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

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 37°, and further incubated for 8 hr at 37°. Either at 4 or 20°, 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 37°, and both CYP3A2 protein and mRNA were not detected. CYP3A1 protein was unaffected by a temperature of 37° 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°, 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° were rapidly restored, when PCLS were further incubated at 37°. 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 37° might be due to a selective targeting (phosphorylation ?) leading to proteolytic disposal by the proteasome.