Bekæmpelsesmiddelforskning fra Miljøstyrelsen, 95 – Virkningsmekanismer for neurotoksisk effekt af mangan og kombinationseffekt af mangan og chlorpyrifos og maneb

Virkningsmekanismer for neurotoksisk effekt af mangan og kombinationseffekt af mangan og chlorpyrifos og maneb

Abstract

Manganese is widely used as an additive to pesticide formulations, and constitutes an integrated and organically bound part of two pesticides: maneb and mancozeb. Manganese can cause neurotoxicity, manganism/parkinsonism, in workers occupationally exposed to manganese. Manganism shares many symptoms with Parkinson's disease. Epidemiological investigations document that workers in agriculture and horticulture, and people living near areas with frequent use of pesticides have increased risk of developing symptoms of Parkinson's disease.

There may be an interaction, even a potentiation, between manganese and pesticides with respect to neurotoxicity. Nothing certain is known about such potential interaction. This project may therefore add knowledge to this question, enabling the results to be used to protect people against occupational and environmental exposure to manganese and pesticides, alone and in combination.

This project hypothesizes an interaction between neurotoxic effects induced by manganese and pesticides, especially in relation to Parkinson's disease. Underlying mechanisms and their potential interaction can be illuminated by an integrated multidisciplinary approach including morphological, immunohistochemical, and neurochemical methods applying the young male Sprague Dawley rat as the model.

In addition to manganese (dosed as manganese chloride), two pesticides were selected for the studies: chlorpyrifos that is widely used and has a documented effect on the cholinergic system, and maneb, a fungicide with organically bound manganese. Dose-range studies were performed for each of these to select proper concentrations in the combination study in which the three chemicals were administered alone, in all combinations with two chemicals, and when all three were administered.

In patients with Parkinson's disease, the dopamine concentration is dramatically reduced in the basal ganglia, including corpus striatum, as a consequence of degeneration of dopaminergic neurons originating in substantia nigra and projecting to corpus striatum. The basal underlying mechanisms are unknown, but induction of oxidative stress and apoptosis seem to be central elements.

The dopamine concentration was selected as an important endpoint, as were the concentrations of noradrenaline, 5-hydroxytryptamine, and various amino acid neurotransmitters [glutamate, taurine and GABA (gamma-aminobutyric acid)] and the activity of acetylcholinesterase. These endpoints were used as indices, biomarkers, of affected related parts of the central nervous system. Additionally, quantitative determination of two relevant proteins α-synuclein and synaptophysin - was applied, as was the activity of the caspase-3 enzyme closely related to apoptotic neurodegeneration. Histochemical stainings were chosen to discriminate between necrotic and apoptotic neurodegeneration and effects on the catecholaminergic nervous system i.e. haematoxylin-eosine, glial fibrillary acidic protein, tyrosine hydroxylase and TUNEL. Focus was placed on corpus striatum and substantia nigra, but also the rest of the brain was investigated.

There were serious problems with the intraperitoneal administration of manganese chloride, because the animals experienced discomfort. This route of administration is often used in investigations, but such symptoms are not reported in literature. The highest dose of manganese chloride was 2.5 mg/kg body weight/day for a maximum of 12 weeks. This increased the manganese concentration in corpus striatum, the rest of the brain and plasma. Administration of manganese chloride for 12 weeks slightly reduced the striatal concentrations of dopamine, glutamate, taurine, GABA, and the acetylcholinesterase activity. These reductions were minimal, and were not regarded as manifested manganese-induced neurotoxicity. The concentrations of α-synuclein and synaptophysin and the histological parameters were not affected.

The dose-range study with chlorpyrifos subcutaneous dosing (2.5, 15 and 30 mg chlorpyrifos/kg bodyweight/week for 12 weeks) revealed increased dopamine concentrations to be induced by the two lowest doses. There was no effect on striatal neurotransmitter amino acids, whereas the acetylcholinesterase activity was reduced in a dose-related manner. The concentrations of α-synuclein and synaptophysin and the histological parameters were not affected.

The dose-range study with maneb intraperitoneal dosing (7.5, 15 and 30 mg maneb/kg bodyweight/week for 12 weeks) demonstrated dose-related increased manganese concentration in corpus striatum. The striatal concentration of 5-hydroxytryptamine increased in a dose related manner as did the 5-hydroxytryptamine concentration in the rest of the brain, indicating early sign of neurotoxicity. Striatal acetylcholinesterase activity was not affected. The concentrations of neurotransmitter amino acids in corpus striatum and the rest of the brain were not changed. The concentrations of α-synuclein and synaptophysin and the histological parameters were not affected. Identification of the speciation in brain tissue of manganese originating from maneb was outside the scope of this project. Consequently, effects could not be interpreted in terms of manganese speciation.

Based on the results in the above studies, in the combinatory study the following doses were chosen: manganese, 2.5 mg Mn/kg body weight/day for 12 weeks as intraperitoneal doses of manganese chloride; chlorpyrifos, 15 mg chlorpyrifos/kg bodyweight/week for 12 weeks as subcutaneous doses; maneb, 10 mg maneb/kg bodyweight/week for 12 weeks as intraperitoneal doses. Beside a control group, manganese, chlorpyrifos and maneb were administered either alone, in all three combinations with two chemicals, and when all three were co-administered i.e. a total of eight groups.

Manganese and maneb increased the striatal concentration of manganese. When administered together data were in accordance with an additive effect on the manganese concentration. Coexposure to chlorpyrifos and maneb decreased striatal dopamine and 5-hydroxytryptamine concentrations. The acetylcholinesterase activity in corpus striatum was reduced in all groups administered chlorpyrifos as when single exposed to maneb and coexposed to manganese and maneb. Remarkably, single exposure to maneb and coexposure to manganese and maneb also reduced striatal concentrations of glutamate, taurine and GABA. The caspase-3 activity was unaffected by all treatments as were the concentrations of α-synuclein and synaptophysin and the histological parameters.

In conclusion, single exposure to manganese, chlorpyrifos, and maneb showed affection of various neurochemical effect parameters of relevance for induction of neurotoxicity related to Parkinson's disease. The combinatory study also revealed affected neurochemistry. However, no consistent pattern of changes emerged, and no obvious synergistic effects, including potentiations, were disclosed under the conditions applied in this project. This does not exclude that another setup for the investigations would not have revealed a consistent pattern of affected biomarkers and synergistic effects. A potential for such effects still exists, which needs to be thoroughly investigated.