Role of temperature on protein and mRNA cytochrome P450 3A (CYP3A) isozymes expression and midazolam oxidation by cultured rat precision-cut liver slices.
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.
Rekka
Eleni
E
Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie (PMNT 7369), Département de Sciences Pharmaceutiques, Université Catholique de Louvain, 73, avenue E. Mounier, 1200 Brussels, Belgium.
Evdokimova
Ekaterina
E
Eeckhoudt
Stéphane
S
Labar
Geoffray
G
Calderon
Pedro Buc
PB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000759:Adjuvants, Anesthesia / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008874:Midazolam / Q000378:metabolism*; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
D000759:Adjuvants, Anesthesia; D007527:Isoenzymes; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione; D008874:Midazolam