Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.
OBJECTIVE
A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model.


METHODS
Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity.


RESULTS
CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities.


CONCLUSIONS
Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.
Goodsaid
Federico M
FM
Department of Genetic and Molecular Toxicology, Schering-Plough Research Institute, PO Box 32, Lafayette, New Jersey 07848, USA. federico.goodsaid@spcorp.com
Palamanda
Jairam R
JR
Montgomery
Diana
D
Mandakas
George
G
Gu
Chunyan
C
Li
Zhiling
Z
You
Xiaoli
X
Norton
Laura
L
Smith
Roger
R
Chu
Inhou
I
Soares
Tony
T
Alton
Kevin
K
Kishnani
Narendra S
NS
Rosenblum
I Y
IY
D000818:Animals; D015153:Blotting, Western; D002994:Clofibrate / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019324:beta-Naphthoflavone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D002994:Clofibrate