Vanessa A. Fitsanakis, Ph.D. - US grants

DESCRIPTION (provided by applicant): In accordance with the mission of the NIEHS, understanding how environment influences human disease etiology and progression, this project will examine the contribution of two high-usage pesticides to general neurotoxicity as well as Parkinson's disease (PD). Numerous reports suggest a link between PD and agricultural environments and/or pesticide usage. Other data indicate that exposure to manganese (Mn) also may be linked to disease onset. Interestingly, many agrochemicals, as well as Mn, impair mitochondrial respiration, resulting in an insufficient quantity of energy production required for cells survival. Furthermore, mitochondrial dysfunction is present in various cell populations in PD patients. The long-term goal of this project is to understand the mechanisms through which pesticides may contribute to cell death in brain regions associate with to PD. Using the nematode Caenorhabditis elegans (C. elegans), the following hypothesis will be tested: Exposure to the popular pesticides Mancozeb (MZ) and TouchDown (TD) leads to neuronal degeneration via mitochondrial inhibition and/or increased oxidative stress. Additionally, it is hypothesized that these pesticides may enter vulnerable neuron through uptake by specific neurotransmitter transporters. These hypotheses will be tested through three specific aims: (1) to establish which mitochondrial complexes are inhibited; (2) to discover the specific reactive oxygen species (ROS) increased following exposure to MZ and/or TD; and (3) to determine if MZ and TD enter neurons through individual neurotransmitter or metal transporters. In order to monitor neurodegeneration, C. elegans with neuronal populations tagged with green fluorescent protein (GFP) will be exposed to varying concentrations of MZ or TD. This will be followed by treatment with various chemical reporters to determine whether mitochondrial function is compromised. Subsequent studies will involve pretreatment of worms with specific neurotransmitter transporter antagonists to determine if assess whether degeneration is decreased following subsequent exposure to MZ or TD. Results from these novel studies will further our understanding of neurotoxic effects related to pesticide exposure. Additionally, this work will provide evidence related to mechanisms involved in intracellular transport of glyphosate-containing herbicides (i.e., TD, RoundUP), or manganese-containing fungicides (i.e., MZ, maneb). It is anticipated that these data will further our understanding not only of general pesticide neurotoxicity, but also of mechanisms through which environmental toxicants may contribute the etiology of idiopathic PD. PUBLIC HEALTH RELEVANCE: These studies are particularly timely and relevant to public health because of the link between Parkinson's disease (PD), the second most-common neurodegenerative disease in the United States, and pesticide exposure. Use of glyphosate-containing herbicides (i.e., RoundUp and TouchDown) is increasing exponentially in the general public and the agricultural community, while Mancozeb, a manganese-containing fungicide, is gaining increased market shares. These pesticides are suspected mitochondrial inhibitors, an observation commonly documented in various cell populations in PD patients.

Project Summary/Abstract In conjunction with the Mission Statement of the National Institute of Environmental Health Sciences (NIEHS), the long-term goal of this research is to determine whether toxicity associated with the widely-used class of fungicides containing Mn/Zn-ethylene-bis-dithiocarbamate (Mn/Zn-EBDC) is exacerbated in people with mutations associated with genes responsible for cellular antioxidant defense systems and mitochondrial homeostasis. Increased amounts of reactive oxygen species (ROS) and mitochondrial inhibition are implicated in many neurodegenerative diseases. Additionally, epidemiology studies indicate that occupational pesticide exposure increases a person?s risk for many diseases associated with the brain, although exposure alone is often insufficient to cause the disease phenotype. On the other hand, specific gene mutations play a role in the etiology of about 20% of the neurodegenerative cases (Parkinson?s disease, Alzheimer?s disease, etc.). Thus, it is likely that the vast majority of patients suffering from these devastating diseases had a genetic background that resulted in a greater vulnerability to a toxic insult (gene-environment interaction). Therefore, the overarching hypothesis of this application is that multiple, low-dose exposures to Mn/Zn-EBDC-containing fungicides, such as mancozeb, may lead to increased neurodegeneration in populations with mutations in LRRK2 (an antioxidant defense system gene), parkin, or PIKN1 (both of which are associated with mitochondrial mitophagy or homeostasis). In order to address our hypothesis, two specific aims will guide the research. The first aim will determine if multiple exposures of Caenorhabditis elegans (C. elegans) to various pesticide concentrations used for fungal control leads to neurodegeneration similar to that observed in single exposure studies published by this lab. The second aim will assess whether exposure to a Mn/Zn-EBDC fungicide leads to greater toxicity in organisms with modified genetic backgrounds. These studies will be completed by treating various strains of C. elegans with environmentally-relevant concentrations of mancozeb. The goal is to characterize the potential increases in lethality, neuronal degeneration, oxidative stress, and mitophagy. These studies are timely and highly relevant to the goals of the NIEHS because they address the potential neurotoxicity of high-use pesticides to which members of the public are routinely exposed (Strategic Plan Theme 2), the interaction of agrochemicals with genes or proteins known to be involved in neurodegneration (Strategic Plan Themes 2 & 3), and to provide education and training to the next generation of scientists and science professionals (Strategic Plan Theme 5).