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Enzyme Research
Volume 2011, Article ID 108395, 8 pages
http://dx.doi.org/10.4061/2011/108395
Research Article

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

1Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6140, South Africa
2Department of Zoology and Entomology, Rhodes University, Grahamstown 6140, South Africa

Received 1 June 2011; Revised 18 August 2011; Accepted 5 September 2011

Academic Editor: Paul Engel

Copyright © 2011 Louise De Almeida et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.