乙醇对CHSE-1细胞中细胞色素P450 2E1的诱导研究

以乙醇为细胞色素P450 2E1的诱导剂,在CHSE-1细胞中研究了该诱导剂与酶活性之间的时间效应关系和剂量效应关系。细胞传代后培养48 h,加入含有不同浓度乙醇的新鲜培养基,孵育诱导24 h,之后加入底物苯胺反应30 min。以苯胺的代谢产物4-氨基酚的生成量来反映CYP2E1活性。苯胺浓度为10～16 mmol/L时CYP2E1活性最大,可达(0.152±0.095)nmol.min-1.mg-1,该浓度范围的苯胺适合用于指示CYP2E1的活性。以Gentox模型对诱导的剂量效应进行拟合,得到一个先升高后降低的曲线,说明乙醇对CHSE-1细胞中CYP2E1有先诱导后抑制作用。拟合方程F(x)=(p0 kx)/(1 〔x/EC50〕nH)参数P0为0.140,nH为2.456,k为0.024,EC50为34.938。CYP2E1酶活性随着诱导时间的增加逐渐增强,24 h内酶活性随时间的增加而增强,可达(0.446±0.092)nmol/(min.mg),呈典型的酶诱导现象,建立了乙醇对CHSE-1中CYP2E1的诱导模型。     

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。     

3 种多环芳烃对条纹锯鮨胚胎发育及早期仔鱼的毒性效应

为探讨不同多环芳烃(PAHs)对海水鱼类早期生长发育的毒性效应及作用机制,分别比较研究了多环芳烃中的3环菲(Phe)、4环芘(Py)、5环苯并芘(Bap)单一暴露和与α-萘黄酮(ANF)联合暴露对条纹锯鮨(Centropristis striata)胚胎发育及早期仔鱼的毒性效应。对其胚胎中的细胞色素酶(EROD)活性、发育畸形(以心脏畸形为准)、孵化率和早期仔鱼死亡率等指标进行测定分析,结果显示:(1)Bap、Py的浓度与EROD活性之间的剂量效应关系比Phe明显,3种PAHs对条纹锯鮨胚胎中EROD活性的诱导能力由高到低依次为Bap、Py、Phe,其活性的最大值均出现在各物质的最高浓度组,其活性分别为对照组的857.52%、514.21%、280.50%;在ANF影响下,EROD的活性诱导被抑制,最高浓度组的活性分别为对照组的189.27%、278.55%、195.40%。(2)综合考虑孵化率、畸形指数和早期仔鱼死亡率等指标,3种PAHs对胚胎发育及早期仔鱼的毒性由高到低依次为Bap、Py、Phe,3种物质的浓度与受精卵的孵化率呈明显负相关,与畸形指数和仔鱼死亡率呈明显正相关;在ANF影响下,Bap和Py处理组的毒性效应得到明显抑制,Phe与ANF联合暴露处理组的毒性效应与单一暴露处理相比无相关性(P0.05)。研究表明,3种PAHs对条纹锯鮨的作用机制存在一定差异,Phe为非特异性麻醉毒性机制,Bap和Py可能通过AHR途径和CYP1 A活性诱导两种机制共同作用。     

草鱼肾细胞中双氟沙星代谢酶的酶动学

细胞色素P450s(CYPs)主要参与动物体内药物代谢。水产养殖中不合理的联合用药常会导致治疗失败,这通常与CYP活性的诱导有关。然而,关于鱼类CYP的诱导却知之甚少。为获得有关CYP诱导的信息,实验采用反相高效液相色谱(RP-HPLC)的方法测定了草鱼肾细胞(CIK)中CYPs的特异性诱导剂对双氟沙星(DIF)的代谢作用及酶动学分析。对照组和β-萘黄酮(BNF)诱导组的酶动学方程分别为1/V=0.1375×1/[S]+0.003和1/V=0.0245×1/[S]+0.0013。DIF与经BNF处理的CIK共孵育后,其代谢量增加了1倍,酶动学参数Clint和Vmax值分别增加了7倍和2倍。BNF是CYP1A的特异性诱导剂,因此,CYP1A可能参与了DIF的代谢。     

草鱼体外培养细胞中β-萘黄酮对CYP1A基因表达的诱导

为了建立以CYP1A cDNA为探针的水环境毒理学细胞模型,以β-萘黄酮(BNF)作为诱导剂,通过半定量PCR技术研究CYP1A在草鱼不同细胞系及其对应组织中的诱导表达情况。在半定量PCR反应参数研究中,对关键的退火温度和循环次数进行了优化,结果显示退火温度为57 ℃,循环次数为30次较为合适,该条件下Gauss迹量的CYP1A/ACT比值能更准确的反映CYP1A的表达水平。对照组和诱导组草鱼细胞中ACT和CYP1A cDNA扩增结果表明,细胞中CYP1A的基础表达量较低,而BNF诱导使GCL、CIK和CO细胞中CYP1A的表达水平得到显著的提高。GCL、CIK和CO细胞三者之间诱导后CYP1A的表达水平有显著差异(P<0.05),诱导表达量大小顺序为GCL>CIK>CO。草鱼细胞系的相应组织中ACT和CYP1A cDNA扩增以及电泳的结果与细胞中较为相似,组织中CYP1A诱导表达量大小顺序也与相应的细胞相同:肝>肾>卵巢。比较分析的结果表明,CYP1A在体内与体外的诱导表达水平具有一定的相关性。     

多氯联苯(PCBl254)对栉孔扇贝消化盲囊和鳃丝EROD、GST酶活力的影响

研究了4种质量浓度（0．5μg／L、1μg／L、10μg／L、50μg／L）PCB1254暴露下,栉孔扇贝（Chlamys ferrari）消化盲囊和鳃丝7-乙氧基-3-异吩唑酮-脱乙基酶（EROD）、谷胱甘肽转移酶（GST）活力的变化。于实验开始后0、0．5d、1d、3d、6d、9d、15d、21d、30d取样测定,结果显示,除鳃丝EROD活力在低质量浓度（0．5μg／L和1．0μg/L）随时间变化不明显（P〉0．05）外,其他各处理组PCB1254对栉孔扇贝消化盲囊和鳃丝的EROD都具有明显的激活作用,而且具有明显的剂量-效应关系。低浓度（0．5μg/L、1．0μg/L）处理下,消化盲囊和鳃丝的GST活力都呈现激活的趋势,而高浓度（10μg/L、50μg/L）下,GST活力都呈现先上升后下降的趋势,且鳃丝GST活力变化幅度较为明显。结果表明,EROD和GST活力的变化在一定程度上能够反应PCBs对栉孔扇贝影响的规律性,是合适的毒理学指标,栉孔扇贝是比较好的评价多氯联苯污染的指示生物,本研究也对栉孔扇贝对多氯联苯的生物转化机制作了初步的探讨。[中国水产科学,2008,15（2）：342-346]     

氟甲喹对异育银鲫细胞色素CYP450主要药酶的影响

在分析了氟甲喹腹腔注射后对异育银鲫(Carassius auratus gibelio)主要药酶活性影响的基础上,从蛋白表达和mRNA转录水平探究了氟甲喹对CYP1A(EROD)的诱导机制.以氟甲喹剂量35 mg/kg一次性腹腔注射异育银鲫24 h后,肝微粒体的7-乙氧基异吩唑酮-O-脱乙基酶(EROD)活性为54.33 pmol/(mg·min).显著高于对照组的34.00pmol/(mg·min)(P<0.01),而红霉索-N-脱甲基酶(ERND)[177.98 pmol/(mg·min)]、氨基比林-N-脱甲基酶(APD)[934.40 pmol/(mg·min)]及7-乙氧基香豆素-O-脱乙基酶(ECOD)[9.84 pmol/(mg·min)]与对照组[分别为140.90pmol/(mg·min)]、850.71 pmol/(mg·min)和8.93 pmol/(mg·min)]相比无显著性差异;除肾组织中ERND活性高于肝脏外,其他CYP亚型药酶活性(APD、EROD和ECOD)均以肝组织中最高.Western-blotting印迹表明,实验组肝脏中CYP1A蛋白含量明显高于对照组,与酶活(EROD)相符合;而肾、肠中没有检测到特异性条带.半定量RT-PCR结果显示,CYPiAmRNA在肝、肾和肠均有表达,但实验组与对照组并无明显差异.体外实验中,不同浓度的氟甲喹与微粒体共孵育,未见EROD活性与浓度、时间的依赖关系.综上推测,氟甲喹对鲫鱼肝CYP1A的诱导是在翻译后水平,可能是加强蛋白的稳定性.本研究旨在为氟甲喹在鱼类中的合理应用提供科学依据.     

草鱼肝细胞脂变模型的建立及脂代谢基因表达分析

为了筛选草鱼肝细胞脂肪变性的最佳诱导剂及浓度,并初步分析脂肪乳剂(lipid emulsions,LE)引起草鱼肝细胞脂肪变性的作用机理,以草鱼(Ctenopharyngodon idellus)正常肝细胞为研究对象,建立草鱼脂肪变性肝细胞模型,以含10%胎牛血清的基础培养液为对照组,处理组为含20%脂肪乳剂0.5~2 m L/L和含20%、50%胎牛血清的诱导培养液,孵育草鱼肝细胞48 h后,定量分析肝细胞内的甘油三酯(TG)含量,观察脂滴积聚情况及肝细胞超微结构的变化,检测细胞培养上清中谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate transaminase,AST)的活性,q RT-PCR技术检测脂代谢关键基因(PPAR?、PPAR?、SREBP-1c、LPL、Lep和UCP2)的转录水平变化,蛋白质印迹技术检测PPAR?、SREBP-1c的蛋白水平变化。结果发现,含1~2 m L/L LE的诱导液组和含20%、50%FBS的诱导液组与对照组相比TG含量均显著上升(P0.05),且20%FBS和各浓度LE诱导组的转氨酶活性与对照组相比差异不显著(P0.05),表明含1~2 m L/L LE的诱导液和含20%FBS的诱导液均可建立草鱼营养性脂肪肝细胞模型。在肝细胞脂变模型中,PPARγ和LPL等脂代谢基因的表达量显著升高(P0.05),而Lep基因表达量显著降低(P0.05),PPARγ和SREBP-1c的蛋白水平升高。结论认为:采用1~2 m L/L LE和20%的FBS均可以在短时间内建立草鱼肝细胞脂肪变性模型,含1 m L/L LE的诱导液诱导效果最佳;肝细胞内脂质的蓄积可能与脂肪代谢关键基因PPARγ、SREBP-1c、LPL及Lep等密切相关。     

氧化乐果对斑马鱼的毒性作用及生物富集性研究

为研究斑马鱼(Danio rerio)作为有机磷农药指示生物的可能性,以斑马鱼为实验对象,采用半静态法,研究浓度为0、2.5、5.0、7.5、10.0 mg/L的氧化乐果对斑马鱼体内蛋白质含量及乙酰胆碱酯酶(Ach E)和过氧化物酶(POD)活性的影响及其对0、2、4、6、8、10 mg/L氧化乐果7 d的富集系数(即BCF7 d),探究氧化乐果对斑马鱼的毒性作用及其生物富集性。结果显示,氧化乐果对斑马鱼的96 h半数致死浓度(LC50)为31.57 mg/L,在1~100mg/L之间,属于高毒药物;蛋白质含量总体表现为先升高、再下降的趋势;Ach E活性受到抑制,总体表现为下降趋势;POD活性表现为诱导-抑制效应;BCF7 d随浓度的增加而减小,为1.54~5.80,且BCF7 d随Ach E活性的降低而降低,毒性作用越大,斑马鱼对氧化乐果的富集作用越弱。研究表明,蛋白质含量和乙酰胆碱酯酶活性与氧化乐果浓度有显著的剂量-效应关系,斑马鱼可以作为有机磷农药污染的指示生物。     

大弹涂鱼仔稚鱼和早期幼鱼的消化酶活性

采用酶学分析的方法，研究了大弹涂鱼前期仔鱼、后期仔鱼、稚鱼和早期幼鱼发育过程中胰腺酶、肠酶和胃蛋白酶活性的变化。结果表明，(1)4种胰腺酶(淀粉酶、胰蛋白酶、糜蛋白酶和羧肽酶A)和8种肠酶(麦芽糖酶、蔗糖酶、乳糖酶、海藻糖酶、纤维二糖酶、碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶)的比活力均在仔鱼期较高，稚鱼期降至最低，早期幼鱼则急剧上升，而每尾鱼酶的总活力却随着幼体的发育而逐渐增加；(2)胃蛋白酶活性在后期仔鱼才开始检测到，此后一直呈显著上升趋势；(3)早期幼鱼肠部位的4种胰腺酶活性分别占其酶总活性的百分比均显著高于稚鱼期；(4) 稚鱼期仅3种肠酶(麦芽糖酶、纤维二糖酶和γ-谷氨酰转肽酶)高度富积在肠刷状缘膜上，而早期幼鱼除蔗糖酶外的7种肠酶在肠刷状缘膜上的富集系数均大于5.1。由此得出结论，(1)在仔鱼期，蛋白质的消化是依靠胰腺酶进行的，进入稚鱼期，胃蛋白酶开始对蛋白质的消化起重要作用；(2)在早期幼鱼，胰腺分泌机制及肠细胞已完全成熟，标志着成鱼的消化模式的形成。     

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.     

Size selectivity of diamond and square mesh codends for hairfin anchovy Setipinna taty in Chinese stow net fisheries

ABSTRACT:   Selectivity experiments of diamond and square mesh codends with different mesh size (30, 35, 40 and 45 mm) for hairfin anchovy Setipinna taty were carried out using the covered codend method in Chinese stow net fisheries. In the present study, 5151 specimens were subsampled and asymmetric and symmetric selectivity curves (Richards and Logistic curves) were fitted to the catch data, using the maximum likelihood method according to the SELECT model. The results show that the Richards curve gives a better fit to the selectivity curve for diamond mesh codends with 30, 35 and 40-mm mesh sizes, whereas the logistic curve gives a better fit for the diamond mesh codend with 45 mm mesh size and square mesh codends with 30, 35, 40 and 45-mm mesh size. With the exception of 45-mm mesh size codends, the L ₅₀ of diamond mesh codends were insignificantly larger than those of square mesh codends. However, the selectivity curves of square mesh codends were steeper than those of diamond mesh codends, except the 30-mm mesh size codends. As for diamond mesh codends, according to the linear relationship between the L ₅₀ and mesh size, 36.5-mm mesh size codends could release 50% juvenile hairfin anchovy (below 1-year old). However, diamond mesh codends with such a mesh size are not selective for other species.     

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).     

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.     

氟离子对西伯利亚鲟胚胎发育的影响

本试验在水温17±1℃的条件下,采用半静态式生物毒性试验方法,探讨了氟离子(F-)对西伯利亚鲟胚胎发育的影响。结果表明,100~600 mg/L氟暴露会导致胚胎孵化延迟,氟暴露组半数孵化时间(MHT)比对照组推迟9~22 h。氟暴露同时导致胚胎死亡率显著增加(P<0.05),出膜仔鱼活力减弱、存活时间缩短、畸形率显著升高(P<0.05),出现卵黄囊畸形、脊椎畸形、眼部充血等畸形症状。试验得出F-致西伯利亚鲟胚胎144 h半致死浓度(LC50)为447.61 mg/L,孵化安全浓度(SC)为4.48 mg/L,导致仔鱼畸形的半数效应浓度(EC50)为536 mg/L。氟对西伯利亚鲟胚胎的安全质量浓度低于我国部分高氟地区地下水、地表水及人为氟污染水域中氟含量,提示天然水体中氟可能会对鱼类胚胎产生负面影响,应引起重视。     

Effects of dietary tea polyphenols on growth,immunity and lipid metabolism of juvenile black carp Mylopharyngodon piceus

An 8‐week feeding experiment was aimed to investigate the effect of dietary tea polyphenols (TP) on growth, immunity and lipid metabolism in juvenile black carp Mylopharyngodon piceus (initial weight 5.90 ± 0.03 g). Tea polyphenols were added at different levels (0, 25, 50, 100 and 500 mg/kg; TP₀, TP₂₅, TP₅₀, TP₁₀₀ and TP₅₀₀). The results are as follows: the highest specific growth rate (SGR) and condition factor (CF) and activity of trypsin (TRS) of intestine in TP₅₀, but SGR and activities of lipase (LPS)and TRS of intestine and content of whole body crude protein in TP₅₀₀ were remarkably lower than TP₀. Compared with TP₀, content of serum superoxide dismutase (SOD) and glutamic oxalacetic transaminase (GOT) remarkably increased (p < .05), but contents of glutathione (GSH), glutathione peroxidase (GSH‐Pox), malondialdehyde (MDA), triglyceride (TG) and low‐density lipoprotein cholesterol (LDL‐C) were significantly decreased (p < .05), content of cortisol was remarkably lower in TP₅₀ and TP₁₀₀ (p < .05), expression of growth hormone (GH) and melanocortin 4 receptor (MC4R) in liver and GH in muscle were remarkably up‐regulated in TP₅₀, but expression of apolipoprotein A‐1 (ApoA1), GH and MC4R of intestine, ApoA1 of liver and MC4R of muscle in TP₅₀₀ were remarkably down‐regulated, contents of complement 3 (C3), complement 4 (C4), high‐density lipoprotein cholesterol (HDL‐C) and LDL‐C were significantly reduced in TP₅₀₀ (p < .05). In conclusion, TP could improve growth performance and oxidative capacity on juvenile black carp, and its optimal dosage was 50 mg/kg.     

甲基异柳磷、水胺硫磷对紫贻贝（Mytilus edulis）生长的影响

本文利用电视显微测量仪作为一种新的光电测试方法，研究了甲基异柳磷和水胺硫磷对贻贝生长的影响。实验表明，当暴露于浓度为０．５６ｍｇ·Ｌ￣（－１）和０．３ｍｇ·Ｌ￣（－１）的甲基异柳磷和水胺硫磷４－６天后，贻贝的生长即可受到明显的抑制。两种农药对贻贝９天ＥＣ＿（５０）值分别为０．４２ｍｇ·Ｌ￣（－１）和０．６０ｍｇ·Ｌ￣（－１）。甲基异柳磷和水胺硫磷还可抑制贻贝的摄食率和滤水率。生长受到严重抑制的贻贝，在转到清洁海水后，６天内生长会恢复到正常水平。