Detection of DNA strand breaks in brown trout ( Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay

Abstract

The single cell gel electrophoresis or `comet' assay was employed to detect DNA strand breaks (SB) induced in isolated brown trout hepatocytes and blood cells incubated in vitro with various sub-cytotoxic concentrations of a range of genotoxic compounds and potential aquatic contaminants. Direct acting agents studied were hydrogen peroxide (H 2O 2; 0–200 μM), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG; 0–50 μM) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX; 0–200 μM). Benzo(a)pyrene (BaP; 0–200 μM), 1-nitropyrene (1-NP; 0–200 μM) and nitrofurantoin (NF; 0–500 μM) were also used to represent polycyclic and nitroaromatic contaminants requiring metabolic activation to exert their effects. The direct damaging agents produced a statistically significant ( P at least <0.05) concentration-dependent increase in the percentage of DNA in the comet tail in both cell types, indicative of increased formation of SB. The percentage of DNA in the tail in both hepatocytes and blood cells respectively (mean values±S.E.M.) was 27.8±4.2 and 30.9±7.8 (200 μM H 2O 2); 60.2±5.6 and 61.3±5.9 (50 μM MNNG); 32.4±2.7 and 28.6±5.1 (200 μM MX). With BaP a statistically significant ( P at least <0.05) concentration-dependent increase was seen at 50 μM and above for hepatocytes but not in blood cells (10.5±2.2 (100 μM BaP)), highlighting the requirement for metabolic activation of this compound. 1-NP and NF also produced concentration-dependent increases in SB in hepatocytes (17.1±4.4 (100 μM 1-NP) and 38.9±7.3 (500 μM NF) with no significant effect in blood cells. All values were compared to control hepatocyte and blood cells (overall mean values 3.6±0.1 and 2.4±0.1 respectively). This study has demonstrated the potential of the comet assay to detect DNA strand breakage in fish cells induced by a range of agents. The technique may provide a sensitive, non-specific end-point of genotoxicity as part of a biomonitoring regime.