Abstract 1334: Benzo(a)pyrene concentration-dependent biotransformation and cytotoxicity in HT-29 colon cells

Abstract

Abstract The majority of sporadic cancer deaths are attributed to environmental factors of which exposure to environmental toxicants is one. Benzo(a)pyrene (BaP), a member of the Polycyclic Aromatic Hydrocarbons (PAH) family of compounds is one such ubiquitous environmental toxicant; found in charcoal broiled/smoked meats, cigarette smoke, automobile exhaust, and industrial emissions. High levels of BaP have been detected in fat-rich foods such as dairy products, barbecued pork sausage, and beef meat. Since diet makes up a substantial amount of BaP intake (∼ 3 – 17 mg/person/day); ingested BaP contributing to gastrointestinal (GI) carcinogenesis deserves attention. Studies conducted using a mouse model in our laboratory has already shown that BaP contributes to colon cancer development. Through metabolic activation, BaP is biotransformed into an array of metabolites. Biotransformation of BaP proceeds through specific biochemical pathways (epoxide and quinone) each one producing specific reactive metabolites that bind with DNA, cause DNA damage and associated changes in cell cycle. The objective of this study was to determine the concentrations of BaP that are most toxic to HT-29 human colon cells and examine if any relationship exist between cellular toxicity and BaP biotransformation. Cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% FBS and antibiotics (penicillin/streptomycin). After revival of cells and growing them to confluence, cells were synchronized overnight by serum starvation method. The HT-29 cells were exposed to 1, 5, 10, 25, 50, and 100μM BaP over the course of 10 days and then counted using a Coulter Counter. Based on a concentration – response study 5, 10, and 25 μM BaP in DMSO (vehicle for BaP; 0.01%) were selected as optimal concentrations to analyze the effect of BaP on cell growth, viability, biochemical, and molecular endpoints 4 days after BaP exposure. The expression of both Phase I and II drug metabolizing enzymes were analyzed by western blot and RT-PCR. Analysis of cell cycle via FACS showed inhibition of the S and G2-phase in cells treated with increasing concentrations of BaP. An increase in CYP 1A1 and 1B1 expression also occurred in a BaP concentration-dependent manner. However, not much difference was seen in Phase II metabolizing enzyme expression levels regardless of the BaP concentrations tested. Our results suggest that BaP cytotoxicity is mediated by metabolic activation (funded by the NIH grants 5T32HL007735-12, 1RO1CA142845-01A1, 1S11ES014156-01A1 and Southern Regional Education Board). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1334. doi:10.1158/1538-7445.AM2011-1334