Abstract

The cytochrome P450 3A (CYP3A)-mediated midazolam oxidation was studied in rat precision-cut liver slices (PCLS) maintained for 20 hr at 4, 20 and 37degrees, and further incubated for 8 hr at 37degrees. Either at 4 or 20degrees, 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 20 hr at 37degrees, and both CYP3A2 protein and mRNA were not detected. CYP3A1 protein was unaffected by a temperature of 37degrees 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 37degrees, 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 20degrees were rapidly restored, when PCLS were further incubated at 37degrees. 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 10-14 hr), the loss of CYP3A2 protein at 37degrees might be due to a selective targeting (phosphorylation ?) leading to proteolytic disposal by the proteasome. (C) 2002 Elsevier Science Inc. All rights reserved.