1: Biochem Pharmacol  2002 Aug 15;64(4):633-43 

Role of temperature on protein and mRNA cytochrome P450 3A (CYP3A) isozymes
expression and midazolam oxidation by cultured rat precision-cut liver slices.

Rekka E, Evdokimova E, Eeckhoudt S, Labar G, Calderon PB.

Unite de Pharmacocinetique, Metabolisme, Nutrition et Toxicologie (PMNT 7369),
Departement de Sciences Pharmaceutiques, Universite Catholique de Louvain, 73,
avenue E. Mounier, 1200 Brussels, Belgium.

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.

PMID: 12167482 [PubMed - indexed for MEDLINE]