Pilot Projects

Project Title: Dissecting the Effects of Chlorpyrifos and Cyfluthrin on CNS-Critical Genes in Human Primary Astrocytes

Principal Investigator: Li Zhang, Ph.D. (CEHNM)

Year: 2005
Award Amount: $25,000

Summary. The goal of this project is to explore the molecular neurotoxic actions of two commonly used pesticides, the organophosphate chlorpyrifos and the type II pyrethroid cyfluthrin. These actions will be investigated in humans using one major type of brain cells—astrocytes. Recent studies by the CCCEH indicated that exposure to chlorpyrifos in fetuses and newborns is associated with adverse neurodevelopmental sequelae. By using microarray gene expression profiling, a group of genes that are known to play important roles in central nervous system (CNS) function were identified. In addition, a group of genes encoding the upstream effectors and downstream targets of the insulin signaling pathway were found to be altered by chlorpyrifos and cyfluthrin, suggesting that insulin signaling pathways may be affected by pesticides. This project will study the effects of cyfluthrin and chlorpyrifos on the expression of CNS-critical genes identified previously by microarray in human fetal astrocytes. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) will be employed to confirm the findings of microarray analysis with regard to the expression levels of identified genes important for CNS function. Western blot analysis will be implemented to assess protein levels of these specific genes and to determine whether the pesticides affect the insulin signaling pathways; it will also examine the dose and time-course responses of selected genes and insulin signaling components to chlorpyrifos and cyfluthrin. Doses relevant to human fetal exposures will be used. Transcript and protein levels of CNS-critical genes and components of the insulin signaling pathway by quantitative real-time RT-PCR and Western blot analysis will be determined.

Results. The Pilot Project was completed as proposed. Real-time quantitative RT-PCR and Western blotting analyses confirmed the major results from microarray gene expression analysis and toxicological analysis were performed to confirm the results. Data using genomic, toxicological and biochemical methods all suggest that cyfluthrin causes more severe effects on human astrocytes than chlorpyrifos in diverse molecular and cellular functions ranging from gene expression to glutamate uptake. In addition, the results show that cyfluthrin and chlorpyrifos up-regulate certain targets of the interferon and insulin signaling pathways, and that they increase the protein levels of activated ERK1/2, a key component of insulin signaling; IL-6, a key inflammatory mediator; and GFAP, a marker of inflammatory astrocyte activation. These results suggest that inflammatory activation of astrocytes may be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.

Outcomes/Publications.

Mense SM, Sengupta A, Lan C, Zhou M, Bentsman G, Volsky DJ, Whyatt RM, Perera FP, Zhang L. 2006. The Common Insecticides Cyfluthrin and Chlorpyrifos Alter the Expression of a Subset of Genes with Diverse Functions in Primary Human Astrocytes. Toxicological Sciences 93:125-35.

Sengupta A, Mense SM, Lan C, Zhou M, Mauro RE, Kellerman L, Bentsman G, Volsky DJ, Louis ED, Graziano JH, Zhang L. Gene expression profiling of human primary astrocytes exposed to manganese chloride indicates selective effects on several functions of the cells. Neurotoxicology, in press, 2007.

Grants Submitted:

Submitted one NIH R21 application, 1R21ES015583-01, "A genomic approach to explore the neurotoxicity of common pesticides," which was not funded, but may be resubmitted after revision.