草鱼肝微粒体的提取及CYP酶活性的测定

CYP酶代谢是药物生物转化的主要途径,其数量和活性大小直接影响药物在体内的活化与代谢。我们对草鱼肝微粒体CYP酶含量及其活性进行了初步研究,以差速离心法提取草鱼肝微粒体,以CO还原差示光谱法测得CYP酶及细胞色素b5含量分别为0.619±0.102 nmol/mg、0.264±0.042 nmol/mg。以7-乙氧异吩噁唑酮-O-脱乙基反应、苯胺-4-羟化反应、氨基比林-N-脱甲基反应作为CYP1A、CYP2E、CYP3A的探针反应,测得EROD酶活为0.043±0.004 nmol/mg/min,ANH酶活为0.028±0.002 nmol/mg/min,AMND酶活为0.207±0.035 nmol/mg/m in。结果表明草鱼肝微粒体中CYP酶发育完好,并且具有参与药物代谢的3种主要亚型活性,其含量与活性大小与其它实验动物相差较大。本实验的方法与结果为草鱼CYP酶的系统研究提供可靠手段,最终为指导水产合理用药提供理论依据。     

黄芩苷与甘草酸对恩诺沙星在异育银鲫体内代谢的影响

将黄芩苷(baicalin,BL)和甘草酸(glycyrrhizin,GZ)口灌异育银鲫(Carassius auratus gibelio),探讨其对恩诺沙星(enrofloxacin,ENR)在体内的代谢和肝微粒体细胞色素氧化酶CYP1A、CYP3A活性的影响。异育银鲫连续7 d分别口灌黄芩苷(100 mg/kg)和甘草酸(100 mg/kg),以口灌玉米油作为对照。末次给药24 h后每组随机取10尾腹腔注射恩诺沙星(10 mg/kg),采用单个动物连续采血,HPLC测定血浆恩诺沙星和其代谢产物环丙沙星(cipmfloxacin,CIP)浓度,分析药动学及其参数;同时每组选取6尾检测肝微粒体CYP1A和CYP3A活性。结果表明:(1)黄芩苷(BL)和甘草酸(GZ)对恩诺沙星的吸收有明显抑制作用,主要表现在恩诺沙星峰浓度(Cmax)降低,曲线下面积(AUC)减少;(2)口灌BL和GZ后,恩诺沙星及其代谢产物环丙沙星的消除半衰期(t1/2z)明显小于对照组(P<0.05),而总体清除率(CLz/F)则增大,说明黄芩苷和甘草酸促进了恩诺沙星和环丙沙星的消除;(3)口灌BL和GZ后,BL组和GZ组的Cmax-CIP/Cmax-ENR比值分别为1.48%、2.22%,对照为0.95%;BL组和GZ组的AUC0-t-CIP/AUC0-t-ENR比值分别为2.16%、1.76%,对照组为1.7%。综合分析代谢产物环丙沙星峰浓度、Cmax-CIP/Cmax-ENR和AUC0-t-CIP/AUC0-t-ENR比值可以得出,黄芩苷和甘草酸对恩诺沙星N-脱乙基具有诱导作用;(4)与对照组相比较,BL组和GZ组的7-乙氧基异吩唑酮-O-脱乙基酶(EROD,CYP1A标志酶)和红霉素-N-脱甲基酶(ERND,CYP3A标志酶)活性显著升高(P<0.05),说明黄芩苷和甘草酸对CYP1A和CYP3A都有诱导作用。结合以上结果,认为黄芩苷和甘草酸加速了恩诺沙星的消除和其代谢产物CIP的生成,很可能与诱导CYP1A和CYP3A活性有关。     

呋喃西林代谢产物在中国对虾(Fenneropenaeus chinensis)体内的消除规律及其对代谢酶活性的影响

中国对虾(Fenneropenaeus chinensis)以30 mg/kg体重剂量投喂呋喃西林药饵5 d后,检测对虾各组织中呋喃西林代谢物氨基脲(Semicarbazide, SEM)浓度和肝胰腺中的药物代谢酶活性。结果显示,肝胰腺中药物起始和达峰浓度最大,远远高于其他各组织。20 d实验结束时,SEM浓度依次为肌肉>鳃>甲壳>血淋巴>肝胰腺,消除半衰期为肌肉(5.89 d)>甲壳(4.66 d) >鳃(2.95 d) >血淋巴(2.70 d) >肝胰腺(1.47 d),肝胰腺中SEM消除最快,肌肉中消除最慢,药物残留时间较长。与对照组相比,给药组肝胰腺中乙氧基香豆素-O-脱乙基酶(ECOD)和氨基比林-N-脱甲基酶(APND)活性均呈现先下降后上升的趋势,停药3 h时,ECOD活性极显著低于对照组(P<0.01),酶活性被最大程度抑制,随后出现上升趋势,并分别于48、72、96、120、240 h时,ECOD活性均极显著高于对照组(P<0.01);APND在停药6 h和9 h时活性最低,被抑制效应最大(P<0.01),240 h时极显著高于对照组(P<0.01),之后APND活性降低并趋向对照组水平。结果表明,SEM在中国对虾体内消除缓慢,消除半衰期较长,并对ECOD、APND酶活性有影响,推测ECOD、APND参与呋喃西林在中国对虾体内的代谢消除。     

诺氟沙星对中国明对虾鳃和血清ECOD、APND和GST活性的影响

诺氟沙星药饵每天2次投喂中国明对虾(Fenneropenaeus chinensis),给药剂量分别为0 mg/kg(对照组)、15mg/kg(LD组)、30 mg/kg(MD组)和60 mg/kg(HD组),连续投喂7 d后,分析诺氟沙星对中国明对虾鳃和血清7-乙氧基香豆素-O-脱乙基酶(ECOD)、氨基比林-N-脱甲基酶(APND)和谷胱甘肽-S-转移酶(GST)活性的影响。结果表明,不同浓度诺氟沙星对中国明对虾鳃和血清GST活性呈现显著抑制作用(P<0.05),且GST活性随着取样时间推移表现出先下降后上升的趋势。其中MD组鳃GST活性在4 h时达到最低,是对照组的8.87%;LD组血清GST活性在24 h达到最低值12.01 U/mL,仅为对照组的25.02%。与对照组相比较,给药组中国明对虾鳃和血清APND和ECOD活性均受到显著抑制(P<0.05),且呈现出一定的时间和剂量效应,随着浓度升高,APND和ECOD活性逐渐降低;而随着时间推移,APND和ECOD活性呈现先下降后上升的趋势。本研究通过探讨诺氟沙星对上述酶类的影响,为诺氟沙星的合理使用及与其他药物的联用提供理论参考。     

草鱼肾细胞中细胞色素P450 3A基因诱导表达及其酶活性分析

采用基因克隆技术获取草鱼(Ctenopharyngodon idellus)细胞色素P450 3A(CYP3A)cDNA序列长1 849 bp,包含完整开放阅读框(open reading frame,ORF)1 542 bp,1个终止密码和含polyA信号3'UTR； ORF编码514个氨基酸,含信号肽(29 aa),2个跨膜螺旋区(23 aa,23 aa),血红素结合域(21 aa)和6个底物识别位点(SRSl-6)；与其他脊椎动物CYP3A氨基酸序列相似度达60％ ～ 92％.用实时定量PCR和分光光度计法分别研究了草鱼肾细胞系(Ctenopharyngodon idellus kidney cell line CIK)中利福平(rifampicin,RIF)诱导CYP3A基因表达和红霉素-N-脱甲基酶(ERND)活性,采用红霉素脱甲基酶活性法测定CYP3A的活性.结果显示,诱导组CIK细胞中CYP3A mRNA表达量在8最高,而CYP3A酶活在10h最高；CYP3A mRNA和酶活性与对照组均具显著差异性（P<0.05）.CYP3A转录水平先于相应酶活变化表达,且转录水平表达显著高于酶活性,这很可能反映了RIF对CYP3A的诱导主要作用于转录水平而不直接作用于酶活,本研究旨为药物对CYP3A转录调控和活性调节提供科学依据,并为渔药代谢酶的研究提供理论依据.     

恩诺沙星对凡纳滨对虾(Litopenaeus vannamei) CYP2基因表达及氨基比林-N-脱甲基酶活性的影响

以凡纳滨对虾(Litopenaeus vannamei)为研究对象,运用RT-PCR方法测定CYP2基因在凡纳滨对虾组织中的表达分布,并分析不同剂量恩诺沙星对凡纳滨对虾肝胰腺中CYP2基因表达和氨基比林-N-脱甲基酶(APND)活性的影响.结果显示,CYP2在肝胰腺、鳃、血淋巴、肌肉、甲壳、肠、胃、心脏和眼柄中均有分布,在肝胰腺中表达量最高,胃次之,血淋巴中表达量最低.口服低(15 mg/kg)、中(30 mg/kg)、高(60 mg/kg)3个剂量恩诺沙星药饵后,凡纳滨对虾肝胰腺中CYP2基因表达和APND活性较对照组均呈现下降趋势;药物浓度越高,基因表达量和酶活性越低,表明恩诺沙星可抑制CYP2在凡纳滨对虾体内的表达.在生产实践中联合用药时,应考虑到因恩诺沙星对CYP2的抑制作用而导致经其代谢的药物在生物体内的蓄积和毒性增强.     

黄芩苷对牙鲆肝CYP1A酶活性及基因表达的影响

本研究以中药黄芩苷为受试药物,研究其在不同剂量和作用时间下对牙鲆(Paralichthys olivaceus)肝CYP1A酶活性和基因表达的影响。采用体内实验,将黄芩苷按低(50mg/kg·d)、中(100mg/kg·d)、高(200mg/kg·d)3个剂量分别口灌牙鲆,连续给药,并分别于给药3d、6d、9d后取样,测定CYP1A酶活性。结果表明,较空白组而言,给药3d,黄芩苷各剂量组鱼肝中EROD酶(CYP1A标志酶)活性没有明显变化(P0.05);给药6d,高剂量组的EROD酶活性极显著增加(P0.01),中剂量组的则显著增加(P0.05);给药9d,高、中剂量组的EROD酶活性均极显著增加(P0.01),低剂量组的EROD酶活性显著增加(P0.05),且黄芩苷对该酶的诱导作用与剂量和药物作用时间均呈正相关。半定量RT-PCR结果表明,各药物剂量组的CYP1A基因表达水平都发生了上调,且这种表达上调的幅度同CYP1A酶的活性一样,随给药时间和剂量的增加而加强,提示黄芩苷作为诱导剂对CYP1A酶活性的作用机制可能与调控CYP1A的转录水平有关。     

异育银鲫肝微粒体体外恩诺沙星N-脱乙基酶酶促反应动力学初步研究

建立了异育银鲫肝微粒体体外孵育恩诺沙星(enrofloxacin,EF)的酶促反应体系,应用RP-HPLC法检测其主要代谢产物环丙沙星(ciprofloxacin,CF),以间接反映恩诺沙星N-脱乙基酶的活性。采用二氯甲烷提取、正已烷去脂提取有效成分,样品回收率均大于81.4%,含CF或EF的肝微粒体样品能在4℃下稳定至少72 h,日内变异系数不超过3.51%,日间变异系数不超过3.71%,EF和CF准确度分别小于8.1%、6.5%,检测限(信超比为3)分别为0.15μmol/L、0.3μmol/L。恩诺沙星在银鲫肝微粒体中代谢符合一级消除方程C=140e-0.072t,消除速率常数(K)为0.072/h,消除半衰期(T1/2)为9.627 h。用Origin Lab软件的Hyperbl模型进行拟和,以方程y=P1.x/(P2+x)表示米氏常数Vmax、Km两者的关系,求得Vmax为0.2602±0.0147nmol/mg.min,Km为53.8985±10.2257μmol/L,内在清除率(Clint)约为0.0048 mL/(min.mg)。恩诺沙星消除率和环丙沙星转化率的研究结果提示,恩诺沙星在银鲫肝微粒体中的代谢以N-脱乙基反应为主,提示可能还通过其它途径生成另外代谢产物。而酶动力学研究数据(Vmax,Clint值)表明,恩诺沙星N-脱乙基酶与底物结合力不强,酶促反应强度处于中等水平。据此,笔者推测恩诺沙星在银鲫肝微粒体中的代谢速度较为缓慢。     

恩诺沙星对凡纳滨对虾(Litopenaeus vannamei)CYP2基因表达及氨基比林-N-脱甲基酶活性的影响

以凡纳滨对虾(Litopenaeus vannamei)为研究对象,运用RT-PCR方法测定CYP2基因在凡纳滨对虾组织中的表达分布,并分析不同剂量恩诺沙星对凡纳滨对虾肝胰腺中CYP2基因表达和氨基比林-N-脱甲基酶(APND)活性的影响。结果显示,CYP2在肝胰腺、鳃、血淋巴、肌肉、甲壳、肠、胃、心脏和眼柄中均有分布,在肝胰腺中表达量最高,胃次之,血淋巴中表达量最低。口服低(15 mg/kg)、中(30 mg/kg)、高(60 mg/kg)3个剂量恩诺沙星药饵后,凡纳滨对虾肝胰腺中CYP2基因表达和APND活性较对照组均呈现下降趋势;药物浓度越高,基因表达量和酶活性越低,表明恩诺沙星可抑制CYP2在凡纳滨对虾体内的表达。在生产实践中联合用药时,应考虑到因恩诺沙星对CYP2的抑制作用而导致经其代谢的药物在生物体内的蓄积和毒性增强。     

外源木聚糖酶对异育银鲫生长、超氧化物歧化酶及溶菌酶活性的影响

在基础饲料中分别添加0 mg/kg、50 mg/kg、100 mg/kg和200 mg/kg的木聚糖酶,饲养初始体重6.71 g左右的异育银鲫(Carassius auratus gibelio)56 d后,研究了木聚糖酶对异育银鲫生长、超氧化物歧化酶(SOD)和溶菌酶活性的影响。结果表明:添加100 mg/kg木聚糖酶组异育银鲫的增重率和特定生长率最大,显著大于对照组(P<0.05),同时该组异育银鲫的饲料系数最低,显著低于对照组(P<0.05)。添加50 mg/kg和100 mg/kg木聚糖酶可显著提高异育银鲫血清、脾脏、肝胰脏、头肾SOD活性以及血清、头肾、脾脏溶菌酶活性。     

五种中药提取物对拟穴青蟹肝微粒体CYP1A的抑制作用

利用肝微粒体体外孵育体系,通过酶促反应动力学研究黄芩、柴胡、连翘、吴茱萸和野菊花5种中药提取物对拟穴青蟹(Scylla paramamosain)肝微粒体CYPIA的抑制作用.方法:5种中草药提取物分别与拟穴青蟹肝微粒体共同孵育,荧光分光光度法测定探针药物7-乙氧基异吩唑酮代谢产物试卤灵的生成量,来反映中药提取物对CY...     

The cytochrome P450 system of atlantic salmon (Salmo salar): I. Basal properties and induction of P450 1A1 in liver of immature and mature fish

The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The K_m for 7-ethoxyresorufin was 0.4 µM, and V_max 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20–30 pmol/min/mg protein). Treatment with the P450 1A1 inducer -naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30–40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (M_r=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at –80°C in 20% glycerol without losing more than 20–40% of its catalytic activity.Parts of this work were presented at the 5th International Symposium on Responses of Marine Organisms to Pollutants, April 1989 in Plymouth, United Kingdom (Larsen and Goksøyr 1989).     

Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain

Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by β-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.     

草鱼孕烷X受体与细胞色素P450 3A mRNA表达相关性初步研究

根据GenBank中斑马鱼(Danio rerio)和虹鳟(Oncorhynchus mykiss)孕烷X受体(pregnane X receptor,PXR)基因序列设计保守区域简并引物,通过PCR扩增获得草鱼(Ctenopharyngodon idellus)PXR cDNA核苷酸序列片段为509 bp,经推导获取169 aa序列。对草鱼与其它物种PXR氨基酸序列进行同源性比较,用以鉴定其在物种中的进化。应用实时荧光定量PCR(qRT-PCR)检测草鱼9种组织PXR和细胞色素P450 3A(cytochrome P450 3A,CYP3A)基因mRNA相对含量,结果表明,其表达丰度依次为:前肠﹥肝脏﹥肾脏﹥鳃﹥肌肉﹥后肠﹥性腺﹥心脏﹥脾脏,CYP3A和PXR基因在草鱼组织中表达分布基本一致,在前肠、肝脏和肾脏高度表达,而在其它组织低度表达。为了进一步研究PXR和CYP3A基因表达相关性,通过细胞,特选用诱导剂利福平(rifampicin,RIF),最佳诱导浓度40μM,孵育1、2、4、6、8、10、12、24 h后,用qRT-PCR检测CYP3A-PXR基因动态表达过程。结果显示,诱导组CYP3A和PXR基因表达量均高于对照组,显示出显著诱导效应。     

Confinement stress and starvation modulate the induction of EROD activity after dietary exposure to PCB 126 in the Mozambique tilapia (Oreochromis mossambicus)

Polychlorinated biphenyls (PCBs) are environmental pollutants, accumulating in the food chain and inducing cytochrome P450 1A (CYP P450 1A) monooxygenase enzymes. This study aimed to investigate the influence of dietary PCB 126 exposure on cytochrome P4501A-associated enzyme activities in head kidney, liver, gill and intestine of unstressed and stressed tilapia (Oreochromis mossambicus) before and after starvation. Fish were fed diets containing 0 μg (control), 0.5 μg (low) or 50 μg (high) PCB126 per kg body weight per day for 7 days. After the PCB-exposure each treatment group was sampled directly, or was subsequently exposed to confinement stress. Replicate groups remained undisturbed, starved for 3 weeks and were thereafter sampled directly or after confinement. Catalytic activity of CYP P4501A was determined as 7-ethoxyresorufin-O-deethylase (EROD) activity. Directly after PCB exposure and after starvation, EROD activities in liver, gill and head kidney increased with higher PCB dose. Intestinal EROD activities were not detectable at any sampling point. Confinement, known to evoke a stress response increased EROD activities in livers (only directly after PCB exposure) and in head kidneys (directly after PCB exposure and after starvation) but only in fish fed the low PCB-diet. We suggest that stressing tilapia induces EROD activities at non-saturating PCB concentrations and that this effect is more pronounced in nourished than in starved fish. These data show that the cytochrome P450 1A response to toxicants in fish is also influenced by non-chemical factors. This should be considered when EROD activities are used as a biomarker for environmental monitoring studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

三种主养草鱼池塘沉积物-水界面碳通量的研究

为了探讨环境因子和底栖配养生物对碳通量的影响,采用实验室培养方法,测定并比较了不同混养模式下沉积物-水界面各形态碳的通量,监测了8月沉积物-水界面上覆水中各形态碳含量的昼夜变化。3种主养草鱼池塘的养殖模式为草鱼、鲢和鳙混养(GSB),草鱼、鲢、鳙和凡纳滨对虾混养(GSBL),草鱼、鲢、鳙和鲤混养(GSBC)。主要实验结果如下:(1)在养殖周期内,3种混养模式沉积物-水界面可溶性无机碳(DIC)通量范围为0.65~16.90 mg/(m²·d),可溶性有机碳(DOC)通量范围为0.16~13.49 mg/(m²·d),颗粒性有机碳(POC)通量范围为-2.29~3.32 mg/(m²·d);GSB和GSBC模式各形态碳通量均以8月最高,GSBL模式则以7月最高。(2)在养殖周期内,3种混养模式沉积物对DIC和DOC均表现为释放,而对POC在4-6月以吸收为主,其余时间表现为释放。(3)在养殖的中后期,3种混养模式的DIC、DOC和POC通量存在一定差异。其中,GSBL和GSBC模式中各形态碳通量值显著高于GSB模式(P<0.05)。(4)8月上覆水中DIC和DOC的含量在4:00达到最大值,POC含量则表现为GSBL模式在4:00达到最大值,12:00达到最小值,而GSBC模式在4:00达到最小值,12:00达到最大值。(5)各形态碳通量的变化与上覆水中的DO、pH呈显著负相关关系,而与水温呈显著正相关关系。     

两种鲤鱼对侵袭性病原体及嗜水气单胞菌抗病力的比较

调查同塘饲养的高寒鲤，散鳞镜鲤当年鱼对侵袭性病原体及人工感染嗜水气单胞菌耐受力进行比较，侵袭性病原体检出8种，其中绦虫只有散鳞镜鲤感染，感染率达到29％，其余种类二种鲤鱼体表，鳃都有寄生，但寄生的程度有所不同，高寒鲤的抵抗力要比散鳞镜鲤强，对人工感染的嗜水气单胞菌的耐受力高寒鲤比散镜鲤高，这可能与品种对病原菌耐性不同有关。     

Effect of blood glucose level on acute stress response of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

Stress has a considerable impact on welfare and productivity of fish, and blood glucose level of fish may be a factor modulating stress response. This study evaluated the effect of blood glucose level and handling on acute stress response of grass carp Ctenopharyngodon idella. Fish were intraperitoneally injected with glucose at 0, 0.2, 0.5, and 1.0 mg g^?1 body mass (BM) and then were exposed to handling for 5 min. Glucose injection resulted in increase of plasma glucose level and liver glycogen content and decrease of plasma lactate level. Handling resulted in increase of plasma levels of cortisol, glucose, and lactate and plasma lactic dehydrogenase (LDH) activity and decrease of liver glycogen content. At 1 h post-stress, the plasma cortisol level was lower in the stressed fish injected with glucose at 0.5 mg g^?1 BM than the stressed fish injected with glucose at 0, 0.2, and 1.0 mg g^?1 BM. No significant differences were found in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase (PK) in the liver between the stressed and unstressed fish, regardless of the dose of glucose injection. At 1 h post-stress, the liver glucose-6-phosphatase (G6Pase) activity was higher in the fish without glucose injection than in the fish injected with glucose. This study reveals that blood glucose level can affect stress response of grass carp by modulating cortisol release and glucose homeostasis through glycogen metabolism and gluconeogenesis in the liver.