DETERMINATION OF SPATIAL PATTERNS OF DNA-DAMAGE AND REPAIR IN INTESTINAL CRYPTS BY MULTICELL GEL-ELECTROPHORESIS

We have developed a method to quantitate DNA strand breaks as a measur e of DNA damage and repair in intact, isolated intestinal crypts. The assay is a modified form of the single-cell gel electrophoresis or 'co met' assay. By maintaining the spatial relationship between the cells we were able to characterise the repair response and the susceptibilit y to DNA damage of cells as a function of their position in the crypt. All cells were equally repair competent over the first 30 minutes of the repair of UV-C and gamma-radiation induced lesions. DNA damage was equally distributed following gamma-radiation hut following incubatio n with the topoisomerase II inhibitor etoposide, damage was greater in the lower crypt with an unusual component to the comet tail which was tapered, implying an incremental change in susceptibility by cell pos ition, This tapered component of the co;net tail resolved rapidly afte r removal of etoposide. The pattern of damage produced by hydrogen per oxide was dose dependent with lower doses producing more strand breaks in the base of the crypt - an effect lost at higher doses, The assay has the ability to detect differences between cells in their susceptib ility to DNA damage and their subsequent repair response which may var y with their proliferative or differentiative status.