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

Table 1 Primer sequences used in the qRT-PCR analysis and NCBI reference sequences

Gene	Forward primer (5’-3’)	Reverse primer (5’-3’)	RefSeq
CYP3A1	CCATCACGGACACAGAAATG	CTTTCCCCATAATCCCCACT	NM013105
CYP3A2	AGTGGGGATTATGGGGAAAG	CAATGATGGGGGAACATCTCC	NM153312
PXR	CAAATCTGCCGTGTATGTGG	GTTTCATGGCCCTTCTGAAA	NM052980
CAR	GAGGAAAGACATGATACTGTCAG	GTCTGGATGAGCTCTTTCTGCT	NM022941
β-actin	GCCACCAGTTCGCCATGGA	TTCTGACCCATACCCACCAT	NM031144

Table 2 Biochemical liver function parameters and Ishak scores

	Sham-operated rats	Mildly cholestatic rats	Severely cholestatic rats
Albumin (g/L)	36.1 ± 3.3	35.8 ± 3.8	31.2 ± 3.7ad
ALT (U/L)	43.7 ± 8.1	44.2 ± 8.8	115.3 ± 32.4cf
AST (U/L)	92.2 ± 41.6	103.5 ± 33.0	402.7 ± 136.6cf
γGT (U/L)	0.1 ± 0.6	0.5 ± 0.5	52.4 ± 34.8be
ALKP (U/L)	133.9 ± 47.1	150.8 ± 38.0	320.1 ± 107.2cf
Conjugated bilirubin (mmol/L)	0.8 ± 0.3	0.7 ± 0.3	133.1 ± 56.9cf
Total bilirubin (mmol/L)	1.1 ± 0.6	1.0 ± 0.3	151.2 ± 64.1cf
Ishak score	0	1-3 (1)	4-6 (5)

The results are given as mean ± SD obtained from 8 rats of each group. Ishak scores are given as range (median).

^aP < 0.05;

^bP < 0.01;

^cP < 0.001 vs sham-operated rats;

^dP < 0.05;

^eP < 0.01;

^fP < 0.001 vs mildly cholestatic rats.

Table 3 Kinetic parameters for 4- and 1’-hydroxylation activities of microsomal preparations obtained from sham-operated and cholestatic rats

	4-OH MDZ		1’-OH MDZ
	Vmax (pmol/mg/min)	Km (μmol/L)	Vmax (pmol/mg/min)	Km (μmol/L)
Sham-operated rats	1009.9 ± 269.4	3.4 ± 1.1	946.7 ± 292.2	2.5 ± 0.9
Mildly cholestatic rats	1116.0 ± 333.7	3.5 ± 1.4	1017.0 ± 317.4	2.8 ± 1.6
Severely cholestatic rats	218.4 ± 88.1cf	6.1 ± 4.7	200.1 ± 68.7cf	4.3 ± 2.6

The results are reported as mean ± SD of 8 rats per group.

^cP < 0.001 vs sham-operated rats;

^fP < 0.001 vs mildly cholestatic rats.