The Use of Cultured Primary Bovine Colon Epithelial Cells as a Screening Model to Detect Genotoxic Effects of Heterocyclic Aromatic Amines in the Comet Assay

Abstract

Isolated epithelial cells from the bovine colon were maintained in dividing monolayer cultures and used as a model system for colon tissue in in vitro toxicological studies. The cytotoxic effects of the heterocyclic aromatic amines 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhiP) were investigated in these cells and IC₅₀ values were determined by inhibition of neutral red uptake into the cultured cells. Although PhiP was not cytotoxic up to concentrations of 500 μM, IQ was cytotoxic above 300 μM. The induction of DNA strand breaks in cultured bovine epithelial colon cells was determined using the alkaline single-cell gel electrophoresis (Comet assay) technique, and subsequently, the DNA damage was used as a determinant of genotoxic effects of the heterocyclic aromatic amines in order to establish this system for detection of adverse effects of chemicals in a model system for the colon. In the absence of an external enzymatic metabolizing system (S9 mix) both amines did not induce DNA strand breaks. When S9 mix was used, PhiP induced DNA strand breaks above 10 μM whereas IQ did not show any significant effect at 300 μM. This cell culture system was found to be a useful screening system for testing of compounds that are considered to affect colonic tissue.

The authors gratefully acknowledge the excellent technical assistance of B. Tibbe, M. Koch, A. Dommermuth, and C. Pütt. This work was supported by grant 0311266 from the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie.