Abstract
The CYP2B6 enzyme is involved in the metabolism of environmental toxins and endogenous compounds. The expression and activity of the cyp2b6 gene is important for the brain’s ability to detoxify harmful compounds. In rats, the Cyp2b1 gene is homologous to the human Cyp2b6 gene and has an isoform, Cyp2b2, with nearly the same sequence. Although the distinction between them is fundamental, most studies report global changes in CYP2B enzymes due to the complexity of the techniques to detect both genes separately. We instrumented a semi-quantitative technique combining multiplex end point RT-PCR with restriction enzyme assays, allowing us to establish a relatively simple, reproducible and affordable method to distinguish Cyp2b1 gene expression from Cyp2b2. Using this technique, we showed differential Cyp2b1 expression after phenobarbital and ethanol induction in the brain and liver and detected increased Cyp2b1 expression after ethanol treatment in the brain striatum that had not been detected before.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Gotoh, O. Evolution of cytochrome p450 genes from the viewpoint of genome informatics. Biol Pharm Bull 35:812–817 (2012).
Ioannides, C. & Lewis, D. F. Cytochromes P450 in the bioactivation of chemicals. Curr Top Med Chem 4: 1767–1788 (2004).
Ingelman-Sundberg, M., Sim, S. C., Gomez, A. & Rodriguez-Antona, C. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects. Pharmacol Ther 116:496–526 (2007).
Wang, H. & Tompkins, L. M. CYP2B6: new insights into a historically overlooked cytochrome P450 isozyme. Curr Drug Metab 9:598–610 (2008).
Xie, H. J. et al. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation. Pharmacogenomics J 3:53–61 (2003).
Ferguson, C. S. & Tyndale, R. F. Cytochrome P450 enzymes in the brain: emerging evidence of biological significance. Trends Pharmacol Sci 32:708–714 (2011).
Upadhya, S. C., Chinta, S. J., Pai, H. V., Boyd, M. R. & Ravindranath, V. Toxicological consequences of differential regulation of cytochrome p450 isoforms in rat brain regions by phenobarbital. Arch Biochem Biophys 399:56–65 (2002).
Zanger, U. M. & Klein, K. Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance. Front Genet 4:24 (2013).
Ravindranath, V. & Strobel, H. W. Cytochrome P450-mediated metabolism in brain: functional roles and their implications. Expert Opin Drug Metab Toxicol 9:551–558 (2013).
Khokhar, J. Y. & Tyndale, R. F. Drug metabolism within the brain changes drug response: selective manipulation of brain CYP2B alters propofol effects. Neuropsychopharmacology 36:692–700 (2011).
Miksys, S., Hoffmann, E. & Tyndale, R. F. Regional and cellular induction of nicotine-metabolizing CYP2B1 in rat brain by chronic nicotine treatment. Biochem Pharmacol 59:1501–1511 (2000).
Agrawal, A. K. & Shapiro, B. H. Phenobarbital induction of hepatic CYP2B1 and CYP2B2: pretranscriptional and post-transcriptional effects of gender, adult age, and phenobarbital dose. Mol Pharmacol 49:523–531 (1996).
Miksys, S. & Tyndale, R. F. Nicotine induces brain CYP enzymes: relevance to Parkinson’s disease. J Neural Transm Suppl:177–180 (2006).
Nuwaysir, E. F., Dragan, Y. P., Jefcoate, C. R., Jordan, V. C. & Pitot, H. C. Effects of tamoxifen administration on the expression of xenobiotic metabolizing enzymes in rat liver. Cancer Res 55:1780–1786 (1995).
Chanyshev, M. D., Kosorotikov, N. I., Titov, S. E., Kolesnikov, N. N. & Gulyaeva, L. F. Expression of microRNAs, CYP1A1 and CYP2B1 in the livers and ovaries of female rats treated with DDT and PAHs. Life Sci 103:95–100 (2014).
Hartley, D. P. & Klaassen, C. D. Detection of chemicalinduced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology. Drug Metab Dispos 28:608–616 (2000).
Morris, D. L. & Davila, J. C. Analysis of rat cytochrome P450 isoenzyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RTPCR). Biochem Pharmacol 52:781–792 (1996).
Streit, S., Michalski, C. W., Erkan, M., Kleeff, J. & Friess, H. Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues. Nat Protoc 4:37–43 (2009).
Gomez-Lira, G., Lamas, M., Romo-Parra, H. & Gutierrez, R. Programmed and induced phenotype of the hippocampal granule cells. J Neurosci 25:6939–6946 (2005).
Cornelison, D. D. & Wold, B. J. Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Dev Biol 191:270–283 (1997).
Roberts, R. J. How restriction enzymes became the workhorses of molecular biology. Proc Natl Acad Sci USA 102:5905–5908 (2005).
Schoedel, K. A., Sellers, E. M. & Tyndale, R. F. Induction of CYP2B1/2 and nicotine metabolism by ethanol in rat liver but not rat brain. Biochem Pharmacol 62: 1025–1036 (2001).
Parmar, D., Dhawan, A. & Seth, P. K. Evidence for Odealkylation of 7-pentoxyresorufin by cytochrome P450 2B1/2B2 isoenzymes in brain. Mol Cell Biochem 189: 201–205 (1998).
Dail, M. B. et al. Spatial distribution of CYP2B1/2 messenger RNA within the rat liver acinus following exposure to the inducers phenobarbital and dieldrin. Toxicol Sci 99:35–42 (2007).
Pustylnyak, V. O., Gulyaeva, L. F. & Lyakhovich, V. V. CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers. Toxicology 216:147–153 (2005).
Lardizabal, M. N. et al. Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity. PLoS One 7:e36323 (2012).
Bonefeld, B. E., Elfving, B. & Wegener, G. Reference genes for normalization: a study of rat brain tissue. Synapse 62:302–309 (2008).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Vanoye-Carlo, A., Gutiérrez-Ospina, G., Marcial-Quino, J. et al. Analysis of Cyp2b1 gene expression in the rat liver and brain by multiplex PCR. Mol. Cell. Toxicol. 11, 407–414 (2015). https://doi.org/10.1007/s13273-015-0043-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13273-015-0043-1