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The effects of copper on P-naphthoflavone (beta NF)-induced ethoxyresorufin O-deethylase (EROD) activity were studied in rainbow trout (Oncorhynchus mykiss) gill filaments (after in vivo exposure) and in gill cells cultured as both primary cultures and as polarised epithelia, i.e. with water in the apical compartment and culture medium in the basolateral compartment. In the in vivo study beta NF and copper were added to the water, in primary cultures both chemicals were added to the culture medium and in cultured epithelia copper was added to the apical water whilst beta NF was added to the basolateral culture medium. In primary cultures this investigation was repeated with and without foetal bovine serum (FBS) supplementation of the culture media. Gill barrier properties, specifically polyethylene glycol (PEG-4000) permeability (i.e. paracellular permeability), sodium efflux and transepithelial electrical resistance (TER) were also investigated in cultured gill cell epithelia after apical treatment with copper. Two micromolar copper had no effect on EROD activity in gill filaments in vivo irrespective of whether EROD was induced by 0.01, 0.1 or 1.0 mu M beta NF Similarly, 0.5-100 mu M copper had no effect on EROD induction in cultured epithelia. In primary cultures copper did reduce EROD induction but the effective concentration was dependent on whether the cells were supplemented with FBS, i.e. EROD activity was reduced by all copper concentrations of 5 and above if FBS was included, but only by 1000 mu M if FBS was omitted. In cultured epithelia PEG-4000 permeability increased, whilst sodium efflux and TER were unaffected following treatment with 75 mu M copper. Based on these results we conclude that the branchial monooxygenase system is a less sensitive target for copper than the barrier properties of the gill. Indeed, these data suggest the apical membrane of the gill epithelial cells minimises the uptake of waterborne copper and therefore protects the intracellular environment, including the CYP1A system. This could enable the freshwater fish gill to retain their potential of first-pass metabolism of waterborne organic compounds whilst simultaneously being exposed to waterborne copper.