Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

Abstract

The interactive effects of an aryl hydrocarbon receptor (AhR) agonist and of a xenoestrogen on biomarker responses were studied in the liver of male mosquitofish (Gambusia holbrooki). Hepatic 7-ethoxyresorufin O-deethylase (EROD) enzymatic activity was measured as a biomarker of exposure to the model AhR agonist beta-naphthoflavone (bNF). Hepatic proteins indicating the exposure of males to the synthetic oestrogen 17alpha-ethynylestradiol (EE2) were monitored by Western blot analysis using immunoserum prepared for this study. After a semi-static exposure only to waterborne EE2, Western blot analysis of liver homogenate revealed the induction of two protein bands (a double band at 205 kDa and a single band at 125 kDa). The interaction between bNF and EE2 was investigated by analysing, on the one hand, EROD activity and, on the other hand, immunoreactivity corresponding to the two oestrogen-dependent protein bands in the liver of fish exposed to different concentrations of bNF for 2 days, then to the same concentrations of bNF plus 0.1 µg l⁻¹ EE2 for 5 days. EE2 changed neither the basal activity of EROD nor its rate of induction with 1.0 and 4.0 µg l⁻¹ bNF. On the other hand, the induction of oestrogen-dependent proteins with 0.1 µg l⁻¹ EE2 was inhibited by exposure to 4.0 µg l⁻¹ bNF. These results together with literature data suggest that field monitoring of xenoestrogen contamination through the analysis of oestrogen-dependent protein in male fish as a biomarker should take into account the possible negative interference of AhR agonists.

Keywords: :

• Fish
• hepatic 7-ethoxyresorufin O-deethylase (EROD)
• oestrogen-dependent protein
• beta-naphthoflavone
• 17alpha-ethynylestradiol
• interaction

Acknowledgements

The authors thank Alexis Fostier (INRA, SCRIBE, Rennes) for fruitful discussions about anti-serum preparation, and Alphonse Quemeneur (INRA, U3EA, Rennes) for providing fish. É. A. and H. R. were equal contributors to this study.