Measurement of DNA strand breakage and DNA repair induced with hydrogen peroxide using single cell gel electrophoresis, alkaline DNA unwinding and alkaline elution of DNA.
Three techniques: single cell gel electrophoresis (SCGE), alkaline elution of DNA (AE), and alkaline DNA unwinding (ADU) were chosen to compare the sensitivity among these methods in detection of DNA damage and repair in human diploid VH10 cell line after short-term exposure to hydrogen peroxide. Using SCGE technique a dose-dependent increase in DNA migration was found in cells exposed to hydrogen peroxide in concentration range from 10 micromol/l to 100 micromol/l. Alkaline DNA unwinding method detected increased level of single strand breaks (ssb) in concentration range from 25 micromol/l to 100 micromol/l of H2O2, and alkaline elution of DNA estimated increased DNA elution rate from concentration 50 micromol/l of H2O2. In a time course study to evaluate the kinetics of DNA repair, both SCGE and ADU techniques showed that the repair of DNA strand breaks is very rapid; the level of ssb in treated cells has returned to near the background level within two hours. After this time damage remaining in the DNA was in the form of oxidised bases as revealed the incubation of treated cells with specific DNA repair endonuclease, formamidopyrimidine-DNA glycosylase.
Gábelová
A
A
Cancer Research Institute, Bratislava, Slovakia.
Slamenová
D
D
Ruzeková
L
L
Farkasová
T
T
Horváthová
E
E
D002648:Child; D002845:Chromatography; D004249:DNA Damage; D004260:DNA Repair; D004586:Electrophoresis; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008297:Male
D006861:Hydrogen Peroxide