Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).
The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.
Sastre
M P
MP
Department of Biology, University of Puerto Rico-Humacao, Humacao, Puerto Rico 00791, USA.
Vernet
M
M
Steinert
S
S
D020552:Comet Assay / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D056890:Eukaryota / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects*; D006861:Hydrogen Peroxide / Q000633:toxicity; D018462:Photobiology; D014466:Ultraviolet Rays / Q000009:adverse effects
D004247:DNA; D006861:Hydrogen Peroxide