Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.     

The microsomal glucose-6-phosphatase enzyme of human gall-bladder

Microsomes isolated from adult human gall-bladders have for the first time been shown to contain specific glucose-6-phosphatase activity. The gall-bladder glucose-6-phosphatase enzyme has the same molecular weight (36,500 daltons) and similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme.     

Action of vasopressin on ATPase activity of microsomal fractions of rabbit heart and liver

Intravenous injection of vasopressin in a dose of 5 pressor units/kg body weight led after 1 h to changes in the ATPase activity of rabbit heart and liver microsomes. These changes differed in direction: Mg- or Ca-activated ATPase activity of the cardiac microsomes was very slightly increased, whereas ATPase activity of the hepatic microsomes was reduced.Laboratory of Molecular Pathology and Biochemistry, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 12, pp. 1433–1434, December, 1976.     

白藜芦醇抑制人肝微粒体代谢色氨酸的研究

目的研究白藜芦醇对人肝微粒体代谢色氨酸生成犬尿氨酸的影响。方法分别考察孵育时间、色氨酸浓度、微粒体蛋白浓度,建立体外人肝微粒体孵育色氨酸代谢体系;采用高效液相色谱-串联质谱法(LC-MS/MS)检测肝微粒体孵育体系中色氨酸和犬尿氨酸浓度。基于体外人肝微粒体孵育色氨酸代谢体系探讨白藜芦醇对色氨酸代谢影响。结果色氨酸在体外人肝微粒体中最佳孵育时间为90 min,色氨酸浓度为8 mg·L^-1,微粒体蛋白浓度为1 g·L^-1;酶反应速率常数K m为(95.91±22.29)μmol·L^-1,最大反应速率V max为(21.34±2.58)μmoL·g^-1·min^-1。与已知的吲哚胺2,3-双加氧酶(IDO)抑制剂Epacadostat相比,结果显示白藜芦醇也能够抑制人肝微粒体中色氨酸代谢生成犬尿氨酸且具有浓度依赖性。结论白藜芦醇具有抑制IDO活性的作用,表明IDO可能是白藜芦醇发挥药理作用的一个潜在靶点,为进一步研究白藜芦醇的药理作用及色氨酸代谢提供重要依据。     

甘草素在体外不同种属肝微粒体中的代谢差异研究

目的对甘草苷的苷元甘草素在体外不同种属肝微粒体中的代谢差异进行比较研究,为甘草苷的进一步研究开发提供参考依据。方法甘草素与不同种属,包括大鼠、小鼠、人、犬及猴的肝微粒体进行孵育,比较其代谢稳定性和代谢转化情况。代谢稳定性通过底物消除法分析甘草素的剩余底物浓度水平随时间的变化,计算体外消除半衰期(t1/2)。进一步对甘草素在肝微粒体孵育后生成的代谢产物谱以及代谢途径进行分析。结果在I相肝微粒体孵育体系中,甘草素在5个种属肝微粒体中发生代谢的t1/2依次为大鼠小鼠人猴犬;在II相肝微粒体孵育体系中,甘草素代谢都非常快,除小鼠外,均在5 min内下降50%以上。甘草素与人肝微粒体进行II相孵育产生的代谢产物谱和猴的相同,其余种属则与人有明显不同。结论甘草素在猴肝微粒体中的代谢稳定性和代谢产物谱均与人最相似,其次为犬肝微粒体,大鼠和小鼠则与人存在明显差异。在进一步的临床前药代和毒理研究中可选用猴或犬作为模型动物。     

An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point

The current study focused on the development of an automated IC(50) cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC(50) determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC(50)s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC(50) assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.     

Aromatase inhibiting and combined estrogenic effects of parabens and estrogenic effects of other additives in cosmetics

There is concern widely on the increase in human exposure to exogenous (anti)estrogenic compounds. Typical are certain ingredients in cosmetic consumer products such as musks, phthalates and parabens. Monitoring a variety of human samples revealed that these ingredients, including the ones that generally are considered to undergo rapid metabolism, are present at low levels. In this in vitro research individual compounds and combinations of parabens and endogenous estradiol (E(2)) were investigated in the MCF-7 cell proliferation assay. The experimental design applied a concentration addition model (CA). Data were analyzed with the estrogen equivalency (EEQ) and method of isoboles approach. In addition, the catalytic inhibitory properties of parabens on an enzyme involved in a rate limiting step in steroid genesis (aromatase) were studied in human placental microsomes. Our results point to an additive estrogenic effect in a CA model for parabens. In addition, it was found that parabens inhibit aromatase. Noticeably, the effective levels in both our in vitro systems were far higher than the levels detected in human samples. However, estrogenic compounds may contribute in a cumulative way to the circulating estrogen burden. Our calculation for the extra estrogen burden due to exposure to parabens, phthalates and polycyclic musks indicates an insignificant estrogenic load relative to the endogenous or therapeutic estrogen burden.     

Design,synthesis, and anticonvulsant activity of some derivatives of xanthone with aminoalkanol moieties

A series of new xanthone derivatives have been synthesized and evaluated for their anticonvulsant properties in the maximal electroshock, subcutaneous metrazole tests and for neurotoxicity in the rotarod in mice, i.p. and rats, p.o. Compound 9 : R,S‐2‐{2‐[(1‐hydroxybutan‐2‐yl]amino)ethoxy}‐9H‐xanthen‐9‐one and compound 12 : R,S‐2‐{3‐[(1‐hydroxybutan‐2‐yl)amino]propoxy}‐9H‐xanthen‐9‐one exerted activity in rats, p.o. 2 and 4 h after administration, respectively. Therefore, metabolic stability of the compounds was evaluated with use of rat microsomes, resulting in half‐life t_1/2 136 and 108 min, respectively, indicating that either the metabolites are very active or the parent compounds exert ADME properties other than metabolism which influence the late onset of activity.     

Enzyme kinetics of imperatorin and its cytochrome P450 phenotyping in liver microsomes北大核心CSCD

OBJECTIVE: To investigate enzyme kinetic characteristics of imperatorin in rat liver microsomes (RLM) or human liver microsomes (HLM), and to identify the reaction phenotyping of human recombinant cytochrome P450 enzyme (CYP) mediated phase I metabolism. METHODS: Imperatorin was incubated at 37°C with HLM or RLM in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH) or uridine 5'-diphosphoglucuronic acid (UDGPA). The concentrations of imperatorin in the incubation systems were determined with LC-MS/MS to evaluate its metabolic stability and enzymatic kinetics. The CYP phenotyping of imperatorin was identified using a panel of human recombinant CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and also using a group of specific inhibitors in HLM. RESULTS: Imperatorin was metabolically eliminated in the presence of NADPH in HLM or RLM. The elimination rates for HLM and RLM in 30 min were 69.7% and 94.5%, respectively, and elimination half-life (t1/2) values were 18.9±0.6 and (2.8±0.4)min, respectively. The extrapolated hepatic clearance parameters (Clh) were 16.9±0.1 and (51.9±0.4)mL·min-1-kg-1. The Michaelism-Menten parameters (Km) were 13.60±0.16 and (14.00± 0.24)umol·L-1 and maximum velocity (Vmax) were (2928±96) and (8434±27)nmol·min-1·g-1, respectively. The metabolic elimination of imperatorin in RLM was quicker than in HLM. The results of CYP phenotyping indicated that CYP1A2, CYP2B6, CYP2C19 and CYP3A4 were the major CYP isoforms involved in the imperatorin metabolism. Their individual contributions assessed using the method of total normalized rate were 20.4%, 7.3%,10.5% and 61.8%, respectively. CONCLUSION: Imperatorin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and CYP3A4 are the major responsible enzymes with a contribution rate above 20%.