A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

• Published in: DNA repair Vol. 4; no. 10; pp. 1129 - 1139
• Main Authors: GRADZKA, Iwona, IWANENKO, Teresa
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 28-09-2005; Elsevier
• Subjects: Animals; Bacteriology; Biological and medical sciences; Cells, Cultured; Comet Assay; DNA - analysis; DNA - drug effects; DNA - radiation effects; DNA Damage; DNA double-strand break induction and rejoining; Electrophoresis, Gel, Pulsed-Field - methods; Fundamental and applied biological sciences. Psychology; Gamma Rays; Growth, nutrition, cell differenciation; Humans; Hydrogen Peroxide - toxicity; Mammalian cells; Mice; Microbiology; Molecular and cellular biology; Molecular genetics; Mutagenesis. Repair; Neutral comet assay; Organic Chemicals - chemistry; Pulse-field gel electrophoresis; SYBR-Gold; X-irradiation; X-irradiation; SYBR-Gold; Pulse-field gel electrophoresis; Neutral comet assay; DNA double-strand break induction and rejoining; Mammalian cells; Gold; Gel electrophoresis; Induction; Mutagenicity testing; Comet assay; Double strand break; Vertebrata; Mammalia; DNA; Irradiation; Repair
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2005.06.001

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0–25
Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA
fragm) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600
Gy of γ-rays. The limit of DSB detection was 0.25
Gy for human G1-lymphocytes and 0.5–1
Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H
2O
2, an inducer of single-strand DNA breaks (SSBs). On the contrary, the H
2O
2 inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5
Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10
Gy, generally proceeded faster than that measured previously after higher (30–50
Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.