Yasuhiro Masubuchi

Th1/Th2 cytokine balance as a determinant of acetaminophen-induced liver injury.

Inflammation is an important pathophysiological event in drug-induced liver injury, which is subsequent to metabolic activation and covalent binding of the reactive metabolites to target proteins. Cytokines are recognized as pro- and anti-inflammatory mediators involved in the progression and regression of the toxicity. We thus hypothesized that disturbed balance of Th1/Th2 cytokines exacerbated the drug-induced hepatotoxicity. Acetaminophen-induced liver injury was investigated in two mouse strains, C57BL/6 and BALB/c, which develop predominantly Th1 and Th2 responses, respectively. More severe liver injury after intraperitoneal administration of acetaminophen was observed in C57BL/6 mice than in BALB/c mice. There was no strain difference in metabolism of acetaminophen into its reactive metabolite, N-acetyl-p-benzoquinone imine, which was assessed by early glutathione consumption. Liver mRNA expression of tumor necrosis factor-alpha (TNF-alpha) and IL-6 were measured as pro- and anti-inflammatory cytokines, respectively. TNF-alpha was highly induced 24 h after administration of acetaminophen in C57BL/6 mice, whereas no change in BALB/c mice. On the other hand, liver IL-6 mRNA expression in BALB/c mice was higher than C57BL/6 mice 24 h after the administration. In addition, treatment of CD-1 mice, another susceptible strain, with an anti-inflammatory polyphenol, resveratrol, protected mice against the acetaminophen-induced liver injury, and the mice with attenuated toxicity revealed lower expression of TNF-alpha and higher expression of IL-6. It is therefore suggested that acetaminophen-induced liver injury is associated with Th1-dominant response in Th1/Th2 cytokine balance, and TNF-alpha may play a pathological role in the toxicity.

Toxicological Significance of Mechanism-Based Inactivation of Cytochrome P450 Enzymes by Drugs

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine.Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes.

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.

Sex difference in susceptibility to acetaminophen hepatotoxicity is reversed by buthionine sulfoximine

Gender is a factor that influences susceptibility of individuals to drug-induced liver injury in experimental animals and humans. In this study, we investigated the mechanisms underlying resistance of female mice to acetaminophen (APAP)-induced hepatotoxicity. Overnight-fasted male and female CD-1 mice were administered APAP intraperitoneally. A minor increase in serum alanine aminotransferase levels was observed in female mice after APAP administration at a dose that causes severe hepatotoxicity in males. Hepatic glutathione (GSH) depleted rapidly in the both genders prior to development of hepatotoxicity, whereas its recovery was more rapid in female than in male mice. This was consistent with higher induction of hepatic glutamate-cysteine ligase (GCL) in females. Pretreatment of mice with L-buthionine sulfoximine (BSO), an inhibitor of GCL, exaggerated APAP hepatotoxicity only in female mice, resulting in much higher hepatotoxicity in female than in male mice. In addition, hepatic GSH was markedly depleted in BSO-pretreated female mice compared with male mice, which supports severe hepatotoxicity in BSO-pretreated females. APAP treatment highly induced multidrug resistance-associated protein 4 (Mrp4) only in female mice. The resulting high Mrp4 expression could thus contribute to decreased hepatic GSH levels via sinusoidal efflux when GCL is inhibited. In conclusion, resistance to APAP hepatotoxicity in female mice and its reversal by pretreatment with BSO could be attributed to sex differences in disposition of hepatic GSH, which may generally determine susceptibility to drug-induced liver injury.

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4‐hydroxylase of 1,2,3,4‐tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean ± se) for hepatic microsomal TIQ 4‐hydroxylase of male Wistar rats were 319.6 ± 26.8 μm and 12.13 ± 1.43 pmol·min–1·mg–1 protein, respectively. When TIQ 4‐hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar > Dark Agouti) and sex (male > female) differences were observed. The microsomal activity toward TIQ 4‐hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (β‐naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4‐hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4‐hydroxylase activity of 64.1 pmol · min–1 · nmol P450–1, which was 3.2‐fold that of microsomes (20.9 pmol · min–1 · nmol P450–1). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4‐hydroxylase activity concentration‐dependently. A male‐specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol·min–1·nmol P450–1), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4‐hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non‐treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4‐hydroxylation in rats pretreated with P448‐inducers.—Suzuki, T.; Fujita, S.; Narimatsu, S.; Masubuchi, Y.; Tachibana, M.; Ohta, S.; Hirobe M. Cytochrome P450 isozymes catalyzing 4‐hydroxylation of parkinsonism‐related compound 1,2,3,4‐tetrahydroisoquinoline in rat liver microsomes. FASEB J. 6: 771‐776; 1992.

Differential selectivity in carbamazepine-induced inactivation of cytochrome P450 enzymes in rat and human liver.

Abstract. Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.

Evaluation of damaged small intestine of mouse following methotrexate administration.

Purpose: Methotrexate (MTX) treatment causes damage to the small intestine, resulting in malabsorption and diarrhea. The active and passive transport capacities of the small intestine are decreased by the treatment. The purpose of this study was to evaluate the damage to the small intestine of mice caused by MTX administration by examining the permeability of the paracellular pathway of the small intestinal epithelium. Methods: MTX was administered orally to male ddY mice once daily for 1–6 days. The permeability of the small intestine to the nonabsorbable markers phenol red (PR) and fluorescein isothiocyanate (FITC) dextrans was examined using everted segments of the intestine. Results: PR and FITC dextran permeation through the small intestine increased significantly in parallel with changes in body weight of the mice, wet weight of the small intestine and chemical composition of the small intestinal epithelium. Conclusions: In addition to changes in permeation through the transcellular pathway reported previously, this study revealed that MTX treatment disorders the paracellular barrier function of the small intestinal epithelium, resulting in increased permeation of nonabsorbable markers via the paracellular pathway of the small intestinal mucosa. The present approach to the examination of the barrier function of the intestinal epithelium could be of great use in evaluating the damage to the small intestine and malabsorption.

Docosahexaenoic acid exhibits a potent protection of small intestine from methotrexate-induced damage in mice

Oral administration of methotrexate (MTX) to mice causes the damage of small intestine. The permeability of poorly absorbable compound fluorescein isothiocyanate (FITC)-labeled dextran (average molecular weight, 4,400) through the small intestine was studied in vitro using everted segments of the small intestine. The permeability of FITC-dextran increased remarkably in the MTX-treated mice and oral administration of docosahexaenoic acid ethyl ester (DHA) protected the small intestine from the increase in the small intestinal permeability induced by the MTX treatment. The MTX treatment decreased retinol concentration in plasma of mice and the coadministration of DHA maintained its concentration to the level of control mice. The present study showed that DHA protected the small intestine of mice from the MTX-induced damage.

Impairment of debrisoquine 4-hydroxylase and related monooxygenase activities in the rat following treatment with propranolol.

The effect of repetitive oral administration of propranolol on hepatic microsomal drug metabolizing enzyme activities in the rat was investigated. Propranolol ring (4-, 5- and 7-)hydroxylase activities were markedly decreased, but, interestingly, N-desisopropylase activity was increased after propranolol administration. A marked decrease in enzyme activity after propranolol pretreatment was also observed with debrisoquine 4-hydroxylation. In addition, a similar decrease was observed with imipramine 2-hydroxylation which co-segregates with debrisoquine/sparteine type polymorphic drug oxidation, but not with imipramine N-demethylation. These results suggest the selective impairment of debrisoquine 4-hydroxylase by propranolol pretreatment.

Down-Regulation of Hepatic Cytochrome P450 Enzymes in Rats with Trinitrobenzene Sulfonic Acid-Induced Colitis

Hepatic cytochrome P450 (P450) enzymes are down-regulated during inflammation. In this study, an animal model of inflammatory bowel disease was subjected to characterization of hepatic P450 expression under inflammatory conditions. Rats were treated intracolonically with 100 mg/kg trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol, and homogenates of colonic mucosa and hepatic microsomes of the rats were prepared. The colitis was accompanied by appearance of higher levels of portal endotoxin, interleukin-6, and nitric oxide metabolites and decreases in contents and activities for hepatic CYP3A2, CYP2C11, and, to a lesser extent, CYP1A2 and CYP2E1. Nimesulide, a preferential COX-2 inhibitor, protected rats with TNBS-induced colitis (TNBS-colitis) against the down-regulation of hepatic CYP3A2. Polymyxin B, which neutralizes endotoxin, curcumin, which has anti-inflammatory properties, and gadolinium chloride, which inactivates macrophages, attenuated the down-regulation of CYP3A2. Similar effects were observed in other P450s such as CYP2C11, but the agents were less effective in attenuating the down-regulation. Our data suggest that endogenous substances leaked from damaged colon in the rats with TNBS-colitis activate Kupffer cells, leading to down-regulation of hepatic P450s with differential susceptibility to the inflammatory stimuli. The colitis model, instead of exogenous administration of lipopolysaccharide or cytokines, could be applied to the study on mechanisms for altered hepatic P450 expression and other liver functions under mild inflammatory conditions.