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

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 gla nd cell mixtures using the model compounds benzo[a]pyrene (BaP), 1-nit ropyrene (1-NP) and nitrofurantoin (NF). Isolated cells were exposed i n vitro to sub-cytotoxic concentrations (50 mu M) of BaP, 1-NP or NF f or 1 h in the dark at 15 degrees C in the absence or presence of vario us cytochrome P-450 inhibitors, antioxidant enzyme inhibitors, the Fre e radical scavenger N-N-t-butyl-alpha-phenylnitrone (PBN), and other m odulators. DNA strand breakage was measured using the comet assay (SB results presented as % tail DNA and was significant for each genotoxic ant at least P < 0.05). SB were seen for all three compounds and diffe rent metabolic pathways of genotoxicity were indicated for the three m odel compounds. BaP-induced strand breakage was indicated to be cytoch rome P-450-catalysed and to occur via the production of BaP quinones b ecause SB were inhibited 94% by 50 mu M clotrimazole (inhibitor of dig estive gland microsomal metabolism of BaP to quinones), stimulated 81% by 25 mu M dicoumarol (inhibitor of DT-diaphorase, EC 1.6.99.2, which metabolises quinones to hydroquinones) and unaffected by 50 mu M alph a-naphthoflavone (inhibitor of digestive gland microsomal metabolism o f BaP to phenols and diols). Involvement of free radical(s) was indica ted in BaP-induced strand breakage (75% SB inhibition by 50 mM PBN), c onsistent with either BaP cation radical formation (i.e. 1-electron ox idation) and/or reactive oxygen species (ROS) generation via BaP quino ne 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 ca talysis by different forms of cytochrome P-450 than for BaP (61% SB in hibition by 50 mu M alpha-naphthoflavone but none by clotrimazole). In contrast to BaP and 1-NP. NF induced strand breakage was indicated no t to involve cytochrome P-450(s) (no SB inhibition by clotrimazole or alpha-naphthoflavone), but to involve free radical(s) (88% SB inhibiti on by 50 mM PBN). consistent with redox cycling of NF and resultant DN A damage via superoxide anion radical (O-2(.-)) and other reactive oxy gen 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. (C) 1998 Elsev ier Science B.V. All rights reserved.