Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg).
Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.
Gielazyn
Michel L
ML
Marine Science Program, University of South Carolina, Columbia, SC 29208, USA. michel.gielazyn@noaa.gov
Ringwood
Amy H
AH
Piegorsch
Walter W
WW
Stancyk
Stephen E
SE
D000818:Animals; D049872:Bivalvia / Q000187:drug effects; D020552:Comet Assay; D004249:DNA Damage; D043382:DNA-Formamidopyrimidine Glycosylase / Q000494:pharmacology*; D006434:Hemocytes / Q000187:drug effects* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D066298:In Vitro Techniques; D008974:Mollusca / Q000187:drug effects*; D010125:Ostreidae / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*
D006861:Hydrogen Peroxide; D043382:DNA-Formamidopyrimidine Glycosylase