Development and Use of Real-Time Reverse Transcription-Polymerase Chain Reaction Assay to Quantify Cytochrome P4501A1 Expression in American Mink

Abstract

The distribution of natural populations of American mink is restricted to locales that are in proximity to aquatic ecosystems. Because of the lipophilicity and persistence of polychlorinated biphenyls (PCBs) and reliance of mink on aquatic-based diets, mink at contaminated locales often bioacccumulate high levels of PCBs. In addition, in controlled laboratory studies, mink are highly sensitive at reproductive and developmental end points to the toxic effects of environmental PCB mixtures. It is believed that most, if not all, toxic effects of PCBs occur through activation of the aryl hydrocarbon receptor (AHR) pathway. Transcription of cytochrome P4501A1 (CYP1A1) by PCBs is also mediated through activation of AHR. Thus, levels of CYP1A1 mRNA provide a quantitative assay of exposure to and early biologic effect of PCBs on mink and may be predictive of toxicity at higher levels of biologic organization. We developed polymerase chain reaction (PCR) primers to amplify CYP1A1 as well as identified a housekeeping gene from mink cDNA. We used real-time reverse transcription-PCR to quantify and compare levels of hepatic CYP1A mRNA among groups of ranched mink kits and juveniles, which were fed diets or exposed in utero to fish that were low in PCBs (Atlantic herring) or to diets that were contaminated with three different levels of PCBs (carp) from Saginaw Bay, Lake Michigan. We found significant differences in CYP1A1 mRNA expression between mink fed the control diet and those fed a PCB-contaminated carp diet at all three treatment levels and exposure times. CYP1A1 mRNA was significantly induced 5.3- to 6.6-fold and 3.7- to 4.7-fold at 6 and 27 weeks, respectively. In previous studies, dietary exposures to PCB-contaminated carp were shown to cause mild to moderate lesions in the mandible and maxilla of these animals. This study demonstrates that hepatic CYP1A1 mRNA may be a sensitive biomarker of exposure of mink to environmentally relevant levels of PCBs and may be predictive of their effects in natural populations.