Resistance to indomethacin-induced down-regulation of hepatic cytochrome P450 enzymes in the mice with non-functional Toll-like receptor 4 Yasuhiro Masubuchi,

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Resistance to indomethacin-induced down-regulation of hepatic cytochrome P450 enzymes in the mice with non-functional Toll-like receptor 4 Yasuhiro Masubuchi, Toshiharu Horie Journal of Hepatology Volume 39, Issue 3, Pages 349-356 (September 2003) DOI: 10.1016/S0168-8278(03)00244-7

Fig. 1 Effects of pretreatment with indomethacin on testosterone 6β-hydroxylation activity in liver microsomes of LPS-sensitive and -resistant mice. LPS-sensitive and -resistant mice were treated with intraperitoneal indomethacin (5 mg/kg per day, 3 days), and the liver microsomes were prepared 24 h after the final administration. Testosterone 6β-hydroxylation activities in the control (open column) and treated (closed column) mice were determined for the index for CYP3A11. Results are means±SE of 5 mice. **, Significantly different from controls (P<0.01); and ##, significantly different from LPS-sensitive mice (P<0.01). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 2 Effects of pretreatment with indomethacin on testosterone 16α-hydroxylation activity in liver microsomes of LPS-sensitive and -resistant mice. Liver microsomes were prepared form mice as described in legend for Fig. 1. Testosterone 16α-hydroxylation activities in the control (open column) and treated (closed column) mice were determined for the index for CYP2D9. Results are means±SE of five mice. **, Significantly different from controls (P<0.01); and ##, significantly different from LPS-sensitive mice (P<0.01). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 3 Effects of pretreatment with indomethacin on phenacetin O-deethylation activity in liver microsomes of LPS-sensitive and -resistant mice. Liver microsomes were prepared form mice as described in legend for Fig. 1. Phenacetin O-deethylation activities in the control (open column) and treated (closed column) mice were determined for the index for CYP1A2. Results are means±SE of five mice. **, Significantly different from controls (P<0.01); and ##, significantly different from LPS-sensitive mice (P<0.01). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 4 Effects of pretreatment with indomethacin on 4-nitrophenol hydroxylation activity in liver microsomes of LPS-sensitive and -resistant mice. Liver microsomes were prepared form mice as described in legend for Fig. 1. 4-Nitrophenol hydroxylation activities in the control (open column) and treated (closed column) mice were determined for the index for CYP2E1. Results are means±SE of five mice. Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 5 Effects of pretreatment with indomethacin on protein expression of CYP3A enzyme in liver microsomes of LPS-sensitive and -resistant mice. Liver microsomes were prepared form mice as described in legend for Fig. 1. The microsomal proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane. The membrane was treated with the antibody to rat CYP3A2 and the immunoblots were developed with the enhanced chemiluminescence detection method. Upper panel was a typical result including control LPS-sensitive mice (A); indomethacin-treated LPS-sensitive mice (B); control LPS-resistant mice (C); and indomethacin-treated LPS-resistant mice (D). The intensities of the stained bands were measured by laser densitometry, and the intensities in the control (open column) and treated (closed column) mice to those of control LPS-sensitive mice were calculated as the relative CYP isozyme contents. Results are means±SE of three mice. *, **, Significantly different from controls (P<0.05, P<0.01); and #, significantly different from LPS-sensitive mice (P<0.05). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 6 Effects of pretreatment with indomethacin on protein expression of CYP2D enzyme in liver microsomes of LPS-sensitive and -resistant mice. Liver microsomes were prepared form mice as described in legend for Fig. 1. Immunoblot analysis of the microsomes was performed as described in legend for Fig. 5, except for using the antibody to rat CYP2D2. Results are means±SE of three mice. **, Significantly different from controls (P<0.01); and #, significantly different from LPS-sensitive mice (P<0.05). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 7 Effects of pretreatment with indomethacin on portal endotoxin concentration in LPS-sensitive and -resistant mice. LPS-sensitive and LPS-resistant mice were treated with intraperitoneal indomethacin (5 mg/kg per day, 3 days), and the blood was collected from portal vein 24 h after the final administration. Serum endotoxin concentrations were measured in the control (open column) and treated (closed column) mice. Results are means±SE of five mice. *, Significantly different from controls (P<0.05); and #, significantly different from LPS-sensitive mice (P<0.05). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 8 In vitro inactivation of microsomal CYPs during oxidative metabolism of indomethacin. Microsomes were preincubated with indomethacin in the absence (open column) or presence (closed column) of NADPH, followed by assay of marker enzyme activities described in Figs. 1–4, which indicate corresponding CYP isozymes (3A, CYP3A11; 2D, CYP2D9; 1A, CYP1A2; 2E, CYP2E1). Relative activities indicate percentage of the control activities obtained from preincubation without indomethacin. **, Significant potentiation of the inhibition as compared with preincubation without NADPH (P<0.01). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)

Fig. 9 Effects of pretreatment with PGN on monooxygenase activities in mouse liver microsomes. Mice were treated with intraperitoneal PGN (4 μg/kg), and the liver microsomes were prepared 24 h after the administration. Testosterone 6β-hydroxylation (3A), 16α-hydroxylation (2D), phenacetin O-deethylation (1A) and 4-nitrophenol hydroxylation (2E) activities of the microsomes were determined. Relative activities indicate percentage of the activities in the control mice. Results are means±SE of five mice. *, Significantly different from controls (P<0.05). Journal of Hepatology 2003 39, 349-356DOI: (10.1016/S0168-8278(03)00244-7)