Abstract
The importance of the cytochrome P450 (CYP) enzyme family in xenobiotic metabolism, as well as their differential expression and activity in response to a wide range of environmental chemicals and pharmaceuticals, is well documented. The objective of this study was to evaluate the specificity of the branched DNA (bDNA) signal amplification technique for the detection of multiple rat CYPs from hepatocellular RNA. Oligonucleotide probe sets were designed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3. The robustness of the bDNA assay was assessed with the CYP2B1/2-specific probe set, and total RNA was isolated from control and phenobarbital (PB)-treated rats. Analysis 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 μg of total RNA). The fidelity of the bDNA assay was evaluated within a single assay and between assays where repeated measurements of a single sample were reproduced reliably. The specificity of individual CYP probe sets was evaluated with five typical CYP-inducing chemicals on the expression of specific hepatic CYP mRNA transcripts. Male Sprague-Dawley rats were administered 3-methylcholanthrene, PB, isoniazid, pregnenolone-16α-carbonitrile, or clofibric acid to induce transcription of CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of chemical-induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1 and CYP1A2 mRNA levels 670- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; pregnenolone-16α-carbonitrile induced CYP3A1/23 expression 34-fold; and clofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data support the use of bDNA signal amplification technology as a robust, reproducible, and efficient means of monitoring the differential expression of multiple isoforms of the CYP enzyme family.
Footnotes
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Send reprint requests to: Curtis D. Klaassen, Ph.D., Dept. of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: cklaasse{at}kumc.edu
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This study was supported by U.S. Public Health Service Grant ES-03192. D.H. was supported by an National Institute of Environmental Health Sciences Training Grant ES-07079 and by the Kansas Health Foundation (KHF-411606).
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↵2 For CYP2B1/2, CYP3A1/23, and CYP4A2/3 oligonucleotide probe sets were not designed to distinguish between these subfamily members (e.g., CYP2B1 and CYP2B2 transcript expression levels were detected together and were not differentiated by the bDNA assay).
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↵3 Capture and label oligonucleotide probes were synthesized with an additional 3′-concatenated nucleotide sequence where the 3′-sequence is constant for either capture probes (ctcttggaaagaaagt) or label probes (aggcataggacccgtgtct) and designates each probe as a capture or label probe. This 3′-nucleotide sequence extension on capture and label probes is separated from the gene-specific oligonucleotide sequence by a stretch of five thymidines (TTTTT). Blocker probes do not contain a nucleotide sequence extension. Each oligonucleotide is listed with the sequence information for each probe in Table 2.
- Abbreviations used are::
- CYP
- cytochrome P450
- bDNA
- branched deoxyribonucleic acid
- GAPDH
- glyceraldehyde 3-phosphate dehydrogenase
- 3MC
- 3-methylcholanthrene
- PB
- phenobarbital
- ISO
- isoniazid
- CLO
- clofibric acid
- PCN
- pregnenolone-16α-carbonitrile
- RLU
- relative luminescence units
- ELISA
- enzyme-linked immunosorbent assay
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription-polymerase chain reaction
- Received September 3, 1999.
- Accepted January 21, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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