Semi-automatic quantitative RT-PCR to measure CYP induction by drugs in human hepatocytes.
An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional liver tissue are incubated with the drugs for 50 h. Total RNA is extracted from culture and the cDNA is prepared by reverse transcription using high fidelity reverse transcriptase. Using appropriate primers, selective amplification of each CYP cDNA is achieved and real-time quantified by SYBR-Green fluorescence measurement. The extent of CYP induction obtained with the tested compounds is compared with the induction obtained with CYP model inducers (methylcholantrene, phenobarbital and rifampicin). This technique can be of value, to considerably simplify the identification of drug candidates with potential CYP inducing ability in man.
Pérez
Gabriela
G
Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda de Campanar 21, E-46009, Valencia, Spain.
Tabares
Beatriz
B
Jover
Ramiro
R
Gómez-Lechón
Maria-José
MJ
Castell
José V
JV
D001681:Biological Assay; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D064420:Drug-Related Side Effects and Adverse Reactions / Q000201:enzymology; D004790:Enzyme Induction; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D015262:Xenobiotics / Q000633:toxicity*
D012333:RNA, Messenger; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System