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Effects of chlorpyrifos and TCP on human kidney cells using toxicity testing and proteomics
Citation:
VanEmon, J., D. Ash, F. van Breukelen, P. Pan, R. Johnson, AND J. Barr. Effects of chlorpyrifos and TCP on human kidney cells using toxicity testing and proteomics. ACS Fall 2015 Meeting, Boston, MA, August 16 - 21, 2015.
Impact/Purpose:
The National Exposure Research Laboratory (NERL) Human Exposure and Atmospheric Sciences Division (HEASD) conducts research in support of EPA mission to protect human health and the environment. HEASD research program supports Goal 1 (Clean Air) and Goal 4 (Healthy People) of EPA strategic plan. More specifically, our division conducts research to characterize the movement of pollutants from the source to contact with humans. Our multidisciplinary research program produces Methods, Measurements, and Models to identify relationships between and characterize processes that link source emissions, environmental concentrations, human exposures, and target-tissue dose. The impact of these tools is improved regulatory programs and policies for EPA.
Description:
An Adverse Outcome Pathway (AOP) is a conceptual framework to apply molecular pathway-based data for use in risk assessment and regulatory decision support. The development of AOPs requires data on the effects of chemicals on biological processes (i.e., molecular initiating events, key intermediate toxicity events, and proteomic changes) to link exposure to effects. Here, we describe the application of morphological, biochemical and proteomics analysis on human cell cultures to support AOP development. HEK293 (ATCC, CRL-1573) cells were cultured in DMEM supplemented with FBS and exposed to chlorpyrifos at 10, 30, 60, and 90 µg/mL, or 3,5,6-trichloro-2-pyridinol (TCP) at 50, 100, 150, and 200 µg/mL. After 24 h of exposure, the cells were removed from the culture plates, stained with trypan blue, counted and tested for cytotoxicity. Flow cytometry on cells stained with Annexin V and propidium iodide was also performed to determine apoptosis. Overall, the cytotoxic effect was in a dose and time dependent manner with the effect becoming more obvious with an increase in time and concentration. Using a proteomic approach, over 1000 peptides were identified and assigned to 372 proteins using high-resolution mass spectrometry. Data evaluation uncovered groups of proteins that consistently appeared to be up-regulated or down-regulated in response to the toxicity of the compounds used. We found that a group of 25 proteins was detected after the cells were exposed to either chlorpyrifos or TCP, including cDNA FLJ54290, ADP-ribosylation factor 3 and Serpinei mRNA binding proteins. A total of 19 proteins were detected after exposure to TCP alone whereas three proteins were detected after exposure to just chlorpyrifos. The addition of proteomic analysis to the study of exposure to TCP on human embryonic kidney cells provided a distinct correlation among cytotoxicity, apoptosis, cytokine expression, and the recovery of damaged cells leading to the discovery of putative biomarkers of exposure that still need to be confirmed and validated.
