Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes

Abstract

The neurotoxicity of high levels of methylmercury (MeHg) is well established both in humans and experimental animals. Astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS). Although the precise mechanisms of MeHg neurotoxicity are ill-defined, oxidative stress and altered mitochondrial and cell membrane permeability appear to be critical factors in its pathogenesis. The present study examined the effects of MeHg treatment on oxidative injury, mitochondrial inner membrane potential, glutamine uptake and expression of glutamine transporters in primary astrocyte cultures. MeHg caused a significant increase in F(2)-isoprostanes (F(2)-IsoPs), lipid peroxidation biomarkers of oxidative damage, in astrocyte cultures treated with 5 or 10 microM MeHg for 1 or 6 h. Consistent with this observation, MeHg induced a concentration-dependant reduction in the inner mitochondrial membrane potential (DeltaPsi(m)), as assessed by the potentiometric dye, tetramethylrhodamine ethyl ester (TMRE). Our results demonstrate that DeltaPsi(m) is a very sensitive endpoint for MeHg toxicity, since significant reductions were observed after only 1 h exposure to concentrations of MeHg as low as 1 microM. MeHg pretreatment (1, 5 and 10 microM) for 30 min also inhibited the net uptake of glutamine ((3)H-glutamine) measured at 1 min and 5 min. Expression of the mRNA coding the glutamine transporters, SNAT3/SN1 and ASCT2, was inhibited only at the highest (10 microM) MeHg concentration, suggesting that the reduction in glutamine uptake observed after 30 min treatment with lower concentrations of MeHg (1 and 5 microM) was not due to inhibition of transcription. Taken together, these studies demonstrate that MeHg exposure is associated with increased mitochondrial membrane permeability, alterations in glutamine/glutamate cycling, increased ROS formation and consequent oxidative injury. Ultimately, MeHg initiates multiple additive or synergistic disruptive mechanisms that lead to cellular dysfunction and cell death.

Figure 1

Effect of MeHg on F₂-IsoPs formation in cultured astrocytes. Rat primary astrocyte cultures were incubated at 37 °C in the absence or presence of MeHg (1, 5, and 10 μM) and F₂-IsoPs levels quantified at 1 and 6 hr, respectively. Data represent the mean ± S.E. from three independent experiments. * p<0.05 versus control by one-way ANOVA followed by Bonferroni multiple comparison tests.

Figure 2

Quantitation of TMRE fluorescent intensities. Cultured astrocytes exposed to MeHg at various concentrations (0, 1, 5, and 10 μM) for 1 hr (A and B) and 6 hr (C and D) and the fluorescent images quantified as described in Section 2. Values are expressed as mean ± S.E.M. of 24 random fields in each group. * p< 0.05, *** p< 0.001 versus control; Δ Δ Δp< 0.001 versus 10μM. note - (2A is image after 1h exposure; 2C is image after 6 h exposure).

Figure 2

Quantitation of TMRE fluorescent intensities. Cultured astrocytes exposed to MeHg at various concentrations (0, 1, 5, and 10 μM) for 1 hr (A and B) and 6 hr (C and D) and the fluorescent images quantified as described in Section 2. Values are expressed as mean ± S.E.M. of 24 random fields in each group. * p< 0.05, *** p< 0.001 versus control; Δ Δ Δp< 0.001 versus 10μM. note - (2A is image after 1h exposure; 2C is image after 6 h exposure).

Figure 2

Quantitation of TMRE fluorescent intensities. Cultured astrocytes exposed to MeHg at various concentrations (0, 1, 5, and 10 μM) for 1 hr (A and B) and 6 hr (C and D) and the fluorescent images quantified as described in Section 2. Values are expressed as mean ± S.E.M. of 24 random fields in each group. * p< 0.05, *** p< 0.001 versus control; Δ Δ Δp< 0.001 versus 10μM. note - (2A is image after 1h exposure; 2C is image after 6 h exposure).

Figure 2

Quantitation of TMRE fluorescent intensities. Cultured astrocytes exposed to MeHg at various concentrations (0, 1, 5, and 10 μM) for 1 hr (A and B) and 6 hr (C and D) and the fluorescent images quantified as described in Section 2. Values are expressed as mean ± S.E.M. of 24 random fields in each group. * p< 0.05, *** p< 0.001 versus control; Δ Δ Δp< 0.001 versus 10μM. note - (2A is image after 1h exposure; 2C is image after 6 h exposure).

Figure 3

Effects of MeHg on glutamine uptake in astrocytes. Rat primary astrocyte cultures were incubated for 30 min at 37 °C in the absence or presence of MeHg (1, 5, and 10 μM) and the net uptake of glutamine (³H-glutamine) was quantified at 1 and 5 min, respectively. MeHg exposure induced a concentration-dependent decrease in glutamine uptake (*** p<0.001 versus control; Δp<0.05; Δ Δ Δp<0.001 versus 10 μM MeHg, n=6–10; mean ± S.E.M.).

Figure 4

Representative agarose gel electrophoresis of RT PCR products for the expression of SNAT3, ASCT2, SNAT1 mRNA (A). Expression of mRNA for SNAT3, ASCT2 and SNAT1 after treatment with MeHg (1 μM, 5 μM, 10 μM). Results are mean ± SD of three independent isolations.*<0.05 vs. control (Mann -Whitney test) (B). Inset: GAPDH expression.

Figure 4

Representative agarose gel electrophoresis of RT PCR products for the expression of SNAT3, ASCT2, SNAT1 mRNA (A). Expression of mRNA for SNAT3, ASCT2 and SNAT1 after treatment with MeHg (1 μM, 5 μM, 10 μM). Results are mean ± SD of three independent isolations.*<0.05 vs. control (Mann -Whitney test) (B). Inset: GAPDH expression.