Manganese-induced reactive oxygen species: Comparison between Mn+2 and Mn+3

NEURODEGENERATION, Vol. 4, pp 329-334 (1995)

Manganese-Induced Reactive Oxygen Species: Comparison Between Mn +2 and Mn +3 Syed E A l i , 1"2'3Helen M. Duhart, 1 Glenn D. Newport, 1 George W. Lipe I and William Slikker, Jr2 ,3 1Neurochemistry Laboratory, Division of Neurotoxicology, National Center for Toxicology Research/FDA, Jefferson, AR 72079-9502; 2Department of Biochemistry and Molecular Biology; 3Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

Manganese (Mn) is an essential element, the deficiency or excess of which is known to cause neurotoxicity in experimental animals and man. The mechanism of action of Mn neurotoxicity is still unclear. The present study was designed to evaluate whether in vitro or in vivo exposure to Mn produced reactive oxygen species (ROS). We also sought to determine if a single injection of Mn produces changes in monoamines concentration in different regions of rat brain. Adult Sprague-Dawley rats were dosed with 0, 50 or 100 mg/kg, ip with either MnC12 (Mn ÷2) or MnOAc (Mn *3) and were sacrificed 1 h after the dose was administered. Brains were quickly removed and dissected for neurochemical analysis. ROS were measured by a molecular probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), and monoamines and their metabolites were measured by HPLC/EC. In vitro exposure to MnCI2 (1-1000 ~M) produced dose-dependent increases of ROS in striatum whereas MnOAc produced similar increases at much lower concentrations (1-100 pM). h~ vivo exposure to MnOAc (Mn ÷3) produced significant increases of ROS in caudate nucleus and hippocampus, whereas MnC12 (Mn ÷2) produced significant effects only in hippocampus. Concentrations of dopamine, serotonin and their metabolites (DOPAC, HVA and 5-HIAA) were not altered with acute injections of either MnCI2 or MnOAc. These data suggest that both divalent and trivalent manganese induce ROS, however, Mn ÷3is an order of magnitude more potent than Mn ÷2.

© 1995 Academic Press Limited

K e y words:

manganese, manganese chloride, manganese acetate, oxidative stress, reactive oxygen species, neurotoxicity

divalent Mn. Although it has been shown for iron that valence state is important for hydroxyl radical formarion, there are no reports on systematically comparing valence states and oxidative stress induction b y Mn (Floyd, 1990; Floyd & Carney, 1993; K u m a r et al., 1995). If iron can produce hydroxyl radicals, it is possible to postulate that Mn +2 and Mn ÷3 m a y also generate hydroxyl radicals b y similar reactions and subsequent neurotoxicity via oxidative stress. Archibald & Tyree (1987) and Seguse-Auguila & Lind (1989) suggest that Mn +3 efficiently oxidized d o p a m i n e compared to Mn +2. Venugopal & Luckev (1978) report that Mn +3 is the biologically active form in m a m m a l s and more active towards chelation in biological systems because of the small Mn +3 ionic radius. Donaldson et al. (1980) have shown that Mn +2 in vitro increases d o p a m i n e

MANGANESE(Mn) is an essential element, but it is also k n o w n to cause neurotoxicity in rodents, monkeys and h u m a n s (Austissier et al., 1982; Bonilla & Diez-Ewald, 1974; Cook et al., 1974; Gianutsos & Murray, 1982; Bird et al., 1984; Donaldson & Barbeau, 1984; Sloot et al., 1994). Chronic exposure to Mn is also k n o w n to cause Parkinson-like s y m p t o m s , although its mechanism(s) of action is still unclear. Mn has several valence states but most of the reported studies have used MnC12,

Correspondence to: Syed F. Ali, Ph.D, Head, Neurochemistry Laboratory, Division of Neurotoxicology, HFT-132, NCTR/FDA, 3900 NCTR Road,Jefferson,AR 72079-9501, USA Received6 February1995;revisedand acceptedfor publication 5 April 1995 © 1995 AcademicPress Limited 1055-8330/95/030329 ÷ 6 $12.00/0 329

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auto-oxidation and p r o p o s e d that Mn in a higher oxidation state m a y efficiently oxidatively attack dopamine. Recently, we demonstrated that MnC12induced ROS formation in vitro was a g e - d e p e n d e n t in rat brain (Soliman et al., 1995). These studies suggest that neonatal rat brain is more susceptible to Mninduced ROS formation as c o m p a r e d to adult rat brain. Several research teams have postulated that Mninduced neurotoxicity m a y be mediated via oxidative stress (Donaldson, 1981; 1984; Donaldson et al., 1982; Wills, 1965; Mishra, 1974; De Rycker & Halliwell, 1978). Their reports suggested that higher valence states of manganese (Mn ÷3, Mn ÷2) p r o d u c e d lipid peroxidation and also enhanced the formation of free radicals. Archibald & Tyree (1987) have s h o w n that in manganese poisoning, Mn .2 might be converted to M n ÷3 complexes which m a y in turn attack catecholamine neurotransmitters. They also suggest that regardless of the state in which Mn enters the brain, it will u n d e r g o spontaneous oxidation and dismutation and convert to M n ÷3, which can efficiently destroy d o p a m i n e b y auto-oxidation. The o x i d a t i o n of d o p a m i n e by M n ÷3 neither p r o d u c e d nor required 02, and Mn ÷3 is far more than efficient Mn ÷2, Mn ÷4, 02 or H202 in oxidizing catecholamines (Archibald & Tyree, 1987). Recently Sloot et al. (1994) reported that intrastriatal injections of M n in rat brain p r o d u c e d selective basal ganglia pathology. They suggested that brain areas with high levels of e n d o g e n o u s iron, Fe binding a n d / o r catecholamines were vulnerable to Mn neurotoxicity. The present s t u d y was u n d e r t a k e n to determine if in vitro or in vivo exposure to MnC12 (Mn +2) or MnOAc (Mn +3) p r o d u c e d a differential effect on ROS, and if alterations in the concentration of m o n o a m i n e s resulted from acute Mn exposure.

Materials and Methods Adult male Sprague-Dawley rats were used from the NCTR breeding colony. The animals were housed three per cage with wood-chip bedding and maintained on a 12L:12D cycle (light, 0700 h; dark 1900 h) in a temperature-controlled (25 + 1°C) room. Food (Purina Laboratory Chow, St. Louis, MO), and tap water were provided ad libitum. Animal care was provided according to the NIH guidelines and the NCTR IACUC.

In vitro experiments Control rat brains were quickly removed and dissected into different regions and P2 fractions were prepared for the ROS assay. For in vitro experiments, different concentrations of MnC12 (Mn ÷2) (1-1000 ~M) and MnOAc (Mn +3) (1-100 ~M) were added to the reaction mixture along with P2 fractions.

In vivo experiments Adult male Sprague-Dawley rats were injected with 0, 50 and 100 mg/kg MnCl2 or MnOAc, ip (1 ml/kg) dissolved in deionized water. Control animals received deionized water, ip (1 ml/kg). One hour after the dose, animals were sacrificed and brains were quickly removed and dissected over ice (Ali et al., 1993). One side of the dissected brain was used to analyse for the formation of ROS, whereas the other side of the brain was dissected and frozen over dry ice and stored at -70°C for monoamine analysis. A s s a y s of reactive oxygen species formation

P2 fractions were prepared from each region of the brain as described by All et al. (1992). Each P2 fraction was diluted 1:10 with 40 mM Tris (pH 7.4) and loaded with 5 ~tM 2',7'dichlorofluorescin diacetate (DCFH-DA) in methanol for 15 min at 37°C, during which time esterase activity resulted in formation of the nonfluorescent compound 2',7'dichlorofluorescin (DCFH) (Bass et al., 1983). Following loading, the fluorescence was recorded prior to (initial) and after an additional 60 min of (final) incubation. The formation of the fluorescent oxidized derivative of DCFH, namely 2',7'-dichlorofluorescein (DCF), was monitored at an excitation wavelength of 488 nm and an emission wavelength of 525 nm on a LS-50 Spectrophotofluorometer (PerkinElmer, Norwalk, CT). The cuvette holder was thermostatically maintained at 37°C. Autofluorescence of P2 fractions was corrected by the inclusion in each experiment of parallel blanks (unloaded P2 fraction). The correction for autofluorescence was always less than 10% of the total fluorescence. ROS formation was quantified from a DCF standard curve in methanol (0.05-1.0 ~M). Determination of monoamine concentrations Concentrations of dopamine (DA), serotonin (5-HT) and their metabolites 3,4-dihydroxyindoleacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined by high performance liquid chromatography (HPLC) c6upled with electrochemical detection (EC) following the method of Ali et al. (1994). Briefly, each region of the brain was weighed and diluted with a measured volume (10% W/V) of 0.2 N perchloric acid containing 100 ng/ml of the internal standard 3,4-dihydroxybenzylamine (DHBA). Brain tissue was then disrupted by ultrasonication, centrifuged (15 000 × g for 7 min) and filtered through a .45 ~tM Nylon-66 microfilter (MF-1 microcentrifuge filter, Bioanalytic System (BAS), W. Lafayette, IN). Aliquots of 25 ~tl representing 2.5 mg of brain tissue were injected directly onto the HPLC/EC system for separation of the neurotransmitters DA, 5-HT, and their metabolites DOPAC, HVA and 5-HIAA. The analytical system included a Waters Associates 510 pump (Milford, MA), a Rheodyne 7125 injector (Rheodyne, Inc., Cotati, CA), a Supelco Supelcosil LC-18, 3 ~tM (7.5 cm x 4.6 mm) analytical column, a LC-4B amperometric detector and LC-17 oxidative flow cell (BAS) consisting of a glassy carbon electrode (TL-5) versus Ag-AgC1 reference electrode maintained at a potential of 0.75 V. The mobile phase con-

Mn-induced oxidative stress

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sisted of 0.07 M potassium phosphate, pH 3.0, 8% methanol and an ion pairing reagent of 1.02 mM 1-Heptane sulfonic acid. Chromatograms were recorded and integrated on a Perkin-Elmer LCI-100 integrator (Perkin-Elmer Corp., Norwalk, CT). The concentration of DA, 5-HT and their metabolites (DOPAC, HVA and 5-HIAA) were calculated using a standard curve. The standard curves were generated by determining in triplicate the ratio between three different known amounts of each amine or its metabolites and a constant amount of the internal standard. Statistical analysis ROS and neurotransmitter concentration data were analysed by analysis of variance (ANOVA) followed where appropriate by Duncan'smultiple range test (Duncan, 1955). A value of P < 0.05 was taken as significant.

ROS at an order of magnitude lower concentration as compared to MnC12. In vivo exposure to MnOAc produced a significant increase of ROS in striatum, and hippocampus (Fig. 2). A similar trend was found in frontal cortex, however, it did not reach statistical significance. Injections of MnC12 in vivo did not produce any significant increase of ROS in striatum, frontal cortex or in hippocampus (Fig. 3). Neither MnC12 nor MnOAc produced any significant alteration in the concentrations of monoamines or their metabolites in any brain regions at I h after dose administration (Tables I and 2).

Discussion Results

In vitro exposure to MnC12 and MnOAc produced a dose-dependent increase of ROS in striatum (Fig. 1B,C). W h e n plotted on a log scale (Fig. 1A), these data show that MnOAc can produce similar increases of

In vitro exposure to either MnCI2 ( M n +2) o r MnOAc (Mn ÷3) produced a dose-dependent increase of ROS in striatum, however, MnOAc produced a significant increase of ROS at much lower concentrations. A significant increase of ROS was observed in rat striat u m and hippocampus after MnOAc injection, but

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Figure 1. Reactive oxygen species formation as measured by DCF formation in P2 fraction prepared from rat striatum following in vitro exposure to MnC12(B) and MnOAc (C) and the same effects on log scale (A). Control value represents zero concentrationof Mn salts. Each value is represented as nM DCF formed/g/min, mean --- SEM (n = 9).

S.F. Ali et al.

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Figure 2. Reactive oxygen species formation as measured b y DCF formation in different regions of rat brain: caudate nucleus (CN), frontal cortex (FC) and hippocampus (HIPP) after intraperitoneal exposure to MnOAc. Each value is represented as nM DCF f o r m e d / g / m i n , mean + SEM (n = 9). *P < 0.05 significantly different from control group.

Figure 3. Reactive oxygen species formation as measured by DCF formation in different regions of rat brain: caudate nucleus (CN), frontal cortex (FC) and hippocampus (HIPP) after in vivo exposure to MnC12. Each value is represented as nM DCF f o r m e d / g / m i n , mean -+ SEM (n -+ 9).

o n l y i n h i p p o c a m p u s a f t e r MnC12. S i n g l e i n j e c t i o n s of t h e s e d i - a n d t r i - v a l e n t s a l t s of m a n g a n e s e p r o d u c e d n o a l t e r a t i o n in m o n o a m i n e c o n c e n t r a t i o n s . R e c e n t l y w e r e p o r t e d t h a t in vitro e x p o s u r e to MnCI2 p r o d u c e d a d o s e - d e p e n d e n t i n c r e a s e of R O S . i n d i f f e r e n t b r a i n r e g i o n s ( S o l i m a n et al., 1995). T h e r e a r e s e v e r a l r e p o r t s

which suggest that trivalent, or higher, valence states o f m a n g a n e s e m a y act as p o w e r f u l o x i d a n t s , a n d e n h a n c e l i p i d p e r o x i d a t i o n (Wills, 1965; M i s h r a , 1974; D e R y c k e r & H a l l i w e l l , 1978; D o n a l d s o n , 1981; D o n a l d s o n et al., 1982; D o n a l d s o n & B a r b e a u , 1985), a n d t h e f o r m a t i o n o f free r a d i c a l s . O u r s t u d y c o n c u r s

Table 1. Effects of MnCl2 on the concentration of dopamine, serotonin and their metabolites (DOPAC, HVA and 5-HIAA) in different regions of rat brain Dopamine

DOPAC

HVA

Serotonin

5-HIAA

1820.11 + 67.13 1926.60 + 67.96 1918.41 - 86.44

189.56 + 26.77 193.93 + 24.50 221.28 -+ 35.18

95.41 +- 5.67 100.45 + 5.27 108.23 -+ 10.34

61.05 + 4.00 65.49 + 1.84 62.59 +- 8.02

13.71 + 2.00 20.63 + 2.82 21.47 +- 4.30

19.36 -+ 2.16 19.75 -+ 2.13 21.46 -+ 2.00

10.16 -+ 1.16 11.83 --- 1.98 14.05 -+ 1.30

51.28 -+ 9.52 59.57 -+ 11.49 59.62 -+ 6.65

17.11 -+ 3.35 18.46 + 6.66 26.75 -+ 2.50

ND ND ND

ND ND ND

28.59 + 3.70 27.59 + 4.85 28.75 + 2.19

27.69 + 5.98 19.05 + 6.64 17.07 + 4.12

60.85 + 11.97 62.96 - 9.86 69.01 --- 10.03

19.29 + 7.64 21.15 + 6.88 31.86 -+ 6.85

CAUDATE N U C L E U S

0 m g / k g M.nC12 50 m g / k g MnC12 100 m g / k g MnC12 FRONTAL CORTEX

0 m g / k g MnC12 50 m g / k g MnC12 100 m g / k g MnC12

57.02 --- 10.42 46.47 -+ 4.68 54.29 -+ 7.66

HIPPOCAMPUS

0 m g / k g MnC12 50 m g / k g MnCI2 100 m g / k g MnCI2 HYPOTHALAMUS 0 m g / k g M-nCI2 50 m g / k g MnC12 100 m g / k g MnC12

ND ND ND 77.56 + 10.94 58.96 + 6.51 67.36 -+ 4.86

22.90 +_ 2.21 18.89 + 3.56 26.40 --. 2.81

5.47 + 0.95 3.95 +-. 0.77 5.25 + 0.88

Each value is expressed as ng/100 mg of wet tissue, mean _+SEM (n = 6-9). See method for detail. ND = not detectable.

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Table 2. Effects of MnOAc on the concentration of dopamine, serotonin and their metabolites (DOPAC, HVA and 5-HIAA) in different regions of rat brain Dopamine

DOPAC

HVA

Serotonin

5-HIAA

1763.68 ± 124.45 1742.06 ± 98.66 1826.22 ± 127.14

152.61+ 10.90 156.31--- 7.04 175.54± 16.49

101.01 _-_ 8.85 105.00 --- 4.89 111.04 ± 13.04

67.70 ± 6.06 65.62 ± 4.49 72.92 + 8.38

16.44 ± 2.57 21.54 ± 2.72 18.28 ± 1.77

30.46 ± 6.13 26.44 ± 1.63 60.42 + 13.56

9.66 + 0.92 11.92 + 0.76 13.76 ± 1.21

9.74 ± 1.82 11.78 __.1.06 12.48 + 1.49

42.52 ± 2.71 52.41 ± 5.09 47.28 ± 3.60

7.04 ± 1.50 11.13 ± 2.28 8.22 ± 1.20

ND ND ND

ND ND ND

ND ND ND

30.19 ± 2.83 29.58 --- 1.52 31.17 ± 1.85

10.23 --- 1.63 10.71 + 3.52 6.80 ± 1.36

14.79 - 0.54 16.01 --- 2.13 18.86 --- 1.57

ND ND ND

64.97 ___5.74 47.04 ___8.50 47.39 ± 5.92

13.45 -+ 2.23 11.65 ± 3.25 17.87 ± 1.99

CAUDATE NUCLEUS

0 mg/kg, MnOAc 50 mg/kg, MnOAc 100 mg/kg, MnOAc FRONTAL CORTEX

0 mg/kg, MnOAc 50 mg/kg, MnOAc 100 mg/kg, MnOAc H1PPOCAMPUS

0 mg/kg, MnOAc 50 mg/kg, MnOAc 100 mg/kg, MnOAc HYPOTHALAMUS

0 mg/kg, MnOAc 50 mg/kg, MnOAc 100 mg/kg, MnOAc

54.81 + 5.62 51.11 --- 6.89 56.17 ± 6.17

Each v a l u e is expressed as n g / 1 0 0 m g of w e t tissue, m e a n +- SEM (n = 6-9). See m e t h o d for detail. N D = not detectable.

with these findings and demonstrate that indeed a higher valence state of manganese (MnOAc, Mn ÷3) p r o d u c e d ROS at lower concentrations than MnCI~ (Mn÷2). The most profound effects of Mn ÷3 were in striatum, a region of high. catecholamine concentration. Archibald & Tyree (1987) reported that M n ÷3, or its pyrophosphate, complexes rapidly and efficiently to carry out four electron oxidation of dopamine, its precursor L-DOPA, and its biosynthetic products epinephrine and norepinephrine. Oxidation of d o p a m i n e b y Mn +3 neither p r o d u c e d nor required oxygen, and Mn ÷3 was more efficient than other valence states of manganese or H202 in oxidizing the catecholamines (Archibald & Tyree, 1987). Recently, Sloot et al. (1994) reported that intrastriatal injection of manganese prod u c e d selective basal ganglia degeneration that is similar to that observed after chronic systemic exposure. In the present study, we found a dose-dependent increase of ROS after both in vitro and in vivo exposure to M n 3. However, w e d i d not find any significant changes in dopamine, serotonin or their metabolite concentrations in striatum or other regions of the brain after acute exposure. These d a t a indicate that Mn÷3produced free radicals but no d a m a g e to the dopaminergic system as defined b y altered d o p a m i n e concentrations at I h after M n administration. Recently, w e reported similar effects b y another neurotoxicant, MPTP, and demonstrated that an acute, high dose of MPTP induced formation of ROS before

it significantly depleted d o p a m i n e concentrations in the striatum (Ali et al., 1994). Therefore, one can speculate that manganese-induced free radical production m a y subsequently affect the dopaminergic system. In the present study, the fact that w e d i d not find any significant changes in d o p a m i n e or its metabolites at I h after administration does not rule out manganeseinduced d o p a m i n e alterations at later time points. Further studies are u n d e r w a y to determine the timecourse of ROS generation after exposure to different manganese valence state salts and the effects on the d o p a m i n e system in striatum at extended time periods. Furthermore, manganese-induced depletion of d o p a m i n e in striatum and other brain regions has been observed after long-term exposure, whereas, short-term exposure to manganese h a d no effect on the concentration of monoamines (Neff et al., 1969; Mustafa & Chandra, 1971; Seth & Chandra, 1984). Recent reports b y Sloot et al. (1994) indicate that intrastriatal injection of MnC12 p r o d u c e d significant depletion of d o p a m i n e only after 3 days. In the current s t u d y animals were sacrificed 1 h after dosing and changes in the formation of ROS were observed without acute d a m a g e to the catecholamine system. In summary, these data suggest that in vitro and in vivo exposure to M n p r o d u c e d d o s e - d e p e n d e n t increases in the formation of ROS. Trivalent m a n ganese (Mn÷3), however, is more potent in this regard than divalent (Mn +2) manganese.

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