title: In vivo DNA damage in gastric epithelial cells.
abstract: A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The 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, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high 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 migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower 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 comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.
authors.affiliation: The Centre for Digestive Diseases, The General Infirmary at Leeds, UK. medsmev@stjames.leeds.ac.uk
authors.forename: S M
authors.initials: SM
authors.lastname: Everett
authors.forename: K L
authors.initials: KL
authors.lastname: White
authors.forename: C J
authors.initials: CJ
authors.lastname: Schorah
authors.forename: R J
authors.initials: RJ
authors.lastname: Calvert
authors.forename: C
authors.initials: C
authors.lastname: Skinner
authors.forename: D
authors.initials: D
authors.lastname: Miller
authors.forename: A T
authors.initials: AT
authors.lastname: Axon
mesh_terms: D001706:Biopsy; D002452:Cell Count; D020552:Comet Assay; D004249:DNA Damage; D004847:Epithelial Cells / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism* / Q000473:pathology; D016481:Helicobacter Infections; D006801:Humans; D007958:Leukocyte Count; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D011402:Pronase; D015203:Reproducibility of Results; D013223:Statistics as Topic; D013272:Stomach Diseases / Q000235:genetics / Q000382:microbiology / Q000473:pathology; D014021:Tissue Preservation
chemical_list: D011402:Pronase