Hypoxia‐induced down‐regulation of CYP1A1/1A2 and up‐regulation of CYP3A6 involves serum mediators

Abstract

1 Acute moderate hypoxia modi®es the catalytic activity and expression of certain isoenzymes of hepatic cytochrome P450 (P450). The aim of this study was to document whether hypoxia a ects hepatic P450 directly or through the release of serum mediators. 2 Rabbits were subjected to a FiO 2 of 8% for 48 h, sacri®ced, and serum and hepatocytes were isolated; hepatocytes from control and rabbits with hypoxia were incubated with serum from control and hypoxic rabbits for 4 and 24 h, and total P450 content, CYP1A1, 1A2 and 3A6 activities and expressions were assessed. Sera were fractionated by size exclusion chromatography and fractions tested for their ability to modify activity and amount of P450, and serum mediators were identi®ed through neutralization experiments. 3 Total serum and fractions with proteins of 15 ± 23 and 65 ± 94 kDa of M r reduced P450 content and expression of CYP1A1, 1A2 and 3A6, as well as CYP1A1, 1A2 and 3A6 mRNA. Total serum and the fraction with 32 ± 44 kDa proteins increased CYP3A6 activity and protein and mRNA. The serum mediators implicated in the decrease in activity and expression of CYP1A1, 1A2 and 3A6 were interferon-g (IFN-g), interleukin-1b (IL-1b) and IL-2. Erythropoietin (Epo) was partly responsible for the increase in P450 content and CYP3A6 expression. 4 In conclusion, acute moderate hypoxia diminishes the activity and expression of CYP1A1, 1A2 and CYP1A1, 1A2 mRNA, and increases CYP3A6 protein, activity and CYP3A6 mRNA. Several mechanisms contribute to these changes in P450, among them the release of cytokines acting as serum mediators.

Animal Models of Acute Moderate Hypoxia Are Associated with a Down-Regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and Up-Regulation of CYP3A6 and P-glycoprotein in Liver

“…To induce hypoxia, rabbits were placed in a Plexiglas chamber (0.75 ϫ 1.20 ϫ 1.25 m 3 ) with a FiO 2 of 8%, adjusted with an oxygen monitor (OM-15; SensorMedics Corp., Yorba Linda, CA) connected to an electrovalve (Asco Valves, Brantford, ON, Canada), which uses nitrogen to displace oxygen from the chamber. This FiO 2 generates a stable arterial partial pressure of O 2 of approximately 35 mm Hg (Fradette et al, 2002;Fradette and du Souich, 2003). Control rabbits were placed into the Plexiglas chamber containing room air (FiO 2 ϭ 21%) for the experiments.…”

“…Proteins in the serum were separated by size exclusion high performance liquid chromatography (HPLC) on a Superose 12 HR column from GE Healthcare BioSciences Inc. (Baie d'Urfé, QC, Canada) as described previously (Fradette et al, 2002). In brief, the flow rate of the mobile phase was set at 0.3 ml min Ϫ1 and column pressure was maintained between 9 and 12 bar with an LKB (Bromma, Sweden) 2150 HPLC pump.…”

“…To prevent the changes in P450 isoform expression induced by the HPLC fractions, the following antibodies were used: a goat anti-rabbit IL-1␤ (anti-IL-1␤) polyclonal antibody, and anti-human IL-2 (anti-IL-2), IFN-␥ (anti-IFN-␥), IL-6 (anti-IL-6), and Epo (anti-Epo) monoclonal antibodies. The antibodies against human proteins were used to neutralize the homologous rabbit proteins because of the known interspecies reactivity of these antibodies (Fradette et al, 2002). An IgG monoclonal antibody to Pseudomonas aeruginosa served as control.…”

“…Aliquots of 2 g of each antibody were added individually to 200 l of the HPLC fractions that were able to decrease the expression of P450 isoforms, and were incubated at 37°C for 1 h. The antibody and the HPLC fractions were incubated with the hepatocytes for 24 h when P450 isoform expression was assessed. The amount of 2 g of antibodies was used because this amount is effective to immunoneutralize IL-1␤, IL-6, and IFN-␥ in the sera of rabbits and of humans with an inflammatory reaction (Fradette et al, 2002).…”

“…Rabbits exposed to a fractional concentration of inspired O 2 (FiO 2 ) of 10% for 24 h demonstrate a reduced clearance of theophylline and decreased expression of CYP1A1 and 1A2, although the expression of CYP3A6 is increased (Kurdi et al, 1999). In rabbits subjected to acute moderate hypoxia, hepatic down-regulation of CYP1A1 and 1A2 is triggered by serum mediators, e.g., interferon-␥ (IFN-␥), interleukin 2 (IL-2), and IL-1␤; the up-regulation of CYP3A6 is, at least in part, associated with erythropoietin (Epo) (Fradette et al, 2002). In vivo, the increase in CYP3A6 by hypoxia is associated with the increased expression and nuclear translocation of hypoxia-inducible factor-1 (HIF-1), activator protein 1 (AP-1), and constitutive androstane receptor (Fradette and du Souich, 2003).…”