我国成人肝微粒体的制备和几种细胞色素P450单加氧酶的活性

7例人肝标本均取自我国成年男性,其中5例来自非疾病死亡者,1例为肝血管瘤手术切除的正常肝组织,1例取自交通事故死亡者。该7例人肝微粒体细胞色素P450和细胞色素b5含量分别为0.36±0.08和0.23±0.05nmol·mg^-1蛋白,氨基比林和乙基吗啡N-脱甲基酶活性分别为1.07±0.23和1.82±0.31nmol·mg^-1·min^-1,7-乙氧基香豆素O—脱乙基酶、硝苯吡啶氧化酶和(-)-吡喹酮羟化酶活性分别为0.30±0.10,0.43±0.18和0.69±0.43nmol.mg^-1·min^-1。     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

吴茱萸次碱在人肝微粒体中对细胞色素P450酶的抑制作用

目的研究吴茱萸次碱(WZY)在人肝微粒体中对细胞色素P450酶的抑制作用。方法对照组和抑制组酶活性均用探针药测定,探针药物及其代谢产物用HPLC进行检测。用代谢产物与母药比值来表达酶的活性。结果加入50μmol·L-1吴茱萸次碱组CYP1A2,CYP2C19,CYP2E1和CYP2D6的活性显著降低,CYP3A4和CYP2C9活性无显著变化。结论吴茱萸次碱对CYP1A2,CYP2C19,CYP2E1和CYP2D6的活性有显著抑制作用,而对CYP2C9和CYP3A4的活性无显著影响。     

中国人肝微粒体中细胞色素P450酶含量和活性的个体间差异(英文)

目的:比较不同中国人肝微粒体中几种重要细胞色素P450(CYP)的酶含量和活性。方法:运用West-ern斑点分析和光密度扫描,对17个汉族、17个壮族和8个苗族受试者肝微粒体中的细胞色素P4501A2(CYP1A2)、2C9及3A4进行定量;非那西丁、甲磺丁脲、异喹胍和奥美拉唑分别用于体外测量CYP1A2、2C9、2D6及3A4的活性。结果:CYP1A2、2C9及3A4的含量和活性具有很大的个体间变异,另外CYP2D6的活性在各样本间也有很大差异;CYP3A4(32％)是中国人肝微粒体中含量最丰富的CYP,CYP2C9(19％)和CYP1A2(16％)的含量也很可观;除了CYP1A2的含量和活性具有一定的种族和性别差异外,未发现其它CYP具有种族和性别差异;CYP1A2、2C9和3A4的酶蛋白含量分别和它们的活性具有很好的相关性。结论:我们的结果为在中国人中进行药物代谢研究提供了非常有价值的信息。     

复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响

目的探讨复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响。方法利用细胞色素P450在铁蛋白的铁原子被还原与一氧化碳形成复合物时出现一特异性吸收峰，在波长大约450nm处呈现最大吸收，450-490nm波长的消光系数经精确测定为91nm^-1cm^-1，可定量测定细胞色素P450。结果复方丹参滴丸能诱导大鼠肝微粒体细胞色素P450的活性，且其作用随给药时间的延长而增强。结论复方丹参滴丸虽然作为OCT药品，但在长期服用或配伍应用时亦应当慎重。     

HPLC测定大鼠肝微粒体P450活性方法学研究进展

近年来，越来越多的研究者意识到使用肝微粒体进行细胞色素P450活性测定的重要性。本文综述了应用高效液相色谱进行大鼠肝微粒体P450活性测定的方法学研究进展，内容包括肝微粒体的制备方法，细胞色素P450代谢酶系的特异性底物及其代谢产物和使用高效液相色谱进行细胞色素P450活性测定的应用条件。     

复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响

目的探讨复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响.方法利用细胞色素P450在铁蛋白的铁原子被还原与一氧化碳形成复合物时出现一特异性吸收峰,在波长大约450 nm处呈现最大吸收,450～490 nm波长的消光系数经精确测定为91 nm-1cm-1,可定量测定细胞色素P450.结果复方丹参滴丸能诱导大鼠肝微粒体细胞色素P450的活性,且其作用随给药时间的延长而增强.结论复方丹参滴丸虽然作为OCT药品,但在长期服用或配伍应用时亦应当慎重.     

复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响

目的探讨复方丹参滴丸对大鼠肝微粒体细胞色素P450含量的影响。方法利用细胞色素P450在铁蛋白的铁原子被还原与一氧化碳形成复合物时出现一特异性吸收峰,在波长大约450 nm处呈现最大吸收,450~490 nm波长的消光系数经精确测定为91 nm-1cm-1,可定量测定细胞色素P450。结果复方丹参滴丸能诱导大鼠肝微粒体细胞色素P450的活性,且其作用随给药时间的延长而增强。结论复方丹参滴丸虽然作为OCT药品,但在长期服用或配伍应用时亦应当慎重。     

柴胡注射液对大鼠细胞色素P450各亚型活性的影响

目的：观察柴胡注射液对大鼠细胞色素P450各亚型活性的影响。方法：24只Wistar大鼠,随机分成3组：对照组,柴胡注射液低、高剂量组。3组分别腹腔注射生理盐水、0.36、0.72mL/kg（相当于原药材0.72、1.44g/kg）柴胡注射液诱导两周后,于d15清晨灌胃给予探针药物咖啡因（10mg/kg）,6h后处死。用HPLC法观察探针药物代谢率的变化来反映柴胡注射液对大鼠CYP1A2活性的影响;West-ernbloting测定大鼠肝微粒体CYP1A2、3A4、4A1蛋白的相对含量。结果：体内、外探针药物代谢率在各剂量组及对照组间均无统计学差异（P〉0.05）,CYP1A2在各剂量组中表达亦无统计学差异。CYP3A4在柴胡注射液高剂量组中表达明显增多（P〈0.05）,CYP4A1在柴胡注射液低、高剂量组表达均明显减少（P〈0.05）。结论：柴胡注射液对大鼠CYP1A2亚型没有影响,但在蛋白质水平,可抑制大鼠肝CYP4A1表达。高剂量柴胡注射液可诱导大鼠肝CYP3A4表达。     

地非三唑对鼠肝微粒体中细胞色素P450的诱导作用

目的 本实验通过用相同剂量地非三唑油溶液对大鼠进行不同天数的腹腔注射，观察地非三唑对鼠肝微粒体中细胞色素P450(CYP)的诱导作用。方法 (1)用CYPIA与CYPIIB的两种特征性最物乙氧基异吩恶唑与7-戊氧基异吩恶唑在经不同天数地非三唑处理的鼠肝微粒体中代谢，用紫外分光光度仪直接测定代谢物异吩恶唑的生成量。从而测定乙氧基异吩恶唑-脱甲基酶(EROD)与7-戊氧基异吩恶唑-脱烷基晦(PROD)的含量，用来表征微粒体中CYPIA与CYPIIB的活性。（2）用CYPIIIA的特征性底物地西泮与经不同天数地非三唑处理的鼠肝微粒体体外共孵育，用氯仿终止反应并提取剩余底物，用反相高效液相(RP-HPLC)内标法测定剩余底物浓度。结果 不同天数地非三唑处理的鼠肝微粒体中EROD的含量比空白组有明显的增加，而且与诱导天数有良好的相关性。而PROD的含量在地非三唑组与空白组之间无明显差异。地西泮在用不同天数地非三唑处理的鼠肝微粒体中则无明显代谢，其代谢程度与诱导的天数没有相关性。结论 地非三唑对CYPIA有诱导作用。     

大鼠肝微粒体细胞色素P4503A参与吡喹酮A环羟化代谢

三乙酰竹桃霉素(TAO)可使地塞米松(DEX)诱导的大鼠肝微粒体内未结合细胞色素P450(P450)含量降低,红霉素(ERY)和乙基吗啡(EMP)N-脱甲基酶活性降低和吡喹酮(PZQ)A环一羟化物生成速率降低。TAO和ERY对PZQA环一羟化物的生成表现为竟争性抑制,且PZQA环一羟化物生成速率与ERY,EMPN-脱甲基酶活性高度相关。结果表明,P4503A(CYP3A)参与了PZQA环的羟化。     

IMMUNOCHEMICAL AND CATALYTICAL CHARACTERIZATION OF THE HUMAN LIVER NADPH-CYTOCHROME P450 REDUCTASE

1. The NADPH-cytochrome P450 reductases (EC 1.6.2.4) from human and rabbit liver have been purified to electrophoretic homogeneity. The human reductase had an apparent monomeric molecular weight of 77,500 and the rabbit enzyme of 76,500. 2. Both flavoproteins exhibited typical flavoprotein spectra and contained equimolar quantities of FAD and FMN. The two reductases were catalytically active in reducing cytochrome c, ferricyanide and dichlorophenolindophenol, and in supporting rabbit liver cytochrome P450 Form 4 metabolism of 2-acetylaminofluorene. 3. An antibody raised in the goat against the human enzyme formed a precipitin line with the human reductase in a double-diffusion assay, but did not react with the rabbit reductase. Similarly, an antibody raised in the goat against the rabbit reductase formed a precipitin line with the rabbit enzyme, but did not cross-react with the human reductase. 4. Both antibodies inhibited cytochrome c reduction by the two reductases suggesting some immunochemical recognition. 5. Immunochemical cross-reactivity was confirmed when both reductases were subjected to the more sensitive immunoblot technique using either anti-human or anti-rabbit reductase IgG. 6. The human and rabbit reductases are essentially similar in amino acid composition, except that the former has larger amounts of serine and glycine.     

氟他胺在大鼠肝微粒体经细胞色素P450 1A2代谢的性别差异

目的体外研究大鼠肝微粒体细胞色素P450 1A2(CYP1A2)对氟他胺(flutamide Flu)代谢的性别差异影响。方法制备正常♀♂大鼠肝微粒体,用CYP1A2抗体与氟他胺(2 mg·L^-1)共同温孵,测定氟他胺主要代谢产物2-羟基氟他胺(2-hydroxyflutamide, HF)和原药的浓度比(HF/Flu),评价氟他胺在大鼠肝微粒体代谢的性别差异。结果在CYP1A2抗体浓度为1∶400,孵育时间为30 min条件下,氟他胺在♂大鼠肝微粒体中的HF/Flu为(1.5±0.6),而♀动物为(0.9±0.4)。不同性别大鼠肝微粒体对氟他胺的代谢存在性别差异(P<0.01)。结论Flu在♂大鼠肝微粒体中代谢快,而在♀大鼠肝微粒体中代谢较慢。♂大鼠体内的CYP1A2酶活性高于♀大鼠。     

黄芩苷对小鼠肝细胞色素P450的选择性诱导

目的　观察黄芩苷对小鼠肝细胞色素P450及其亚家族的影响。方法　用紫外分光光度法分别测定小鼠肝微粒体细胞色素P450与b5含量及氨基比林N-脱甲基酶(ADM)、7-乙氧基香豆素O-脱乙基酶(ECD)、苯并芘羟化酶(AHH)活性。用蛋白印迹杂交技术鉴定细胞色素P450同功酶。结果　黄芩苷可使小鼠肝微粒体细胞色素P450含量显著增加,并使ADM,ECD及AHH 3种酶活力显著增强。对6种P450同功酶的鉴定结果显示,黄芩苷可选择性诱导1A1,2B1及2C11 3种同功酶,对细胞色素b5含量及3A2,2D1和2E1 3种同功酶无诱导作用。结论　黄芩苷对小鼠肝细胞色素P450有选择性诱导作用。     

Multiple forms of cytochrome P450

The microsomal monooxygenase system is characterized by its broad substrate specificity which includes endogenous substrates as well as lipophilic drugs and chemicals. From in vitro investigations it was known that the relative reactivities and the pattern of products varied greatly with species, sex, age, diet or pretreatment with drugs of the animal. The suggestion that this was possibly due to a variety of cytochrome P450 enzymes rather than a single monooxygenase was recently confirmed by the isolation of several cytochrome P450 species with different although overlapping substrate specificities. In view of the consequences of a genetic and environment-dependent pattern of monooxygenases for drug metabolism and drug-mediated toxicity the methods of a quantitative assessment of the various forms are discussed.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977This work was in part supported by the Deutsche Forschungsgemeinschaft, SonderforschungsbereichThis work was in part supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich     

磷酸川芎嗪与大鼠肝微粒体相互作用的研究

Sprague-Dawley6大鼠ig50、100和300mg/kg磷酸川芎嗪,对肝微粒体细胞色素P-450酶系无显著性影响。苯巴比妥诱导P-450b后再ig磷酸川芎嗪,其体内血药浓度明显低于正常对照组,说明川芎嗪在体内代谢可能和肝微粒体细胞色素P-450酶系统中的P-450b同功酶有关。     

丹红注射液对大鼠肝微粒体5种CYP亚型酶活性的影响

摘要目的研究丹红注射液对5种细胞色素P450亚型酶活性的影响,为临床合理用药提供参考。方法采用大鼠体外肝微粒体孵育法,分别以非那西丁、甲苯磺丁脲、右美沙芬、氯唑沙宗、睾酮为CYP1A2、CYP2C9、CYP2D6、CYP2E1、CYP3A4的探针药物,在大鼠肝微粒体孵育体系中孵育,用高效液相色谱（HPLC）法测定相应的代谢产物,比较空白对照组和丹红注射液低、中、高剂量组之间探针药物代谢率的差异,评价丹红注射液对各亚型酶活性的影响。结果在体外肝微粒体孵育体系中,丹红注射液低剂量组中CYP1A2 和CYP2C9活性与空白对照组相比,差异无统计学意义（P＞0.05）;中和高剂量组中CYP1A2和CYP2C9活性与空白对照组相比降低,差异有统计学意义（P＜0.05或P＜0.01）;丹红注射液低、中、高剂量组中CYP2D6、CYP2E1、CYP3A4的活性与空白对照组相比,差异无统计学意义（P＞0.05）。丹红注射液对大鼠肝微粒体CYP1A2酶活性的半数抑制浓度（IC50）和抑制常数（Ki）分别为0.54%和0.226%。结论丹红注射液对大鼠肝微粒体CYP1A2酶活性有抑制作用,且为混合型抑制;对CYP2C9有弱抑制作用;对CYP2D6、CYP2E1、CYP3A4酶活性无明显影响。     

Changes in cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver and kidney

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2α-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was <10%. Furthermore, immunoblotting showed that 1,1-DCE (≥400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6β-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40–70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid ω-hydroxylase (LAOH), and testosterone 7α-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60–66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was␣observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (∼1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE. Received: 28 January 1997 / Accepted: 18 August 1997     

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally with BPA daily for 4 days, at doses of 10, 20, and 40 mg/kg. Among the P450-dependent monooxygenase activities, testosterone 2α-hydroxylase (T2AH) and testosterone 6β-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40 mg/kg BPA. The levels of the control activities were 13 and 50%, respectively. Furthermore, immunoblotting showed that BPA (20 or 40 mg/kg) significantly reduced CYP2C11/6 and CYP3A2/1 protein levels in rat liver microsomes. In addition, estradiol 2-hydroxylase (ED2H) and benzphetamine N-demethylase (BZND) activities were significantly decreased by BPA at 20 and 40 mg/kg (by 19–73%). The K _m values for T2AH and T6BH in 20 and 40 mg/kg BPA-treated rats were significantly high compared with that in control rats. The V _max for T2AH was dose-dependently decreased by BPA treatment, whereas that of T6BH was only decreased by BPA at 40 mg/kg. On the other hand, lauric acid ω-hydroxylase (LAOH) activity was significantly increased by BPA at 20 and 40 mg/kg (1.5- and 1.7-fold, respectively). Immunoblot analysis showed that 20 and 40 mg/kg BPA induced CYP4A1/2 protein expression. However, the activities 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), chlorzoxazone 6-hydroxylase (CZ6H), erythromycin N-demethylase (EMND), and testosterone 7α-hydroxylase (T7AH) were not affected by BPA at any dose. These results suggest that BPA affects male-specific P450 isoforms in rat liver, and that these changes closely relate to the toxicity of BPA. Received: 26 January 1998 / Accepted: 26 February 1998