Title

Identification and characterization of P450 2E1-like enzyme in winter flounder

Date of Completion

January 1997

Keywords

Biology, Molecular|Health Sciences, Toxicology|Environmental Sciences

Degree

Ph.D.

Abstract

This study was undertaken to determine if winter flounder possess an enzyme with characteristics of mammalian cytochrome P450 2E1. Winter flounder, a commercially important species endemic to coastal Northeast United states, are often used in pollution monitoring studies. Such studies have noted a correlation between incidence of hepatic anomalies in winter flounder and elevated levels of contaminants. Cytochrome P450s, a superfamily of Phase I detoxification enzymes, are induced by and their activity linked to toxic and carcinogenic effects of contaminants, predominantly organics. The mammalian isoform, 2E1, is induced by and its activity linked to detrimental effects of small volatile organics and nitrosamines.^ In mammals chlorzoxazone (CZX) is the best known 2E1-specific substrate and hydroxylation by Winter flounder ranged from 0.06 to 0.22 nmol/mg/min, the same order of magnitude as in rats and humans. The reaction was characteristic of P450-catalyzed reactions in that it occurred almost exclusively in microsomal fractions, required NADPH, and was inhibited by carbon monoxide. The following 2E1-like characteristics were also observed: Km and Vmax were approximately 280 $\mu$M and 101 pmol/mg/min; the reaction was significantly inhibited by dithiodiethylcarbamate, aniline and methanol; 1A-specific inhibitors, 7,8-benzoxone and ethoxyresorufin had minimal effect on the reaction; troleandomycin, a 3A-specific inhibitor, had no effect; fasting significantly increased overall liver CZX turnover, and exposure to ethanol increased specific activity, though not significantly; EROD activity did not correlate with CZX hydroxylation.^ Acclimation temperature was found to influence temperature optimum, and seasonal observations and effects of simulating seasonal temperature change indicate a compensatory effect. Results from Boston Harbor winter flounder suggest that CZX metabolism may serve as a useful biomarker of exposure to small volatile organics, otherwise difficult to detect in the aquatic environment. Results from brown bullhead catfish, a freshwater equivalent of winter flounder, support this supposition in that liver microsomal activity in fish from a trichloroethylene contaminated site were 3x higher than those from a reference site. Taken together these data support the existence of a 2E1-like enzyme in fish, provide the foundation for a new biomarker and suggest a potential mechanism of hepatic damage in fish exposed to complex mixtures of contaminants. ^