Rat brain endothelial cells are a target of manganese toxicity

Abstract

Manganese (Mn) is an essential trace metal; however, exposure to high Mn levels can result in neurodegenerative changes resembling Parkinson's disease (PD). Information on Mn's effects on endothelial cells of the blood-brain barrier (BBB) is lacking. Accordingly, we tested the hypothesis that BBB endothelial cells are a primary target for Mn-induced neurotoxicity. The studies were conducted in an in vitro BBB model of immortalized rat brain endothelial (RBE4) cells. ROS production was determined by F(2)-isoprostane (F(2)-IsoPs) measurement. The relationship between Mn toxicity and redox status was investigated upon intracellular glutathione (GSH) depletion with diethylmaleate (DEM) or L-buthionine sulfoximine (BSO). Mn exposure (200 or 800 microM MnCl(2) or MnSO(4)) for 4 or 24h led to significant decrease in cell viability vs. controls. DEM or BSO pre-treatment led to further enhancement in cytotoxicity vs. exposure to Mn alone, with more pronounced cell death after 24-h DEM pre-treatment. F(2)-IsoPs levels in cells exposed to MnCl(2) (200 or 800 microM) were significantly increased after 4h and remained elevated 24h after exposure compared with controls. Consistent with the effects on cell viability and F(2)-IsoPs, treatment with MnCl(2) (200 or 800 microM) was also associated with a significant decrease in membrane potential. This effect was more pronounced in cells exposed to DEM plus MnCl(2) vs. cells exposed to Mn alone. We conclude that Mn induces direct injury to mitochondria in RBE4 cells. The ensuing impairment in energy metabolism and redox status may modify the restrictive properties of the BBB compromising its function.

Figure 1

Figure 1 A and B – Viability of RBE4 cells exposed to MnCl₂ or MnSO₄ during 4h (200 and 800 µM), after a 24h DEM 600 µM and BSO 1mM treatment. Values are expressed as means ± SEM from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001 compared to control; There are no statistical significant differences among the treatment groups.

Figure 1

Figure 1 A and B – Viability of RBE4 cells exposed to MnCl₂ or MnSO₄ during 4h (200 and 800 µM), after a 24h DEM 600 µM and BSO 1mM treatment. Values are expressed as means ± SEM from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001 compared to control; There are no statistical significant differences among the treatment groups.

Figure 2

Figure 2 A and B – Viability of RBE4 cells exposed to MnCl₂ or MnSO₄ during 24h (200 and 800 µM), after a 24h DEM 600 µM and BSO 1mM treatment. Values are expressed as means ± SEM from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001 compared to control; _ΔΔΔ p< 0.001 compared to Mn 200 in Mn 200 µM group or Mn 800 in Mn 800 µM group, respectively.

Figure 2

Figure 2 A and B – Viability of RBE4 cells exposed to MnCl₂ or MnSO₄ during 24h (200 and 800 µM), after a 24h DEM 600 µM and BSO 1mM treatment. Values are expressed as means ± SEM from 3 independent experiments. * p< 0.05, ** p< 0.01, *** p< 0.001 compared to control; _ΔΔΔ p< 0.001 compared to Mn 200 in Mn 200 µM group or Mn 800 in Mn 800 µM group, respectively.

Figure 3

Quantitation of F2-IsoPs levels in RBE4 cells exposed to MnCl₂ 200 and 800 µM for either 4 or 24h. * p<0.05 compared to control.

Figure 4

Figure 4 A and B – Quantitation of TMRE fluorescent intensities. Cultured RBE4 cells exposed to Mn at various concentrations (200 and 800 µM) with/without DEM for 2h. Values are expressed as mean±SEM of 12 random fields in each group from 3 independent experiments. *** p<0.001 vs. control; Δ p<0.05, ΔΔ p<0.01, ΔΔΔ p<0.001 vs. DEM; ▲▲p<0.01 vs. Mn 200 or Mn 800 µM.

Figure 4

Figure 4 A and B – Quantitation of TMRE fluorescent intensities. Cultured RBE4 cells exposed to Mn at various concentrations (200 and 800 µM) with/without DEM for 2h. Values are expressed as mean±SEM of 12 random fields in each group from 3 independent experiments. *** p<0.001 vs. control; Δ p<0.05, ΔΔ p<0.01, ΔΔΔ p<0.001 vs. DEM; ▲▲p<0.01 vs. Mn 200 or Mn 800 µM.