Beneficial effects of histidine and carnosine on ethanol-induced chronic liver injury.

Alleviative effects of histidine and carnosine in mice against ethanol-induced oxidative and inflammatory was examined. After chronic alcoholic liver injury was induced, histidine and carnosine at 0.5, 1, 2g/L were added to the drinking water for 3 weeks. Results showed that the post-intake of histidine or carnosine markedly decreased alanine aminotransferase and aspartate aminotransferase activities (P<0.05). Ethanol treatment increased malondialdehyde (MDA) level, decreased glutathione (GSH) content and catalase and glutathione peroxidase (GPX) activities, and increased cytochrome P450 2E1 (CYP2E1) activity in liver (P<0.05). The post-intake of histidine and carnosine significantly decreased MDA formations, increased GSH content, enhanced catalase and GPX activities, and suppressed CYP2E1 activity (P<0.05), in which the effects on catalase and CYP2E1 activities were dose-dependent (P<0.05). Ethanol treatment elevated hepatic levels of c-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) (P<0.05), the post-intake of histidine and carnosine significantly and dose-dependently diminished the release of CRP, IL-6, and TNF-alpha (P<0.05). Ethanol treatment caused down-regulation in both catalase and GPX mRNA expression, and up-regulated both IL-6 and TNF-alpha mRNA expression (P<0.05). Histidine and carnosine post-treatments significantly and dose-dependently upregulated catalase mRNA, and down-regulated mRNA expression of IL-6 and TNF-alpha (P<0.05). Based on the observed anti-oxidative and anti-inflammatory effects, the supplement of histidine or carnosine might be helpful for the treatment of chronic alcoholic liver injury.     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响(英文)

目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶。方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学。鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶。结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征。当底物浓度为氟西汀25μmol/L和100μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r_1=0.821,P_1=0.001;r_2=0.668,P_2=0.013),当底物浓度为氟西汀100μmol/L时,N-FLU的生成率与S-美芬妥因4’-羟化酶活性显著相关(r=0.717,P=0.006)。PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73％vs 45％,P<0.01)。结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主。     

地非三唑对大鼠肝细胞细胞色素P450 CYP1A1/2的诱导作用

目的 试从mRNA表达水平阐明地非三唑对鼠肝微粒体中细胞色素P450 CYP1A1/2的诱导机制。方法 给SD大鼠腹腔注射地非三唑，采用Trizol法提取大鼠肝脏RNA,用RT-PCR测定经地非三唑处理1, 2及4 d的鼠肝中细胞色素P450 CYP1A1, CYP1A2 mRNA的表达水平。结果 地非三唑处理不同时间的鼠肝细胞中细胞色素P450 CYP1A1, CYP1A2 mRNA的表达水平比空白对照组明显增加，空白对照组CYP1A1吸光度比值为0.270±0.040, 诱导1, 2及4 d的吸光度比值分别为0.343±0.055, 0.417±0.045及0.603±0.083；空白对照组的CYP1A2吸光度比值为0.613±0.189, 而诱导1，2及4 d的吸光度比值分别为1.510±0.226, 3.057±0.518及4.120±0.458。随着诱导时间的增加，细胞色素P450 CYP1A1及CYP1A2 mRNA的表达也逐步增加，诱导时间与表达水平之间存在一定的线性关系,相关系数分别为0.9984和0.9563。结论 地非三唑对细胞色素P450 CYP1A1/2 mRNA表达具有诱导作用。     

Effects of the antimycobacterial compound 2-phenoxy-1-phenylethanone on rat hepatocytes and formation of metabolites

Context: Neolignans are usually dimers formed by oxidative coupling of allyl and propenyl phenols, and the neolignan analogue, 2-phenoxy-1-phenylethanone (LS-2) is a promising antimycobacterial compound showing very weak cytotoxicity in mammalian cells and lack of acute toxicity in murine models.

Objectives: To investigate the mechanism of action of LS-2 in rat hepatocytes by evaluating the activity levels of enzymes related to oxidation status and drug-metabolizing activity.

Materials and methods: Hepatocytes were treated with LS-2 from 0.05 up to 1 mM, for 24 and 48 h, and reduced glutathione (GSH), lipid peroxidation and cytochrome P450 enzyme (CYP450) activity were assayed. A homologous series of phenoxazone ethers were used as substrates to measure the enzymatic profile. The biotransformation of LS-2 was studied in hepatocytes by gas chromatography-mass spectrometry (GC-MS) for detection and analysis of possible metabolites.

Results: Hepatocytes treated with LS-2 up to 1 mM for 24 or 48 h did not induce the formation of GSH and lipid peroxidation. O-Dealkylation activities of the isoenzymes CYP4501A1, CYP4501A2, CYP4502B1 and CYP4502B2 were also not detected in the hepatocytes treated with LS-2 for 24 or 48 h.

Discussion and conclusion: The results indicate that LS-2 or its two detected metabolites, 2-phenoxy-1-phenylethanol and 2,4-(2-hydroxy-2-phenylethoxy)phenol, are not cytotoxic to rat hepatocytes. These compounds maintain a balance between the production of pro-oxidant agents and their respective antioxidant systems. The data show that enzymes related to oxidation status and drug-metabolizing activities are not involved in the mechanism of action of LS-2.     

Differential detoxifying responses to crude oil water-accommodated fraction in Hyallela curvispina individuals from unpolluted and contaminated sites

Sublethal effects of water-accommodated fraction (WAF) from crude oil of Neuquén basin, Northern Patagonia-Argentina, were examined on both antioxidant and detoxification system of Hyalella curvispina adults collected in Los Barreales (LB) lake and in an oil-polluted stream (DS). The effects of WAF exposure during 6, 24 and 48 h were evaluated in the glutathione content (GSH) and glutathione S-transferase (GST), catalase (CAT) and cytochrome P450 (CYP450) activities. Populations from DS and LB showed not only different basal GSH content and enzyme activities but also different behavior to WAF exposure. LB population exposed to WAF showed a significant increase in GSH content, CAT and CYP450 activities, compared to control group. DS population presented high basal levels in CAT and CYP activity compared with LB population, but their response to WAF exposure was minor. Amphipods from DS, chronically exposed to hydrocarbons, were adapted to their environment.     

阿司匹林联合华法林对HepG2细胞中CYP3A4酶的抑制作用

目的研究阿司匹林联用华法林对肝药物代谢酶CYP3A4活性的影响及其机制。方法不同质量浓度的阿司匹林、华法林及联合用药处理Hep G2细胞48 h,采用MTT法检测细胞存活率;通过荧光素酶报告基因技术检测各组对PXR转录酶活性和酶CYP3A4活性的影响;采用荧光定量PCR法和Western blotting法检测Hep G2细胞的酶CYP3A4的m RNA和蛋白表达水平。结果与对照组比较,阿司匹林组、联合用药组细胞的孕烷X受体(PXR)转录酶活性、酶CYP3A4活性均显著降低(P0.01),CYP3A4 m RNA和蛋白表达水平显著降低(P0.05、0.01),华法林组均无显著差异。结论阿司匹林联用华法林能够抑制药物代谢酶CYP3A4活性,其机制可能是通过抑制PXR受体的m RNA和蛋白表达实现的。     

心肌缺血再灌注损伤对大鼠肝细胞色素P450酶代谢的影响

目的探讨心肌缺血再灌注状态下,大鼠肝代谢功能和相关的氧化/抗氧化能力变化。方法雄性SD大鼠随机分为5组,除假手术组外,制备在体心肌缺血再灌注模型,并于缺血40min、再灌注15,60和180min分别处死大鼠,检测血浆丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性,肝匀浆丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性;以红霉素N-脱甲基酶、五氧基异噁唑O-脱乙基酶和苯胺羟化酶法为探针测定肝细胞色素P450(CYP)3A,CYP2B1和CYP2E1催化功能;RT-PCR法检测肝Ⅰ相药物代谢酶CYP3A1,CYP2B1/2,CYP2E1,以及Ⅱ相解毒酶NAD(P)H醌氧化还原酶(NQO1)及其上游因子NF-E2相关因子(Nrf2)mRNA水平。结果再灌注60 min,肝匀浆MDA含量升高(P<0.05),SOD活力下降(P<0.01);再灌注180 min时,血浆ALT和AST活性升高(P<0.05)。Nrf2基因于再灌注60 min时显著激活(P<0.05),下游因子NQO1 mRNA于再灌注180 min时明显上调(P<0.05)。CYP3A催化功能和mRNA水平分别于再灌注60和180 min开始明显降低(P<0.05);CYP2B1/2 mRNA和催化功能水平分别于再灌注15和180 min开始明显降低(P<0.05);CYP2E1催化功能无明显改变。结论大鼠心肌缺血再灌注可引起肝组织氧化应激及并导致功能损伤。在再灌注早期,具有抗氧化功能的NQO1在转录水平显著上调,其机制可能与上游因子Nrf2被激活相关;CYP3A和CYP2B催化功能在转录和(或)转录后水平明显下调。     

灯盏花素对张氏肝细胞中CYP3A4和CYP2C19的影响和机制

目的：观察灯盏花素对人张氏肝细胞中孕烷X受体（PXR）及细胞色素P450 3A4（CYP3A4）和细胞色素P450 2C19（CYP2C19） mRNA和蛋白表达的影响,初步探讨灯盏花素对CYP3A4和CYP2C19的影响及其作用机制。方法： MTT法检测不同浓度的灯盏花素对张氏肝细胞活力的影响;采用实时荧光定量PCR（RT-qPCR）检测不同浓度灯盏花素对张氏肝细胞中PXR、CYP3A4及CYP2C19 mRNA的表达;采用Western-blot检测不同浓度的灯盏花素对CYP3A4和CYP2C19蛋白的表达影响。结果： MTT结果表明不同浓度的灯盏花素处理张氏肝细胞24 h、48 h后的IC₅₀分别为9 768.8、1 372.8 μmol·L^-1,IC₁₀分别为2 689.2、154.4 μmol·L^-1;RT-qPCR结果表明灯盏花素对CYP3A4和PXR mRNA的影响呈抑制作用,对CYP2C19 mRNA的影响呈不同程度的促进作用;Western-blot结果表明灯盏花素对CYP3A4蛋白的影响呈抑制作用,对CYP2C19蛋白的影响呈促进作用。结论：灯盏花素对CYP3A4的抑制作用可能通过抑制PXR途径;对CYP2C19的促进作用可能不经PXR通路。     

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

Inhibition of the metabolism of brotizolam by erythromycin in humans: in vivo evidence for the involvement of CYP3A4 in brotizolam metabolism

AIMS: To obtain in vivo evidence for the involvement of cytochrome P450 (CYP) 3A4 in the metabolism of brotizolam. METHODS: Fourteen healthy male volunteers received erythromycin 1200 mg day(-1) or placebo for 7 days in a double-blind randomized crossover manner. On the 6th day they received a single oral 0.5-mg dose of brotizolam, and blood samplings were performed for 24 h. RESULTS: Erythromycin treatment significantly increased the peak plasma concentration (P < 0.05), total area under the plasma concentration-time curve (P < 0.01), and elimination half-life (P < 0.01) of brotizolam. CONCLUSIONS: The present study provides in vivo evidence for the involvement of CYP3A4 in brotizolam metabolism.     

Effect of butylated hydroxytoluene,curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

1.?The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes.

2.?Treatment of human hepatocytes for 72 h with 2–200 µM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 µM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 µM BHT.

3.?In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels.

4.?The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6β-hydroxylase activity.

5.?In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.     

淫羊藿苷对不同月龄大鼠肝微粒体细胞色素P450的影响

目的 揭示淫羊藿苷(Ica)对大鼠肝微粒体细胞色素P450的含量及部分亚型的影响,并比较月龄的差异.方法 ig给予6月龄和18月龄的♂SD大鼠Ica( 60 mg· kg -1),4周后取肝脏,用钙沉淀法提取肝微粒体,BCA法测定微粒体蛋白浓度;用一氧化碳还原差示光谱法测定CYP450的含量;用ELISA法测定CYP1 A1、CYPb5的含量;用比色法测定苯胺羟化酶(反映CYP2E1活性)和红霉素-N-脱甲基酶(反映CYP3A活性)的活性;用real - time RT - PCR检测CYP1 A1、CYP2A3、CYP2E1、CYP3A1、CYP3A2和CYP4B1 mRNA的表达.结果 60 mg· kg-1 Ica明显增加了CYP450的总酶和CYP1 A1的含量、CYP3A的活性及CYP1 A1、CYP3A1、CYP3A2 mRNA的表达,降低了CYP2E1的活性及其mRNA的表达;但Ica对上述各指标的诱导或抑制作用在大鼠月龄方面差异不明显;Ica对CYPb5的含量及CYP2A3、CYP4B1 mRNA的表达未见明显影响.结论 Ica对大鼠肝微粒体CYP450总酶、CYPI A1和CYP3A具有诱导作用,对CYP2E1具有抑制作用,该作用未见明显月龄差异.     

Deactivation of anti-cancer drug letrozole to a carbinol metabolite by polymorphic cytochrome P450 2A6 in human liver microsomes

1. Cytochromes P450 (P450) involved in letrozole metabolism were investigated. Among 13 recombinant P450 forms examined, only P450 2A6 and 3A4 showed activities in transforming letrozole to its carbinol metabolite with small K_m and high V_max values yielding apparent V_max/K_m values of 0.48 and 0.24 nl min^?1 nmol^?1 P450, respectively.

2. The metabolic activities of individual human liver microsomes showed a significant correlation with coumarin 7-hydroxylase activities (P450 2A6 marker) at a letrozole concentration of 0.5 μM, while a good correlation was also seen with testosterone 6β-hydroxylase activities (P450 3A4 marker) at 5 μM substrate concentration with different inhibition by 8-methoxypsolaren.

3. Significantly low carbinol-forming activities were seen in human liver microsomes from individuals possessing CYP2A6*4/*4 (whole CYP2A6 gene deletion) at a letrozole concentration of 0.5 μM. A V_max/K_m value measured for CYP2A6.7 (amino acid substitution type) in human liver microsomes, in the presence of anti-P450 3A4 antibodies, was approximately seven-fold smaller than that for CYP2A6.1 (wild-type).

4. These results demonstrate that P450 2A6 and 3A4 catalyse the conversion of letrozole to its carbinol metabolite in vitro at low and high concentrations of letrozole. Polymorphic variation of CYP2A6 is considered to be relevant to inter-subject variation in therapeutic exposure of letrozole.

     

NADPH-cytochrome P-450 reductase is involved in flunitrazepam reductive metabolism in Hep G2 and Hep 3B cells

Flunitrazepam (FNTZ), like other benzodiazepines, has a high affinity for the benzodiazepine receptor within the gama-aminobutyric acid (GABA) complex. These affinities correlate with the pharmacological and therapeutic potencies of the drug. FNTZ is a drug commonly abused by young adults. In humans, FNTZ is oxidized to the major metabolites N-demethylflunitrazepam (DM FNTZ) and 3-hydroxyflunitrazepam (3-OH FNTZ) and reduced to 7-aminoflunitrazepam (7A FNTZ). Human CYP2C19 and CYP3A4 are the principal P-450 cytochromes involved in DM FNTZ and 3-OH FNTZ formation. However, it is not clear which enzyme is responsible for the reduction of FNTZ to 7-aminoflunitrazepam (7A FNTZ). In this study, the involvement of NADPH-cytochrome P-450 reductase in the conversion of FNTZ to 7A FNTZ was investigated in two human hepatoma cell lines, human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human HADPH-cytochrome P-450 reductase. Significantly more FNTZ was converted to 7A FNTZ in Hep G2 than in Hep 3B cells, and this difference was associated with the catalytic activity and protein levels of NADPH-cytochrome P-450 reductase in these cells. In Hep G2 cells, conversion of FNTZ to 7A FNTZ was effectively inhibited by alpha-lipoic acid, an NADPH-cytochrome P-450 reductase inhibitor. In addition, formation of 7A FNTZ by the microsomal fraction of Hep G2 cells was specifically inhibited by antibody against NADPH-cytochrome P-450 reductase. Under hypoxia (N2 85%; CO2 5%; H2 10%), human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human NADPH-P-450 reductase catabolized FNTZ to 7A FNTZ in a concentration-dependent manner. These results suggest that NADPH-cytochrome P-450 reductase is involved in the reductive metabolism of FNTZ to 7A FNTZ under hypoxic conditions.     

Tissue-specific alterations in the 6-hydroxylation of chlorzoxazone following traumatic brain injury in the rat.

Interest in the non-neuronal alterations following traumatic brain injury (TBI) has led to research evaluating hepatic metabolism following injury. Several models of injury demonstrate tissue-specific alterations in cytochrome P450 activity. This study examined tissue-specific alterations in cytochrome P450-mediated hydroxylation in the rat model of TBI. Male Sprague-Dawley rats received anesthesia alone, craniotomy, or craniotomy plus TBI. Rats were sacrificed at 24 and 48 h. Liver, kidney, and brain cortex microsomes were isolated. Total liver P450 content, 6-hydroxychlorzoxazone formation rate, and CYP2E1 protein were evaluated. In liver microsomes, spectral P450 was decreased to 86 +/- 5% (p < 0.05) of control at 24 h following injury, and 6-hydroxychlorzoxazone formation rate decreased to 74 +/- 18% of control (p < 0.05) at 48 h following injury. In kidney microsomes, 6-hydroxychlorzoxazone formation rate was increased to 154% of control (p < 0.01) 24 h following injury. 6-Hydroxychlorzoxazone formation rate was unaffected by TBI in brain cortical microsomes. The CYP2E1 inhibitor, 4-methylpyrazole, inhibited the formation of 6-hydroxychlorzoxazone in brain, kidney, and liver microsomes. These data demonstrate that tissue-specific alterations in 6-hydroxychlorzoxazone formation rate occur following TBI.     

细胞色素P450 CYP4A的组织和细胞表达特异性

目的确定细胞色素P450 CYP4A在组织和细胞水平表达是否具有特异性。方法采用Western印迹法分析细胞色素P450 CYP4A蛋白在培养的牛肺动脉内皮细胞、平滑肌细胞及牛肾动脉内皮细胞的表达;采用原位杂交法检测细胞色素P450 CYP4A mRNA在大鼠肺支气管内皮和血管内皮细胞的表达;以14C标记的花生四烯酸(AA)作为反应底物,用荧光高效液相色谱法(HPLC)通过检测牛肺动脉内皮细胞催化外源性AA生成20-羟基廿碳四烯酸(20-HETE)的反应,确定肺动脉内皮细胞是否存在细胞色素P450 CYP4A;将肺动脉内皮细胞破碎并用乙酸乙酯萃取,用荧光物质标记萃取物,用荧光HPLC检测内源性20-HETE,从代谢产物水平确定细胞色素P450 CYP4A的功能蛋白质表达。结果①Western印迹法分析结果表明,细胞色素P450 CYP4A蛋白在牛肺动脉内皮细胞和平滑肌细胞中均有表达,在肺动脉内皮细胞中的表达明显高于平滑肌细胞(积分吸光度值分别为10182±279,5249±167);在牛肾动脉内皮细胞中也有表达,但明显低于肺动脉内皮细胞的表达(积分吸光度值分别为12173±171,17863±207)。②原位杂交结果显示,细胞色素P450 CYP4A mRNA可在大鼠肺支气管内皮细胞和大鼠肺血管内皮均有表达。③用HPLC在牛肺动脉内皮细胞中检测到20-HETE,表明细胞色素P450 CYP4A在肺动脉内皮细胞存在。结论细胞色素P450 CYP4A有其特定的组织和细胞分布,在牛和大鼠肺脏动脉内皮细胞有高表达。     

In vitro characterization of the biotransformation of thiocoraline, a novel marine anti-cancer drug

Thiocoraline is a potent new marine anti-cancer drug in vitro, which will be tested in phase I clinical studies shortly. To assess the biotransformation and the potential implications for human pharmacology and toxicology, the in vitro metabolism of thiocoraline was characterized using human plasma, human liver preparations, cytochrome P450 (CYP) and uridine diphosphoglucuronosyl transferase (UGT) supersomes and human cell lines.Thiocoraline is significantly metabolized by enzymes present in human plasma; t (1/2) shifted from 25.2 h in phosphate buffered saline to 4.3 h in human plasma. Using CYP supersomes it was shown that thiocoraline is mainly metabolized by CYP3A4, with CYP1A1, CYP2C8 and CYP2C9 playing a minor role in the biotransformation (<3%). Only minor glucuronidation was observed for thiocoraline by UGT1A1 and UGT1A9 and no glucuronidation was observed in human liver S9 fraction. In addition, no glucosidation and sulfation were observed for thiocoraline in human liver cytosol and S9 fraction. However, the metabolites formed by cytochrome P450 were further conjugated by UGT, glutathione-S-transferase (GST) and sulfotransferase (ST). In contrast to the CYP metabolism observed in supersomes, no effect could be observed from the CYP3A4 inhibitors on the cytotoxicity of thiocoraline in Hep G2 cells. However, this could be due to low CYP expression levels in the Hep G2 and IGROV-1 cell line.These results provide evidence that human CYP3A4 plays a major role in the metabolism of thiocoraline in vitro and that the metabolites formed by CYP are conjugated by the phase II enzymes UGT, ST and GST.