Detection of DNA strand breaks in isolated mussel (Mytilus edulis L. ) digestive gland cells using the "Comet" assay.
Isolated mussel (Mytilus edulis L.) digestive gland cells were analyzed using the single-cell gel electrophoresis or "comet" assay to assess the ability of potential aquatic contaminants to induce DNA strand breaks (SBs) and to investigate the potential application of this technique as part of an aquatic biomonitoring regime. Freshly prepared cell suspensions from digestive gland were exposed in vitro to hydrogen peroxide (H2O2, 0-200 microM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX, 0-200 microM), benzo[a]pyrene (BaP, 0-200 microM), 1-nitropyrene (1-NP, 0-250 microM) and nitrofurantoin (NF, 0-1000 microM) for 1 h in the dark at 15 degreesC in the presence of the DNA repair inhibitor cytosine-beta-D-arabinofuranoside (araC). DNA strand breakage was measured using the comet assay. There were significant concentration-dependent increases in the percentage of DNA in the comet tail (mean values+/-SD) for all doses compared with controls (P<0.05) with H2O2 (up to 61.4+/-5.1% at 100 microM), MX (up to 34. 3+/-2.2% at 200 microM), BaP (up to 24.7+/-5.1 at 100 microM), 1-NP (up to 54.7+/-5.0% at 200 microM), and NF (up to 68.1+/-4.5% at 500 microM). There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 microM H2O2 and BaP only. This study has demonstrated the potential of the comet assay to detect DNA strand breakage at subcytotoxic concentrations of a range of agents, some of which require metabolic activation. This may provide a sensitive, but nonspecific, molecular biomarker of genotoxicity.
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
C L
CL
Mitchelmore
C
C
Birmelin
D R
DR
Livingstone
J K
JK
Chipman
D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D049872:Bivalvia / Q000378:metabolism*; D003561:Cytarabine; D004247:DNA / Q000032:analysis*; D004249:DNA Damage / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000032:analysis*; D004064:Digestive System / Q000737:chemistry / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D005663:Furans / Q000633:toxicity; D006861:Hydrogen Peroxide / Q000633:toxicity; D066298:In Vitro Techniques; D009152:Mutagenicity Tests; D011721:Pyrenes / Q000633:toxicity; D014874:Water Pollutants, Chemical / Q000633:toxicity
D004277:DNA, Single-Stranded; D005663:Furans; D011721:Pyrenes; D014874:Water Pollutants, Chemical; D003561:Cytarabine; D001564:Benzo(a)pyrene; C054121:3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone; D004247:DNA; D006861:Hydrogen Peroxide; C032668:1-nitropyrene