Development and application of a real-time quantitative PCR assay for determining expression of benzo-apyrene- Inducible cytochrome P450 1A in Nile tilapia (Oreochromis niloticus)

  • AAI Hassanin
  • Y Kaminishi
  • MMM Osman
  • ZH Abdel-Wahad
  • MAH El-Kady
  • T Itakura
Keywords: Benzo-a-pyrene, oreochromis, real-time PCR, internal control.

Abstract

Cytochrome P4501A’s (CYP1A) constitute a ubiquitous family of proteins associated with the detoxification of organic compounds such as PCB (polychlorinated biphenyl), PAH (polyaromatic hydrocarbons) and dioxin. These compounds are documented to induce the CYP1A gene in a variety of tissues of many fish species. Consequently, changes in CYP1A gene expression have been used as a biomarker for contaminant exposure in fish populations using a variety of techniques. Of all of these
methods, quantitative PCR appears to be the most sensitive. It has been used to assess impact of environmental pollution in marine ecosystems using different fish models. Subsequently, for measuring benzo-a-pyrene (BaP) induction of CYP1A mRNA in different organs of tilapia (Oreochromis niloticus), ribosomal protein large P0-like protein (RPLP0-like protein) and -actin genes as internal controls were selected based on previous studies to assess their expression variability. Real-time polymerase chain
reaction (real-time PCR) analysis of liver, intestine, gills and kidney revealed a distinct induced expression in liver and intestine (127.1 and 79.3 in liver, 26 and 56.1 in intestine using RPLP0 and -actin genes respectively as internal controls) with no detectable expression in the other organs studied.
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