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  • The most significant finding was the inhibition of EROD

    2019-07-08

    The most significant finding was the inhibition of EROD and BFCOD activity by CLO. Inhibition of EROD and BFCOD were reversible, as pre-incubation did not enhance CLO inhibitory potency, and no reductions in IC50 values were observed. We further characterized the kinetic pattern of inhibition using six substrate concentrations and found that EROD activity was non-competitively inhibited by CLO (decreased Vmax, similar Km). Most studies investigating the inhibitory mechanism of imidazole fungicides such as propiconazole, prochloraz, and CLO on CYP1A1 in fish have suggested differing inhibiting mechanisms, such as non-competitive (Snegaroff and Bach, 1989, Levine et al., 1999), competitive (Snegaroff and Bach, 1989, Miranda et al., 1998), and non-competitive mixed type mechanisms (Levine and Oris, 1999). This variability might be due to the variation in substrate and inhibitor concentrations used in the studies and the fact that the imidazoles have different modes of inhibition. The role of inter-species and individual variations in the response to the inhibitor should also be considered. Another family of CYP450 that is responsible for the biotransformation of a large number of xenobiotics in fish liver is CYP3A (Hegelund and Celander, 2003). According to Stresser et al. (2000), CLO is a submicromolar inhibitor of CYP3A in microsomal protein prepared from wild-type baculovirus infected insect Apramycin Sulfate weight using 7-benzyloxy-4-trifluoromethylcoumarin (BFC) as a substrate. We demonstrated a similar interaction between CLO and product formation from BFC. Kinetic analysis showed a competitive mechanism of inhibition (similar Vmax, increased Km), suggesting that both CLO and BFC are metabolized by the same isoform. It is possible that both inhibitors used in the present study are metabolized by the investigated enzymes, especially CYP3A. Thus, it is likely that the observed decrease in specific substrate metabolism is due to competition between CLO and the substrate for the active site of the enzyme and is not indicative of inhibition of overall specific CYP450 activity. The Ki values for CLO inhibition of EROD and BFCOD activity were relatively low, suggesting that ability of CYP1A and CYP3A to metabolize xenobiotics may be reduced in the presence of CLO. To our knowledge, neither in vivo or in vitro inhibition potential of CLO and DEX on PNPH activity in fish has been studied, whereas studies on human cell lines have demonstrated that CLO is a uncompetitive inhibitor of CYP2E1 (Tassaneeyakul et al., 1998). Unlike EROD and BFCOD, which were both inhibited by CLO at concentrations ranging from 1 to 100μM, PNPH activity was reduced only in the presence of the highest tested CLO concentration. Although vascular parenchyma inhibition did not exceed 50%, there is a possibility that this is sufficient to be of physiological significance. Thus, the potential effects of this inhibition require further research. A possible limitation of our study was that two concentrations of the inhibitor CLO used for the kinetic analyses were above the calculated Ki values. These concentrations were Apramycin Sulfate weight chosen from a preliminary study using several concentrations of CLO to reduce enzyme activity by 50% for EROD and by 80–90% for BFCOD. It could be argued that these conditions may not be sufficient to determine precise Ki values, yet, we believe that this set of experiments allows for accurate determination of inhibition mode. This is the first demonstration of non-competitive inhibition of EROD activity by CLO in hepatic microsomes of rainbow trout. BFCOD activity was inhibited by CLO competitively. Therefore, a controlled in vivo study is warranted to further investigate the effect of CLO on activity of these enzymes. The presence of DEX in the incubations did not affect either EROD or BFCOD activity. Neither CLO nor DEX affected PNPH activity in hepatic microsomes from rainbow trout.
    Acknowledgements