PMID- 12167482 OWN - NLM STAT- MEDLINE DCOM- 20020912 LR - 20191227 IS - 0006-2952 (Print) IS - 0006-2952 (Linking) VI - 64 IP - 4 DP - 2002 Aug 15 TI - Role of temperature on protein and mRNA cytochrome P450 3A (CYP3A) isozymes expression and midazolam oxidation by cultured rat precision-cut liver slices. PG - 633-43 AB - The cytochrome P450 3A (CYP3A)-mediated midazolam oxidation was studied in rat precision-cut liver slices (PCLS) maintained for 20hr at 4, 20 and 37 degrees, and further incubated for 8hr at 37 degrees. Either at 4 or 20 degrees, midazolam was oxidised by PCLS at similar rates to that observed in freshly cut slices. Moreover, PCLS kept a regioselectivity since 4-hydroxylation was more important than 1'-hydroxylation. Conversely, PCLS totally lost their capacity to oxidise midazolam after 20hr at 37 degrees, and both CYP3A2 protein and mRNA were not detected. CYP3A1 protein was unaffected by a temperature of 37 degrees but its mRNA was totally lost. By blocking transcription with actinomycin D, the decay of both CYP3A mRNAs followed the same profile at either 20 or 37 degrees, indicating that temperature affected the CYP3A2 protein stability. Cell functionality was not involved in such an impairment since the low values of ATP, GSH and protein synthesis rates observed at 4 and 20 degrees were rapidly restored, when PCLS were further incubated at 37 degrees. The use of rat supersomes expressing either CYP3A1 or CYP3A2, strongly supported the hypothesis that 4-hydroxymidazolam was mainly formed by CYP3A2. These results suggest that: (1) CYP3A1 protein is constitutive and largely expressed in rat liver slices; (2) regioselective midazolam oxidation appears to be mainly CYP3A2 dependent; and (3) since CYP3A isoforms have similar half-lives (about 10-14hr), the loss of CYP3A2 protein at 37 degrees might be due to a selective targeting (phosphorylation ?) leading to proteolytic disposal by the proteasome. FAU - Rekka, Eleni AU - Rekka E AD - Unite de Pharmacocinetique, Metabolisme, Nutrition et Toxicologie (PMNT 7369), Departement de Sciences Pharmaceutiques, Universite Catholique de Louvain, 73, avenue E. Mounier, 1200 Brussels, Belgium. FAU - Evdokimova, Ekaterina AU - Evdokimova E FAU - Eeckhoudt, Stephane AU - Eeckhoudt S FAU - Labar, Geoffray AU - Labar G FAU - Calderon, Pedro Buc AU - Calderon PB LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - Biochem Pharmacol JT - Biochemical pharmacology JID - 0101032 RN - 0 (Adjuvants, Anesthesia) RN - 0 (Isoenzymes) RN - 0 (RNA, Messenger) RN - 8L70Q75FXE (Adenosine Triphosphate) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.14.14.1 (Aryl Hydrocarbon Hydroxylases) RN - EC 1.14.14.1 (Cyp3a23-3a1 protein, rat) RN - EC 1.14.14.1 (Cytochrome P-450 CYP3A) RN - EC 1.5.- (Oxidoreductases, N-Demethylating) RN - GAN16C9B8O (Glutathione) RN - R60L0SM5BC (Midazolam) SB - IM MH - Adenosine Triphosphate/metabolism MH - Adjuvants, Anesthesia/metabolism MH - Animals MH - *Aryl Hydrocarbon Hydroxylases MH - Blotting, Western MH - Cytochrome P-450 CYP3A MH - Cytochrome P-450 Enzyme System/*biosynthesis/genetics/metabolism MH - Glutathione/metabolism MH - In Vitro Techniques MH - Isoenzymes/*biosynthesis/genetics/metabolism MH - Liver/*enzymology/metabolism MH - Male MH - Midazolam/*metabolism MH - Oxidation-Reduction MH - Oxidoreductases, N-Demethylating/*biosynthesis/genetics/metabolism MH - RNA, Messenger/biosynthesis MH - Rats MH - Rats, Wistar MH - Reverse Transcriptase Polymerase Chain Reaction MH - Temperature EDAT- 2002/08/09 10:00 MHDA- 2002/09/13 10:01 CRDT- 2002/08/09 10:00 PHST- 2002/08/09 10:00 [pubmed] PHST- 2002/09/13 10:01 [medline] PHST- 2002/08/09 10:00 [entrez] AID - S0006295202012583 [pii] AID - 10.1016/s0006-2952(02)01258-3 [doi] PST - ppublish SO - Biochem Pharmacol. 2002 Aug 15;64(4):633-43. doi: 10.1016/s0006-2952(02)01258-3.