Expression and Induction of Cyp1A1 in Black Seabream (Spondyliosoma Cantharus) Hepatocyte Cultures: Effects of Heavy Metals

Abstract

We developed a new method, which allowed black seabream hepatocytes to be maintained in primary-culture for several days. This method was shown to be suitable for studying sea fish CYPs expression and regulation following xenobiotic treatment. After isolation, hepatocytes were directly mixed with a type I collagen gel and culture medium, seeded in 6-well tissue culture plates (2.10⁶ cells/35 mm well) and incubated under atmospheric air at 20°C. After 24 hrs, the cells were exposed for 1, 2 and 3 days to 3-methylcholanthrene (3MC), heavy metals (Cd(II), Cu(II), Pb(II), Zn(II), or 3MC with increasing concentrations of these metals. Cytotoxicity was assessed by the neutral red test: the sequence was Cd(II) > Cu(II) > Pb(II) > Zn (II) (EC₅₀: 57.234, 544 and 722 μM respectively) and appeared to be correlated with the metal solubility. CYP1A1 expression was monitored by EROD activity as well as by Western and Northern blots. 3MC induced the CYP1A1-related EROD activity in a time-and dose-dependent manner. These data were confirmed by Western blot (intense band of 55–60 KDa) and Northern blot analysis. Maximal induction (17-fold over control) was obtained at 1 μM after 72 hrs of exposure. This induction was inhibited by simultaneous exposure to Cd(II), Cu(II), Pb(II) or Zn(II). This effect was dose-dependent and EROD activity was decreased to less than 50% of the control (1 μM 3MC), with EC₅₀ ranging from 1.3 μM for Cd(II) to 16 μM for Zn(II). CYP1A1 protein and mRNA levels were also reduced. CYP1A induction response in fish liver is increasingly being used in ecotoxicological studies. Our results lead to the conclusion that heavy metals significantly affect CYPs expression and therefore have to be taken into consideration in biomarker monitoring.