Inhibitory effects of heavy metals on cytochrome P4501A induction in permanent fish hepatoma cells

Abstract

The interactions in vitro of heavy metals Cd(II), Co(II), Cu(II), Ni(II), Pb(II), and Zn(II) with cytochrome P4501A (CYP1A) induction response and enzyme activity were studied in fish hepatoma cells PLHC-1. Cells were simultaneously exposed to heavy metals and to 3-methylcholanthrene (3-MC), an inducer of CYP1A. Heavy metals were added to the cells in different concentrations. Cytotoxicity were measured in the neutral red (NR) assay, relative CYP1A protein contents in an enzyme-linked immunosorbent assay (ELISA), and CYP1A activities in the ethoxyresorufin-O-deethylase (EROD) assay. All metals had a more pronounced effect on EROD activity than on CYP1A protein content and cytotoxicity. For the most active metal Cd(II), a 50% inhibition of EROD activity was observed at significantly lower concentrations (2.2 · 10⁻⁵ M) than a 50% reduction of CYP1A protein (5.3 · 10⁻⁵ M), and a 50% cytotoxicity (1.4 · 10⁻⁴ M). The inhibitory potency of the metals had the following order: Cd(II)>Ni(II)>Cu(II)>Co(II)=Zn(II)>Pb(II). In a second set of experiments, lysates of 3-MC—induced cells were exposed to heavy metals. Cd(II) and Cu(II) caused a 50% inhibition of EROD activity at significantly lower concentrations than in the experiments with living cells, at 8.2 · 10⁻⁶ M and 1.3 · 10⁻⁵ M, respectively, whereas the effect by Co(II) occurred at a significantly higher concentration (8.2 · 10⁻⁴ M). The results indicate that Cd(II) and Cu(II) in particular may affect the CYP1A system of the liver of fish at low concentrations through direct inhibition of the CYP1A enzyme activity. CYP1A induction response in fish liver is increasingly being used in biomonitoring programs. In the environment, interactions of CYP1A-inducing and CYP1A-inhibiting components (such as heavy metals) can be expected and must be taken into consideration.