Manganese Oxidation State and Its Implications for Toxicity - Chemical

Chemistry and Biochemistry, and Environmental Toxicology, University of California, Santa Cruz, California 95064. Chem. Res. Toxicol. , 2002, 15 (9), ...
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Chem. Res. Toxicol. 2002, 15, 1119-1126

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Articles Manganese Oxidation State and Its Implications for Toxicity Stephen H. Reaney,*,†,‡ Catherine L. Kwik-Uribe,‡ and Donald R. Smith*,‡ Chemistry and Biochemistry, and Environmental Toxicology, University of California, Santa Cruz, California 95064 Received February 28, 2002

Manganese (Mn) is ubiquitous in mammalian systems and is essential for proper development and function, though it can also be toxic at elevated exposures. While essential biologic functions of Mn depend on its oxidation state [e.g., Mn(II), Mn(III)], little is known about how the oxidation state of elevated Mn exposures affect cellular uptake, and function/toxicity. Here we report the dynamics of EPR measurable Mn(II) in fresh human plasma and cultured PC12 cell lysates as a function of exposure to either manganese(II) chloride or manganese(III) pyrophosphate, and the effects of exposure to Mn(II) versus Mn(III) on total cellular aconitase activity and cellular Mn uptake. The results indicate that Mn(II) or Mn(III) added in vitro to fresh human plasma or cell lysates yielded similar amounts of EPR measurable Mn(II). In contrast, Mn added as Mn(III) was significantly more effective in inhibiting total cellular aconitase activity, and intact PC12 cells accumulated significantly more Mn when exposures occurred as Mn(III). Collectively, these data reflect the dynamic nature of Mn speciation in simple biological systems, and the importance of Mn oxidation/speciation state in mediating potential cellular toxicity. This study supports concern over increased environmental exposures to Mn in different oxidation states [Mn(II), Mn(III), and Mn(IV)] that may arise from combustion products of the gasoline antiknock additive methycyclopentadienyl manganese tricarbonyl (MMT).

Introduction Manganese (Mn) is ubiquitous in mammalian systems and is essential for proper development and function (1). It can exist in the (II), (III), and (IV) oxidation states, although the latter has not been found in mammalian systems. Mn(II) exhibits chemistry similar to Ca(II) (2) and Mg(II) (3), while Mn(III) is similar to Fe(III) (4). The various functions of Mn in biologic systems depend in large part on its oxidation state chemistry. For example, Mn(II) is an essential cofactor in glutamine synthetase (5) and a number of transferases (6) and hydrolases (7), while the role of Mn in mitochondrial superoxide dismutase (8) is dependent on interconversion between the Mn(II) and Mn(III) oxidation states. While trace amounts of Mn provide important essential functions in biology, elevated Mn exposures have been shown to cause significant toxicity. Neurotoxic effects of elevated Mn have been shown in a number of laboratory studies (9-13), consistent with epidemiologic studies that have suggested an association between elevated environmental Mn exposure and Parkinsonism (14-18). While the exact mechanisms of these effects remain unclear, these studies collectively suggest that elevated * To whom correspondence should be addressed. E-mail: (S.H.R.) [email protected] or (D.R.S.) [email protected]. † Chemistry and Biochemistry. ‡ Environmental Toxicology.

environmental exposures to Mn may be sufficient to exacerbate the emergence of neurological diseases such as Parkinsonism (9, 10). There is a pressing need to elucidate the neurotoxic mechanisms of moderate level Mn exposures, in light of the incorporation of the antiknock additive methycyclopentadienyl manganese tricarbonyl (MMT) into gasoline in Canada, the U.S., and elsewhere (19-21). Widespread use of MMT in gasoline may lead to increased environmental exposures to MMT combustion products that contain Mn in a variety of oxidation states [e.g., Mn(II), Mn(III), and Mn(IV)] (22, 23). In addition, the physiologic impact of increased exposure may be exacerbated in populations such as the elderly, since compensatory physiological mechanisms that may function to moderate the toxic effects of metals at the cellular level could be compromised in the normal aging process, rendering aged individuals more susceptible to toxicity (24, 25). Manganese is believed to exert toxicity via a number of mechanisms, including the direct or indirect formation of reactive oxygen species (ROS) (26, 27), the oxidation of biological molecules (28), and the disruption of cellular calcium (12) and cellular iron homeostasis (29). Recent studies have demonstrated in vitro that elevated Mn leads to inhibition of complexes within the mitochondrial electron transport chain (13, 30-32), consistent with resultant increased oxidative stress (33). Further, Archibald and Tyree (28) showed in vitro that Mn(III) is more

10.1021/tx025525e CCC: $22.00 © 2002 American Chemical Society Published on Web 08/29/2002

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effective at oxidizing dopamine than Mn(II), which can lead to the generation of damaging radical quinones. Nonetheless, the role of elevated Mn levels in the direct formation of ROS in vivo remains unclear, since it has been suggested that the redox potential of the Mn(III)/ Mn(II) couple precludes a role for Mn in Fenton-like chemistry reactions (34, 35). Thus, there is a need to elucidate the role of Mn oxidation state as a potential mediator of cellular toxicity. Therefore, our objective was to systematically investigate how the oxidation state of Mn exposure may affect Mn toxicity. Specifically, we investigated (1) the suitability of various inorganic Mn(III) compounds for use in Mn exposure studies; (2) the dynamics of electron paramagnetic resonance (EPR) measurable Mn oxidation state in biological samples as a function of the Mn oxidation state of exposure [i.e., Mn(II) versus Mn(III)]; and (3) the effects of exposure to Mn(II) versus Mn(III) on several outcomes of cellular function. The need for these studies is justified by the fact that while the vast majority of Mn toxicity studies have used manganese(II) chloride as a source of Mn(II) exposure, they have used a variety of different sources of Mn(III) (e.g., pyrophosphate, acetate, transferrin complex), or ignored Mn(III) sources altogether. Further, few if any studies have systematically evaluated the integrity of their Mn(III) source, or the resulting oxidation state of Mn postexposure. Together, the results of this study show the importance of Mn oxidation state/speciation in mediating potential cellular toxicity, they demonstrate the dynamic nature of Mn speciation in simple biological systems, and they indicate that manganese(III)-pyrophosphate is the most suitable source of inorganic Mn(III) exposures that we evaluated.

Experimental Section Reagents. Materials were obtained from the following vendors: undifferentiated PC12 (rat pheochromocytoma) cells, American Type Culture Collection (ATCC, Manassas, VA); fetal bovine serum (FBS), ATCC; heat-inactivated horse serum and fungizone (250 units/mL), Life Technologies, Inc.; RPMI 1640 Medium, ATCC; potassium hydroxide (KOH) and optima grade nitric acid (HNO3),Fisher Scientific; EPR tubes, Wilmad Glass; collagen coated plates, BD glassware; penicillin (10000 units/ mL)/streptomycin (100 mg/mL), and all other reagents were from Sigma Aldrich. Stability of Mn(III) Source. Mn(III)P2O7 (manganesepyrophosphate), Mn(III)(CH3CO2)3‚2H2O (manganese-acetate dihydrate), and Mn(III)C6H8O7 (manganese-citrate) were explored as possible sources of Mn(III). For the exposure studies, manganese-pyrophosphate solutions were prepared by a modification of the method of Kenton et al. (36). Briefly, approximately 1 g of manganese-acetate dihydrate was dissolved in 20 mL of Milli-Q ultrapure water. The resulting hydrated Mn(IV) complex precipitate was collected and washed with ethanol and Milli-Q water, respectively. The collected precipitate was added to 20 mL of 100 mM sodium pyrophosphate, pH 7.4, to yield a ∼92 mM Mn solution. Manganese(II)-chloride was then added to this solution to give 12 mM final concentration of added manganese(II)-chloride. The resultant manganese(III)pyrophosphate solution was then filtered (0.2 µm) to remove the remaining precipitate. The absence of measurable Mn(II) in the final solution was confirmed by EPR, as described below. Manganese(III)-acetate stock solutions were prepared fresh daily to 50 mM concentration in 100% ethanol. After stirring for several minutes, the solution was filtered (0.2 µm) and then centrifuged at 10000g for 10 min to remove any undissolved Mn particulates. This solution was then diluted to 25 mM Mn using

Reaney et al. dimethyl sulfoxide (DMSO, final concentration 50% v/v), and used immediately for the experiments. Manganese(III)-citrate was prepared using a manganous-permanganate reaction in the presence of excess ligand (citrate) (37). The manganous-permanganate reaction is a specific case of the general reaction: Mn(VII) + 4Mn(III) + 5L f 5Mn(III)L, using manganese(II)chloride as the source of Mn(II) and manganous-permanganate as the source of Mn(VII). The total Mn concentration for all solutions was determined using atomic absorption spectrometry (AAS) using a PerkinElmer 2380 instrument. Mn(II) concentrations in the manganesepyrophosphate and manganese-acetate solutions were determined by EPR, as detailed below. Concentrations of the manganese(III)-pyrophosphate and manganese(III)-citrate complexes were determined independently by UV-vis spectroscopy using their respective extinction coefficients [at λ ) 258 nm,  ) 6.2 mM-1 cm-1 (36), and at λ ) 340 nm,  ) 310 M-1 cm-1, respectively (37)]. The content of these various Mn(III) complexes were evaluated by determining the relative enrichment of Mn(III) versus Mn(II) in the final solution, and the stability of the Mn(III) solutions was evaluated over time (weeks to months). The stability of manganese(III)-acetate and manganese(III)pyrophosphate solutions for Mn exposure cell culture studies was evaluated. Manganese(III)-acetate or manganese(III)-pyrophosphate was spiked into RPMI 1640 medium to a final concentration of 100 µM Mn and sampled immediately (time zero) and again after incubating (37° C, 5% CO2) in RPMI medium for 5 h. At each time point (0 and 5 h) two aliquots of each RPMI-spiked solution were taken. One aliquot was centrifuged at 10000g for 5 min, and the supernatant was removed and acidified to 1 N with nitric acid. The second aliquot was acidified without centrifugation. This entire procedure was performed in triplicate for both the manganese(III)-acetate and manganese(III)-pyrophosphate-spiked RPMI solutions. Samples were then analyzed for total Mn by Zeeman graphite furnace AAS (GFAAS), using a Perkin-Elmer 4100ZL instrument. The scattering of light by medium containing manganese(III)-acetate, manganese(III)-pyrophosphate, or manganese(II)chloride was spectrophotometrically examined to semiquantify the formation of any Mn precipitates following addition of those Mn complexes to cell culture medium. For this, RPMI 1640 was spiked to a final concentration of 100 µM manganese(III)acetate, manganese(III)-pyrophosphate, or manganese(II)chloride and incubated for 1 h at 37 °C. The scattering of light was quantified at 340 nm wavelength after scanning over the range of 200-800 nm. Further, aliquots of the above solutions were centrifuged at 10000g for 5 min after incubation, and absorbance (light scattering) again measured to further confirm the presence of a precipitate. Media containing no Mn was used as the reference for all samples. Measurement of Mn Oxidation State by EPR. All samples were prepared for EPR analyses by adding glycerol to fresh samples (20% glycerol v/v final), and then aliquoting the samples into EPR tubes and rapidly flash freezing in liquid nitrogen. Sample concentrations of EPR measurable paramagnetic Mn(II) were determined using a Bruker Electron Spin Resonance ESP300 spectrometer (X-band). Samples of manganese(III)citrate complex could not be measured by EPR because the properties of the solution did not reliably allow freezing in EPR tubes. Sample Mn(II) concentrations were quantified against a seven point Mn(II) standard curve. Mn(II) standards were prepared from a certified Mn reference solution over the concentration range of 0 to 59 µM or 0 to 1000 µM Mn(II) in 2% ultrapure HNO3/20% glycerol. EPR spectra peak intensities were quantified by taking the difference between the peak of the first (low field) hyperfine and the trough of the fifth hyperfine. This approach gave comparable or superior results to quantitation by area integration of the five hyperfine peaks. The EPR instrument parameters used in all analyses were as follows: 160 mW power, 9.41 GHz frequency (X-band), 121 K analysis

Manganese Oxidation State and Toxicity temperature, 26.5 G modulation amplitude, 100 kHz modulation frequency, 40.96 ms conversion time, and a 163.84 ms time constant. Data were summed from 8 to 16 scans for each sample spectra, depending on the Mn concentration range of the experiment. Measurable Mn Oxidation State in Simple Aqueous Samples. The effect of pH on EPR measurable Mn(II) in aqueous solution was determined under oxic and anoxic conditions. For the oxic experiment, a 20 µM manganese(II)-chloride in 20% glycerol (v/v) pH 2.3 parent solution was prepared in an open-air environment. Aliquots of this solution were adjusted to pH 2.3 (no KOH added), 4.3, 6.8, and 8 using 10 M KOH. Sub-aliquots of these latter solutions were immediately flash frozen for EPR analyses of Mn(II), while the remaining fractions were used to determine the solution pH and total Mn concentration by GFAAS. A similar experiment was performed under anoxic nitrogen atmosphere conditions, using a 50 µM manganese(II)-chloride parent solution and a Vac Atmosphere model HE493 glovebox. Sixteen separate aliquots were taken, and pH was adjusted as above. Sub-aliquots of these latter solutions were immediately flash frozen for EPR analyses of Mn(II), while the remaining fractions were used to determine the solution pH and total Mn concentration. Measurable Mn Oxidation State in Biological Samples. Manganese(II)-chloride or manganese(III)-pyrophosphate were added to fresh human plasma, or PC12 cellular homogenates to achieve a final Mn concentration of 14.5 µM (plasma) or 1250 µM (cellular homogenates). Following the addition of Mn, samples were incubated for 30 min (cell homogenates) at 25° C, after which glycerol was added (final glycerol 20%, v/v) and the samples then flash frozen for measurable Mn(II) analyses by EPR and total Mn analyses by GFAAS. Total cellular homogenates were prepared as described below in the “Measurement of aconitase activity” section. To investigate the effect of Mn incubation time of the sample on the amount of EPR measurable Mn(II), manganese(II)chloride and manganese(III)-pyrophosphate were added separately to fresh human plasma to a final Mn concentration of 14.5 µM and incubated for 5, 90, or 540 min at 25° C in a glovebag purged with argon gas. Following incubation, glycerol was added to the samples and they were immediately flash frozen in liquid nitrogen, as noted above. Total Aconitase Activity in PC12 Whole Cell Homogenates. (1) Cell Culture and Preparation of Cell Homogenates. PC12 cells were utilized here because they are a widely used model of catecholaminergic cells that produce both dopamine and norepinephrine, and because previous studies have demonstrated specific effects of elevated Mn exposure on these cells (e.g., refs 29 and 30). Undifferentiated PC12 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 50 units/ mL penicillin, 50 mg/mL streptomyocin, and 1 unit/mL fungizone. Cells were cultured on collagen coated plates in a humidified environment at 37 °C with 5% CO2. All experiments were restricted to cell passages four to seven. Cells were harvested when 70 to 80% confluent. Cells were shown to be mycoplasma-free, based on the hoechst stain method. Sprague-Dawley rat (Simonson Labs, Gilroy, CA) primary mixed glial cultures containing type 1 and type 2 astrocytes were derived from day 2-3 postnatal rats, using previously described methods (38). Cell homogenates from the PC12 and primary astrocyte cell culture models were prepared by suspending fresh cells in homogenization buffer (20 mM Hepes, pH 7.5, 1 mM dithiothreitol, 2 mM citrate, 0.7 mg/mL pepstatin and 0.5 mg/ mL leupeptin; 5 × 106 cells/1.5 mL of buffer), and sonicating using an ultrasonic processor (six pulses at 60 000 Hz). Cell homogenates were then frozen at -80 °C until use. All procedures related to animal care were in accordance with the guidelines in the Guide for the Care and Use of Laboratory Animals (NRC, 1996).

Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1121 (2) Measurement of Aconitase Activity. To determine the effect of Mn(II) and Mn(III) on total cellular aconitase activity, cell homogenates were thawed and 100 µL aliquots of the homogenate were mixed with manganese(II)-chloride or manganese(III)-pyrophosphate to achieve final Mn concentrations of 100 to 3860 µM, and incubated for 30 min at 25° C. Subsequently, 300 µL of reaction buffer (150 mM triethanolamine buffer, 1.5 mM magnesium chloride, 1.5 mM NADP+, and 1.5 units/mL of isocitrate dehydrogenase, pH 8) was added following a modification of the method of Rose (39), and the NADPH formation reaction was monitored at 340 nm over 4 min at 37° C using a Beckman DU650 spectrophotometer equipped with a Fisher Scientific Isotemp circulator. The mean of analytical duplicates was determined for individual samples, and each Mn exposure level was analyzed in triplicate. Cellular Accumulation of Mn during Mn(II) or Mn(III) Exposure. PC12 cells were cultured as described above. Subsequently, cells were exposed to medium (RPMI + 5% horse serum/fetal bovine serum) not supplemented with Mn (control) or supplemented with 100 µM or 500 µM manganese(II) chloride or manganese(III) pyrophosphate for 24 h. Each exposure level was conducted in triplicate. Following exposure, the cells were harvested, pelleted (200g for 10 min), and the cell pellet washed once with 5 mM ethylenediaminetetraacetic acid (EDTA) and twice with phosphate-buffered saline to remove surficial (nonaccumulated) Mn from the intact cell sample. The cell pellet was then lysed via hypo osmotic shock and sonication in Milli-Q water, and an aliquot was taken for protein determination. Cellular Mn levels were determined by Zeeman GFAAS, using a modified procedure of Smith et al. (40). Assessment of Cytotoxicity. Cytotoxicity was determined by measuring the lactate dehydrogenase (LDH) activity of the medium following exposure. LDH activity was determined by monitoring the disappearance of NADH at 340 nm (41). Given that the medium contains inherent LDH activity, an aliquot of medium not used for the exposures was collected and the inherent activity of the medium subtracted from the activity measured in the medium following Mn exposure. Activity was expressed as a function of sample protein content. All protein concentrations were determined using the BioRad assay kit (BioRad Labs). Statistical Analyses. Treatment and pairwise comparisons were performed using analysis of variance (ANOVA) and Tukey’s post-hoc comparison tests. Pairwise comparisons using the nonparametric Kruskal-Wallis test were performed when sample variances were significantly unequal. P-values less than 0.05 were considered statistically significant for all tests. All analyses were conducted using SYSTAT (SPSS Inc., 10th ed., 2000).

Results To systematically evaluate the effects of exposure to Mn(II) versus Mn(III) in simple biological systems, a number of studies were conducted. First, we investigated various inorganic Mn(III) complexes for their suitability as a source of Mn(III) exposure. Second, we investigated the oxidation state of Mn in vitro in both simple aqueous and complex biological systems following exposure to Mn(II) or Mn(III) and how this outcome varied as a function of solution pH and the presence of dissolved oxygen. The presence of dissolved oxygen and solution pH are important factors that affect Mn oxidation state. Finally, we investigated in vitro how Mn oxidation state [i.e., Mn(II) versus Mn(III)] affected total cellular aconitase activity and cellular Mn accumulation in cell lysates and intact cells. Stability of Mn(III) Source. Manganese(III)-pyrophosphate proved to be the most reliable and suitable source of Mn(III) for use in the studies reported here.

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Figure 1. Stability of manganese(III)-pyrophosphate (solid bars) and manganese(III)-acetate (hatched bars) in RPMI culture medium. Mn-spiked medium (100 µM) was incubated for 0 h (black bars and white hatched bars) or 5 h (solid and hatched gray bars) at 37 °C, and then acidified and total Mn measured by GFAAS (no centrifugation), or centrifuged (10000g for 10 min) and the resulting supernatant acidified and Mn measured by GFAAS. Values are expressed as the mean percent of total Mn added (n ) 3). Error bars are standard deviation (SD).

Prepared solutions of manganese(III)-pyrophosphate in water (100 µM total Mn concentration) contained no EPR measurable Mn(II). These data corroborated the stable levels of manganese(III)-pyrophosphate measured by UV-vis spectrometry, and total Mn levels measured by AAS (data not shown). Manganese(III)-pyrophosphate added to RPMI cell culture medium also showed no detectable precipitate, even when added at concentrations (1000 µM Mn) 5-fold higher than the cell culture exposures conducted here. In contrast, neither manganese(III)-acetate nor manganese(III)-citrate proved acceptable for these studies. Manganese(III)-acetate remained soluble at stock concentrations of ∼25 mM when dissolved in ethanol/DMSO (50:50 v/v). However, a visible Mn precipitate rapidly formed when this stock was diluted to concentrations as low as 100 µM in water or RPMI cell culture medium (evaluated within minutes of mixing, and again after 5 h incubation). This loss of Mn(III) was presumably due to disproportionation to Mn(II) and Mn(IV) (oxide precipitate), and the formation of manganese(III)-hydroxide or oxide precipitates. This was substantiated by an increase in light scattering in manganese(III)-acetate/ RPMI solutions, which was greatly reduced upon centrifugation, and by the significant (90%) loss of total Mn in the supernatant of centrifuged RPMI samples (10000g for 10 min) (Figure 1). This was not observed in manganese(III)-pyrophospate/RPMI solutions. Manganese(III) citrate proved unacceptable for these studies due to a poor yield in its preparation ( 7 (Figure 2). Measurable Mn Oxidation State in Biological Samples. Manganese(II)-chloride or manganese(III) pyrophosphate was added separately to fresh human plasma at a concentration of 14.5 µM Mn, to further evaluate the effects of sample composition on the oxidation state of added Mn. Results show that the oxidation state of added Mn had no measurable effect on the amount of EPR measurable Mn(II) in the plasma samples (p > 0.4). More specifically, EPR measurements of plasma samples spiked separately with manganese(II)-chloride or manganese(III)-pyrophosphate suggest a rapid convergence of Mn redox activity, such that ∼50% of the added Mn existed as EPR measurable Mn(II) after only 5 min of sample incubation regardless of whether Mn(II) or Mn(III) was added (Figure 3). A similar convergence of EPR measurable Mn(II) was observed when manganese(II) chloride or manganese(III) pyrophosphate (1250 µM total Mn concentration) was added to fresh PC12 cell lysates. However, in this case, only ∼20 to 25% of the added Mn

Manganese Oxidation State and Toxicity

Figure 3. Effect of adding 14.5 µM manganese(II)-chloride (squares) versus 14.5 µM manganese(III)-pyrophosphate (circles) on EPR measurable Mn(II) levels in fresh human plasma, following incubation for 5, 90, or 540 min. Measured Mn(II) mean values are expressed as the percent of total sample Mn (n ) 3, (SD). Two-way ANOVA analyses indicated no measurable effect of time or the oxidation state of added Mn on the measured Mn(II) (p > 0.4).

Figure 4. Effect of added Mn, Co, and Fe on total cellular aconitase activity in PC12 cell lysates (mean ( SD, n ) 3). Cell homogenates were incubated for 30 min with micromolar concentrations of manganese(II)-chloride (hatched bars), manganese(III)-pyrophosphate (filled bars), Co, or Fe. There was a significant overall inhibitory effect of Mn (p < 0.001, ANOVA), as well as a significantly greater effect of Mn(III) versus Mn(II) (p < 0.001) on total aconitase activity. “a, b, c” ) Mn treatment means with different letters were significantly different from one another (p < 0.05 Tukey’s test).

remained as measurable Mn(II) (data not shown). Notably, the measurable Mn(II) was slightly higher in samples spiked with Mn(II) (26% of total Mn) compared to samples spiked with Mn(III) (21% of total Mn), though this difference was only marginally significant (p ) 0.046). These cell lysates correspond to the samples in which total cellular aconitase activity was measured (1250 µM Mn exposure level, Figure 4). Total Aconitase Activity in PC12 Whole Cell Homogenates. Total cellular aconitase activity was assessed as a predictable outcome of elevated Mn exposure (29, 30). Cellular aconitase is present in two forms, cytosolic aconitase (c-acontitase) and mitochondrial aconitase (m-aconitase). m-Aconitase functions as an enzyme in the Krebs cycle responsible for the conversion of citrate to isocitrate. c-Aconitase also exhibits catalytic activity, though its primary role is in regulating cellular iron (Fe) homeostasis as iron regulatory protein 1 (IRP1). Both proteins contain a 4Fe - 4S cluster central to their catalytic/regulatory function and both are known to be affected by elevated Mn exposures (29, 30). There was a significant overall inhibitory effect of Mn, as well as a significantly greater effect of Mn(III) versus Mn(II) on total aconitase activity in PC12 cell lysates (p

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< 0.001). The in vitro addition of manganese(II)-chloride to PC12 cell lysates over an exposure range of 500 to 2500 µM caused a significant dose-dependent inhibition of total cellular aconitase activity (p < 0.001), with a reduction of activity to ∼75 and ∼60% of control values at the 1250 and 2500 µM doses, respectively (Figure 4). The addition of manganese(III)-pyrophosphate caused a significantly greater inhibition of total aconitase activity compared to equimolar doses of Mn(II) (p < 0.001, Figure 4). For example, the 500 µM and 1250 µM doses of Mn(III) [the latter being the highest Mn(III) dose investigated here] caused significant reductions to ∼38 and ∼5% of control (p < 0.001), while the same exposures of Mn(II) caused reductions to 98% (nonsignificant) and ∼75% (p < 0.001) of control (Figure 4). Finally, addition of 1040 µM cobalt (Co), a metal thought to replace Fe in the Fe-S centers of aconitase (42) caused an expected significant reduction in total aconitase activity to ∼40% of control values (p < 0.001). The addition of 335 µM Fe did not significantly increase total aconitase activity (p > 0.05). The latter results with Co are consistent with the suggestion that Mn replaces Fe in the Fe-S center of aconitase, while the results with Fe indicate that the inherent activity of aconitase in the assay was not Fe limited. Similar studies of Mn inhibition of total aconitase activity conducted in primary rat mixed astrocyte cell lysates showed a comparable overall dose-effect of Mn (p < 0.001), with a significantly greater effect of Mn(III) compared to Mn(II) (p < 0.001, comparing the 500 µM dose for each). Addition of 1040 µM Co caused the expected significant reduction in aconitase activity to ∼60% of control (p ) 0.014). These data fully corroborate the effects of Mn(II) and Mn(III) observed in PC12 cell lysates. Cellular Accumulation of Mn During Mn(II) or Mn(III) Exposure. Total PC12 cellular Mn levels increased dramatically relative to control values (∼0.29 µmol of Mn/g of protein), with increases of ∼70-fold following exposure to 100 µM manganese(II)-chloride for 24 h and increases of >3000-fold following exposure to 500 µM manganese(III)-pyrophosphate (Figure 5). Notably, cells exposed to 100 µM manganese(III)-pyrophosphate accumulated ∼8-fold more Mn (166 µmol of Mn/g of protein) compared to cells exposed to equimolar amounts of manganese(II)-chloride (20 µmol of Mn/g of protein) (p < 0.001, Figure 5). There was no significant difference in total accumulated cellular Mn between Mn(II) and Mn(III) exposures of 500 µM. This latter outcome may be attributed to a significant cytotoxicity of Mn at these higher exposure levels, based on significant decreases in cell viability (trypan blue exclusion) and significant increases in exposure medium LDH levels. For example, the 100 µM Mn(II) and Mn(III) exposures produced moderate increases in medium LDH of 106% and 135% of control, while the 500 µM Mn(II) and Mn(III) exposures produced increases in LDH of 170 and 224% of control, respectively (Figure 5, inset).

Discussion This study showed the dynamic nature of Mn speciation in simple biological systems, and the potential importance of Mn oxidation/speciation state in mediating cellular toxicity. Our results suggest that redox processes and ligand binding are the predominant factors controlling the EPR measurable Mn(II) in the plasma and cell

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Figure 5. Effect of exposure to manganese(II)-chloride (hatched bars) and manganese(III)-pyrophosphate (filled bars) in culture medium on total Mn accumulation by PC12 cells. “/” Cells exposed to 100 µM Mn(III) accumulated significantly more total Mn than cells exposed to Mn(II) (p < 0.001). Inset shows the effect of Mn exposure on LDH activity measured in culture medium as an indicator of cytotoxicity (see text). All bars are mean values ((SE, n ) 3).

lysate samples. The importance of redox processes is best demonstrated by the fact that significant amounts of EPR measurable Mn(II) were detected following addition of Mn(III) in fresh plasma [i.e., ∼50% of sample Mn measured as Mn(II), Figure 3] and in PC12 cell lysates [∼25% of sample Mn measured as Mn(II), text]. Further, we observed a complementary “loss” of EPR measurable Mn(II) on the order of ∼50% (plasma) and ∼75% (cell lysates) upon addition of Mn(II). The observation that only ∼25% of added Mn(II) was detected as EPR measurable Mn(II) in the cell lysate samples, compared to ∼50% in the fresh plasma samples, may be due to a greater oxygen content and hence more oxidizing conditions in the cell lysates. This assumption is based on the fact that the cell lysate samples were processed under ambient atmosphere conditions, which was logistically necessary to perform the aconitase activity assay, while the plasma samples were processed under low oxygen conditions in an argon gas-purged glovebag. Further, the change in EPR measurable Mn(II) in these samples occurred rapidly (within 5 min), such that incubation time was not a factor. Taken together, these observations suggest that rapid redox processes in the plasma and cell lysate samples led to the reduction of added Mn(III) f Mn(II), and presumably also to the oxidation of added Mn(II) f Mn(III). The suggestion of redox processes in governing the oxidation of added Mn is substantiated by effects of sample oxygen content and pH on added Mn(II) in simple aqueous systems. We observed a significant ∼85% loss of EPR measurable Mn(II) in water under oxic (ambient atmosphere) conditions as the sample pH approached neutrality (Figure 2). In contrast, 80 to 90% of added Mn(II) remained as EPR measurable Mn(II) under anoxic nitrogen atmosphere conditions at comparable pH. These latter results also indicate that at pH ∼7 very little Mn(II) is lost to hydroxo bridging of paramagnetic Mn(II) centers, which results in a loss of EPR signal. The substantial loss of added Mn(II) under oxic conditions did not yield a detectable precipitate in the sample, suggest-

Reaney et al.

ing that added Mn(II) was oxidized to Mn(III) and not to insoluble Mn oxides or hydroxides. The above observations are consistent with the known behavior of Mn in both aqueous and biological systems (4). Williams and others (2-4) have noted that Mn(II) generally forms relatively weak complexes with biomolecules as does Ca(II) and Mg(II), whereas Mn(III) forms much stronger complexes with ligands, similar to Fe(III). The latter is perhaps best illustrated by the binding of Mn(III) to the Fe(III) transport protein transferrin (43). Thus, it is plausible that some essential biological functions may be more sensitive to disruption from elevated Mn exposures of different oxidation states. Our observation that added Mn(III) was a significantly more potent inhibitor of total cellular aconitase activity than added Mn(II) and that intact cells accumulated significantly more Mn when exposures occurred as Mn(III) substantiates the above suggestion that Mn oxidation state may be an important factor mediating toxicity. Notably, these outcomes seemingly contradict the EPR measurements in plasma and cell lysates noted above, in which the EPR measurable Mn(II) converged to similar values regardless of whether the samples were spiked with Mn(II) or Mn(III). The basis for this apparent contradiction is not yet clear. Nor is it clear what mechanism(s) may underlie the greater effect of added Mn(III), though it does not appear to be mediated by the EPR measurable Mn(II) fraction of total sample Mn. Evidence suggesting that aconitase activity may be altered by elevated Mn first surfaced more than three decades ago (44). Several mechanisms can explain the inhibitory effect of Mn on aconitase activity, in which Mn may (1) allosterically alter aconitase by binding to secondary (i.e., nonsubstrate) sites on the enzyme (44); (2) bind to citrate (45), the substrate of aconitase, thereby reducing citrate availability to the enzyme; or (3) displace Fe in the Fe-S cluster of aconitase (46). Currently, it is unclear which if any of these mechanisms may be operant in vivo. Our observations are also consistent with the few existing studies on the toxicity of Mn(III). For example, Archibald and Tyree (1987) found that in vitro manganese(III)-pyrophosphate was a more effective oxidizer of dopamine (catecholamine) than manganese(II)-chloride or Mn(IV)-oxide. This suggests that Mn(III) may be more toxic than Mn(II) through direct oxidation of biological molecules in vivo. Further, Suarez et al. (1995) reported that manganese(III)-pyrophosphate was more toxic than manganese(III)-transferrin complex to cultured neuroblastoma cells. In that study, the authors suggested that the manganese(III)-transferrin complex, while facilitating the transport of Mn into the cells, also protected the cells from manganic ion (Mn(III)) damage. More recently, a study by Zheng and colleagues (46) suggested that manganese(III)-acetate is more toxic to PC12 cells than manganese(II)-chloride. A common suggestion in these studies is that the greater relative toxicity of Mn(III) is related to its greater oxidative reactivity, consistent with the large reduction potential of “free” Mn(III) (E° ) +1.51 V). However, while the role of elevated Mn(III) serving as a general oxidant is an attractive hypothesis, the extent to which this occurs in vivo where one would expect Mn(III) to be largely if not completely coordinated with biologic ligands remains to be shown. The suitability of various Mn(III) complexes as sources of Mn(III) exposure was investigated, because it has been

Manganese Oxidation State and Toxicity

suggested that Mn(III) complexes can vary substantially in their stability and chemical behavior (4, 37, 47). Also, understanding the integrity of a defined chemical challenge is a fundamental property of exposure assessment that is important in interpreting the measured outcomes, yet it is often overlooked. Here we found that only manganese(III)-pyrophosphate, and not manganese(III)acetate or citrate, was suitable as a stable and reliable source for the cell lysate and intact cell exposure studies (e.g., Figure 1). In particular, we found that a precipitate was rapidly formed upon mixing manganese(III)-acetate (as a 1:1 ethanol:DMSO stock) in either water or cell culture medium, indicating that it is very unstable under neutral aqueous conditions (Figure 1). These latter results suggest that manganese(III)-acetate-disproportionated to a great extent, yielding a mixture of soluble Mn(II) and insoluble Mn(IV). Our results also substantiate previous studies showing that manganese(III)-pyrophosphate complexes are more stable at neutral pH than complexes of manganese(III)-citrate or Mn(III)-EDTA (37). In summary, this study showed that the relative amount of EPR measurable Mn(II) in our simple biological systems (fresh human plasma, PC12, and primary rat mixed astrocyte cell lysates) was similar regardless of whether Mn was added as Mn(II) or Mn(III). In contrast, however, Mn(III) was a more effective inhibitor of total cellular aconitase enzyme activity and was more readily accumulated by intact PC12 cells than was Mn(II). While the accurate assessment of Mn oxidation state in situ requires additional study, the EPR method utilized here provides useful information on the degree to which Mn oxidation state is controlled by sample environment. The extent that the effects observed here are operative in vivo remains to be shown, though studies are now underway to evaluate this important question.

Acknowledgment. We would like to thank Dr. Colin Burns, Dr. Glen Millhauser, and Joe McNulty for their discussions pertaining to the EPR measurements and Dr. Roberto Gwiazda for assistance in these measurements. We would also like to thank Dr. Pradip Mascharak for his insightful discussions. This work was supported by the National Institutes of Environmental Health Sciences (Grant ES/NS05936) and by the University of California Toxic Substances Research and Teaching Program.

References (1) Underwood, E. J. (1977) Trace Elements in Human and Animal Nutrition, Fourth ed., Academic Press, New York. (2) Andersson, M., Malmendal, A., Linse, S., Ivarsson, I., Forsen, S., and Svensson, L. A. (1997) Structural basis for the negative allostery between Ca(2+)- and Mg(2+)- binding in the intracellular Ca(2+)-receptor calbindin D9k. Protein Sci 6, 1139-47. (3) Vermote, C. L. M., Vipond, I. B., and Halford, S. E. (1992) Ecorv Restriction Endonuclease - Communication Between Dna Recognition and Catalysis. Biochemistry 31, 6089-97. (4) Silva, J. R. R. F. d., and Williams, R. J. P. (1991) The biological chemistry of the elements: the inorganic chemistry of life, Oxford University Press, Oxford. (5) Wedler, F. C., and Denman, R. B. (1984) Glutamine synthetase: the major Mn(II) enzyme in mammalian brain. Curr. Top. Cell Regul. 24, 153-69. (6) Hendrickson, T. L., and Imperiali, B. (1995) Metal ion dependence of oligosaccharyl transferase: implications for catalysis. Biochemistry 34, 9444-50. (7) Kodama, H., Shimojo, N., and Suzuki, K. T. (1991) Distribution of manganese in rat pancreas and identification of its primary binding protein as pro-carboxypeptidase B. Biochem. J. 278, 85762.

Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1125 (8) Hsieh, Y., Guan, Y., Tu, C., Bratt, P. J., Angerhofer, A., Lepock, J. R., Hickey, M. J., Tainer, J. A., Nick, H. S., and Silverman, D. N. (1998) Probing the active site of human manganese superoxide dismutase: the role of glutamine 143. Biochemistry 37, 4731-9. (9) Witholt, R., Gwiazda, R. H., and Smith, D. R. (2000) The neurobehavioral effects of sub chronic manganese exposure in the presence and absence of pre-parkinsonism. Neurotoxicol. Teratol. 22, 851-61. (10) Gwiazda, R. H., Lee, D., Sheridan, J., and Smith, D. R. (2002) Low Cumulative Manganese Exposure Affects Striatal GABA but not Dopamine. Neurotoxicology (in press). (11) Olanow, C. W., Good, P. F., Shinotoh, H., Hewitt, K. A., Vingerhoetes, F., Snow, B. J., Beal, M. F., Calne, D. B., and Perl, D. P. (1996) Manganese intoxication in the rhesus monkey: A clinical, imaging, pathologic, and biochemical study. Neurology 46, 4928. (12) Gavin, C. E., Gunter, K. K., and Gunter, T. E. (1990) Manganese and calcium efflux kinetics in brain mitochondria. Relevance to manganese toxicity. Biochem. J. 266, 329-34. (13) Brown, S., and Taylor, N. L. (1999) Could mitochondrial dysfunction play a role in manganese toxicity? Environ. Toxicol. Pharmacol. 7, 49-57. (14) Barbeau, A. (1984) Manganese and extrapyramidal disorders (a critical review and tribute to Dr. George C. Cotzias). Neurotoxicology 5, 13-35. (15) Corrigan, F. M., Murray, L., Wyatt, C. L., and Shore, R. F. (1998) Diorthosubstituted polychlorinated biphenyls in caudate nucleus in Parkinson’s disease. Exp. Neurol. 150, 339-342. (16) Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., Kortsha, G. X., Brown, G. G., and Richardson, R. J. (1997) Occupational exposures to metals as risk factors for Parkinson’s disease. Neurology 48, 650-658. (17) Rybicki, B. A., Johnson, C. C., Uman, J., and Gorell, J. M. (1993) Parkinsons Disease Mortality and the Industrial Use of Heavy Metals in Michigan. Movement Disord. 8, 87-92. (18) Yamada, M., Ohno, S., Okayasu, I., Okeda, R., Hatakeyama, S., Watanabe, H., Ushio, K., and Tsukagoshi, H. (1986) Chronic manganese poisoning: a neuropathological study with determination of manganese distribution in the brain. Acta Neuropathol. 70, 273-8. (19) Lynam, D. R., Roos, J. W., Pfeifer, G. D., Fort, B. F., and Pullin, T. G. (1999) Environmental effects and exposures to manganese from use of methylcyclopentadienyl Manganese Tricarbonyl (MMT) in gasoline. Neurotoxicology (Little Rock) 20, 145-150. (20) Abbott, P. J. (1987) Methylcyclopentadienyl manganese tricarbonyl (MMT) in petrol: the toxicological issues. Sci. Total Environ. 67, 247-55. (21) Wallace, L., and Slonecker, T. (1997) Ambient air concentrations of fine (PM2.5) manganese in U.S. national parks and in California and Canadian cities: the possible impact of adding MMT to unleaded gasoline. J. Air Waste Manage. Assoc. 47, 64252. (22) Ressler, T., Wong, J., Roos, J., and Smith, I. L. (2000) Quantitative speciation of Mn-bearing particulates emitted from autos burning (methylcyclopentadienyl)manganese tricarbonyl-added gasolines using XANES spectroscopy. Environ. Sci. Technol. 34, 950-8. (23) Ter Haar, G. L., Griffing, M. E., Brandt, M., Oberding, D. G., and Kapron, M. (1975) Methylcyclopentadienyl Manganese Tricarbonyl as an Antiknock: Composition and Fate of Manganese Exhaust Products. J. Air Pollut. Control Assoc. 25, 858-60. (24) Cory-Slechta, D. A. (1990) Lead exposure during advanced age: alterations in kinetics and biochemical effects. Toxicol. Appl. Pharmacol. 104, 67-78. (25) Silbergeld, E. K. (1991) Neurological perspective on lead, CRC Press, Boca Raton, FL. (26) Brouillet, E. P., Shinobu, L., McGarvey, U., Hochberg, F., and Beal, M. F. (1993) Manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism. Exp. Neurol. 120, 89-94. (27) Ali, S. F., Duhart, H. M., Newport, G. D., Lipe, G. W., and Slikker, W., Jr. (1995) Manganese-induced reactive oxygen species: comparison between Mn+2 and Mn+3. Neurodegeneration 4, 32934. (28) Archibald, F. S., and Tyree, C. (1987) Manganese poisoning and the attack of trivalent manganese upon catecholamines. Arch. Biochem. Biophys. 256, 638-50. (29) Kwik-Uribe, C. L., Reaney, S., Zhu, Z., and Smith, D. (2002) Alterations in the cellular abundance of IRP-dependent proteins and the size of the labile iron pool as a consequence of in vitro manganese exposure in undifferentiated PC12 cells. J. Biol. Chem. (submitted for publication).

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Chem. Res. Toxicol., Vol. 15, No. 9, 2002

(30) Zheng, W., and Zhao, Q. (2001) Iron overload following manganese exposure in cultured neuronal, but not neuroglial cells. Brain Res. 897, 175-9. (31) Gavin, C. E., Gunter, K. K., and Gunter, T. E. (1999) Manganese and calcium transport in mitochondria: implications for manganese toxicity. Neurotoxicology 20, 445-53. (32) Galvani, P., Fumagalli, P., and Santagostino, A. (1995) Vulnerability of mitochondrial complex I in PC12 cells exposed to manganese. Eur. J. Pharmacol. 293, 377-83. (33) Turrens, J. F. (1997) Superoxide production by the mitochondrial respiratory chain. Biosci. Rep. 17, 3-8. (34) McCay, P. B., Gibson, D. D., and Hornbrook, K. R. (1981) Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat-labile factor not glutathione peroxidase. Fed. Proc. 40, 199-205. (35) Halliwell, B. (1984) Manganese ions, oxidation reactions and the superoxide radical. Neurotoxicology 5, 113-7. (36) Kenton, R. H., and Mann, P. J. G. (1955) The oxidation of manganese by illuminated chloroplast preparations. Biochem. J. 61, 279-86. (37) Klewicki, J. K., and Morgan, J. J. (1998) Kinetic behavior of Mn(III) complexes of pyrophosphate, EDTA, and citrate. Environ. Sci. Technol. 32, 2916-22. (38) Golub, M. S., Han, B., and Keen, C. L. (1999) Aluminum uptake and effects on transferrin mediated iron uptake in primary cultures of rat neurons, astrocytes and oligodendrocytes. Neurotoxicology 20, 961-70. (39) Rose, I. A., and O’Connell, E. L. (1967) Mechanism of aconitase action. I. The hydrogen transfer reaction. J. Biol. Chem. 242, 1870-9.

Reaney et al. (40) Smith, D. R., Osterloh, J. D., Niemeyer, S., and Flegal, A. R. (1992) Stable isotope labeling of lead compartments in rats with ultralow lead concentrations. Environ. Res. 57, 190-207. (41) Wroblewski, F., and LaDue, J. S. (1955) PSEBM 10, 210-13. (42) Faridoon, K. Y., Zhuang, H. Y., and Sykes, A. G. (1994) Kinetic Studies On the Reaction of M2+ Ions With Aconitase Fe3s4(0) to Give Fe3ms4(2+) Clusters (M)Fe, Mn, Co). Inorg. Chem. 33, 2209-12. (43) Aisen, P., Roland, A., and Refield, A. (1969) The chromium, manganese, and cobalt complexes of transferrin. J. Biol. Chem. 244, 4628-4633. (44) Villafranca, J. J., and Mildvan, A. S. (1971) The mechanism of aconitase action. II. Magnetic resonance studies of the complexes of enzyme, manganese(II), iron(II), and substrates. J. Biol. Chem. 246, 5791-8. (45) Villafranca, J. J., and Mildvan, A. S. (1972) The mechanism of aconitase action. 3. Detection and properties of enzyme-metalsubstrate and enzyme-metal-inhibitor bridge complexes with manganese(II) and iron(II). J. Biol. Chem. 247, 3454-63. (46) Chen, J. Y., Tsao, G. C., Zhao, Q., and Zheng, W. (2001) Differential cytotoxicity of Mn(II) and Mn(III): special reference to mitochondrial [Fe-S] containing enzymes. Toxicol. Appl. Pharmacol. 175, 160-8. (47) Suarez, N., Walum, E., and Eriksson, H. (1995) Cellular neurotoxicity of trivalent manganese bound to transferrin or pyrophosphate studied in human neuroblastoma (SH-SY5Y) cell cultures. Toxicol. In Vitro 9, 717-21.

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