Robson Luiz Puntel

Manganese in Health and Disease

Manganese is an important metal for human health, being absolutely necessary for development, metabolism, and the antioxidant system. Nevertheless, excessive exposure or intake may lead to a condition known as manganism, a neurodegenerative disorder that causes dopaminergic neuronal death and parkinsonian-like symptoms. Hence, Mn has a paradoxal effect in animals, a Janus-faced metal. Extensive work has been carried out to understand Mn-induced neurotoxicity and to find an effective treatment. This review focuses on the requirement for Mn in human health as well as the diseases associated with excessive exposure to this metal.

Protective effect of Melissa officinalis aqueous extract against Mn-induced oxidative stress in chronically exposed mice.

Manganese (Mn) is an essential element for biological systems; however occupational exposure to high levels of this metal may lead to neurodegenerative disorders, resembling Parkinsons disease (PD). While its mechanisms of neurotoxicity have yet to be fully understood, oxidative stress plays a critical role. Thus, the main goal of this study was to investigate the efficacy of aqueous extract of Melissa officinalis in attenuating Mn-induced brain oxidative stress in mice. Sixteen male mice were randomly divided into two groups and treated for 3 months: the first group consumed tap water (control group) and the second group was treated with Mn (50 mg/kg/day for habituation during the first 15 days followed by 100 mg/kg/day for additional 75 days) in the drinking water. After 3 months both groups were sub divided (n=4 per group) and treated for additional 3 months with Mn and/or M. officinalis in the drinking water. The first group (control) was treated with water and served as control; the second group (M. officinalis) was treated with M. officinalis (100 mg/kg/day); the third group was treated with Mn (100 mg/kg/day); the fourth group (Mn+M. officinalis) was treated with both Mn and M. officinalis (100 mg/kg/day each). Mn-treated mice showed a significant increase in thiobarbituric acid reactive species (TBARS) levels (a marker of oxidative stress) in both the hippocampus and striatum. These changes were accompanied by a decrease in total thiol content in the hippocampus and a significant increase in antioxidant enzyme activity (superoxide dismutase and catalase) in the hippocampus, striatum, cortex and cerebellum. Co-treatment with M. officinalis aqueous extract in Mn-treated mice significantly inhibited the antioxidant enzyme activities and attenuated the oxidative damage (TBARS and decreased total thiol levels). These results establish that M. officinalis aqueous extract possesses potent antioxidative properties, validating its efficacy in attenuating Mn-induced oxidative stress in the mouse brain.

Modulation of methylmercury uptake by methionine: prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism.

Methylmercury (MeHg) is an ubiquitous environmental pollutant which is transported into the mammalian cells when present as the methylmercury-cysteine conjugate (MeHg-Cys). With special emphasis on hepatic cells, due to their particular propensity to accumulate an appreciable amount of Hg after exposure to MeHg, this study was performed to evaluate the effects of methionine (Met) on Hg uptake, reactive species (RS) formation, oxygen consumption and mitochondrial function/cellular viability in both liver slices and mitochondria isolated from these slices, after exposure to MeHg or the MeHg-Cys complex. The liver slices were pre-treated with Met (250 μM) 15 min before being exposed to MeHg (25 μM) or MeHg-Cys (25 μM each) for 30 min at 37 °C. The treatment with MeHg caused a significant increase in the Hg concentration in both liver slices and mitochondria isolated from liver slices. Moreover, the Hg uptake was higher in the group exposed to the MeHg-Cys complex. In the DCF (dichlorofluorescein) assay, the exposure to MeHg and MeHg-Cys produced a significant increase in DFC reactive species (DFC-RS) formation only in the mitochondria isolated from liver slices. As observed with Hg uptake, DFC-RS levels were significantly higher in the mitochondria treated with the MeHg-Cys complex compared to MeHg alone. MeHg exposure also caused a marked decrease in the oxygen consumption of liver slices when compared to the control group, and this effect was more pronounced in the liver slices treated with the MeHg-Cys complex. Similarly, the loss of mitochondrial activity/cell viability was greater in liver slices exposed to the MeHg-Cys complex when compared to slices treated only with MeHg. In all studied parameters, Met pre-treatment was effective in preventing the MeHg- and/or MeHg-Cys-induced toxicity in both liver slices and mitochondria. Part of the protection afforded by Met against MeHg may be related to a direct interaction with MeHg or to the competition of Met with the complex formed between MeHg and endogenous cysteine. In summary, our results show that Met pre-treatment produces pronounced protection against the toxic effects induced by MeHg and/or the MeHg-Cys complex on mitochondrial function and cell viability. Consequently, this amino acid offers considerable promise as a potential agent for treating acute MeHg exposure.

Complex methylmercury-cysteine alters mercury accumulation in different tissues of mice

Methylmercury (MeHg) can cause deleterious effects in vertebrate tissues, particularly in the central nervous system. MeHg interacts with sulfhydryl groups from low and high molecular weight thiols in the blood, which can facilitate MeHg uptake into different tissues. The purpose of this study was to examine the effect of MeHg-Cysteine (MeHg-Cys) complex administration on Hg-uptake in cerebral areas (cortex and cerebellum), liver and kidney of adult mice. Animals were divided into four groups: control (1 mL/kg distilled water), MeHg (2 mg/kg), Cys (2 mg/kg) and MeHg-Cys complex (0.8 molar ratio). Mice received one intraperitoneal injection per day for 60 consecutive days. Treatment with MeHg significantly increased mercury concentrations in all tissues analysed when compared with the control group. The accumulation of mercury in brain and in liver was further increased in animals that received MeHg-Cys complex when compared with the MeHg alone group. However, renal Hg decreased in MeHg-Cys treated mice, when compared with the group treated only with MeHg. In summary, the transport of MeHg-Cys complex was tissue-specific, and we observed an increase in its uptake by liver and brain as well as a decrease in kidney.

Apocynin prevents vascular effects caused by chronic exposure to low concentrations of mercury.

Mercury increases the risk of cardiovascular disease and oxidative stress and alters vascular reactivity. This metal elicits endothelial dysfunction causing decreased NO bioavailability via increased oxidative stress and contractile prostanoid production. NADPH oxidase is the major source of reactive oxygen species (ROS) in the vasculature. Our aim was to investigate whether treatment with apocynin, an NADPH oxidase inhibitor, prevents the vascular effects caused by chronic intoxication with low concentrations of mercury. Three-month-old male Wistar rats were treated for 30 days with a) intramuscular injections (i.m.) of saline; b) HgCl2 (i.m. 1st dose: 4.6 µg/kg, subsequent doses: 0.07 µg/kg/day); c) Apocynin (1.5 mM in drinking water plus saline i.m.); and d) Apocynin plus HgCl2. The mercury treatment resulted in 1) an increased aortic vasoconstrictor response to phenylephrine and reduced endothelium-dependent responses to acetylcholine; 2) the increased involvement of ROS and vasoconstrictor prostanoids in response to phenylephrine, whereas the endothelial NO modulation of such responses was reduced; and 3) the reduced activity of aortic superoxide dismutase (SOD) and glutathione peroxidase (GPx) and increased plasma malondialdehyde (MDA) levels. Treatment with apocynin partially prevented the increased phenylephrine responses and reduced the endothelial dysfunction elicited by mercury treatment. In addition, apocynin treatment increased the NO modulation of vasoconstrictor responses and aortic SOD activity and reduced plasma MDA levels without affecting the increased participation of vasoconstrictor prostanoids observed in aortic segments from mercury-treated rats. Conclusions: Mercury increases the vasoconstrictor response to phenylephrine by reducing NO bioavailability and increasing the involvement of ROS and constrictor prostanoids. Apocynin protects the vessel from the deleterious effects caused by NADPH oxidase, but not from those caused by prostanoids, thus demonstrating a two-way action.

Role of Calcium and Mitochondria in MeHg-Mediated Cytotoxicity

Methylmercury (MeHg) mediated cytotoxicity is associated with loss of intracellular calcium (Ca2+) homeostasis. The imbalance in Ca2+ physiology is believed to be associated with dysregulation of Ca2+ intracellular stores and/or increased permeability of the biomembranes to this ion. In this paper we summarize the contribution of glutamate dyshomeostasis in intracellular Ca2+ overload and highlight the mitochondrial dysfunctions induced by MeHg via Ca2+ overload. Mitochondrial disturbances elicited by Ca2+ may involve several molecular events (i.e., alterations in the activity of the mitochondrial electron transport chain complexes, mitochondrial proton gradient dissipation, mitochondrial permeability transition pore (MPTP) opening, thiol depletion, failure of energy metabolism, reactive oxygen species overproduction) that could culminate in cell death. Here we will focus on the role of oxidative stress in these phenomena. Additionally, possible antioxidant therapies that could be effective in the treatment of MeHg intoxication are briefly discussed.

The influence of Bauhinia forficata Link subsp. pruinosa tea on lipid peroxidation and non-protein SH groups in human erythrocytes exposed to high glucose concentrations

ETHNOPHARMACOLOGICAL RELEVANCE Bauhinia forficata (BF) has been traditionally used as tea in folk medicine of Brazil for treatment of Diabetes mellitus (DM). AIM OF THE STUDY To evaluate the effects of BF leaf tea on markers of oxidative damage and antioxidant levels in an experimental model of hyperglycemia in human erythrocytes in vitro. MATERIALS AND METHODS Human erythrocytes were incubated with high glucose concentrations or glucose and BF tea for 24h and 48h. After incubation lipid peroxidation and non-protein SH levels were analyzed. Moreover, quantification of polyphenols and flavonoids, iron chelating property, scavenging of DPPH, and prevention of lipid peroxidation in isolated lipids were also assessed. RESULTS A significant amount of polyphenols and flavonoids was observed. The main components found by LC-MS analysis were quercetin-3-O-(2-rhamnosyl) rutinoside, kaempferol-3-O-(2-rhamnosyl) rutinoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. BF tea presents important antioxidant and chelating properties. Moreover, BF tea was effective to increase non-protein SH levels and reduce lipid peroxidation induced by high glucose concentrations in human erythrocytes. CONCLUSION The antioxidant effects of BF tea could be related to the presence of different phenolic and flavonoids components. We believe that these components can be responsible to protect human erythrocytes exposed to high glucose concentrations against oxidative damage.

Mitochondrial electron transfer chain complexes inhibition by different organochalcogens.

Mitochondrial dysfunction plays a pivotal role in the cell toxicology and death decision. The aim of the present study was to investigate the effect of three organocompounds (ebselen [Ebs], diphenyl diselenide [(PhSe)(2)] and diphenyl ditelluride [(PhTe)(2)]) on mitochondrial complexes (I, II, I-III, II-III and IV) activity from rat liver and kidney to determine their potential role as molecular targets of organochalcogens. All studied organochalcogens caused a statistically significant inhibition of the mitochondrial complex I activity. Ebs and (PhTe)(2) caused a statistically significant inhibition of the mitochondrial complex II activity in both hepatic and renal membranes. Hepatic mitochondrial complex II activity was practically unchanged by (PhSe)(2), whereas it significantly inhibited renal complex II activity. Mitochondrial complex IV activity was practically unchanged by the organochalcogens. Furthermore, organochalcogens inhibited the mitochondrial respiration supported by complex I or complex II substrates. The inhibitory effect of Ebs, (PhSe)(2) and (PhTe)(2) on mitochondrial complex I was prevented by NADH, but it was not prevented by catalase (CAT) and/or superoxide dismutase (SOD). Additionally, the organochalcogens-induced inhibition of complex I and II was completely reversed by reduced glutathione (GSH). In conclusion, Ebs, (PhSe)(2) and (PhTe)(2) were more effective inhibitors of renal and hepatic mitochondrial complex I than complex II, whereas complexes III and IV were little modified by these compounds. Taking into account the presented results, we suggest that organochalcogen-induced mitochondrial complexes I and II inhibition can be mediated by their thiol oxidation activity, i.e., Ebs, (PhSe)(2) and (PhTe)(2) can oxidize critical thiol groups from mitochondrial complexes I and II. So, mitochondrial dysfunction can be considered an important factor in the toxicity of Ebs, (PhSe)(2) and (PhTe)(2).

N-methyl-D-aspartate Receptors are Involved in the Quinolinic Acid, but not in the Malonate Pro-oxidative Activity in vitro

Oxidative stress plays a significant role in the neurotoxicity of a variety of agents that interact with the N-methyl-D-aspartate (NMDA) receptors. Here we investigated in a comparative way the pro-oxidative effects of quinolinic acid (QA) and malonate, two neurotoxic substances that act through distinct primary molecular mechanisms on the production of thiobarbituric acid reactive species (TBARS) by brain homogenates. In fact, QA is thought to activate directly the NMDA receptor, whereas malonate seems to act primarily by inhibiting oxidative metabolism. The malonate-induced TBARS formation was not modified by cyanide (CN−) or 2,4-dinitrophenol. MK-801 did not reduce basal or malonate induced-TBARS production in fresh tissues preparations. However, in heat-treated preparations a significant effect of MK-801 against basal TBARS production was observed, but not on the malonate induced-TBARS production. QA induced-TBARS production was significantly prevented by MK-801 either in fresh or heat-treated preparations. The antioxidant effect of MK-801 on basal and QA-induced TBARS production increased as the temperatures used to treat S1 were increased. Succinate dehydrogenase (SDH) was inhibited by malonate but not by QA. Malonate was able to chelate iron(II) and the malonate-iron complex(es) is(are) active as measured by its(their) activity on deoxyribose degradation assay. These findings indicate that direct interactions of malonate with NMDA receptors are not involved in malonate pro-oxidative activity in vitro. QA pro-oxidative activity in vitro was related, at least in part, to its capability in stimulate NMDA receptors. Taken together, these findings indicated that malonate pro-oxidative activity in vitro could be attributed to its capability of changing the ratio Fe2+/ Fe3+, which is essential to TBARS production.

The antioxidant properties of different phthalocyanines.

Oxidative stress is involved in the etiology of several chronic diseases, including cardiovascular disease, diabetes, cancer, and neurodegenerative disorders. From this perspective, we have evaluated the possible antioxidant capacities of five different phthalocyanines (PCs), consisting of four metallophthalocyanines (MPCs) and one simple phthalocyanine (PC) in order to explore, for the first time, the potential antioxidant activities of these compounds. Our results show that all PCs tested in this study have significant antioxidant activity in lipid peroxidation assay, providing protection from sodium nitroprusside -induced oxidative damage to supernatant from the homogenized liver, brain, e rim of mice. Compared to the non-induced control, the PCs were generally more efficient in reducing malondialdehyde levels in all assays on lipid peroxidation induced by sodium nitroprusside; the order of approximate decrease in efficiency was as follows: manganese-PC (better efficiency)>copper-PC>iron-PC>zinc-PC>PC (worst efficiency). Furthermore, the copper-PC and manganese-PC compounds exerted a significant protective effect in deoxyribose degradation assays, when employing Fe(2+), Fe(2+)+H(2)O(2), and H(2)O(2) solutions. In conclusion, all PCs tested here were shown to be promising compounds for future in vivo investigations, because of their potential antioxidant activities in vitro.