Detection of chemical-induced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology

The importance of the cytochrome P450 (CYP) enzyme family in xenobiotic met abolism, as well as their differential expression and activity in response to a wide range of environmental chemicals and pharmaceuticals, is well doc umented. The objective of this study was to evaluate the specificity of the branched DNA (bDNA) signal amplification technique for the detection of mu ltiple rat CYPs from hepatocellular RNA. Oligonucleotide probe sets were de signed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3. The robustness o f the bDNA assay was assessed with the CYP2B1/2-specific probe set, and tot al RNA was isolated from control and phenobarbital (PB)-treated rats. Analy sis of these RNA samples by bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magnitude (0.1-100 mg of total RNA). The fidelity of the bDNA assay was evaluated wit hin a single assay and between assays where repeated measurements of a sing le sample were reproduced reliably. The specificity of individual CYP probe sets was evaluated with five typical CYP-inducing chemicals on the express ion of specific hepatic CYP mRNA transcripts. Male Sprague-Dawley rats were administered 3-methylcholanthrene, PB, isoniazid, pregnenolone-16 alpha-ca rbonitrile, or clofibric acid to induce transcription of CYP1A1, CYP1A2, CY P2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of che mical- induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1 and CYP1A2 mRNA levels 6 70- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; preg nenolone-16 alpha-carbonitrile induced CYP3A1/23 expression 34-fold; and cl ofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data supp ort the use of bDNA signal amplification technology as a robust, reproducib le, and efficient means of monitoring the differential expression of multip le isoforms of the CYP enzyme family.