Evaluation of cytoprotective effects of compounds isolated from Copaifera langsdorffii Desf. against induced cytotoxicity by exposure to methylmercury and lead.

Copaifera langsdorffii L. is one of the most known medicinal species in Brazil. Its leaves are rich in phenolic compounds with potential biological activities as an antioxidant and chelating agent. This paper reports the isolation of four compounds from the hydroalcoholic extract of the leaves of C. langsdorffii and the investigation of their possible cytoprotective effects against heavy metal poisoning. Quercitrin (1), afzelin (2), 3,5-di-O-(3-O-methyl galloyl) quinic acid (3) and 4,5-di-O-(3-O-methyl galloyl) quinic acid (4), were associated with toxic doses of methylmercury and lead and evaluated by Alamar blue cell viability assays in HepG2 and PC12. The compounds displayed significant cytoprotective effect for the HepG2 cell line against both metals. Compounds 1-4 did not protect PC12 cells against methylmercury induced-cytotoxicity, but at lower concentrations, they protected against lead induced-cytotoxicity. The evaluated compounds showed a promising cytoprotection effect against exposure to heavy metals and should be further investigated as protective agents.

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.

Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS).

Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05-10 microM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.

Cipura paludosa extract prevents methyl mercury-induced neurotoxicity in mice.

Cipura paludosa (Iridaceae), a native plant widely distributed in the north of Brazil, is used in traditional medicine as an anti-inflammatory and analgesic agent, against tuberculosis and gonorrhoea and for regulation of menstrual flow. However, scientific studies on the pharmacological properties of C. paludosa are scarce. We have examined the potential protective effects of the ethanolic extract of C. paludosa against methyl mercury (MeHg)-induced neurotoxicity in adult mice. MeHg was diluted in drinking water (40 mg/l, freely available) and the ethanolic C. paludosa extract (CE) was diluted in a 150 mM NaCl solution and administered by gavage (10 and 100 mg/kg body weight, respectively, twice a day). Because treatment lasted for 14 days and each animal weighed around 40 g, the total dosage of plant extract given to each mouse was 5.6 and 56 g, respectively. After the treatment period, MeHg exposure induced a significant deficit in the motor coordination, which was evident by a reduction (90%) in the falling latency in the rotarod apparatus. Interestingly, this phenomenon was completely recovered to control levels by CE co-administration, independent of dosages. MeHg exposure inhibited cerebellar glutathione peroxidase (mean percentage inhibition of 42%) - an important enzyme involved in the detoxification of endogenous peroxides - and this effect was prevented by co-administration of CE. Conversely, MeHg exposure increased cerebellar glutathione reductase activity (mean percentage inhibition of 70%), and this phenomenon was not affected by C. paludosa co-administration. Neither MeHg nor CE changed the cerebellar glutathione levels. This study has shown for the first time, the in vivo protective effects of CE against MeHg-induced neurotoxicity. In addition, our findings encourage studies concerning the beneficial effects of C. paludosa on neurological conditions related to excitotoxicity and oxidative stress.

Additive pro-oxidative effects of methylmercury and ebselen in liver from suckling rat pups.

Oxidative stress has been pointed as an important molecular mechanism for liver injury in methylmercury (MeHg) poisoning. Ebselen, a seleno-organic compound that possesses anti-oxidant properties, is a useful therapeutic agent used in clinical situations involving oxidative stress. Here, we examined the possible in vivo protective effect of ebselen against the pro-oxidative effects of MeHg in liver from suckling rat pups. The effects of MeHg exposure (subcutaneous injections of methylmercury chloride: 2 mg/kg) on the hepatic levels of thiobarbituric acid reactive substances (TBARS) and non-ptotein thiols (NPSH), and on liver glutathione peroxidase (GSHPx) activity, as well as the possible antagonist effect of ebselen (10 mg/kg; subcutaneously) against MeHg effects, were evaluated during the post-natal period. In addition, the possible in vitro interaction between ebselen, glutathione (GSH) and MeHg was investigated by light/UV spectroscopy, with particular attention to the formation of complexes involving ebselen selenol intermediate and MeHg. After in vivo exposure, MeHg and ebselen alone increased hepatic TBARS levels. Moreover, simultaneous treatment with both compounds caused a higher increase in hepatic TBARS levels when compared to the treatments with individual compounds. Liver NPSH decreased after treatments with MeHg and ebselen alone. A significant negative correlation between hepatic TBARS and NPSH was observed. MeHg alone decreased liver GSHPx activity and ebselen, which did not affect this variable per se, reverted this inhibitory effect of MeHg. Light/UV spectroscopy showed that ebselen and GSH form a chemical intermediate that regenerates ebselen after MeHg addition. The presented results show that ebselen abolished the MeHg-induced inhibition on liver GSHPx activity, but did not prevent the oxidative effects of MeHg on liver lipids and NPSH. MeHg affects the in vitro interaction between ebselen and GSH and this phenomenon seems to be responsible for its inhibitory effect toward thiol-peroxidase activity. Additionally, ebselen presents pro-oxidative effects on rat liver, pointing to thiol depletion as a molecular mechanism related to ebselen-induced hepatotoxicity.

Methylmercury increases glutamate release from brain synaptosomes and glutamate uptake by cortical slices from suckling rat pups: modulatory effect of ebselen.

During the early postnatal period the brain is extremely sensitive to external agents. Here, we examined the effect of subcutaneous injections of methylmercury (MeHg; 2 mg/kg) during the suckling period (postnatal days [PND] 3-10, 3-17, or 3-24) on glutamate release from brain synaptosomal preparations and on glutamate uptake by brain cortical slices of rat pups. The possible antagonist effect of ebselen against MeHg effect was also examined at PND 24. MeHg increased the basal (but not K+-stimulated) glutamate release and glutamate uptake at PND 24. A strong tendency of increase in the basal glutamate release from synaptosomes (p= 0.088) was observed at PND 17. Ebselen, which did not affect glutamate release and uptake per se, prevented both effects of MeHg. This study indicates that (1) the effect of MeHg on glutamate release could be involved in its toxicity; (2) the increase in the glutamate uptake could represent a pathophysiological response to MeHg-induced glutamate release; (3) the inhibitory effect of ebselen on MeHg-induced glutamate release could be related to its reported neuroprotective effects.