Abstract 224: Effect of Agent Orange and Agent Blue derived carcinogens on bladder cancer cell lines and a murine bladder cancer model
Authors: Rajendron Ganeshkumar , Abbott Erika , Thompson Jeffery , Patel Shachi , Barchowsky Aaron , Dennis Katie , Woolbright Benjamin L. , Taylor John A. 
Published: June 2022

Abstract
Agent Blue and Agent Orange were defoliants used in high quantities during Vietnam era conflicts in Southeast Asia. The US Department of Veterans Affairs has recently recognized that exposure to both Agent Blue and Agent Orange can potentially increase risk of bladder cancer (BCa) and has added BCa to the list of diseases for which Veterans can seek military benefits. Agent Blue contained significant quantities of organic arsenic and although arsenic is a recognized carcinogen, the understanding of how arsenic affects BCa is still unclear in the literature. Moreover, the effects of the Agent Orange contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on BCa remains largely unstudied, especially in populations that were exposed during this period. We hypothesized chronic exposure to carcinogens TCDD or organic arsenic in the form of cacodylic acid (CA) would enhance cancer associated phenotypes. We treated benign urothelial cells (UROtsa and SV-HUC-1 cells) with low doses of cacodylic acid (0.05-5µM) or TCDD (0.03 - 3nM) for up to 6 months. While we found minor differences in proliferation rates there was no consistent trend across samples in either cell line nor was cellular migration affected. Because benign urothelial cells represent a transformed cell line and not true urothelial cells, we next wanted to assess how CA affected BCa formation in mice using the validated N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced BCa model. TCDD was omitted from this step as its carcinogenicity in mice is understood to dramatically exceed human levels and thus is not relevant to human data. To approximate early-life exposure akin to what Vietnam Era Veterans may have experienced, we treated C57Bl/6J mice with BBN for 16 weeks or 20 weeks with or without increasing concentrations of cacodylic acid (5, 10, 50ppm) for 4 weeks during BBN treatment, starting at week 4 and ending at week 8, and evaluated outcomes associated with BCa formation and progression. While previous reports have found CA promotes BBN induced BCa, we found co-treatment with CA had no effect at 16 weeks post BBN, but reduced both bladder weights (a surrogate for tumor volume) and tumor stage after 20 weeks. RNA-sequencing was used to determine how CA affected the transcriptome of BBN induced tumors. Differential gene expression and Reactome analysis indicated the predominant changes were associated with alterations in RNA processing and translation, EGFR signaling and more. Our work thus far indicates that the ability of TCDD/CA exposure to promote BCa formation is likely context dependent. Future studies will enhance our understanding of how CA interacts with BBN acutely, and attempt to define mechanisms of how arsenic may protect against BBN induced BCa in experimental models, and whether this is clinically relevant for Vietnam Era Veterans.
Citation Format: Ganeshkumar Rajendron, Erika Abbott, Jeffery Thompson, Shachi Patel, Aaron Barchowsky, Katie Dennis, Benjamin L. Woolbright, John A. Taylor. Effect of Agent Orange and Agent Blue derived carcinogens on bladder cancer cell lines and a murine bladder cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 224.