Rate and capacity of hepatic microsomal ring-hydroxylation of phenol to hydroquinone and catechol in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol at 11 and 25 degreesC by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and catechol (CAT). An HPLC method with integrated ultraviolet and electrochemical detection was used for metabolite identification and quantification at low (pmol) formation rates found in fish. The Michaelis-Menten saturation kinetics for the production of HQ and CAT over a range of phenol concentrations were determined at trout physiological pH, The apparent Km's for the production of HQ and CAT at 11 degreesC were 14 +/- 1 and 10 +/- 1 mM, respectively, with Vmax's of 552 +/- 71 and 161 +/- 15 pmol/min per mg protein. The kinetic parameters for HQ and CAT at 25 degreesC were 22 +/- 1 and 32 +/- 3 mM (Km) and 1752 +/- 175 and 940 +/- 73 pmol/min per mg protein (Vmax), respectively. The calculated increase in metabolic rate per 10 degreesC temperature rise (Q(10)) was 2.28 for HQ and 3.53 for CAT production. These experiments assess the potential for metabolic bioactivation in fish through direct quantification of putative reactive metabolites at the low, but toxicologically significant, chemical concentrations found in aquatic organisms. This work initiates a series of studies to compare activation pathway, rate, and capacity across fish species, providing a basis for development of biologically-based dose response models in diverse species. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.