Same-sample analysis of ethoxyresorufin-O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes

被引：37
作者：
Head, Jessica A.
Kennedy, Sean W. [1 ]
机构：
[1] Univ Ottawa, Dept Biol, Ctr Adv Res Environm Genom, Ottawa, ON K1N 6N5, Canada
[2] Environm Canada, Natl Wildlife Res Ctr, Ottawa, ON K1A 0H3, Canada
来源：
ANALYTICAL BIOCHEMISTRY | 2007年 / 360卷 / 02期
关键词：
Q-PCR; CYP1A4; CYP1A5; EROD; hepatocyte; biomarker; avian; degraded RNA;
D O I：
10.1016/j.ab.2006.10.033
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin-O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibnzo-p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample. Crown Copyright (c) 2006 Published by Elsevier Inc. All rights reserved.
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页码：294 / 302
页数：9
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