Many human pharmaceuticals are weak inhibitors of the cytochrome P450 system in rainbow trout (Oncorhynchus mykiss) liver S9 fractions

Abstract

Introduction: Pharmaceutical residues are widely detected in aquatic environment and can be taken up by nontarget species such as fish. The cytochromes P450 (CYP) represent an important detoxification mechanism in fish, like in humans. In the present study, we assessed the correlation of the substrate selectivities of rainbow trout CYP1A and CYP3A homologues with those of human, through determination of the half-maximal inhibitory concentrations (IC₅₀) of a total sixteen human pharmaceuticals toward CYP1A-like ethoxyresorufin O-deethylase (EROD) and CYP3A-like 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (RT-S9).

Methods: The inhibitory impacts (IC₅₀) of atomoxetine, atorvastatin, azelastine, bimatoprost, clomethiazole, clozapine, desloratadine, disulfiram, esomeprazole, felbinac, flecainide, orphenadrine, prazosin, quetiapine, sulpiride, and zolmitriptan toward the EROD and BFCOD activities in RT-S9 were determined using the IC₅₀ shift assay, capable of identifying time-dependent inhibitors (TDI). Additionally, the nonspecific binding of the test pharmaceuticals to RT-S9 was assessed using equilibrium dialysis.

Results: Most test pharmaceuticals were moderate to weak inhibitors of both EROD and BFCOD activity in RT-S9, even if most are noninhibitors of human CYP1A or CYP3A. Only bimatoprost, clomethiazole, felbinac, sulpiride, and zolmitriptan did not inhibit either activity in RT-S9. EROD inhibition was generally stronger than that of BFCOD and some substances (atomoxetine, flecainide, and prazosin) inhibited selectively only EROD activity. The strongest EROD inhibition was detected with azelastine and esomeprazole (unbound IC₅₀ of 3.8 ± 0.5 µM and 3.0 ± 0.8 µM, respectively). None of the test substances were TDIs of BFCOD, but esomeprazole was a TDI of EROD. Apart from clomethiazole and disulfiram, the nonspecific binding of the test pharmaceuticals to the RT-S9 was extensive (unbound fractions <0.5) and correlated well (R ² = 0.7135) with their water-octanol distribution coefficients.

Discussion: The results indicate that the P450 interactions in RT-S9 cannot be explicitly predicted based on human data, but the in vitro data reported herein can shed light on the substrate selectivity of rainbow trout CYP1A1 and CYP3A27 in comparison to their human homologues. The IC₅₀ concentrations are however many orders of magnitude higher than average environmental concentrations of pharmaceuticals. The time-dependent EROD inhibition by esomeprazole could warrant further research to evaluate its possible interlinkages with hepatotoxic impacts on fish.

Keywords: bioaccumulation; cytochrome P450; ecotoxicology; enzyme inhibition; pharmaceuticals; rainbow trout.

FIGURE 1

The nonlinear regression analyses of the residual EROD activities at different concentrations of the test pharmaceuticals in RT-S9, when the test substance and RT-S9 (1 mg/mL) were preincubated for 30 min without any cofactors (Series A), with 1 mM NADPH (Series B), or with all cofactors, including 1 mM NADPH, 1 mM UDPGA, 0.05 mM PAPS, and 2.5 mM glutathione (Series C), prior to initiation of the EROD marker reaction (10 min). On the basis of EROD inhibition, the test substances were grouped between (A) those that did not significantly inhibit EROD activity within the tested concentration range, (B) those that had apparent EROD inhibition of ≤50% at the highest test concentration, and (C) those that had >50% EROD inhibition at the highest test concentration. Each datapoint in each of the Series A-C represents an average of n = 2 replicate incubations.

FIGURE 2

The nonlinear regression analyses of the residual BFCOD activities at different concentrations of the test pharmaceuticals in RT-S9, when the test substance and RT-S9 (1 mg/mL) were preincubated for 30 min without any cofactors (Series A), with 2 mM NADPH (Series B), or with all cofactors, including 2 mM NADPH, 1 mM UDPGA, 0.05 mM PAPS, and 2.5 mM glutathione (Series C), prior to initiation of the BFCOD marker reaction (20 min). On the basis of BFCOD inhibition, the test substances were grouped between (A) those that did not significantly inhibit BCFOD activity within the tested concentration range and (B) those that had apparent BFCOD inhibition of ≤50% at the highest test concentration. Each datapoint in each of the Series A-C represents an average of n = 2 replicate incubations.

FIGURE 3

Correlation of the test substances’ nonspecific binding (unbound fractions) in RT-S9 with their water-octanol distribution coefficients (LogD_OW at the pH 7.8, for basic and acidic substances) or partitioning coefficients (LogK_OW for neutral substances). The unbound fractions (f_U,RT-S9) represent the experimental values (n = 3 replicate assays with each test substance at 10 µM concentration).The LogD_OW and LogK_OW values are from Chemaxon and PubChem databases, respectively. The regression line represents the linear correlation (R ² = 0.7135) with 95% confidence interval.