The effect of high dose endotoxin on CYP3A2 expression in the rat.
OBJECTIVE
The purpose of our research was two-fold: 1) to further characterize the downregulation of CYP3A2 mRNA, protein, and activity during an acute phase response (APR); 2) most importantly, to relate the time-dependent activation of nuclear proteins to putative DNA binding sequences within the CYP3A2 5'-flanking region, with the loss in CYP3A2 expression.


METHODS
Rats were injected (2.0 mg/animal, i.p.) with LPS and sacrificed at 1, 2, 4, 6, 8, 24, 48, and 72 hours. Hepatic nuclear protein was isolated and analyzed for binding activity to AP-1, NFkappaB, and NF-IL6 consensus sequences. Hepatic CYP3A2 mRNA levels were determined by solution hybridization and CYP3A2 protein, CYP3A2 activity, and total P450 were measured in hepatic microsomes.


RESULTS
Computer analysis of the 5'-flanking region of CYP3A2 revealed the presence of 5 NF-IL6 and 4 AP-1 putative DNA binding sites. The strongest increase in AP-1 binding activity occurred between 6 and 24 hr, and the alteration in binding complexes to an NF-IL6 oligonucleotide occurred between 4 and 24 hr. Maximum loss in CYP3A2 mRNA occurred at 8 hr post-LPS injection and remained lowered at the 24 hr timepoint. CYP3A2 protein was significantly decreased at 24, 48, and 72 hours post-LPS treatment with corresponding decreases in CYP3A2 activity and total P450.


CONCLUSIONS
The changes in NF-IL6 and AP-1 binding after LPS treatment, which appears to correlate with the changes in CYP3A2 mRNA, combined with the presence of putative NF-IL6 and AP-1 sites located in the CYP3A25'-flanking region, may indicate a potential role for NF-IL6 and AP-1 in CYP3A2 downregulation during an APR.
Roe
A L
AL
Graduate Center for Toxicology, University of Kentucky, Lexington, USA.
Warren
G
G
Hou
G
G
Howard
G
G
Shedlofsky
S I
SI
Blouin
R A
RA
D000210:Acute-Phase Reaction / Q000378:metabolism; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C022657:steroid hormone 6-beta-hydroxylase