Methylmercury-induced cytotoxicity and oxidative biochemistry impairment in dental pulp stem cells: the first toxicological findings.

BACKGROUND: Methylmercury (MeHg) is a potent toxicant able to harm human health, and its main route of contamination is associated with the consumption of contaminated fish and other seafood. Moreover, dental amalgams are also associated with mercury release on human saliva and may contribute to the accumulation of systemic mercury. In this way, the oral cavity seems to be the primary location of exposure during MeHg contaminated food ingestion and dental procedures but there is a lack of literature about its effects on dental tissues and the impact of this toxicity on human health. In this way, this study aimed to analyze the effects of different doses of MeHg on human dental pulp stem cells after short-term exposure. METHODS: Dental pulp stem cells from human exfoliated deciduous teeth (SHED) were treated with 0.1, 2.5 and 5 µM of MeHg during 24 h. The MeHg effects were assessed by evaluating cell viability with Trypan blue exclusion assay. The metabolic viability was indirectly assessed by MTT reduction assay. In order to evaluate an indicative of antioxidant defense impairment, cells exposed to 0.1 and 5 µM MeHg were tested by measuring glutathione (GSH) level. RESULTS: It was observed that cell viability decreased significantly after exposure to 2.5 and 5 µM of MeHg, but the metabolic viability only decreased significantly at 5 µM MeHg exposure, accompanied by a significant decrease in GSH levels. These results suggest that an acute exposure of MeHg in concentrations higher than 2.5 µM has cytotoxic effects and reduction of antioxidant capacity on dental pulp stem cells.

Assessment of the cytoprotective effect of the homeopathic compound Canova® on African green monkey kidney (VERO) cell line exposed to the drug dipyrone sodium.

Dipyrone or metamizole is one of the most frequently used analgesic worldwide. Despite its widespread use, this drug may exert genotoxic and cytotoxic effects on lymphocytes. Therefore, studies with therapeutic agents that may provide protection against these effects are important. The homeopathic compound Canova® (CA) appears to be a beneficial candidate for preventing DNA damage and cellular lethality, since this compound acts as an immunomodulator associated with cytoprotective actions. Hence, the aim of the present investigation was to determine the potential cytoprotective effects of CA using cell line VERO as a model. VERO cells were incubated with sodium dipyrone and subsequently subject to the comet, apoptosis and immunocytochemistry assays. Data demonstrated that sodium dipyrone induced an increase in DNA damage index (DI) employing the comet assay. However, when VERO cells were co-treated with CA at the three concentrations studied, a significant reduction in DI was observed, indicating an antigenotoxic effect attributed to CA. Further dipyrone induced an elevation in %apoptosis at 24 and 48 hr. However, when dipyrone was co-incubated with CA, a significant reduction in %apoptosis was noted at the three concentrations of CA employed. Results from immunocytochemical analysis showed a rise in the expression of caspase 8 and cytochrome C when cells were exposed to dipyrone. In contrast, co-treatment of dipyrone and CA significantly reduced the effect of dipyrone. Therefore, evidence indicated that CA acted as an anticytotoxic and antigenotoxic agent counteracting damage induced by dipyrone.

Genotoxic and cytotoxic effects of the drug dipyrone sodium in African green monkey kidney (Vero) cell line exposed in vitro.

Dipyrone or metamizole is one of the most used analgesics, mainly due to its low financial cost. However, in some countries, the sale of dipyrone is prohibited due to reported severe cases of agranulocytosis as a result of its use. Despite its high use, studies showing genotoxic and cytotoxic effects of dipyrone in mammalian cells are scarce. Therefore, in the present study, we assessed cell viability, genotoxic effects, cytotoxic effects (by apoptosis and necrosis induction), and the induction of reactive oxygen species (ROS) in Vero cells (a cell line obtained from the red kidney of green monkey) exposed to dipyrone. Our results showed a significant reduction in viability of cells exposed to dipyrone by the MTT assay. A significant increase in damage index evaluated by a comet assay was also observed, which indicates its genotoxic effects. In which concerns the cytotoxic effects of dipyrone, we observed a significant increase in the number of apoptotic cells using fluorescent dyes after 24 h and 48 h of treatment with the drug. Our results also showed that there was no significant difference in the induction of ROS generation after treatment of the cells with the drug assessed by the DCFH-DA assay. Thus, our work showed that dipyrone is both a genotoxic and cytotoxic drug to Vero cells in the assessed conditions.

In Vitro Assessment of Cytoprotective Effects of CANOVA against Cell Death Induced by the Anti-malarial Artesunate - A Preliminary Experiment.

BACKGROUND: Artesunate (ATS) is a semi-synthetic compound derived from artemisinin, which is widely accepted in the treatment of malaria. However, there is evidence that ATS, under certain in vitro conditions, induces several impairments to normal cell functions. Canova (CA) is a Brazilian homeopathic formulation indicated for patients with depressed immune system. CA shows both in vitro and in vivo protective effects against mutagenic/carcinogenic compounds. Therefore, we aimed to assess in vitro the cytoprotective effects of CA against the cytotoxicity of ATS in Vero cells. METHODS: Viability of Vero cells exposed to ATS was assessed by MTT assay, whereas the anti-cytotoxic effect of CA was evaluated by apoptosis and necrosis quantification with fluorescent dyes. RESULTS: After 24 hours of ATS treatment, a reduction in cell viability was observed at 32 and 64 µg/mL, the latter being statistically significant (p < 0.05) in relation to the negative control. The concentration of 64 µg/mL was chosen for the subsequent experiments. ATS significantly induced both apoptosis and necrosis in Vero cells in relation to controls (p < 0.01). We also observed a statistically significant decrease in the number of apoptotic cells observed in the CA 16% + ATS co-treatment compared with ATS treatment (p < 0.01). Treatment with CA alone also had no influence on either type of cell death. CONCLUSION: Our results demonstrated that ATS is cytotoxic in the assessed conditions. However, such cytotoxicity was attenuated when the cells were treated simultaneously with ATS and CA.

Markers of oxidative-nitrosative stress induced by artesunate are followed by clastogenic and aneugenic effects and apoptosis in human lymphocytes.

Artesunate (ARS) is a semi-synthetic derivative of artemisinin, used as an outstanding antimalarial drug, which also displays antitumor, anti-inflammatory and immunosuppressive effects. In spite of the numerous reports showing the antitumor activity of ARS, the particular mechanisms associated with its cytotoxicity and genotoxicity in non-neoplastic human cells remain unclear. Here we aimed to verify the specific chromosome damages and the changes in markers of oxidative-nitrosative stress and apoptosis triggered by ARS exposure in human peripheral blood lymphocytes. Cultures were incubated in the presence of ARS and the number of binucleated cells was determined. To discriminate between micronuclei (MN) containing a whole chromosome or an acentric chromosome, the MN test was employed in combination with the fluorescence in situ hybridization assay. Alterations in the levels of superoxide anion (O2- ) and nitric oxide (NO) were measured by the nitroblue tetrazolium and Griess assay, respectively. Changes in the expression of the apoptotic markers were assessed by immunocytochemistry. We found that ARS induced a significant formation of both centromere-positive MN (C+ MN) and centromere-negative MN (C- MN). These alterations were accompanied by an increase in both cellular levels of O2- and total NO production, and a remarkable enhancement in the expression of the apoptotic markers cytochrome c and caspases 8 and 9. Together these findings reveal that ARS induces changes in the oxidative-nitrosative status of human lymphocytes, which are followed by apoptosis and clastogenic and aneugenic effects.

Cytotoxic and Genotoxic Effects of Fluconazole on African Green Monkey Kidney (Vero) Cell Line.

Fluconazole is a broad-spectrum triazole antifungal that is well-established as the first-line treatment for Candida albicans infections. Despite its extensive use, reports on its genotoxic/mutagenic effects are controversial; therefore, further studies are needed to better clarify such effects. African green monkey kidney (Vero) cells were exposed in vitro to different concentrations of fluconazole and were then evaluated for different parameters, such as cytotoxicity (MTT/cell death by fluorescent dyes), genotoxicity/mutagenicity (comet assay/micronucleus test), and induction of oxidative stress (DCFH-DA assay). Fluconazole was used at concentrations of 81.6, 163.2, 326.5, 653, 1306, and 2612.1µM for the MTT assay and 81.6, 326.5, and 1306µM for the remaining assays. MTT results showed that cell viability reduced upon exposure to fluconazole concentration of 1306µM (85.93%), being statistically significant (P<0.05) at fluconazole concentration of 2612.1µM (35.25%), as compared with the control (100%). Fluconazole also induced necrosis (P<0.05) in Vero cell line when cells were exposed to all concentrations (81.6, 326.5, and 1306µM) for both tested harvest times (24 and 48 h) as compared with the negative control. Regarding genotoxicity/mutagenicity, results showed fluconazole to increase significantly (P<0.05) DNA damage index, as assessed by comet assay, at 1306µM versus the negative control (DI=1.17 vs DI=0.28, respectively). Micronucleus frequency also increased until reaching statistical significance (P<0.05) at 1306µM fluconazole (with 42MN/1000 binucleated cells) as compared to the negative control (13MN/1000 binucleated cells). Finally, significant formation of reactive oxygen species (P<0.05) was observed at 1306µM fluconazole vs the negative control (OD=40.9 vs OD=32.3, respectively). Our experiments showed that fluconazole is cytotoxic and genotoxic in the assessed conditions. It is likely that such effects may be due to the oxidative properties of fluconazole and/or the presence of FMO (flavin-containing monooxygenase) in Vero cells.