High throughput ribonuclease protection assay for the determination of CYP3A mRNA induction in cultured rat hepatocytes.
1. A rapid 96-well plate based method for the determination of CYP3A mRNA induction in primary rat hepatocytes has been developed which has substantial advantages over current technologies including the ability to test the effect of relatively large numbers of new chemical entities on the expression of CYP3A mRNA in hepatocytes. 2. The ribonuclease protection assay detects changes in mRNA levels in small numbers of hepatocytes by the utilization of a radiolabelled antisense riboprobe that will hybridize CYP3A1 and CYP3A23. Using in situ hybridization techniques in conjunction with Amersham 96-well Cytostar-T scintillating microplates, there is no need for isolation of mRNA. A simple ribonuclease digestion step allows quantitative data to be generated easily within 1 week of hepatocyte isolation. 3. Rat hepatocytes were cultured for 48 h post-isolation on the Cytostar plates coated with a basal matrix of Matrigel. Prototypical CYP3A inducers (dexamethasone and pregnenolone 16alpha-carbonitrile) have been studied using various treatment periods from 0.5 to 24 h. Methylclofenapate and beta-naphthoflavone, prototypical inducers of CYP4A and CYP1A respectively, have been used as controls to show specificity of the [33P]-labelled riboprobe for the CYP3A family. 4. Time-dependent increases in CYP3A mRNA were demonstrated following exposure of hepatocytes to prototypical CYP3A inducers, but not for methylclofenapate or beta-naphthoflavone, so demonstrating specificity for CYP3A mRNA over CYP1A and CYP4A. Analysis of the 24-h induction data demonstrates that significant differences from controls can be determined and that induction potential can be assessed. The system has the potential to screen for overall CYP3A mRNA induction in response to compounds at an early stage in drug research.
Surry
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Department of Drug Metabolism and Pharmacokinetics, Neuroscience Research Centre, Merck Research Laboratories, Harlow, UK. Dominic_Surry@Merck.com
McAllister
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Meneses-Lorente
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Evans
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DC
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D002992:Clofenapate / Q000494:pharmacology; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D017403:In Situ Hybridization / Q000379:methods*; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000187:drug effects / Q000235:genetics; D010089:Oxidoreductases, N-Demethylating / Q000187:drug effects / Q000235:genetics*; D011285:Pregnenolone Carbonitrile / Q000494:pharmacology; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases / Q000378:metabolism; D013778:Tetrazolium Salts / Q000737:chemistry; D018675:Toxicity Tests / Q000379:methods; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D015347:RNA Probes; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D011285:Pregnenolone Carbonitrile; D002992:Clofenapate; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D042926:Cytochrome P-450 CYP4A; D010089:Oxidoreductases, N-Demethylating; D012260:Ribonucleases