After being exposed to the reagents, the liver slices were homogenized in buffer I (1 mL), and an aliquot of 10 μL (50 μg/protein, Peterson, 1977) of both the homogenate of the liver slices and the homogenate of the isolated mitochondria was added to 3 mL of buffer III (containing 5 mM glutamate and 5 mM Sotrastaurin research buy succinate). After 10 s, 10 μM (DCHF–DA) (prepared in ethanol) was added to the mixture; and the fluorescence intensity from DCF was measured
for 300 s and expressed as a percentage of the untreated control group. The oxygen consumption of the liver slices was measured using an oxymeter (Hansatech model with a Clark-type electrode) at 30 °C. Two slices, weighting approximately 30 μg (30 ± 2 μg) each, were selected and placed in 2 mL Krebs–Ringer buffer. Fifteen minutes after methionine addition, glutamate/succinate (5 mM each) was placed in the medium to increase the respiratory state. After 30 min, either the MeHg solution or the MeHg–Cys complex solution was added. The respiratory ratio and oxygen consumption were determined
and compared among groups. Cell viability and mitochondrial activity were measured by dehydrogenase activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Mosmann, 1983). check details After 30, 60 and 120 min of exposure to the respective treatment, four liver slices were selected and incubated with MTT (5 μg/mL) for 20 min. The MTT reduction reaction was stopped by the addition of 1.5 mL of dimethylsulfoxide (DMSO). buy Cobimetinib The formazan color and the colorimetric intensity were determined by the difference in absorbance readings at 570–630 nm, using an UV 2450 Shimadzu spectrophotometer. The ratio values were standardized to protein content and expressed as a percentage of the untreated control group. All experiments were standardized to protein concentrations (Peterson, 1977), and, when appropriate, were expressed as a percentage of untreated control values.
Data were analyzed statistically by one-way ANOVA, followed by Duncan’s multiple range tests when appropriate. The significance between the respiratory rates (Table 1) was analyzed statistically by t-test. Differences between groups were considered to be significant when P < 0.05. The first set of experiments was designated to analyze the Hg content in liver slices and mitochondria isolated from liver slices. The Fig. 1 shows that treatment with MeHg alone caused a significant increase in the Hg concentration in both liver slices (A) and in mitochondria isolated from liver slices (B) and that the content of Hg was further increased in the group exposed to the MeHg–Cys complex when compared to the group treated with MeHg alone (Figs. 1A and B). The data in Fig. 1 also reveal that pre-treatment with Met was effective in reducing the Hg levels of the slices exposed to MeHg or the MeHg–Cys complex (Fig. 1A).