Full Length Research Paper

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

Abeer A. I. Hassanin¹, Yoshino Kaminishi²*, Mohamed M. M. Osman³, Zamzam H. Abdel-Wahad⁴, Mohamed A. H. El-Kady⁵ and Takao Itakura⁶

^1,2,5,6Laboratory of Marine Biotechnology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan.

^1,3Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt.

⁴Department of Animal Hygiene, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt.

⁵The National Institute of Oceanography and Fisheries, Alexandria, Egypt.

*Corresponding author. E-mail: kaminisi@fish.kagoshima-u.ac.jp. 

Tel: 81-99-286-4221. Fax: 81-99-286-4015.

Accepted 11 June, 2009

   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 β-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 β-actin genes respectively as internal controls) with no detectable expression in the other organs studied.

Key words: Benzo-a-pyrene, oreochromis, real-time PCR, internal control.