Multiple mechanisms in indomethacin-induced impairment of hepatic cytochrome P450 enzymes in rats  Yasuhiro Masubuchi, Emi Masuda, Toshiharu Horie  Gastroenterology  Volume 122, Issue 3, Pages 774-783 (March 2002) DOI: 10.1053/gast.2002.31886 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Inactivation of CYP3A2 but not CYP2C11 during incubation of microsomes with indomethacin in the presence of NADPH. Liver microsomes from untreated male rats were preincubated without (○) or with (●, 100 μmol/L; ▵(, 500 μmol/L; ▴, 1 mmol/L) indomethacin in the presence of NADPH, followed by assay of (A) testosterone 6β-hydroxylation and (B) 16α-hydroxylation activities. Remaining activities are represented as percent of the activity obtained without the preincubation. Time-dependent decreases in the activities were shown as semilogarithmic plots, and pseudo–first-order kinetic constants for the inactivation (k) were obtained for each indomethacin concentration. The reciprocal of k was plotted against the reciprocal of the indomethacin concentration (A, inset). Results are means ± SE of 3 different microsomes. Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Lack of effect of preincubation of microsomes with indomethacin on monooxygenase activities in rat liver microsomes. Liver microsomes were preincubated without (○) or with (●) indomethacin (500 μmol/L) in the presence of NADPH, followed by assay of (A) ethoxyresorufin O-deethylation, (B) pentoxyresorufin O-depentylation, and (C) 4-nitrophenol hydroxylation activities. Remaining activities are represented as percentage of the activity obtained without the preincubation. Results are means ± SE of 3 different microsomes. Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Time course of covalent binding of 14C-labeled materials to liver microsomal proteins after incubation of microsomes with [14C]indomethacin and NADPH. Liver microsomes from untreated male Wistar rats were incubated with [14C]indomethacin (25 μmol/L) in the presence (●) or absence (○) of NADPH. After the mixture was washed as described in Materials and Methods, remaining radioactivities were measured to determine the amounts of covalent bound materials to liver microsomal proteins. Results are means ± SE of 4 different microsomes. *P < 0.05, significantly different from control. Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of incubation conditions on covalent binding of 14C-labeled materials to liver microsomal proteins after incubation of microsomes with [14C]indomethacin and NADPH. Liver microsomes from untreated male Wistar rats were incubated with [14C]indomethacin (25 μmol/L) in the presence (+) or absence (−) of NADPH. The microsomes were also coincubated with SKF-525A (100 μmol/L), troleandomycin (TAO; 1 mmol/L), and reduced glutathione (GSH; 1 mmol/L) in the presence of [14C]indomethacin and NADPH. After the mixture was washed as described in Materials and Methods, remaining radioactivities were measured to determine the amounts of covalent bound materials to liver microsomal proteins. Results are means ± SE of 3–6 different microsomes. *P < 0.05, **P < 0.01, significantly different from NADPH(+). Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of pretreatment of rats with GdCl3 and aminoguanidine on indomethacin-induced decreases in testosterone 6β-hydroxylation and testosterone 16α-hydroxylation activities in vivo. Rats were pretreated with GdCl3 (7.5 mg/kg) or aminoguanidine (AG; 100 mg · kg−1 · day−1 for 3 days) and were treated with indomethacin (▨, 10 mg/kg) or vehicle (□) 24 hours after the final dose of each compound. Liver microsomes were prepared 24 hours after the indomethacin challenge, followed by assay of (A) testosterone 6β-hydroxylation and (B) testosterone 16α-hydroxylation activities. Results are means ± SE of 3–6 rats. *P < 0.05, **P < 0.01, significantly different from control (no indomethacin). Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of pretreatment of rats with GdCl3 and aminoguanidine (AG) on indomethacin-induced decreases in phenacetin O-deethylation and 4-nitrophenol hydroxylation activities. Rats were treated in the same manner as described in Figure 5. After treatment, liver microsomes were subjected to the assay of (A) phenacetin O-deethylation and (B) 4-nitrophenol hydroxylation activities. Results are means ± SE of 3–6 rats. *P < 0.05, **P < 0.01, significantly different from control (no indomethacin). #P < 0.05, ##P < 0.01, significantly different from control (no GdCl3 or aminoguanidine). Gastroenterology 2002 122, 774-783DOI: (10.1053/gast.2002.31886) Copyright © 2002 American Gastroenterological Association Terms and Conditions