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

Abstract

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.

Fig. 1

Effects of Met pre-treatment on Hg uptake in rat liver slices (A) and mitochondria (B) exposed to MeHg or the MeHg–Cys complex. Slices were pre-treated for 15 min with Met (250 μM) and then exposed for 30 min to MeHg (25 μM) or the MeHg–Cys complex (25 μM each); (^*Indicates p<0.05 from control; ⁺Indicates p<0.05 from MeHg; ^#Indicates p<0.05 from the MeHg–Cys complex; n=6 mean±S.E.).

Fig. 2

Effects of Met pre-treatment on DFC-RS production in rat liver slices (A) and mitochondria (B). Slices were pre-treated for 45 min with Met (50, 100 and 250 μM). The tracings of figure are representative lines of 3 independent experiments.

Fig. 3

Effects of exposure to MeHg or the MeHg–Cys complex on DFC-RS production in rat liver slices (A) and mitochondria (B). Slices were exposed for 30 min to MeHg (25 μM) or the MeHg–Cys complex (25 μM each). (^*Indicates p<0.05 from control; ⁺Indicates p<0.05 from MeHg; n=5 mean±S.E.). The tracings of Figs. 3A and B are representative and averaged lines respectively.

Fig. 4

Effects of Met pre-treatment on DFC-RS production in mitochondria exposed to MeHg or the MeHg–Cys complex. Slices were pre-treated for 15 min with Met (250 μM) and then exposed for 30 min to MeHg (25 μM) or the MeHg–Cys complex (25 μM each). Insets in Fig. 4 represent statistical analysis. (^*Indicates p<0.05 from control; ⁺Indicates p<0.05 from MeHg; ^#Indicates p<0.05 from MeHg–Cys complex; n=6 mean±S.E.). The tracings of Fig. 4 are representative lines.

Fig. 5

Effects of exposure to MeHg or the MeHg–Cys complex on oxygen consumption in rat liver slices (A). Effects of Met pre-treatment on oxygen consumption in rat liver slices (B) exposed to MeHg or the MeHg–Cys complex. Slices were pre-treated for 15 min with Met (250 μM) and then exposed for 30 min to MeHg (25 μM) or the MeHg–Cys complex (25 μM each); (n=5 mean±S.E.).

Fig. 6

Effects of Met pre-treatment on mitochondrial function of cells exposed to MeHg or the MeHg–Cys complex. Slices were pre-treated for 15 min with Met (250 μM) and then exposed for 30, 60 or 120 min to MeHg (25 μM) or the MeHg–Cys complex (25 μM each) (Figs. 6A, B, and C respectively). (^*Indicates p<0.05 from control; ⁺Indicates p<0.05 from MeHg; ^#Indicates p<0.05 from MeHg–Cys complex; n=6 mean±S.E).