Induced Cytochrome P450 in Winter Flounder (Pseudopleuronectes americanus) from Coastal Massachusetts Evaluated by Catalytic Assay and Monoclonal Antibody Probes

Abstract

Levels of hepatic microsomal ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities and sensitivity to inhibition by α-naphthoflavone in winter flounder (Pseudopleuronectes americanus) from Deer Island flats in Boston Harbor, off Plymouth Beach, off Nantucket, and at the outer New Bedford Harbor in Buzzards Bay, Massachusetts, suggested induction of cytochrome P450 by environmental chemicals. Levels of activity were higher in fish from Boston Harbor and Plymouth Bay than from Nantucket and Buzzards Bay. In fish from Buzzards Bay the levels of EROD and AHH activities were closely correlated, with some fish there apparently uninduced. Immunoblot analysis of flounder liver microsomes with a monoclonal antibody (1-12-3) against β-naphthofiavone-inducible scup cytochrome P450E revealed a single cross-reacting protein in untreated fish from all four field sites. A similar protein was induced by β-naphthoflavone treatment. Levels of this flounder protein correlated positively with levels of AHH and EROD activity in Buzzards Bay fish, consistent with a conclusion that some fish there were uninduced. The results demonstrate the induction of a P450E counterpart in flounder in Massachusetts waters and indicate its identity as the AHH and EROD catalyst. The results also establish the use of monoclonal anti-P450E in analysis of environmental induction.