A microgel electrophoresis technique for the direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides.
We demonstrate by single-cell microgel electrophoresis that the 2 main techniques, trypsinization and scraping, used to collect normal diploid mammalian cells cultured in monolayer induce DNA damage. To minimize this potential interference with studies on DNA damage and repair, we have standardized the single-cell gel electrophoretic (SCG) technique for the in situ quantitation of DNA single-strand breaks and alkali-labile sites in cultured human-fibroblasts. To demonstrate the utility of this technique, human neonatal foreskin-derived fibroblasts were allowed to attach to frosted microscope slides and then either irradiated with X-rays (25-200 rad) or treated for 1 h with hydrogen peroxide (2.2-140.8 mumoles). Treatment with either agent induced a dose-dependent increase in DNA migration. At equal levels of DNA damage, cell-to-cell variability in DNA migration was more heterogeneous for hydrogen peroxide-treated cells than for X-irradiated cells. A time course study to evaluate the kinetics of DNA repair for X-ray (200 rad)-induced damage indicated that the damage was completely repaired within 2 h. Applications of this technique for in vitro toxicology are discussed.
Singh
N P
NP
Department of Biology, Eastern Washington University, Cheney 99004.
Tice
R R
RR
Stephens
R E
RE
Schneider
E L
EL
D002448:Cell Adhesion; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D004586:Electrophoresis / Q000379:methods*; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D013696:Temperature; D014965:X-Rays / Q000009:adverse effects
D006861:Hydrogen Peroxide