this explanation. A more physiologically relevant form of MnOz is the loose hydrous matrix produced by precipitation and oxidation of aqueous Mn(OH)2 to Mn(OH)3 and MnOz in response to weakly alkaline conditions. While a white [Mn(OH),], precipitate showed no ability to oxidize dopamine, a preparation which had turned dark brown due to the spontaneous formation of Mn(OH)3, followed by its oxidation to MnOz, did oxidize dopamine to a limited extent (Fig. 3). Is Mn3+ or Mn4+ the proximal oxidizing species when dopamine is exposed to reagent MnOz? The addition of pyrophosphate greatly enhanced the dopamine oxidizing activity of the MnOz granules and dust as well as the hydrous brown Mn precipitate (Fig. 3). Since MnOz + pyrophosphate + MnClz disproportionates to Mn3+-pyrophosphate and pyrophosphate will solubilize the Mn3+ of Mn(OH)3, are these stimulations by pyrophosphate due to its ability

VI

BETWEEN THE PRESENCE OF MANGANESE COMPLEXES, SUPEROXIDE, AND HYDROGEN PEROXIDE AND THE OXIDATION OF DOPAMINE

RELATIONSHIPS

AODd X lo3 min-’ Reaction mixture, Reaction mixture, pyrophosphate Reaction mixture, oxidase Reaction mixture, Reaction mixture, acetaldehyde Reaction mixture, Reaction mixture, Reaction mixture, EDT-4 (1:l) Reaction mixture, EDTA (1:l) + Reaction mixture, Reaction mixture, Mn*+ + 100 U Reaction mixture, Mnzf + 100 U Reaction mixture, Mn2+ + 20 mM

complete less

0.3
less xanthine
less acetaidehyde less + 0.4 mM Mn*+ + 100 U SOD + 100 U catalase + 10 PM Fe3+-


+ 10 PM Fe3+20 mM mannitol + 0.4 mM Mn*+ + 0.4 mM SOD + 0.4 mM catalase + 0.4 mM mannitoi

0.5 3.5 0.9 3.7 2.4

Note. Reaction mixture: 50 mM K+-phosphate, 10 mM acetaldehyde, 1.6 X 10-a units of xanthine oxidase ml-‘, 5 mM Na+-pyrophosphate, and 0.1 mM dopamine. Assays were run at 25°C pH 7.8, in a calibrated Giiford 2000 spectrophotometer.

TABLE RELATIONSHIPS

BETWEEN THE PRESENCE OXIDATION

Reaction mixture Reaction mixture less xanthine oxidase Reaction mixture plus 0.4 mM Mn2+ Reaction mixture plus 0.4 mM Mn’+ + 100 U SOD Reaction mixture pius 0.4 mM Mn2+ + 100 U catalase Reaction mixture plus 0.4 mM Mn2+ + 20 nM mannitol Note. Reaction mixtures and procedures dopamine was replaced by 0.1 m&i DL-dopa,

VII

OF MANGANESE,

OF DL-DOPA,

Assay

TYREE

EPINEPHRINE,

(A0D4r6

SUPEROXIDE, AND HYDROGEN AND NOREPINEPHRINE

DL-dopa X lo3 mini)

Norepinephrine (AODns X lOa min-‘)

PEROXIDE,

AND THE

Epinephrine (AODds X lo3 mini)

0.3
0.6
6.5
0.9

0.5

1.5

2.5

2.4

-

2.3

2.3

-

were identical norepinephrine,

to those used in Table or epinephrine.

VI, except

that

the 0.1 mM

MANGANESE

AND

CATECHOLAMINE

647

OXIDATION

480 nm absorbances and the absorbance ratio of the 50009 supernatant. To produce equal amounts of Mn3+ pyrophosphate using MnClz and MnOs dust required 12 h (see Methods and Materials). A control substituting 100 mM Na+-orthophosphate (pH 7.2) for pyrophosphate showed uncletectable (G4.4 pM) Mn3+ in the 5000g supernatant. DISCUSSION

0

4

8

12

16

TIME (mid

FIG. 2. Effect of MnOz dust and granules on dopamine oxidation. Reactions employed MnOz dust (part.icle size 0.5-5.0 pm) and MnOz granules (particle size 30-60 pm). The MnOz granules were prewashed in aliquots of the reaction buffer, 10 mM K+-phosphate, pH 7.2, to remove dust. Dopamine was present at 1.0 mM and its oxidation was followed by measuring the appearance of the absorbance peak at 472 nm. During the reaction the tubes were periodically agitated to minimize MnOz settling. In the trials employing MnOa dust, samples were centrifuged (12,000g) for 30 s before measuring absorbance. The trials were 200 mM MnOz dust (0); 200 mM granular MnOa (0); 4 mM MnOI dust (n); 4 mM granular MnOa (0); and 200 mM granular MnOz preexposed to dopamine for 12 min, then rinsed and used (w); complete system less MnOz (+).

Parkinsonism, manganism and MPTP poisoning appear to exhibit related symptoms because in all three there is insufficient dopamine in the brain stem due to the destruction of clopaminergic cells, dopamine receptors, or dopamine itself. In this report we have demonstrated that Mn3+ in a pyrophosphate complex (probably Mn3+[pyrophosphate]3) has the capacity to rapidly destroy the principal brain catecholamines and their precursor, dopa, oxidizing them to at best useless and at worst toxic products. The general reaction seen in vitro (Tables VI and VII) is given in Eq. [3]: acetaldehyde

02

2H202

x0 acetic

acid )i

4t.tn3+

dopamine

4MIl A

dopachrome

4H H202+02-

4op

x

[31 to stabilize and increase the available Mn3+? When 1.0 mM o-dianisidine (3,3’-dimethoxy-4,4’-diaminobiphenyl) was exposed to Mn3+ -pyrophosphate, it immediately formed an intense orange oxidation product (X = 446 nm, t mM = 9.24), indicating that it was a good detector of Mn3+. Addition of Na+-pyrophosphate to granular MnOB or to the hydrous brown Mn precipitate resulted in a greatly increased rate of o-dianisidine oxidation to the orange product. Likewise, pyrogallol (1,2,3trihydroxybenzene) was oxidized to a yellow product by both Mn3+-pyrophosphate and MnOz in the presence of pyrophosphate (not shown). Finally, 1.5 ml of 100 mM Na+pyrophosphate (pH 7.2) was added to 0.2 ml of the washed hydrous brown Mn precipitate (Mn(OH)3 + MnOB) and incubated for 15 min, yielding 14.0 mM Mn3+ pyrophosphate, as determined by the 259 and

Mn3+-pyrophosphate activity could be mistaken for peroxidase activity, as it readily oxidized the three common peroxiclase assay substrates tried, namely guaiacol, o-dianisidine, and pyrogallol. It is worth noting that the nigral area of the brain reportedly has high peroxidatic activity (14), but unless cyanide or catalase negative controls are run, part or all of the observed “peroxidase” could as easily be Mn3+-mediated activity (Eq. [2b]). If the quinone oxidation products of dopa, dopamine, and 6-OHDA exert toxicity via covalent binding to cell proteins (11, 13, 19, 20, 47) and via evolution of oxyradicals (ll-13), and autooxidation and polymerization of polyphenols (e.g., neuromelanins) often proceed via one-electron transfer reactions and HzOz-evolving monoamine oxidase is very active in the

648

ARCHIBALD

AND TYREE

following

reactions

(44,45):

Mn2+ + 2 OH- + Mn( OH)2l 2 Mn( OH)2 + f O2 + H20 + 2 Mn( OH)3l

[41 [5]

Given such a precipitate, or one in which some or all of the Mn3+ has been further oxidized to Mn02, we have shown that the formation of a Mn3+-pyrophosphate chelate will occur. The amount of Mn3+ chelate thus formed is dependent on the initial concentrations of Mn2+ and Mn4+, the pH, 0 3 6 and competing chelators. Third, peroxiTIME (mid dases capable of specifically oxidizing FIG. 3. Effect of Na+-pyrophosphate on the oxidation Mn2+-pyrophosphate to Mn3+--pyrophosof 1.0 mM dopamine by MnOa. Buffer-washed granular phate are known from both plants (horseMnOs was the same as for Fig. 2. The hydrous brown radish peroxidase) (48) and fungi (the MMn precipitate was prepared and washed as described 2 peroxidase of Phanerochaete chrysospwunder Methods and Materials. The reaction was perium (49). Thus it would not be surprising formed as for Fig. 2 at 23”C, pH 7.2, in 10 mM K+if the high reported peroxidatic (or monophosphate buffer and measured at 472 nm. In the pyrophosphate trials, K+-phosphate was reduced to 6.7 amine oxidase) activity of the substantia mM and 3.3 mM Na+-pyrophosphate was added. The nigra (14) could, especially with the aid of trials were hydrous brown Mn precipitate (10 ~1 in 3 a phenolic or two, carry out the same reml) (m); the same precipitate in the presence of 3.3 action. Finally, we have clearly shown that mM Na+-pyrophosphate (0); 200 mM washed granular a flux of 0; (which is to be expected in the MnOa (0); 200 mM washed granular MnOa + 3.3 mM substania nigra) efficiently drives the oxiNa+-pyrophosphate (0); complete system + 3.3 mM dation of Mn2+ to Mn3+ in a pyrophosphate Na+-pyrophosphate, less Mn (0). complex as well as in other complexes (33). Chelating species capable of facilitating stable Mn3+ complex formation and which nigral area, it is very likely that accu- may be available in viva include citrate, mulations of o-quinone products result pyrophosphate, polyphosphates, malate, in significant fluxes of 02, H202, and gluconate, and fumarate (31,33,35), as well OH - production in the substantia nigra (2, as the catecholamines themselves. While 10,12,13). While in some biological systems free aqueous Mn3+ ions have a standard rethe presence of Mn2+ chelates or complexes dox potential 1.52 eV positive of hexaquo would act as a protectant by scavenging Mn2+ (and can split Hz0 directly), com0, and preventing lipid peroxidation, in the plexation greatly reduces the energy substantia nigra it would only exacerbate needed to form Mn3+, increases the stabilthe situation by producing more Mn3+ ity of the Mn3+ complex formed, and inwhich, in turn, would oxidize more dopa- creases the selectivity of the Mn3+-memine. How likely is it that Mn3+ is present diated oxidations carried out; e.g., reduced in the brain during manganese poisoning? glutathione but not NADH or NADPH (28), There are at least four reasonable mechand dopa but not tyrosine are oxidized by anisms. First, it has been noted that the Mn3+-pyrophosphate. It is worth noting that what human most toxic manganese dusts are braunite (Mn203 and Mn2Si03) and “old” dusts in brain catalase there is tends to be localized which Mn is principally in the +3 and +4 in the cell bodies of catecholamine neurons (25), thereby further stabilizing any Mn3+ valences (4). Second, when Mn2+ accumulates in a neutral or alkaline aerobic complexes in the area (see (33) and Eq. [2c]), aqueous system it tends to form a darkas would any deposits of MnOz. As we have ening precipitate due to the readiness with shown, the only compound of Mn4+ known to occur in vivo, Mn02, does react with dowhich Mn3’ (as [Mn(OH)3JJ is spontaneously formed from soluble Mn2+ via the pamine, but its primary reaction mecha-

MANGANESE

AND

CATECHOLAMINE

nism is via the production of Mn3+. In the absence of pyrophosphate, dopamine itself apparently is a good chelator for Mn3+, but when pyrophosphate is present, Mn3+-pyrophosphate production from MnOz is greatly favored as the standard Mn3+ preparation procedure demonstrates. The $5, +6, and +7 oxidation states of Mn, known primarily from the permanganate ion, MnO; and other compounds produced under extreme conditions, are not known to be formed in biological systems and MnO; is a sufficiently powerful oxidant to have very little selectivity or stability. Thus the findings presented here indicate that several of the earlier hypotheses to explain Mn toxicity in the brain stem are unlikely. Direct destruction of dopamine or its precursors by 0, or H,O, is unlikely as tyrosine, DL-dopa, and dopamine are relatively stable in the presence of a large flux of xanthine oxidase generated 02 and Hz02. At relatively high concentrations, as may be found in intracellular vesicles, norepinephrine and dopamine reportedly react with 0; (42), but at least in the xanthine oxidase Oi-generating system this reaction is far slower than the reaction of 02 with Mn2+, Mn3+-pyrophosphate, epinephrine, or cytochrome c,, (Tables VI, VII). Toxicity due to oxyradicals generated directly by Mn deposits or precipitates in tissue is unlikely as Mn2+ and Mn3+ will not generate 0, or OH. Mn02 efficiently catalyzes the conversion of H202 to water and 02, and Mn2+ is only known to generate H202 in the process of scavenging 0;. This tendency of Mn to scavenge rather than generate oxy radicals probably accounts for the ability of Mn to block oxy-radical-mediated lipid peroxidation in the brain and elsewhere (10,2’7-29), and not exacerbate it as Cu and Fe salts and complexes often do (34, 36). Manganese is unlikely to exacerbate the production of 6-OHDA, because Mn3+ rapidly further oxidized 6-OHDA and other catecholamines with adjacent hydroxyl groups, and Mn2+-pyrophosphate showed no significant effect on 6-OHDA or its precursor dopamine. However, should Mn2+ be present when 6-OHDA autooxidation releases O;, it would further hasten 6-OHDA destruction by forming Mn3+. Based on the

649

OXIDATION

data presented here, the most attractive hypothesis for manganese neurotoxicity is that Mn in the brain stem, whether originally entering in the +2, +3, or +4 state, will, via spontaneous oxidation and dismutation (favored by the slightly alkaline highly aerobic CNS milieu), peroxidatic activity, or Oi-mediated oxidation, give rise to Mn3+ which in a simple complex (perhaps with the catecholamines themselves) will efficiently oxidatively destroy dopamine, epinephrine, norepinephrine, and their precursor dopa. If the resulting 0-quinones evolve 02, then further Mn3+ forms and the process is accelerated; and if the quinones are cytotoxic, then as the dopaminergic cells die off and the level of dopamine falls, a higher and higher proportion of the remaining dopamine would be destroyed by a constant concentration of tissue manganese. Finally, it is worth repeating Cotzia’s speculation that Parkinsonism is a form of chronic manganism (4). Many researchers in Parkinsonism are now convinced by studies following identical twins, understanding of the neurotoxic effects of MPTP, and other data that Parkinsonism has an important environmental component, i.e., some substance or class of substances to which some people are exposed significantly accelerates the natural rate of nigral dopaminergic cell death. Average soils contain 0.02 to 0.15% Mn, with some substantially higher. If even a few years of inhalation of dusts from high Mn containing ores frequently results in the severe nigral and pallidal cell destruction of manganism, why shouldn’t many decades of inhaling soil dust with low percentages of Mn result in a slower, milder, and more localized but similar effect, a predisposition to Parkinsonism? REFERENCES

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J. (1837)

Brit.

J. Ann

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Phamacol.

1, 41-42. 2. DONALDSON, J., AND BARBEAU, A. (1985) in Metal Ions in Neurology and Psychiatry, pp. 259-285, Alan R. Liss, New York. 3. BARBEAU, 4. COTZIAS,

A. (1984) G. (1958)

Neurotoxicology

Physiol.

5, 13-36.

Rev. 38, 503-532.

650

ARCHIBALD

5. BERNHEIMER, H., BIRKMAYER, W., HORNYKIEWICZ, O., JELLINGER, K., AND SEITELBERGER, F. (1973) J. New-01 Sci 20,415-425. 6. LANGSTON, J. W., BALLARD, P., TETRUD, J. W., AND IRWIN, I. (1983) Science 219.979-980. 7. COHEN, G., AND MYTLINEOU, C. (1984) Science 225, 529-531. 8. MENA, I., MARIN, D., FUENZALIDA, S., AND COTZIAS, G. C. (1967) Neurology 17,128-136. 9. GREENHOUSE, A. H. (1982) Clin. Neuropharmacol. 5, 45-92. 10. DONALDSON, J., MCGREGOR, D., AND LABELLA, F. S. (1982) Canad J. Physiol. Pharmacol. 60, 1398-1405. 11. GRAHAM, D. G. (1984) Newotoxicology 5,83-96. 12. COHEN, G., AND HEIKKILA, R. E. (1974) J. Biol. Chem. 249,2447-2452. 13. GRAHAM, D. G., TIFFANY, S. G., BELL, W. R., JR., AND GUTNECHT, W. F. (1978) Mol. Pharmacol. 14,644-653. 14. AMBANI, L. M., WOERT, M. H., AND MURPHY, S. (1975) Arch. Neural. 32,114-118. 15. HEILBRONN, E., ERIKSSON, H., AND HAGGBLAD, J. (1982) Neurobehav. Toxic01 Teratol. 4,655-658. 16. BONILLA, E., AND DIETZ-EWALD, M. D. (1974) J. Neurochem. 22,297-299. 17. JONSSON, G. (1976) Med Biol. 54.406-420. 18. GRAHAM, D. G. (1978) Mol. Phawnacol. 14, 633643. 19. TRANZER, J. P., AND THOENEN, H. (1974) Expelientia 29(3), 314-315. 20. SANER, A., AND THOENEN, H. (1971) Mel Pharmo,coL 7,147-154. 21. ROTMAN, A., DALY, J. W., CREVELING, C. R., AND BREAKEFIELD, X. 0. (1976) B&hem. Pharmad 25,383-388. 22. AUTISSIER, N., ROCHETTE, L., DUMAS, P., BELEY, A., LOIREAU, A., AND BRALET, J. (1982) To&cology 24,175-182. 23. CUTLER, R. G. (1984) in Free Radicals in Molecular Biology, Aging and Disease (Armstrong, D., Sohal, R. S., Cutler, R. G., and Slater, T. F. Eds.), pp. 235-273, Raven Press, New York. 24. TOLMASOFF, J. M., ONO, T., AND CUTTER, R. G. (1980) Proc. Natl. Acad Sci. USA 77,2777-2781. 25. MCKENNA, O., ARNOLD, G., AND HOLTZMAN, E. (1976) Brain Res. 117,181-194. 26. DONALDSON, J., LABELLA, F. S., AND GESSER, D. (1980) Neurotwicolcgy 2,53-64. 27. CHOM, B., SEEMAN, P., DAVIS, A., AND MADRAS, B. K. (1982) Eur. J. Phurmucol. 81,111-116.

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