Optimization of a Cytochrome-P450-Monooxygenase-1A-Mediated EROD Assay in the Cape Hake Species Merluccius capensis and Merluccius paradoxus (Pisces).

Louise De Almeida,William Froneman,Brett Pletschke

doi:10.4061/2011/108395

Abstract

Cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds, for example, polychlorinated biphenyls (PCBs) and the induction of this protein is often measured in terms of CYP1A-mediated 7-ethoxyresorufin-O-deethylase (EROD) activity. This study was aimed at developing this assay in the Cape hake species Merluccius capensis and Merluccius paradoxus (considered one stock). Microsomal fractions were obtained from frozen fish liver samples by differential centrifugation. Fluorimetric and spectrophotometric analysis of the EROD assay resulted in the spectrophotometric (at 572 nm) detection method being selected, as this method resulted in a lower degree of variability and demonstrated higher reproducibility. The activity in the EROD assay was enhanced in the presence of NADPH, and the addition of dicumarol (phase II enzyme inhibitor) to the reaction mixtures prevented the underestimation of this assay by the inhibition of DT-diaphorase. In summary, an EROD assay was established for use in Cape hake species.

Highlights

In recent years the increased production and release of organic trace pollutants, for example, herbicides, metals, polychlorinated biphenyls, alkylphenols, insecticides and industrial effluent mixtures into the marine environment have increased concern and awareness into the bioaccumulation, bioconcentration, and biomagnification of these pollutants in marine organisms [1,2,3]
A higher response signal was observed in fractions (d) and (e), which led to the assumption that higher concentrations of Cytochrome P450 monooxygenase 1A (CYP1A) were present in these two fractions
Both spectrophotometric (Figure 3(a)) and fluorimetric (Figure 3(b)) results indicated that the highest EROD activity was present in pellet 2 (93 pmol/min/mg and 22 pmol/min/mg, resp.)

Summary

Introduction

In recent years the increased production and release of organic trace pollutants, for example, herbicides, metals, polychlorinated biphenyls, alkylphenols, insecticides and industrial effluent mixtures into the marine environment have increased concern and awareness into the bioaccumulation, bioconcentration, and biomagnification of these pollutants in marine organisms [1,2,3]. The production and release of these pollutants have been directly linked to the reduction in successful reproduction and increased mortality in several fish species [4,5,6]. A biomarker is defined as a biological response (molecular, physiological, or behavioral) which can be traced back to the exposure or the toxic effect of environmental pollutants. These effects can be measured in body fluids, cells, or tissues [9]. The use of biomarkers has several advantages over the use of analytical chemistry for the detection of pollutants in the aquatic environment. The use of analytical techniques can often be very expensive and requires specialized training [10]

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