Mixture effects between different azoles and β-naphthoflavone on the CYP1A biomarker in a fish cell line

• Prochloraz and nocodazole inhibit EROD activities in situ.
• Prochloraz and nocodazole induce CYP1A mRNA levels.
• EROD inhibitors triggers enhanced mixture effects with an AhR ligand.
• Mixture effects on the EROD biomarker are concentration and time-dependent.
• EROD is induced in cells pre-treated with a microtubule disassembling agent.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) – CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist β-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2–4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48 h, whereas exposure to the omeprazole for 48 h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50 = 1.3 – 7.7 μM), whereas omeprazole had no effect on this activity (IC50 = 72 μM). Exposure to 10 μM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10 μM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24 h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24 h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24 h at concentrations that are known to disassemble microtubules at 24 h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24 h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.