In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric ca ncer, but studies of DNA damage in gastric epithelial cells are limited. Th e comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determin ed optimal conditions fur biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endosc opy were digested using combinations of pronase and collagenase, ethylenedi aminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell susp ension was assessed for cell concentration and epithelial cell and leukocyt e content. A score for DNA damage, the comet %, was derived from the cell s uspension, and the effect of various digestion conditions was studied. Cell s were incubated with H2O2 and DNA damage was assessed. Pronase and collage nase provided optimum digestion conditions, releasing 1.12 X 10(5) cells pe r biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had h igh inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migr ation. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H2O2 (p = 0.005). Serial 25-min digestions we re performed on biopsies from 13 patients to release cells from successivel y deeper levels in the crypt, Levels of DNA migration were significantly lo wer with each digestion (r = - 0.94, p < 0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The come t assay is a reproducible measure of DNA damage in gastric epithelial cells . Damage accumulates in older, more superficial cells, and can be induced b y oxidative stress. (C) 2000 Elsevier Science B.V. All rights reserved.