Detection of DNA Strand Breaks in Isolated Mussel (Mytilus edulisL.) Digestive Gland Cells Using the “Comet” Assay

Abstract

Isolated mussel (Mytilus edulisL.) 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 exposedin vitroto hydrogen peroxide (H2O2, 0–200 μM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX, 0–200 μM), benzo[a]pyrene (BaP, 0–200 μM), 1-nitropyrene (1-NP, 0–250 μM) and nitrofurantoin (NF, 0–1000 μM) for 1 h in the dark at 15°C in the presence of the DNA repair inhibitor cytosine-β-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 μM), MX (up to 34.3±2.2% at 200 μM), BaP (up to 24.7±5.1 at 100 μM), 1-NP (up to 54.7±5.0% at 200 μM), and NF (up to 68.1±4.5% at 500 μM). There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 μM H2O2and 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.