Developmental exposure to the organochlorine insecticide endosulfan damages the nigrostriatal dopamine system in male offspring

Abstract

The contribution of environmental toxicants to the etiology and risk of Parkinson's disease (PD) has been clearly established, with organochlorine insecticides routinely shown to damage the nigrostriatal dopamine pathway. Although PD is generally considered an adult onset disease, it has been postulated that exposure to environmental contaminants or other factors early in life during critical periods of neurodevelopment could alter the dopaminergic circuit and predispose individuals to developing PD. Recent epidemiological evidence has found exposure to the organochlorine insecticide endosulfan to be a risk factor for PD. However, the specific dopaminergic targets or vulnerable developmental time points related to endosulfan exposure have not been investigated. Thus, we sought to investigate dopaminergic neurotoxicity following developmental exposure to endosulfan as well as following an additional challenge with MPTP. Our in vitro findings demonstrate a reduction in SK-N-SH cells and ventral mesencephalic primary cultures after endosulfan treatment. Using an in vivo developmental model, exposure to endosulfan during gestation and lactation caused a reduction in DAT and TH in the striatum of male offspring. These alterations were exacerbated following subsequent treatment with MPTP. In contrast, exposure of adult mice to endosulfan did not elicit dopaminergic damage and did not appear to increase the vulnerability of the dopamine neurons to MPTP. These findings suggest that development during gestation and lactation represents a critical window of susceptibility to endosulfan exposure and development of the nigrostriatal dopamine system. Furthermore, these exposures appear to sensitize the dopamine neurons to additional insults that may occur later in life.

Keywords: Dopamine; Endosulfan; GABA; Neurodevelopment; Nigrostriatal; Parkinson's disease.

Figure 1

Treatment with endosulfan reduces cell viability in SK-N-SH dopaminergic neuroblastoma cells. SK-N-SH cells were grown to confluency and treated with increasing concentrations of endosulfan for 72 hrs. Alterations in cell viability were then evaluated using the WST-1 cell proliferation assay. A significant reduction in cell viability was observed at each concentration of endosulfan. Columns represent percent change from DMSO control. Data represent the mean ± SEM of 12 experimental replicates per treatment group performed over 3 separate experiments. ***Values significantly different from control (p < 0.001).

Figure 2

Exposure of ventral mesencephalic dopamine neurons to endosulfan caused a reduction in the number of TH+ neurons. Treatment of mesencephalic cultures with endosulfan caused significant reduction in TH+ neurons beginning with 20 µM. The number of TH+ neurons was further reduced with treatment of 25 and 30 µM of endosulfan. Images below represent TH+ neurons following exposure to DMSO or 30 µM endosulfan for 24hrs. Green = TH+ neurons. Blue = DAPI nuclear stain. Columns represent the percent change from DMSO. Data represent the mean ± SEM of 4 experimental replicates per treatment group performed across 3 separate experiments. ***Values significantly different from DMSO control (p < 0.001).

Figure 3

GABA_A receptors colocalize with dopamine neurons in the substantia nigra pars compacta. The expression of GABA_A receptors on dopaminergic neurons in the SNpc was evaluated using immunofluorescence. Neurons from control animals were stained for the GABA_A 2α receptor subunit as well as TH, showing a clear colocalization of these proteins in the SNpc. A lack of overlap between these two proteins can be seen in the substantia nigra pars reticulata, which is enriched in GABAergic neurons but not dopaminergic neurons. Green = TH. Red = GABA_A 2α receptor subunit.

Figure 4

Developmental endosulfan exposure caused reductions in the DAT and TH in the striatum of male offspring. Female mice were administered 0 (control) or 1 mg/kg endosulfan throughout gestation and lactation and striatal DAT (A and C) and TH (B and C) protein expression was evaluated by immunoblot in male offspring at 16 weeks of age. Data represent mean ± SEM (6–8 animals each from a different litter per treatment group). *Values for animals that are significantly different from controls (p < 0.05). **Values for animals that are significantly different from controls (p < 0.01).

Figure 5

Developmental exposure to endosulfan increases the vulnerability of dopamine neurons to MPTP. Female mice were administered 0 (control) or 1 mg/kg endosulfan throughout gestation and lactation. At 16 weeks of age, male mice were administered 2 × 10 mg/kg MPTP and evaluated 7 days later for alterations to the nigrostriatal dopamine system. (A) Treatment with MPTP caused a significant reduction in striatal DAT expression that was further exacerbated in the endosulfan exposure group. (B) A similar reduction of striatal TH was also seen. Data represent mean ± SEM (6–8 animals each from a different litter per treatment group). ***Values significantly different from control or endosulfan only group (p < 0.001). ^$Values significantly different from endosulfan group (p < 0.001). ^#Values significantly different from control + MPTP group (p < 0.05).

Figure 6

Developmental exposure to endosulfan and challenge with MPTP does not affect the expression of GABAergic or glutamatergic markers in the striatum. Female mice were administered 0 (control) or 1 mg/kg endosulfan throughout gestation and lactation. At 16 weeks of age, male mice were administered 2 × 10 mg/kg MPTP and evaluated 7 days later for alterations to the striatum. (A) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal GAT1. (B) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal GAD67. (C) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal vGlut. Data represent mean ± SEM of 6 animals.

Figure 7

Adult exposure to endosulfan does not alter expression of DAT and TH in the striatum. Two month old male mice were exposed to 0 (control) or 1 mg/kg/day of endosulfan for 30 days. A subset of mice were administered 2 × 10 mg/kg MPTP and evaluated 7 days later for alterations to the nigrostriatal dopamine system. (A) Exposure to endosulfan did not cause reduction in striatal DAT. Treatment with MPTP significantly reduced expression of DAT in the striatum that were not dependent upon exposure to endosulfan. (B) Exposure to endosulfan did not cause reduction in striatal DAT. Treatment with MPTP significantly reduced expression of TH in the striatum that were not dependent upon exposure to endosulfan. Data represent mean ± SEM of 6 animals. **Values for animals that are significantly different from respective control or endosulfan only group (p < 0.01). ***Values significantly different from respective control or endosulfan only group (p < 0.001).

Figure 8

Adult exposure to endosulfan does not alter expression of GABAergic or glutamatergic proteins in the striatum. Two month old male mice were exposed to 0 (control) or 1 mg/kg/day of endosulfan for 30 days. A subset of mice were administered 2 × 10 mg/kg MPTP and evaluated 7 days later for alterations to the striatum. (A) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal GAT1. (B) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal GAD67. (C) Exposure to endosulfan and MPTP did not cause alterations in the expression of striatal vGlut. Data represent mean ± SEM of 6 animals.