Effect of synthetic antioxidants on hydrogen peroxide formation, oxyferro cytochrome P-450 concentration and oxygen consumption in liver microsomes

Synthetic antioxidants lead in vitro to increased H2O2 formation in rat liver and lung microsomes and in guinea pig and hamster liver microsomes. Butylated hydroxyanisole and ethoxyquin are more potent than propyl-, octyl-, and dodecyl gallate; butylated hydroxytoluene is only weakly active. Extra production of H2O2 is maximal at antioxidant concentrations between 50 and 500 microM and is dependent on the concentration of NADPH. It is paralleled by increased microsomal oxygen consumption and decreased concentration of oxycytochrome P-450 and is enhanced by pretreatment of the animals with phenobarbital. Both the endogenous and the antioxidant-stimulated H2O2 production are inhibited by metyrapone. In vivo administration of ethoxyquin and butylated hydroxyanisole in the diet leads to decreased oxycytochrome P-450 concentrations but not to increased H2O2 formation in liver microsomes. No extra production of H2O2 was observed in a glucose oxidase or xanthine oxidase system; rather, inhibition occurred in the latter system. Our data suggest that antioxidants enhance the oxidase function of cytochrome P-450. This effect is discussed in view of the known toxicity of these food additives.     

大鼠正常肝脏和癌前病变肝脏微粒体谷胱甘肽 S-转移酶的纯化及其鉴定

本文通过 DE_(52)-纤维素和羟基磷灰石柱层析及 GSH-Sepharose-4B 亲和层析法,分离纯化了正常大鼠肝脏和癌前病变肝脏微粒体 GST,得到了三个微粒体 GST 同工酶组分,分别命名为A_1,A_2,A_3.用 SDS 聚丙烯酰胺凝胶电泳法测得其亚基分子量分别为25000D,26000D,43000D.以 CDNB 为底物按 Habig 方法测定 GST 活性,发现大鼠肝脏癌前病变微粒体同工酶 A_1和 A_2的活性显著增高,而同工酶 A_3在正常和癌前病变大鼠之间无显著差异.本实验结果表明,大鼠肝脏癌前病变组织的微粒体 GST 同工酶组分中 A_1和 A_2显著增高,可作为肝脏癌前病变的酶学指标.     

Involvement of multiple cytochrome P450 isoforms in naproxen O-demethylation

Objective: A series of studies was undertaken to determine the cytochrome P450 isoform(s) involved in naproxen demethylation and whether this included the same isoforms reported to be involved in the metabolism of other NSAIDs. Methods: (S)-Naproxen was incubated with human liver microsomes in the presence of a NADPH-generating system and the formation of desmethylnaproxen was measured by high-performance liquid chromatography (HPLC). To further clarify the specific isoforms involved, experiments were conducted with preparations expressing only a single P450 isoform (vaccinia virus-expressed cells and microsomes derived from a lymphoblastoid cell line, each transfected with specific P450 cDNAs) as well as inhibition studies using human liver microsomes and putative specific P450 inhibitors. Results: In human liver microsomes (n=7), desmethylnaproxen formation was observed with a mean k_M of 92 (21) μmol · l⁻¹, V_max of 538 pmol · min⁻¹ · mg⁻¹ protein and C_int2 (reflective of a second binding site) of 0.36 μl · min⁻¹ · mg⁻¹ protein. This C_int2 term was added since Eadie-Scatchard analysis suggested the involvement of more than one enzyme. Studies using putative specific P450 inhibitors demonstrated inhibition of this␣reaction by sulfaphenazole, (apparent Ki= 1.6 μmol · l⁻¹), warfarin (apparent Ki=27 μmol · l⁻¹), piroxicam (apparent Ki=23 μmol · l⁻¹) and tolbutamide (apparent Ki=128 μmol · l⁻¹). No effect was observed when α-naphthoflavone and troleandomycin were employed as inhibitors, but reaction with furafylline produced, on average, a maximum inhibition of 23%. At a naproxen concentration of 150 μmol · l⁻¹, formation of desmethylnaproxen was observed in cells expressing P450 1A2, 2C8, 2C9 and its allelic variant 2C9R144C. To further characterize these reactions, saturation kinetics experiments were conducted for the P450s 1A2, 2C8 and 2C9. The k_M and V_max for P450 1A2 were 189.5 μmol · l⁻¹ and 7.3 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Likewise, estimates of k_M and V_max for P450 2C9 were 340.5 μmol · l⁻¹ and 41.4 pmol · min⁻¹ · pmol⁻¹ P450, respectively. Reliable estimates of k_M and V_max could not be made for P450 2C8 due to the nonsaturable nature of the process over the concentration range studied. Conclusion: Multiple cytochrome P450 isoforms (P450 1A2, 2C8 and 2C9) appear to be involved in naproxen demethylation, although 2C9 appears to be the predominant form. Received: 16 September 1996 / Accepted in revised form: 20 December 1996     

Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine

Summary Human hepatic microsomes were isolated from wedge biopsies of the liver from 13 patients undergoing abdominal surgery. Ultrasonic homogenisation was used to increase the yield of microsomal monooxygenase activity (7-ethoxycoumarin O-deethylase, NADPH-cytochrome c reductase), resulting in a 30% higher total enzyme activity per g liver than preparation by other techniques. In 4 individual microsomal preparations the influence of cimetidine and ranitidine on Michaelis-Menten kinetics of O-deethylation and of reductase activity were studied. Without the H₂-receptor blocking drugs, enzyme kinetics of O-deethylation with a K_m of 51.0±16.4 µM (n=3) were obtained using Lineweaver-Burke plots. Both, cimetidine and ranitidine inhibited the O-deethylation; cimetidine had a five-fold higher inhibitory affinity (K_i 1.01 and 3.94 mM) to the monooxygenase than ranitidine (K_i 4.96 and 17.70 mM) in the uninduced liver. However, in liver from a patient with induced enzyme activity (K_m=478.0 µM), the K_i of ranitidine was similar to that of cimetidine (K_i ran 3.57 versus K_i cim 2.49 mM). The reductase activity was not inhibited by ranitidine and only marginally so by cimetidine.The results suggest that in human hepatic microsomes oxidative drug metabolism is inhibited by both H₂-receptor antagonists. However, the inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes. Thus, while cimetidine is an relatively nonspecific enzyme inhibitor, ranitidine might more selectively inhibit induced drug metabolizing enzymes.     

Comparison of the metabolism of the three antidepressants amitriptyline,imipramine, and chlorimipramine in vitro in rat liver microsomes

The metabolism of the tricyclic antidepressants amitriptyline (AMI), imipramine (IMI), chlorimipramine (CMI) and some of their metabolites was studied in vitro in isolated liver microsomes of female Spraque-Dawley rats. Nine metabolites of AMI, seven metabolites of IMI, and 11 metabolites of CMI were quantitatively determined with high-performance liquid chromatography. The main metabolic reactions, mediated by an NADPH generating system, were hydroxylation, demethylation, and N-oxidation. The ratio of these reactions was different for the three drugs. AMI was hydroxylated more than CMI and CMI more than IMI. The order for demethylation was CMI>AMI=IMI, the order for N-oxidation IMI>CMIAMI. The substrate dependence of metabolism was investigated. Demethylation and N-oxidation increased proportionally to increasing substrate concentrations, whereas formation of hydroxylated metabolites became saturated (in the concentration range of 10^–6–10^–5 M). The in vitro metabolism was compared with the in vivo metabolism in humans, reflected by the plasma concentrations of these drugs and their metabolites. A good agreement in metabolic pathways was found.     

Monoamine oxidase B oxidizes a novel multikinase inhibitor KW-2449 to its iminium ion and aldehyde oxidase further converts it to the oxo-piperazine form in human

(E)-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}piperazine (KW-2449) is a novel multikinase inhibitor. During our clinical study, we found that KW-2449 is mainly metabolized to its oxo-piperazine form (M1). An inhibition study suggested that monoamine oxidase-B (MAO-B) oxidizes KW-2449 to an iminium (intermediate) and aldehyde oxidase (AO) then metabolizes the intermediate to M1. The conversion of KW-2449 to the iminium (intermediate) by MAO-B was confirmed by the formation of its cyanide adduct. This cooperative metabolic pathway by MAO-B and AO was newly identified in the metabolism of piperazine. The clearance of KW-2449 by MAO-B and AO in human was estimated based on the kinetic analysis with in vitro-in vivo extrapolation. The systemic clearance in human was similar to the calculated value, indicating that the extrapolation approach was applicable to KW-2449 metabolism. Finally, we found that (E)-3-amino-1-{4-[2-(1H-Indazol-3-yl)vinyl]benzoyl}-pyrrolidine (Compound A) as a stable compound against MAO-B and AO. The total body clearance of Compound A was reduced to one tenth of KW-2449, demonstrating that preventing the metabolism of MAO and AO led to more preferable pharmacokinetic profiles. As piperazine is often introduced to drug candidates to improve lipophilicity of the compound to get more hydrophilic nature, the results of this study provide useful information for future drug development.     

Identification and interspecies characterization of UDP-glucuronosyltransferase isoforms catalyzing acacetin glucuronidation using recombinant UGT enzymes and microsomes

ObjectiveTo explore the glucuronic acid metabolism of acacetin in human liver and intestinal microsomes to better characterize human uridine 5′-diphospho (UDP)-glucuronosyltransferase (UGT) isoforms. In addition, interspecies comparisons were performed to identify the most appropriate experimental animal model for an in vivo study.MethodsLiquid chromatography tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) were used to confirm the successful biosynthesis of acacetin-7-O-glucuronide. Human isoforms of UGT and isozyme-specific chemical inhibitors were used for recombinant assays. Acacetin glucuronidation kinetics were assessed by combining acacetin with recombinant human UGT isoforms or with microsomes from humans or experimental animals. Kinetic differences between species were assessed in vitro using the same approach.ResultsWe identified multiple UGT isoforms that facilitated acacetin glucuronidation, and found that UGT1A1 was the major isoform that catalyzed this process. Acacetin-7-O-glucuronide formation exhibited clear substrate inhibition kinetics when combined with recombinant UGTs or with liver/intestinal microsomes derived from humans, monkeys, rats, mice, dogs, or pigs. Intrinsic metabolic clearance values of human intestinal microsomes were two-fold greater than those of human liver microsomes. Among the evaluated species, the K_m value of dog microsomes (0.86 μM) was greatest in acacetin glucuronidation, while mice exhibited the highest CL_int value, 5.05 mL/min/mg. The CL_int values of microsomes derived from monkeys and minipigs were 1.99 mL/min/mg and 2.12 mL/min/mg, respectively, exhibiting similar intrinsic metabolic clearance activity to that observed in humans.ConclusionMonkey may represent a suitable model for experimental studies of acacetin pharmacokinetics owing to a high sequence homology of UGT1A1 and similar UGT1A1 glucuronidation activity to humans.     

芫花主要黄酮成分对肝微粒体UGTs及UGT1A1活性的体外抑制作用

目的 考察芫花主要黄酮成分芫花素和芹菜素对尿苷二磷酸葡萄糖醛酸转移酶（UGTs）及UGT1A1活性的影响。方法 采用体外肝微粒体孵育模型,以4-硝基酚（4-nitrophenol,4-NP）为底物检测UGTs活性,胆红素为底物检测UGT1A1活性;利用UV及UPLC-MS/MS测定底物或代谢产物的含量。结果 对UGTs,在大鼠肝微粒体、小鼠肝微粒体以及人肝微粒体孵育体系中,芫花素和芹菜素均能不同程度地抑制UTGs活性;抑制强弱顺序：在大鼠肝微粒体温孵体系中,芫花素>芹菜素;在小鼠肝微粒体以及人肝微粒体温孵体系中,芹菜素>芫花素。对UGT1A1,在人肝微粒体孵育体系中,芫花素和芹菜素均表现为中等强度的竞争性抑制作用,抑制强弱顺序：芹菜素（IC₅₀=12.40 μmol·L^-1）>芫花素（IC₅₀=23.21 μmol·L^-1）。结论 芫花素和芹菜素对不同肝微粒体孵育体系中UGTs及UGT1A1均可产生显著抑制作用且存在种属差异。芫花素及芹菜素可能存在基于UGT酶的药物相互作用。     

In vitro cytotoxicity of pyrazine-2-diazohydroxide: specificity for hypoxic cells and effects of microsomal coincubation

Summary The antitumor drug pyrazine-2-diazohydroxide exhibits cytotoxicity to A204 tumor cells in vitro under acid conditions. The IC₅₀ with a 1 hr drug exposure at pH of 7.4 was 61 g/ml and at pH of 6.0 it was 31 g/ml. It is suggested that the increased cytotoxicity is due to the acid catalyzed formation of a reactive pyrizinyldiazonium ion from pyrazine-2-diazohydroxide. Pyrazine-2-diazohydroxide is also more cytotoxic to A204 cells under hypoxic conditions in the presence of glucose with an IC₅₀ at pH 7.4 of 22 g/ml. The increased cytotoxicity of pyrazine-2-diazohydroxide under acid and hypoxic conditions may favor selective toxicity to solid tumors in vivo. Coincubation with rat hepatic microsomes increased the cytotoxicity of pyrazine-2-diazohydroxide to A204 cells. The effect did not require NADPH and was not due to formation of metabolites. There was an increased rate of degradation of pyrazine-2-diazohydroxide in the presence of microsomes, presumably with formation of the pyrizinyldiazonium ion. The final degradation product 2-hydroxypyrazine was not cytotoxic to A204 cells. The effect of microsomes on pyrazine-2-diazohydroxide cytotoxicity is probably of little in vivo significance.     

Biosynthesis of retinal phospholipids by base exchange reactions

Bovine retinal microsomes possess high activity of the enzymes that catalyze the exchange of free serine, choline, and ethanolamine with an endogenous lipid substrate to yield the products phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine. These enzymes are most active at pH 7·5–8, are Ca²⁺-dependent and do not require metabolic energy. Apparent K_m values for serine, choline and ethanolamine are 148, 616 and 63 μm, respectively, with apparent V_max values of 24, 18 and 32 nmol/hr/mg, respectively. These values are similar to those reported for brain.