Multiple Pesticide Exposure: Immunotoxicty and Oxidative StressEPA Grant Number: U915639
Title: Multiple Pesticide Exposure: Immunotoxicty and Oxidative Stress
Investigators: Rabideau, Christine L.
Institution: Virginia Polytechnic Institute and State University
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 1999 through August 1, 2001
Project Amount: $58,756
RFA: STAR Graduate Fellowships (1999) RFA Text | Recipients Lists
Research Category: Fellowship - Toxicology , Academic Fellowships , Health Effects
The objective of this research project is to examine the relationship of pesticide mixtures and immunotoxic risk in vitro, as it pertains to the role of free radicals. Humans and other animals are exposed to a myriad of chemicals, either concurrently or sequentially, throughout their lifetimes. Often, the metabolism of these chemicals results in the formation of unstable, free radical intermediates or reactive oxygen species (ROS). Studies have indicated that these ROS play a role in chemical metabolism and immune-cell response and regulation. The hypothesis is that ROS and/or other free radical intermediates, generated during the metabolism of various pesticides, mediate the immunotoxic effects of environmental toxicants in mixture.
Our approach is first to determine the extent of immune response by C57BL/6 young adult, male mice splenocytes, in vitro, to three different pesticides and mixtures of those pesticides. To do this, splenocytes are isolated and treated with piperonyl butoxide (PBO), lindane, or malathion to obtain time- and dose-response curves. Two assays have been chosen to assess the concentration of each pesticide that yields approximately 25 percent immunotoxicity. The alamarBlue Cytotoxicity Assay uses the fluorogenic redox indicator dye, alamarBlue, to monitor metabolically active cells. The In Situ Cell Death Assay labels nicks in the DNA of cells undergoing cell death, and is analyzed via flow cytometry. After identifying the pesticide concentration for 25 percent cytotoxicity, splenocytes can be treated with mixtures of two pesticides (PBO and malathion, lindane and malathion, and each pesticide alone at one concentration).
Secondly, we will examine the role of ROS in immunotoxicity, by assessing the levels of oxidative enzymes in splenocytes treated with pesticides and mixtures of pesticides. Often, when cells or organisms are exposed to chemicals, ROS are generated, and enzyme induction can offer cellular protection against these ROS. Thus, oxidative enzyme levels can be used as indicators of the oxidative status of cells. The enzymes of interest are superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Quantitation of each enzyme in the cytosolic fraction of the cells, treated with various pesticides, will be monitored using colorimetric techniques.