Abstract 2118: Particulate chromate induces persistent DNA double strand breaks resulting in disruption of homologous recombination repair in human lung cells

Abstract

Abstract Hexavalent chromium (VI) is a well-documented respiratory toxicant and carcinogen. The most carcinogenic form is the particulates, which produce numerous types of DNA damage including DNA double strand breaks. The aim of this study is to determine the genotoxicity of particulate chromate and investigate cellular responses of repair proteins after chronic exposure. We used zinc chromate as a representative hexavalent chromium compound because it has been shown to be carcinogenic and induce tumors in epidemiology studies and experimental animals. We investigated the DNA double strand break formation using both comet assay and immunofluorescence staining. We exposed cells to zinc chromate for 24, 48, 72 96 and 120 hours and found it induced concentration-dependent increases in DNA double strand breaks by both measures. We further investigated the kinetics of repair protein after chronic zinc chromate exposure. We developed human lung cells expressing green fluorescent protein-tagged 53BP1 (GFP-53BP1) and generated time-lapse videos of chromate treated cells. We observed residual focal formation of 53BP1 after chronic zinc chromate exposure, which indicate some DNA double strand breaks persist. There are two repair pathways that can be utilized to repair DNA double strand breaks. Non-homologous end joining (NHEJ) is active throughout the cell cycle while homologous recombination (HR) is active in late S and G2 phases. We first investigated the sub-cellular distribution of HR proteins including MRE11, phophorylated ATM and Rad51. We found that the nuclear fraction of HR repair proteins increased in 24 hours indicating HR is active. However, it largely decreased over time. We also observed concentration- and time-dependent decrease in Rad51 foci formation, which strongly suggest that HR is inhibited. Future work will focus on elucidating NHEJ repair pathway and identifying key proteins involved in transitions between two repair pathways. This work was supported by NIEHS grant ES016893 (J.P.W.), cooperative agreement #EP-08-01 through the Maine Space Grant Consortium (J.P.W.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2118. doi:1538-7445.AM2012-2118