Evidence for cytochrome P-450 catalysis and free radical involvement in the production of DNA strand breaks by benzo[a]pyrene and nitroaromatics in mussel ( Mytilus edulis L.) digestive gland cells

Abstract

The mechanisms involved in the production of DNA strand breaks (SB) by model polycyclic aromatic hydrocarbon and nitroaromatic contaminants were investigated in isolated mussel ( Mytilus edulis L.) digestive gland cell mixtures using the model compounds benzo[a]pyrene (BaP), 1-nitropyrene (1-NP) and nitrofurantoin (NF). Isolated cells were exposed in vitro to sub-cytotoxic concentrations (50 μM) of BaP, 1-NP or NF for 1 h in the dark at 15°C in the absence or presence of various cytochrome P-450 inhibitors, antioxidant enzyme inhibitors, the free radical scavenger N- N- t-butyl- α-phenylnitrone (PBN), and other modulators. DNA strand breakage was measured using the comet assay (SB results presented as % tail DNA and was significant for each genotoxicant at least P<0.05). SB were seen for all three compounds and different metabolic pathways of genotoxicity were indicated for the three model compounds. BaP-induced strand breakage was indicated to be cytochrome P-450-catalysed and to occur via the production of BaP quinones because SB were inhibited 94% by 50 μM clotrimazole (inhibitor of digestive gland microsomal metabolism of BaP to quinones), stimulated 81% by 25 μM dicoumarol (inhibitor of DT-diaphorase, EC 1.6.99.2, which metabolises quinones to hydroquinones) and unaffected by 50 μM α-naphthoflavone (inhibitor of digestive gland microsomal metabolism of BaP to phenols and diols). Involvement of free radical(s) was indicated in BaP-induced strand breakage (75% SB inhibition by 50 mM PBN), consistent with either BaP cation radical formation (i.e. 1-electron oxidation) and/or reactive oxygen species (ROS) generation via BaP quinone formation and redox cycling. 1-NP-induced SB was indicated to occur via free radical mechanism(s) (84% SB inhibition by 50 mM PBN) and catalysis by different forms of cytochrome P-450 than for BaP (61% SB inhibition by 50 μM α-naphthoflavone but none by clotrimazole). In contrast to BaP and 1-NP, NF induced strand breakage was indicated not to involve cytochrome P-450(s) (no SB inhibition by clotrimazole or α-naphthoflavone), but to involve free radical(s) (88% SB inhibition by 50 mM PBN), consistent with redox cycling of NF and resultant DNA damage via superoxide anion radical (O 2 ·−) and other reactive oxygen species production. NF was more effective in producing SB compared to equimolar concentrations of BaP and 1-NP, possibly reflecting the greater direct redox cycling capacity of this compound.