Pregnane X receptor and constitutive androstane receptor modulate differently CYP3A-mediated metabolism in early- and late-stage cholestasis

Abstract

Aim: To ascertain whether cholestasis affects the expression of two CYP3A isoforms (CYP3A1 and CYP3A2) and of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

Methods: Cholestasis was induced by bile duct ligation in 16 male Wistar rats; whereas 8 sham-operated rats were used as controls. Severity of cholestasis was assessed on histological examination of liver sections, and serum concentrations of albumin, AST, ALT, GGT, ALPK and bilirubin. Gene and protein expressions of PXR, CAR, CYP3A1 and CYP3A2 were assessed by means of qRT-PCR and Western blot, respectively. Alterations in CYP3A activity were measured by calculating the kinetic parameters of 4-OH and 1'-OH-midazolam hydroxylation, marker reactions for CYP3A enzymes.

Results: The mRNA and protein expression of CYP3A1 increased significantly in mild cholestasis (P < 0.01). At variance, mRNA and protein expression of CYP3A2 didn't change in mild cholestasis, whereas the expression and activity of both CYP3A1 and CYP3A2 decreased dramatically when cholestasis became severe. Consistently with these observations, the nuclear expression of both PXR and CAR, which was measured because they both translocate into the cell nucleus after their activation, virtually disappeared in the late stage of cholestatic injury, after an initial increase. These results indicate that early- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently, probably as consequence of the different activation of PXR and CAR.

Conclusion: Early- and late-stage cholestasis affects CYP3A-mediated drug metabolism differently. PXR and CAR might be targeted therapeutically to promote CYP3A-mediated liver detoxification.

Keywords: CYP3A; Cholestasis; Constitutive androstane receptor; Drug metabolism; Pregnane X receptor.

Figure 1

Histological analyses of rat livers. Representative photomicrographs of liver sections taken from sham-operated (A), mildly cholestatic (B) and severely cholestatic rats (C), stained with van Gieson to detect liver fibrosis (magnification × 5).

Figure 2

mRNA expression of CYP3A isoforms in rat livers. Gene expression of CYP3A1 (A) and CYP3A2 (B). Results are mean ± SEM, obtained from 8 rats of each group. ^aP < 0.05, ^bP < 0.01 vs sham-operated rats; ^eP < 0.01 and ^fP < 0.001 vs rats with mild cholestasis.

Figure 3

Microsomal protein expression of CYP3A isoforms in rat livers. A representative Western blot experiment showing CYP3A1 (A) and CYP3A2 (B) protein expression in microsomal fractions obtained from sham-operated and cholestatic rats. Densitometric analysis of CYP3A1 (C) and CYP3A2 (D) protein bands. Results are mean ± SEM obtained from 8 rats of each group. Each experiment was conducted in triplicate. ^aP < 0.05, ^bP < 0.01 vs sham rats; ^eP < 0.01, ^fP < 0.001 vs mildly cholestatic rats.

Figure 4

Enzymatic activity of CYP3A in microsomes obtained from sham-operated, mildly and severely cholestatic rats. Kinetics of 4- and 1’- hydroxylation activities of liver microsomes obtained from sham-operated, mildly cholestatic and severely cholestatic rats. Results are means ± SEM of data obtained from 8 rats per group. For each rat, enzymatic activity was tested in duplicate.

Figure 5

mRNA expression of nuclear receptors in rat livers. Pregnane x receptor (A) and constitutive androstane receptor (B) gene expression in sham-operated and cholestatic rats reported as fold variations compared with sham rats. Results are mean ± SEM obtained from 8 rats in each group. ^aP < 0.05, ^cP < 0.001 vs sham rats; ^dP < 0.05, ^fP < 0.01 vs mildly cholestatic rats.

Figure 6

Nuclear protein expression of nuclear receptors in rat livers. Representative Western blot membrane showing pregnane x receptor and constitutive androstane receptor protein expression in the cytoplasmic and nuclear fractions of sham and cholestatic rats (A). GAPDH and Histone H3 were used to assess the purity of the cytoplasmic and nuclear fractions, respectively. Densitometric analysis of proteins in the nuclear fraction representing the PXR (B) and CAR (C) nuclear expression. Results are expressed as mean ± SEM obtained from 8 rats in each group. ^aP < 0.05, ^cP < 0.001 vs sham rats; ^dP < 0.05, ^fP < 0.001 vs mildly cholestatic rats.