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. 2013 Mar:35:121-8.
doi: 10.1016/j.neuro.2013.01.002. Epub 2013 Jan 11.

The effect of manganese on dopamine toxicity and dopamine transporter (DAT) in control and DAT transfected HEK cells

Jerome A Roth  1 , Zhezheng Li, Swetha Sridhar, Habibeh Khoshbouei
Affiliations

The effect of manganese on dopamine toxicity and dopamine transporter (DAT) in control and DAT transfected HEK cells

Jerome A Roth et al. Neurotoxicology. 2013 Mar.

Abstract

Chronic exposure to Mn results in the development of a neurological disorder known as manganism characterized by neurological deficits resembling that seen in Parkinsonism. Although dopaminergic neurons within the nigrostriatal pathway appear intact, Mn-induced irregularities in DA transmission have been observed including decreased amphetamine-induced DA release and loss of the dopamine transporter (DAT). Results of studies to evaluate the effect of Mn and DA on cell viability in control and DAT-transfected HEK cells reveal that Mn is equally toxic to both cell lines whereas DA was only toxic to cells containing DAT. DA toxicity was saturable suggesting that transport may be rate limiting. When Mn and DA were added simultaneously to the media, cell toxicity was similar to that produced by Mn alone suggesting that Mn may suppress DA uptake in the DAT containing cells. Preincubation of DA prior to the addition of Mn resulted in cell death which was essentially additive with that produced independently by the two agents. Mn was also shown to decrease DA uptake and amphetamine-induced DA efflux in DAT containing cells. Time-lapsed confocal microscopy indicates that Mn can promote trafficking of cell surface DAT into intracellular compartments which may account for the decrease in DA uptake and DA efflux in these cells. Mn-induced internalization of DAT may provide an explanation for disruption in DA transmission previously reported in the striatum.

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Conflict of interest statement

There is no conflict of interest which effects objectivity in regard to publishing this paper.

Figures

Fig. 1
Fig. 1
(A) Concentration dependence for Mn toxicity in HEK-293 and HEK YFP-DAT cells. HEK-293 and HEK YFP-DAT cells were treated with 25 – 400 μM of Mn for 24 hrs. Toxicity was measured by the MTT assay and compared to the control samples treated with no Mn. Data are presented as the mean ± S.E.M for three independent experiments. There was no significant difference (p=0.997, Two way ANOVA) in cellular viability between HEK-293 and HEK YFP-DAT cells. (B) Time course for Mn toxicity in HEK-293, and HEK YFP-DAT cells. HEK-293 and HEK YFP-DAT cells were treated with 200 μM of Mn for 6, 12, and 24hrs. Toxicity was measured by MTT assay and compared to control samples treated with Mn for few seconds. Data are presented as the mean ± S.E.M for three independent experiments. There was no significant difference (p=0.533, Two Way ANOVA) in cellular viability between HEK-293 and HEK YFP-DAT cells.
Fig. 2
Fig. 2
(A) Concentration dependence for DA toxicity in HEK-293 and HEK YFP-DAT cells. HEK-293 and HEK YFP-DAT cells were treated with 25 – 200 μM of DA for 24 hrs. Toxicity was measured by the MTT assay and compared to the control samples treated with no DA. Data are presented as the mean ± S.E.M for three independent experiments. There was significant difference (p < 0.001) in cellular viability between HEK-293 and HEK YFP-DAT cells. (B) Time course for DA toxicity on HEK-293, and HEK YFP-DAT cells. HEK-293 and HEK YFP-DAT cells were treated with 100 μM dopamine for 6, 12, 24 and 48 hrs. Toxicity was measured by the MTT assay and compared to the control samples treated with no DA. Data are presented as the mean ± S.E.M for three independent experiments. There was significant difference (p < 0.01) in cellular viability between HEK-293 and HEK YFP-DAT cells.
Fig. 3
Fig. 3
(A) Mn and DA-induced toxicity when co-incubated in HEK cells. A. Mn and DA co-incubation with HEK-293 cells. DA (50 μM) was preincubated for 24 hrs. prior to the addition of Mn. Toxicity was measured by the MTT assay and compared to the control samples treated with no DA. Data presents the mean ± S.E.M for three independent experiments. There was no significant difference in cellular viability between Mn treated and Mn/DA treated in HEK-293 cells. (B) Mn and DA-induced toxicity when co-incubated in HEK YFP-DAT cells. DA (50 μM) was preincubated for 24 hrs. prior to the addition of Mn. Toxicity was measured by the MTT assay and compared to the control samples treated with no DA. Data presents the mean ± S.E.M for three independent experiments. There was no significant difference in cellular viability between Mn treated and Mn/DA treated in HEK YFP-DAT.
Fig. 4
Fig. 4
Mn-induced toxicity in HEK YFP-DAT cells pre-incubation with DA. Cells were pre-incubated with 100 μM DA for 12 hrs. and then with 50 and 100 μM of Mn for an additional 24 hrs. Toxicity was measured by MTT assay and compared to the control samples treated with no Mn and no DA. Data are presented as the mean ± S.E.M of three independent experiments. For all these experiments, there was significant difference in cellular viability between manganese treated and manganese/dopamine treated HEK YFP-DAT cells (p-value for Mn and Mn + DA 50 or 100 μM respectively: p < 0.001, p < 0.001). There was a significant difference between cell viability for Mn only treatment in comparison to the no Mn control (50 μM Mn, p < 0.0005; 100 μM Mn, p < 0.001). The cell viability for DA only for 36 hr. exposure was 65 percent.
Fig. 5
Fig. 5
Mn-induced toxicity in HEK YFP-DAT cells initially pre-incubation with DA for 12 hrs. Cells were pre-incubated with 100 μM DA for 12 hrs., then the DA was removed and incubation continued with 50 and 100 μM of Mn for another 24 hrs. Toxicity was measured by MTT assay and compared to the control samples treated with no Mn and no DA. Data are presented as the mean ± S.E.M of three independent experiments. For the 50 μM experiment, there was significant difference in cellular viability between manganese treated and manganese/dopamine treated HEK YFP-DAT samples. However, for the 100 μM experiment there was no significant difference in cell viability between Mn treated and Mn/dopamine treated HEK YFP-DAT. (p-value for Mn and Mn + DA 50 and 100 μM respectively: (p < 0.001, p = 0.2). There was a significant difference between cell viability for Mn only treatment in comparison to the no Mn controls (50 μM Mn, p < 0.001, 100 μM Mn, p < 0.001). The cell viability for DA only control for 12 hr. washed exposure is 66 percent.
Fig. 6
Fig. 6
Effect of Mn on the redistribution of DAT in HEK YFP-DAT cells. DAT-HEK cells were grown overnight on chambered dishes in DMEM before being changed to OPTIMEM prior to imaging. A single position on the dishes was selected using the confocal software and baseline images were taken prior to addition of Mn. Cells were treated with vehicle or 0.3 mM Mn and incubated on the stage for 60 min. prior to imaging on a Zeiss LSM-510 Meta NLO laser scanning confocal microscope. The data is average of 15–20 cells from five independent experiments.
Fig. 7
Fig. 7
Changes is current-voltage (I(V)) relationships produced by Mn on DAT-mediated DA efflux. DAT mediated DA efflux was measured at +60 to −100 mV membrane potentials when 0, 10 and 100 μM after Mn was dialyzed into the cell. Addition of 50 μl (pipette volume) of a 100 μM Mn solution to the bath solution did not affect DAT-mediated DA efflux at these membrane potentials (data not shown). DAT-mediated DA efflux was isolated by subtracting the current produced in the presence of the selective DA uptake inhibitor, GBR12935, from the baseline current (current produced in the absence of AMPH). The AMPH-induced DA efflux was defined as the current recorded in the presence of the AMPH, minus the current recorded after addition of GBR12935 to the bath with substrate still present.
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