PMC

Western blot analysis of LAT-1 from different sources. Homogenates from rat glioma C6 cells, rat hippocampus (hippo; positive control) or mouse hearth (negative control) were submitted to SDS polyacrylamide gel electrophoresis, electroblotted and incubated overnight with primary polyclonal antibody against LAT-1 (1:500). β-Actin bands are also shown (protein load control).

Dose–response curve of mercurial-induced cell toxicity. Rat glioma C6 cells (~80% confluence) were treated with (A) MeHg or MeHg-Cys, (B) EtHg or EtHg-Cys in HBSS. After 30 min of mercurial’s treatment, HBSS was replaced by fresh DMEM and the cells were incubated for additional 24 h at 37 °C in a humidified atmosphere of 5% CO₂. Results of the MTT assays were expressed as percentage of control values (non treated cells). *p < 0.05 and ***p < 0.001 when compared to controls by one-way ANOVA followed by the Tukey’s HSD test. Data are expressed as mean ± SEM (N = 3 independent experiments).

Effect of L-methionine on mercurial-induced cell toxicity. Rat C6 glioma cells were pre-treated with L-Met (at final concentrations 1000 fold greater than those of the respective Hg-compound) for 15 min before treatment with (A) MeHg (4.5 μM) or MeHg-Cys (12 μM), (B) EtHg (6 μM) or EtHg-Cys (10 μM) in HBSS. Thus, the concentrations of L-Met were 4.5 mM, 12 mM, 6 mM or 10 mM in wells treated with MeHg, MeHg-Cys, EtHg or EtHg-Cys, respectively. After the exposure to mercurials (30 min), HBSS was replaced by fresh DMEM and the cells were incubated for additional 24 h. Results of the MTT assays were expressed as percentage of control values (dashed line). The asterisks (*) mean significant differences (p < 0.001) when compared to control and the symbol + means significant difference (p < 0.001) when compared to cells treated only with the respective mercurial (without Cys) by two-way ANOVA followed by the Bonferroni test. Data are expressed as mean ± SEM (N = 6–9 independent experiments).

Glutathione levels after mercurial exposure. Rat C6 glioma cells were treated with MeHg, MeHg-Cys (A), EtHg, or EtHg-Cys (B) in HBSS (at concentrations approximating their respective EC₅₀ value in the toxicity assay – ). L-Methionine was added to the incubation medium (HBSS) 15 min before the mercurial’s addition at a final concentration 1000 fold greater than the Hg-compounds. After 30 min, HBSS was replaced by fresh DMEM and the cells were incubated for additional 4 h at 37 °C in a humidified atmosphere of 5% CO₂. GSH concentration in control C6 cells (non treated) was 69.0 ± 2.2 nmol of GSH/mg of protein. GSH levels are expressed as percentage of control values (100%, dashed line). The asterisks (*) mean significant differences (p < 0.01) when compared to control and the symbol + means significant difference (p < 0.05) when compared to cells treated only with the respective mercurial (without Cys) by two-way ANOVA followed by the Bonferroni test. Data are expressed as mean ± SEM (N = 4 independent experiments).