Effects of heavy metals and 3-methylcholanthrene on expression and induction of CYP1A1 and metallothionein levels in trout (Oncorhynchus mykiss) hepatocyte cultures

Induction of both CYP1A1 and metallothioneins (MTs) in fish liver is increasingly being used in ecotoxicological studies. The interaction of heavy metals (Cd, Cu, Zn, Pb) with the CYP1A induction response and MT levels was studied in trout (Oncorhynchus mykiss) hepatocyte cultures. Cells were exposed to 3-methylcholanthrene (3-MC) or to increasing heavy metal concentrations or to a mixture of both (3-MC and one heavy metal). Metal cytotoxicity was assessed by the neutral red test. Ranking of toxicity was Cd(II) > Cu(II) > Zn(II) > Pb(II) (EC50: 45, 222, 873, and 945 muM, respectively). CYP1A1 expression was monitored by ethoxyresorufin-O-deethylase (EROD) activity as well as by Western and Northern blots. As expected, 3-MC induced EROD activity in a time- and dose-dependent manner (maximal induction 5 times that of the control at 0.5 muM and after a 72-h exposure period). These data were confirmed by Western blot (intense band of 55-60 KDa) and Northern blot analyses. Induction caused by 0.5 muM 3-MC was reduced to less than 50% of control by the concomitant exposure to Cd, Cu, Pb, or Zn (EC50: from 1 muM for Cd(II) to 18 muM for Pb(II)). The MTs were significantly induced in hepatocytes exposed to heavy metals for 24 h. In the presence of 3-MC (0.5 muM), MT levels were significantly lower than those found in cells treated with metals alone at 24 h only. Our results lead to the conclusion that heavy metals significantly affect CYP expression and that a CYP1A1 inducer (3-MC) can modulate the induction of MTs. These data have to be taken into consideration in biomarker monitoring.