ascorbic-acid and Manganese-Poisoning

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Cell Survival; Chromatography, High Pressure Liquid; Dopamine; Dopamine Agents; Drug Synergism; Flow Cytometry; Levodopa; Manganese Poisoning; Oxidative Stress; PC12 Cells; Rats

Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), glutathione (GSH), ascorbic acid (AA), dehydroascorbic acid (DHAA) and uric acid (UA) were determined in the striatum and/or in the brainstem of 3-month-old male Wistar rats after subchronic oral exposure to MnCl2 (20 mg kg-1 daily) alone or associated to buthionine (S,R)sulphoximine-ethyl ester (BSO-E), an inhibitor of GSH synthesis. The NA, DA, DOPAC, GSH and glutathione disulphide (GSSG) concentrations were also determined in PC12 cells incubated with Mn alone or associated with either BSO-E or AA. When PC12 cells were incubated with AA, cellular AA and DHAA concentrations were also determined. It was found that BSO-E: (a) decreased GSH levels in the striatum and in the brainstem; (b) potentiated the Mn-induced increase in AA oxidation and uric acid formation in both brain regions; and (c) potentiated the Mn-induced DA and NA depletion in the brainstem. Moreover, the changes in striatal DA metabolism induced by the BSO-E association with Mn (decrease in DA, DOPAC and HVA levels and in the DOPAC + HVA/DA ratio) are consistent with the hypothesis of a loss of dopaminergic neurons. In PC12 cells, BSO-E decreased GSH and GSSG levels and potentiated the Mn-induced decrease-in DA and NA concentrations. On the contrary, AA antagonised the Mn-induced DA and NA depletion. AA antagonised also the Mn- and MN+ BSO-induced decrease in PC12 cells viability. In conclusion, the impairment of neuronal antioxidant system activity plays a permissive role in the oxidative stress-mediated Mn neurotoxicity.

Topics: Animals; Ascorbic Acid; Brain Stem; Chlorides; Dopamine; Glutathione; Male; Manganese Compounds; Manganese Poisoning; Methionine Sulfoximine; Neostriatum; PC12 Cells; Rats; Rats, Wistar

A deficiency of striatal dopamine (DA) is generally accepted as an expression of manganese (Mn) toxicity in experimental animals. Since compromised cellular defence mechanisms may be involved in Mn neurotoxicity, we investigated the response of the neuronal antioxidant system [ascorbic acid (AA) oxidation, glutathione (GSH) and uric acid levels] and neurochemical changes in the striatum in aged rats exposed to Mn. Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), AA, dehydroascorbic acid (DHAA), GSH and uric acid were determined after subchronic oral exposure to MnCl2 200 mg/kg (3-month-old rats) and 30-100-200 mg/kg (20-month-old-rats). Aged rats had basal levels of striatal DA, DOPAC, HVA, 5-HT, 5-HIAA, GSH and AA lower than those of young rats. In the striatum of aged rats, Mn induced biphasic changes in the levels of DA, DOPAC, HVA (an increase at the lower dose and a decrease at the higher dose) and DHAA (opposite changes). Mn decreased GSH levels and increased uric acid levels both in the striatum and in synaptosomes in all groups of aged rats. All of these parameters were affected to a lesser extent in young rats. In conclusion, the response of cellular defence mechanisms in aged rats is consistent with a Mn-induced increase in the formation of reactive oxygen species. An age-related impairment of the neuronal antioxidant system may play an enabling role in Mn neurotoxicity.

Topics: Aging; Animals; Ascorbic Acid; Dopamine; Glutathione; Male; Manganese Poisoning; Neostriatum; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Serotonin; Synaptosomes; Uric Acid

By monitoring dopamine metabolism in rat pheochromocytoma derived PC12 cell cultures during extended treatment with manganese chloride, we assessed the functional changes occurring in a dopaminergic system during the development of manganese-induced damage. Besides eliciting a specific toxic effect on PC12 cells, manganese induced the complete disappearance of extracellular (free) but not intracellular (vesicle stored) dopamine and its metabolite 3,4-dihydroxyphenylacetic acid. This effect was observed also using low manganese concentrations (1 microM) and mainly occurred by non-enzymatic catechol oxidation since it was still evident in a cell free medium and it was fully prevented by ascorbic acid but not by reduced glutathione. The possibility of a mere "non-biological" action was ruled out by the observation of an irreversible effect of manganese as manifested by the cells' apparent inability to release dopamine or 3,4-dihydroxyphenylacetic acid into the culture medium even after complete manganese removal (post-manganese incubation). That a free radical mechanism was not involved in the genesis of this irreversible effect was shown by the fact that neither ascorbic acid, catalase, superoxide dismutase nor glutathione-peroxidase were able to prevent the decrease in catecholamine levels in the "post-manganese" incubation medium when added at the same time as the manganese. The results establish this PC12 cell system as an in vitro model for studying interactions between manganese and catechols and provide a detailed description of the nature of the neurochemical alterations that this heavy metal can induce in a dopaminergic system.

Topics: Adrenal Gland Neoplasms; Animals; Ascorbic Acid; Cell Survival; Chlorides; Culture Media; Dopamine; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Manganese Compounds; Manganese Poisoning; Oxidation-Reduction; Pheochromocytoma; Tumor Cells, Cultured; Zinc

Topics: Ascorbic Acid; Dopamine; Dopamine Antagonists; Humans; Manganese Poisoning; Oxidation-Reduction; Parkinson Disease, Secondary

Albino rats were given intraperitoneally manganese chloride (Mn2+, 4mg/kg) daily for a period of 30 days. Manganese significantly inhibited the lipid peroxidation potential of treated rat brain without altering the contents of iron and ascorbic acid, the two prooxidant factors. In vitro lipid peroxidation studies in the fresh and heated brain homogenates showed almost a non-enzymatic mechanism of inhibition by this metal ion. 30 micrometers Mn2+ concentrations completely inhibited the formation of malonaldehyde (MDA) at 3 hours of incubation. Iron was found to reverse, to some extent, the effect of manganese on in vitro lipid peroxide formation in the mitochondrial fraction of brain and at concentrations of 5 micrometers Fe2+ the amount of MDA formed is comparable to that observed with 1 micro meter Fe2+ in the mitochondrial fraction without manganese. These observations suggest that the central nervous system toxicity of manganese may not be associated with accelerated in vivo lipid peroxidation. However, the mechanism of iron induced reversal on in vitro inhibition of lipid peroxidation by manganese is not understood, at present.

Topics: Animals; Ascorbic Acid; Brain; Iron; Lipid Peroxides; Male; Malondialdehyde; Manganese; Manganese Poisoning; Mitochondria; Rats