Abstract 4801: Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner.

Abstract

Abstract Subchronic dosing of BHT in mice is a well-known model used to discretely study the tumor promotion stage of lung adenocarcinoma. Using this model, we have previously shown that functional toll-like receptor 4 (TLR4) is protective against inflammatory sequelae and primary tumor formation in the mouse lung. Gap junctional intercellular communication (GJIC) has been shown to regulate the signal transduction pathways of cells, and inhibition of this communication by toxicant exposure has been linked to dysregulation and eventual carcinogenic endpoints. As part of our research on the protective nature of TLR4 during tumor promotion, we utilized the BHT subchronic model (4 weekly i.p. doses of BHT) to examine the early effects on BALF in both BALB/c (BALB; Tlr4 sufficient) and C.C3H-Tlr4Lps-d mice (BALBLps-d-d; Tlr4 mutant). Analysis of the BALF 24 hours after the final dose for protein content and cytology was indicative of an inflammatory profile in all mice treated with BHT. Magnitude of response to BHT was highest in the BALBLps-d mice; the BALB appeared resistant. In an effort to further characterize the response of the pulmonary epithelium, the BALF samples were used to treat cultured C10 cells, a murine immortalized line derived from BALB/c alveolar type II cells, which are a precursor cell type for adenocarcinoma. Using the scrape-load dye transfer assay, a well-defined method to measure GJIC, we demonstrated marked inhibition of cellular communication after a 4 hour exposure to cell-free BALF that is both treatment and strain dependent. The direction and magnitude of effect is in parallel with that of the direct analysis of the BALF, i.e. we observed more inhibition in BALF from BALBLps-d compared to BALB mice. In addition, quantitative PCR (QPCR) analysis of C10 cells exposed to cell-free BALF for 24 hours indicates alterations in the transcription patterns in genes of innate immunity that are also treatment and strain dependent. These novel findings suggest endocrine signaling from the inflammatory milieu are complicit in the inhibition of GJIC during the promotion phase of carcinogenesis. Furthermore, these data establish a link between active gap junctions, tumor promotion, and the protective nature of TLR4 in an ex vivo model which will facilitate a mechanistic examination of these protective effects at the molecular level. Funded by ACS RSG-10-162-01-LIB (AKB). Citation Format: Thomas Hill, Carla-Maria Alexander, Alison K. Bauer. Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2013-4801