Functional analysis of a metal response element in the regulatory region of flounder cytochrome P450 1A and implications for environmental monitoring of pollutants.
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AbstractCytochrome P450 1A (CYP 1A) is a member of a multigene family of xenobiotic metabolizing enzymes. CYP 1A is highly inducible by numerous environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) and is widely used in biomonitoring studies. Therefore, understanding the regulation of this gene is important for accurate interpretation of biomarker data. We describe here the functional role of a metal response element (MRE) in the European flounder CYP 1A promoter region. To help elucidate the potential role of this MRE, reporter gene constructs, with or without site-directed mutagenesis, were used in conjunction with a dual-luciferase assay. The electrophoretic mobility shift assay (EMSA) was also used to investigate potential protein binding at this MRE site. Treatment with the prototypical PAH 3-methylcholanthrene (3MC) (1.0 microM) produced a dose-dependent response at the CYP 1A promoter, whereas treatment with cadmium (0-1.0 microM) produced little transcriptional activity at either the wild-type or mutated promoter. Cotreatment with cadmium (1.0 microM) and 3MC (1.0 microM) reduced induction at this promoter to 1.83-fold compared to 3MC treatment alone (4.0-fold induction). Mutation of the MRE site resulted in abolishment of this cadmium-related loss of 3MC-dependent activity. Furthermore, a retarded band was observed in the EMSA when the MRE was used as a probe and incubated with liver nuclear protein from flounder treated with cadmium. The results not only add to knowledge of the diversity in vertebrate CYP 1A regulation but also raise the complexity of interpretation of CYP 1A induction in monitoring studies that involve mixtures of PAHs and metals.
CitationToxicol. Sci. 2006, 92 (2):387-393
DescriptionBiomarker: CYP 1A gene expression. Exposure/effect represented: 3-methylcholanthrene (3MC) / induction of CYP 1A gene expression. Tissue/biological material/sample size: PLHC-1 cells (Poeciliopsis lucida) Method of analysis: CYP 1A induction was measured as the normalized firefly to Renilla luciferase ratio. The firefly and Renilla luciferase activity was measured using a luminometer (Tecan, Weymouth, UK) and the Dual-Luciferase Assay kit (Promega). Firefly luciferase expression was normalized to that of Renilla, and data were expressed as mean fold induction over DMSO controls. The DNA-binding abilities of the wild-type and mutated MRE fragments were compared using the gel shift assayImpact on outcome (including dose-response): Treatment with the prototypical PAH 3-methylcholanthrene (3MC) (1.0 microM) produced a dose-dependent response at the CYP 1A promoter, whereas treatment with cadmium (0-1.0 microM) produced little transcriptional activity at either the wild-type or mutated promoter. Cotreatment with cadmium (1.0 microM) and 3MC (1.0 microM) reduced induction at this promoter to 1.83-fold compared to 3MC treatment alone (4.0-fold induction). Mutation of the MRE site resulted in abolishment of this cadmium-related loss of 3MC-dependent activity. Furthermore, a retarded band was observed in the EMSA when the MRE was used as a probe and incubated with liver nuclear protein from flounder treated with cadmium. KEYWORDS - CLASIFFICATION: analysis;Animals;Aryl Hydrocarbon Hydroxylases;Biological Markers;biomarkers of exposure & effect: method development & validation;biosynthesis;Cadmium;Cell Line,Tumor;Cyprinidae;drug effects;Environmental Monitoring;enzymology;Flounder;Gene Expression Regulation,Enzymologic;Genes,Reporter;genetics;hydrocarbons;Liver;method development & validation;methods;Methylcholanthrene;Mutagenesis,Site-Directed;Mutation;Promoter Regions (Genetics);Research;Response Elements;toxicity;Transfection;validation;Water;Water Pollutants;Water Pollutants,Chemical;
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