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 i nduction in primary rat hepatocytes has been developed which has substantia l advantages over current technologies including the ability to test the ef fect 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 smal l numbers of hepatocytes by the utilization of a radiolabelled antisense ri boprobe that will hybridize CYP3A1 and CYP3A23. Using in situ hybridization techniques in conjunction with Amersham 96-well Cytostar-T scintillating m icroplates, there is no need for isolation of mRNA. A simple ribonuclease d igestion 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 pl ates coated with a basal matrix of Matrigel. Prototypical CYP3A inducers (d examethasone and pregnenolone 16 alpha-carbonitrile) have been studied usin g various treatment periods from 0.5 to 24 h. Methylclofenapate and beta-na phthoflavone, prototypical inducers of CYP4A and CYP1A respectively, have b een used as controls to show specificity of the [P-33]-labelled riboprobe f or the CYP3A family. 4. Time-dependent increases in CYP3A mRNA were demonstrated following expos ure of hepatocytes to prototypical CYP3A inducers, but not for methylclofen apate or beta-naphthoflavone, so demonstrating specificity for CYP3A mRNA o ver 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 overa ll CYP3A mRNA induction in response to compounds at an early stage in drug research.