Same-sample analysis of ethoxyresorufin-O-deethylase activity and cytochrome P4501A mRNA abundance in chicken embryo hepatocytes.
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-tetrachlorodibenzo-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.
Head
Jessica A
JA
Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ont., Canada K1N 6N5.
Kennedy
Sean W
SW
D000818:Animals; D002642:Chick Embryo; D002645:Chickens; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1