Commercially Available Pesticides Cause Synergistic Or Additive Damages in Dopaminergic Cells: Relevance for Parkinson’S Disease Pathology
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Central Annals of Neurodegenerative Disorders Bringing Excellence in Open Access Research Article *Corresponding author Y. Hwan Kim, Delaware State University, Dover, DE19901- 2277, USA, Tel: 1-302-857-6524; Fax: 1-302-857-7378; Commercially Available Email: Submitted: 28 July 2016 Pesticides Cause Synergistic Accepted: 06 September 2016 Published: 09 September 2016 Copyright or Additive Damages in © 2016 Kim et al. Dopaminergic Cells: Relevance OPEN ACCESS Keywords • Additive toxicity for Parkinson’s Disease • Synergistic damage • Pesticide • Oxidative stress Pathology • Parkinson’s disease Eric M. Janezic, Janae E. Caviness, Xenia D. Davis, Sambee G. Kanda, and Yong-Hwan Kim* Department of Biological Sciences, Delaware State University, USA Abstract Although several genetic mutations have been identified to cause Parkinson’s disease (PD), the vast majority of cases are considered to be sporadic or multifactorial. Interestingly, epidemiological studies have shown that PD is more prevalent amongst farmers and rural populations. Thus, it has been suggested that exposure to pesticides and other environmental toxins may increase the risk of PD. In support of this notion, it has been shown that the herbicide, paraquat, and the fungicide, maneb, can cause motor deficits individually, as well as cause synergistic damage in mice when used together. Here we tested commercially available pesticides for causing synergistic or additive damage in an in vitro PD model, when used together. We exposed rat dopaminergic N27 cells to commercially-used pesticides such as acephate, alachlor, atrazine, diuron, 2-methyl-4-chlorophenoxyacetic acid (MCPA), and mecoprop at varying concentrations and measured cell viability using MTT assays. After identifying the non-toxic concentrations of single pesticide treatments, we measured the cell viability with the exposure of two different combinations of these pesticides. When tested individually, only high concentrations of diuron (14.4 and 28.8 µM) caused a significant decrease in cell viability. However, when we examined the effect of the combined pesticides at concentrations that did not show damage individually, we identified four combinations that cause synergistic loss, and five that cause additive loss in cell viability. Our results suggest that exposure to multiple combinations of pesticides may cause dopaminergic toxicity and further lead to sporadic PD pathology. Furthermore, we found that apoptosis was the mechanism of cell death in at least one of the pesticide combinations. Our results can bring more public awareness to the detrimental effects of combined pesticide usage in PD pathology. INTRODUCTION of dopaminergic neurons in the Substantia Nigra pars compacta Parkinson’s disease (PD) is an age-related disorder and the these only account for up to 10 percent of cases [1]. A majority of most common motor neurodegenerative disorder, affecting cases(SNpc). are Many known genetic as sporadicfactors have or idiopathicbeen identified Parkinson’s to cause diseasePD, but more than 1% of the population over the age of 60. Motor (iPD): cases with unknown causes. symptoms of PD include tremors, loss of balance, and trouble initiating movement. Non-motor symptoms associated with PD are depression, trouble sleeping, hyposmia, and constipation or among farmers and rural populations, as well as an increase in diarrhea [1,2]. The pathological hallmark of the disease is the the Epidemiologicalprevalence of cases studies since identified the rise ofan industrialization increase in PD [3-6].cases formation of protein aggregates, known as Lewy Bodies, and loss The increased risk of developing PD in farmers is thought to be Cite this article: Janezic EM, Caviness JE, Davis XD, Kanda SG, Kim YH (2016) Commercially Available Pesticides Cause Synergistic or Additive Damages in Dopaminergic Cells: Relevance for Parkinson’s Disease Pathology. Ann Neurodegener Dis 1(2): 1010. Kim et al. (2016) Email: Central Bringing Excellence in Open Access due to the exposure to high concentrations of pesticides [7,8]. Table 1: A list of pesticides tested in this study and their common uses. Additionally a study from Baldi et al., [9], reported a higher Chemical Name Common Use likelihood of PD amongst farmers in France if they had been exposed to high levels of pesticides. As further support, in a case Acephate Citrus trees and golf courses study in Germany, iPD was more prevalent in workers that had Alachlor Corn, soybean, peanuts, and weed control been exposed to pesticides and other environmental toxins [10]. Corn, grain, sugarcane, Christmas trees, and weed Atrazine Some iPD patients have an increased level of organochlorine control pesticides in their brain upon post mortem autopsy [11]. Diuron Fruit trees, cotton, oats, wheat, and home ponds Interestingly, the rates of PD have decreased in Guam after MCPA Cereals, trees, turf, and grassland reducing the amount of environmental toxins used in farming Mecoprop Wheat, oat, barley, turf, and drainage banks [12]. The pesticides we tested have been commercially available and used for various purposes in agricultural settings. can lead to PD symptoms. As an example, the pesticide, rotenone, Studies in animal models have identified that some pesticides causes PD symptoms in mice, and has been used as a common vitro experiments. The cells were cultured in RPMI 1640 medium with 100 units/mL penicillin/streptomycin, and 10% fetal bovine is a mitochondrial complex I inhibitor, damaging dopaminergic serum (Life Technologies, Carlsbad, CA). Cells were incubated in model of PD in flies and others [13]. More specifically, rotenone neurons [14,15]. Additionally, subcutaneously administered 5% CO at 37° C, as described [29]. rotenone leads to Lewy Body formation in a PD mouse model 2 [16]. Dieldrin, another pesticide, also leads to dopaminergic cell Pesticides loss in primary cultures [17]. We selected pesticides that are commercially used in farming The herbicide paraquat is another toxin that causes motor and weed control. The pesticides screened in this study include: dysfunction, induced by dopaminergic loss in the SNpc of acephate, alachlor, atrazine, diuron, MCPA, and mecoprop (Table mice [18]. Paraquat also causes cell death by inhibition of the 1). All chemicals and pesticides were purchased from Sigma- mitochondrial complex I, inducing mitochondrial dysfunction Aldrich (St. Louis, MO). For in vitro experiments, the pesticides were dissolved in dimethyl sulfoxide (DMSO), followed by mixing protein aggregation in mice after weekly exposures [20]. Maneb, ain fungicide,the striatum also [19]. causes In addition, slight motor it causes dysfunction α-synuclein-mediated in mice [21]. 0.1% in cell culture media. However, the combination of paraquat and maneb leads to in media, so that the final concentration of DMSO was less than synergistic damage, greater than the sum of the two pesticides Pesticide combination individually [22,23]. Since this discovery, paraquat and maneb Cells were cultured in 96-well plates as described previously. have been banned for use in the United States and European Union. Except for three positive control wells, one pesticide was added However, farmers still use other pesticides to protect their crops to every well at a concentration that did not cause damage, as (see Table 1 for the list of pesticides and their uses), even though shown from our initial screening. The second pesticide was these pesticides may cause detrimental side effects based on a added, in triplicate, to their designated wells at increasing number of reports. For example, long-term exposure to mecoprop concentrations. A yellow colored reagent, 3-(4,5-dimethylthiazol- leads to the development of cancer in the urinary tract in mice [24]. 2-yl)-2,5-diphenyltetrazolium bromide (MTT) was converted to Chronic exposure to low doses of multiple herbicides, including formazan, a purple product, via mitochondrial enzyme activity alachlor and diuron, causes changes in metabolites involved in for measuring viable cells with single pesticide exposure as oxidative stress in mice [25]. Additionally, long-term exposure described [22]. A total of fourteen combinations were screened to alachlor on its own modulates the activity of the oxidative for cell viability. stress regulating enzymes: superoxide dismutase, catalase, and glutathione S-transferase [26]. Damage in cholinergic neurons Cell Viability Assay Cells were plated in a 96-well plate (5,000 cells per well) and grown for 24 h. The media was then aspirated, and the pesticides Filipovhas been et identifiedal., [28] found from a wilddecrease squirrels in dopamine and birds levels after in entering striatal slicesa field after that applicationhad been sprayed of the herbicidewith acephate atrazine. [27]. In Interestingly, our current (< 0.1% DMSO in media) were added to each well in triplicates. study, we tested six commercially-used pesticides and showed Each plate contained three wells with vehicle (DMSO) as a positive that multiple combinations including mecoprop/acephate, control. Following another 24 h of incubation, an MTT assay was diuron/acephate, diuron/atrazine, and diuron/alachlor can lead performed to measure cell viability. The plate was emptied of to synergistic or additive cell death in N27 rat dopaminergic plate was incubated for 3 hours in the condition described above. media, and 20 μL of MTT reagent was added to each well, and the additive toxicity such as apoptosis from those toxic pesticide combinations.cells. Furthermore,