基于斑马鱼模式动物的心脑静片急性毒性研究

目的 ：利用斑马鱼模式动物考察心脑静片急性毒性。方法 ：心脑静片3份样品提取液分别给药斑马鱼胚胎处理,以野生型为阴性对照,主要检测其对斑马鱼胚胎致畸、致死的作用。结果 ：心脑静片3份测试样品在所测浓度范围内对斑马鱼早期发育具有明显的毒性和致死作用,其半数中毒剂量分别为0.0448 mg/ml、0.0857 mg/ml、0.1275 mg/ml,半数致死剂量分别为0.2123 mg/ml、0.2193 mg/ml、0.2511 mg/ml,半数异常剂量分别为0.0321 mg/ml、0.0419 mg/ml、0.0437 mg/ml。低浓度的胚胎异常主要为发育滞后,停药后表型和恢复接近正常。结论：心脑静片不同样品具有类似的胚胎发育毒性,并显示出急性致死效应,且其致死浓度窗口较窄。     

骨刺片对斑马鱼胚胎发育的急性毒性研究

目的 研究两批骨刺片样品对斑马鱼胚胎发育的急性毒性作用。方法 以斑马鱼胚胎为试验对象,观察骨刺片样品A（0.01、0.05、0.10、0.20、0.30、0.50、1.00 mg/mL）、样品B（0.01、0.05、0.10、0.50、1.00、2.00、10.00 mg/mL）对斑马鱼胚胎发育的影响,包括胚胎致畸、致死检测;同时采用超高效液相色谱法-四极杆飞行时间-质谱（UPLC-QTOF-MS）对两批样品所含化学成分进行定性分析。结果 两批样品对斑马鱼胚胎发育的影响相似,主要表现为高浓度下的致死作用,低浓度下主要以发育滞后表型为主;但二者作用浓度存在一定差异,样品A的半数致畸剂量（TD₅₀）为225.4 mg/mL,半数致死剂量（LD₅₀）为292.0 mg/mL;样品B的TD₅₀为60.3 mg/mL,LD₅₀为1.382 mg/mL。UPLC-QTOF-MS分析结果显示,两批样品所含主要化学成分基本一致,但某些成分含量存在一定差异;初步分析显示既是有效成分又是毒性成分的士的宁含量与斑马鱼胚胎试验结果呈正相关。结论 斑马鱼胚胎模型可用于评价骨刺片样品的急性毒性,两批骨刺片样品对斑马鱼胚胎发育影响均表现为高浓度下的致死作用,低浓度下,特别是样品B表现为轻度非正常表型,且均可逆。     

甘遂不同炮制品及提取物对斑马鱼的急性毒性研究

目的以模式生物斑马鱼为实验对象,评价甘遂不同炮制品及提取方法的急性毒性。方法采用回流提取方法制备甘遂不同炮制品的水提液和醇提液;将它们的提取液按几何级数设置浓度梯度,添加到鱼生活的水中,观察给药后96 h鱼只的死亡情况,以此为判断待测药物毒性大小的依据,采用SPSS Statistics 17软件计算不同炮制品水提液和醇提液对斑马鱼的半数致死浓度(LC50)。结果斑马鱼对甘遂不同炮制品的水提液和醇提液均表现出急性毒性反应,且毒性作用呈现出明显的量-毒关系;不同炮制品水提液LC50明显高于相应醇提液;同一提取方法不同炮制品的急性毒性大小顺序为甘遂生品>清炒品>醋润品>醋炙品。结论以斑马鱼作为实验动物,甘遂生品的醇提液急性毒性最强、甘遂醋炙品的水提液急性毒性最低。本实验为进一步认识与评价甘遂毒性及醋炙减毒机制提供了依据。     

毒结清丸对斑马鱼的急性毒性及其肝脏毒性评价

目的：开展毒结清丸对斑马鱼的急性毒性试验及其肝脏毒性评价,为毒结清丸在临床用药安全方面提供实验依据。方法：将3 dpf野生型AB品系斑马鱼暴露在不同浓度毒结清丸水溶液中,绘制最佳"浓度-死亡率"效应曲线,计算毒结清丸对斑马鱼的最大非致死浓度（MNLC）和LC₁₀,并对其急性毒性进行评价;通过分析斑马鱼肝脏面积、肝脏不透光度、卵黄囊面积和肝脏病理切片来评价毒结清丸对斑马鱼的肝脏毒性。结果：毒结清丸对斑马鱼的MNLC为674 μg·mL^-1,LC₁₀为741 μg·mL^-1,毒性靶器官为肝脏;毒结清丸对斑马鱼肝脏面积和肝脏不透光度均没有明显影响,674 μg·mL^-1和741 μg·mL^-1质量浓度组可诱发斑马鱼卵黄囊吸收延迟;肝脏病理学分析显示,给药组与正常对照组相似,未见明显异常。结论：在MNLC和LC₁₀的浓度下,毒结清丸仅引起卵黄囊吸收延迟;斑马鱼肝脏面积、肝脏不透光度和肝脏病理切片未见明显异常。     

舒筋定痛片对斑马鱼胚胎发育的急性毒性研究

目的 采用斑马鱼胚胎模型评价舒筋定痛片样品的急性毒性。方法 以模式生物斑马鱼胚胎为实验对象,以不同硼砂含量的舒筋定痛片样品给药处理,野生型为阴性对照,于不同发育时间点观察给药后胚胎的发育情况,包括胚胎致畸、致死检测。结果 舒筋定痛片4组测试样品在不同浓度下对斑马鱼胚胎发育有明显影响,高浓度时胚胎毒性以导致胚胎发育停滞为主,中浓度时可致发育滞后并出现心脏、脑、躯干等主要器官畸形,低浓度时大多数胚胎发育接近正常,少数胚胎出现胚胎发育中轻度的滞后。结论 舒筋定痛片测试样品对斑马鱼胚胎发育的影响主要表现为高、中浓度下的胚胎发育停滞、滞后;同时,研究表明同一企业样品毒性作用与其中所含硼砂存在一定剂量依赖性正相关。     

基于斑马鱼的香青兰总黄酮整体发育急性毒性评价研究

目的 利用模式生物斑马鱼研究香青兰总黄酮（TFDM）整体发育急性毒性。方法 采用发育至48 h的斑马鱼暴露于5、10、20、40、42、44、46、48、50、100 μg·mL^-1的TFDM,分别于暴露后24、48 h（24、48 hpe）,计算死亡率、半数死亡浓度（LC₅₀）值;测量每组斑马鱼幼鱼的体长,进行形态学观察并评分;显微镜下观察斑马鱼心脏形态并拍照,记录心率,使用Image-Pro Plus 5.1测量斑马鱼静脉窦-动脉球（SV-BA）距离;显微镜下观察各组斑马鱼是否有体侧水肿来判断TFDM对肾脏的影响并拍照;应用肝脏标记绿色荧光的转基因斑马鱼Tg （L-FABP∶EGFP）,通过检测肝脏荧光强度和面积,观察TFDM对肝脏毒性的影响。结果 TFDM的24 hpe LC₅₀为50 μg·mL^-1,48 hpe LC₅₀为48 μg·mL^-1,100 μg·mL^-1 TFDM组斑马鱼幼鱼全部死亡。与空白对照组比较,20 μg·mL^-1以下的TFDM对斑马鱼的形态和心、肝、肾各脏器无影响;20 μg·mL^-1浓度的TFDM处理斑马鱼48 h导致个别斑马鱼鱼鳔体积减小或缺失,对其他脏器无显著影响;40 μg·mL^-1的TFDM导致斑马鱼出现轻微的心包水肿,处理48 h以后斑马鱼体长显著减小（P＜0.01）,形态评分显著下降（P＜0.01）,斑马鱼的肝脏形态出现轻微变化,但肝脏荧光强度和肝脏荧光面积无显著性变化,对肾脏无影响;暴露在50 μg·mL^-1的TFDM中24 h,斑马鱼出现心包水肿,心率显著下降（P＜0.05）,肝脏荧光强度和面积显著减小（P＜0.05）,肾脏无明显变化。结论 TFDM对斑马鱼的毒性较小,低浓度（≤10 μg·mL^-1）的TFDM对斑马鱼无毒性;中浓度（20 μg·mL^-1）下TFDM对斑马鱼的毒性微弱,仅导致部分斑马鱼鱼鳔体积减小或缺失,对其他各脏器无毒性;高浓度（≥40 μg·mL^-1及以上）下有轻微的心脏毒性和肝脏毒性,在临床应用中有必要合理控制用量。     

斑马鱼胚胎急性毒性和小鼠急性经口毒性测试方法的相关性研究

传统的毒理学安全性评价方法主要采用大鼠、小鼠和兔子等作为受试实验动物来进行体内实验,获取相关毒理学毒性数据.然而,啮齿类动物毒性检测的周期长、花费高,且随着动物实验3R原则的推广,逐渐限制了现代生物医药和食品行业中大规模使用动物试验来进行毒理学安全性评价.     

河豚毒素实验疫苗对小鼠口服河豚毒素中毒的免疫保护效应

目的　开发河豚毒素 (TTX)的抗毒疫苗。方法　TTX与载体蛋白中国鲎血蓝蛋白 (TTH)、破伤风类毒素 (TT)和牛血清白蛋白 (BSA)化学偶联 ,分别制成免疫抗原TTX TTH和TTX TT ,检测抗原TTX BSA。经腹腔或皮下注射免疫原 (用Freund佐剂 ) ,免疫BALB/c小鼠 ;每组 12只 ;5个月内接受免疫原累计量 375 μg/鼠。定期采集动物血清 ,ELISA法监测血清中抗体质量 (滴度及亲和力 )。经igTTX的生理盐水攻击免疫鼠 ,以检验抗毒效价 ;首次剂量6 30 μg·kg- 1,间隔 2～ 3周后提高剂量再次攻击活存动物 ,观察攻毒实验后症状 ,记录 2 4h存活率及死亡鼠的存活时间。结果　所试两种人工抗原的抗毒效应无明显差异。免疫鼠经ig 6 30 ,80 0 ,12 0 0 ,15 0 0 ,2 0 0 0 μg·kg- 1TTX攻毒时 ,存活率分别为10 0 % ,95 % ,90 % ,70 %和 4 5 % ;测得半数死亡剂量约为 2 0 0 0 μg·kg- 1。免疫鼠经 2～ 5次igTTX重复攻毒 ,86 %动物累积耐受剂量高于 3.5mg·kg- 1;4 3%动物累积耐受剂量高于 5 .5mg·kg- 1。中毒对照动物ig 6 0 0 μg·kg- 1TTX全部死亡。结论　研制的TTX的化学实验疫苗可高效预防TTX口服攻毒 ,免疫预防是对抗TTX中毒很有希望的途径     

河豚毒素处方前初步研究

目的 考察河豚毒素（TTX）在不同溶剂中的溶解性及稳定性,以及温度和pH值对稳定性的影响。方法 配制TTX不同介质的溶液,采用高效液相色谱法（HPLC）测定其在不同温度、不同pH缓冲液中的浓度,分析计算其溶解度及稳定性。结果 TTX在pH值为3.5时溶解度最大,随着pH值增加其溶解度逐渐降低。TTX在强碱条件下降解最为迅速,在70 ℃条件下、0.1 mol/L的氢氧化钠溶液中,20 min即完全降解。稳定性试验结果同样证明TTX在碱性条件下的稳定性最差,在37 ℃、pH值=7.4的磷酸缓冲盐溶液（PBS）中,TTX浓度在1~10 h时开始持续降低,28天降解率为88.07±0.27%。结论 TTX易溶于pH值=3.5的酸性水溶液,几乎不溶于碱性水溶液。其稳定性与温度、介质pH值密切相关,在酸性水溶液中较为稳定,在碱性条件下易降解,温度升高会加速其降解过程。     

河豚毒素的生物合成

介绍了国内外河豚毒素研究的最新动态-利用海洋细菌进行河豚毒素生物合成的研究。包括河豚毒素的来源，产河豚毒素的菌种及性质，培养条件及影响因素。代谢合成机理及未来的研究展望。     

Aquatic toxicology of cypermethrin. I. Acute toxicity to some freshwater fish and invertebrates in laboratory tests

Cypermethrin is a synthetic pyrethroid. Its acute toxicity to ten freshwater invertebrates (Daphnia magna, Asellus aquaticus, Gammarus pulex, Cloeon dipterum, Gyrinus natator, Chironomus thummi, Aedes aegypti Cheoborus crystallinus, Corixa punctata, and Piona carnea) was determined in the laboratory using 24-h static water tests. The 24-h EC₅₀ values (based on reduced motility) ranged from 0.02 μg·l^?1for A. aquaticus and P. carnea to 2 μg·l^?1 for D. magna, and for seven of the species the EC₅₀ values were < 0.1 fig μg·l^?1 The 24-h LC₅₀values for G. natator, C. thummi, and C. punctata were > 5 μg·l^?1 For the seven more susceptible species the 24-h LC₅₀values ranged from 2 μg·l^?1 to 0.05 μg·l^?1The acute toxicity of cypermethrin to some species of fish (Cyprinus carpio. Scardinius erythrophthalmus, Salmo gairdneri, Salmo trutta and Tilapia nilotica) was determined using 96-h continuous-flow tests. The 96-h LC₅₀ values obtained were within the range 0.4–2.2 μg·l^?1.The solubility of cypermethrin within the range of test temperatures (15–25°C was estimated to be in the range 5–10 μg·l^?1.     

Estimation of toxicity to marine species with structure-activity models developed to estimate toxicity to freshwater fish

Structure-activity models which were developed to estimate toxicity of chemicals to freshwater fish were tested for use with an estuarine fish (Cyprinodon variegatus) and mysids (Mysidopsis bahia). Significant linear and polynomial relationships that correlated well existed between reported 96-h LC₅₀ values for each marine species and log P (log octanol/water partition coefficient). Good linear relationships were obtained when the 96-h LC₅₀ values for C. variegatus and M. bahia were regressed on water solubility (μmol/l). These models were compared to models developed for freshwater fish using log P and log S. Models using log P to estimate acute toxicity for two freshwater fish produced 96-h LC₅₀ values similar to those measured for C. variegatus and M. bahia, whereas, those models developed with water solubility produced 96-h LC₅₀ values similar to those for C. variegatus, but not for M. bahia. The data indicated that models developed with log P for freshwater fish can be used to estimate toxicity to C. variegatus for a minimum of 58% of the chemicals, whereas models using water solubility estimated toxicity to C. variegatus for a minimum of 77% of the chemicals within an order of magnitude for screening purposes. The calculated 96-h LC₅₀ values were compared to the measured values for each marine species and those measured for Pimephales promelas (fathead minnow) and Poecilia reticulata (guppy). Tests indicated generally that calculated 96-h LC₅₀ values were overestimates of the measured 96-h LC₅₀ values when models for freshwater fish were used to estimate toxicity to each marine species. More data are required for marine species to determine if highly significant relationships between marine and freshwater fish exist with comparisons using larger sample sizes.     

河豚毒素对4种急性疼痛模型的镇痛效应比较研究

目的评估河豚毒素(tetrodotoxin,TTX)对4种急性疼痛模型的镇痛效果,为其合理应用提供实验支持。方法动物肌内注射1 mg/kg盐酸吗啡或不同剂量TTX,TTX剂量为0、0.5、1、2、4、8μg/kg,给药后40 min,分别进行醋酸扭体实验、福尔马林刺激实验、热板实验和甩尾实验,记录动物疼痛反应或痛阈,计算疼痛抑制率;取动物血清,Elisa法测定花生四烯酸含量。结果盐酸吗啡对4种急性疼痛模型均有显著镇痛效应;TTX可减少醋酸诱导的小鼠扭体次数,降低福尔马林诱导的大鼠Ⅰ相和Ⅱ相疼痛反应,对两种疼痛模型的最高疼痛抑制率均达到80.00%以上;TTX在甩尾实验和热板实验中有一定的镇痛作用,最高疼痛抑制率分别为25.00%、19.79%。醋酸和福尔马林均能导致动物血清花生四烯酸升高,但是TTX对花生四烯酸无显著抑制作用。结论 TTX对醋酸和福尔马林诱导的化学性刺激疼痛模型具有良好的镇痛效果,而对热诱导(热板和热水)的物理性刺激疼痛模型的镇痛效果较弱,TTX可能通过阻断炎性介质介导的疼痛反应产生镇痛效果。     

Malathion toxicity: In vivo inhibition of acetylcholinesterase in the fish Brachydanio rerio (Cyprinidae)

Acetylcholinesterase (AChE) activity in the nervous tissue (brain) of the fish Brachydanio rerio was significantly inhibited by exposure to the organophosphorus (OP) pesticide, malathion. The inhibition was dose- as well as time-dependent. The fish survived even when the activity of the enzyme was inhibited by 90%. There was a significant recovery in the activity of AChE when malathion stress was lifted.     

Attenuating effects of natural organic matter on microcystin toxicity in zebra fish (Danio rerio) embryos -- benefits and costs of microcystin detoxication

To contribute to the understanding of joined factors in the environment, impact of pure microcystins (-RR and -LF) on zebra fish (Danio rerio) embryos were investigated individually and in combination with a natural organic matter (NOM). The applied NOM was a reverse osmosis isolate from Lake Schwarzer See (i.e., Black Lake, BL-NOM). Teratogenic effects were evaluated through changes in embryonic development within 48 h of exposure. Detoxication activities were assessed by the activities of phase II biotransformation enzymes, soluble and microsomal glutathione S-transferase (s, mGST). Oxidative stress was assessed by determining both the production of hydrogen peroxide and by analyzing the activities of the antioxidative enzymes, guajacol peroxidase (POD), catalase (CAT), glutathione peroxidase (GPx), and the glutathione restoring enzyme glutathione reductase (GR). Energetic costs were evaluated by determining contents of fat, carbohydrates, and proteins in both exposed and control embryos. BL-NOM attenuated toxic effects of MC-LF and MC-RR verified by less pronounced teratological effects within 24 h, in particular, as well as less rise in the activity of s-GST, when compared with embryos exposed to either pure toxins or in combination with organic matter. BL-NOM also diminished oxidative effects caused by MC-LF; however, it failed to attenuate oxidative stress caused by MC-RR. Content of lipids was significantly reduced in exposed embryos following a trend similar to that obtained with teratological and enzymatic assays confirming the attenuating effect of BL-NOM. Physiological responses to microcystins and NOM required energetic costs, which were compensated to the expense of the energy resources of the yolk, which in turn might affect the normal development of embryos.     

钙纳米粒子急性毒性及长期毒性试验

近年来纳米粒子的研制及应用已成为一个研究热点[1].该文观察了钙纳米粒子对小鼠的急性毒性和对大鼠的长期毒性,以评价钙纳米粒子的用药安全性.     

麝香接骨丸的限量试验研究

目的　通过临床前毒理学试验 -急性毒性试验(限量试验)考察麝香接骨丸临床用量的急性毒性情况。方法　取健康符合实验要求的小鼠 4 0只,随机分成两组,实验组 2 0只给予麝香接骨丸悬浮液(2 0 g·kg-1,0 4g·ml-1),另 2 0只作对照组,给予同体积灭菌水,药后观察 14d,记录试验动物在试验前、后的表现以及期间的体重。结果　观察期内,试验动物未出现异常反应。结论　麝香接骨丸临床用量不会引起急性毒性反应。     

银杏露急性毒性及长期毒性试验

目的：观察小鼠一日内给予较大剂量的银杏露时的急性中毒及死亡情况。及连续重复给予银杏露对大鼠所产生的毒副作用情况,为拟定人用安全剂量提供参考。方法：急性毒性试验为测定小鼠最大给药量方法,长期毒性试验为大鼠连续以银杏露60、30、15g(生药）/ml灌胃给药,观察小鼠各项有利生理,病理指标的方法。结果：银杏露对小鼠一日内灌胃的最大药量为240g(生药）/kg,大鼠长期毒性试验结果显示银杏露对大鼠的体重,活动情况,进食量,外表体征,血象,血液生化,脏器系数,病理检查等指标等均无不良影响。结论：银杏露按拟定临床剂量及疗程服用是安全的。     

Matrix metalloproteinase-13 is required for zebra fish (Danio rerio) development and is a target for glucocorticoids.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade the proteins of the extracellular matrix (ECM). Expression and activity of the MMPs are essential for embryogenesis, where MMPs participate in the normal ECM remodeling that occurs during tissue morphogenesis and development. Studies have demonstrated that MMP gene expression is inhibited by glucocorticoids in mammalian cell culture systems and that exposure to glucocorticoids causes developmental abnormalities in several species. Therefore, we proposed that glucocorticoids impede normal development through alteration of MMP expression. Zebra fish (Danio rerio) were used as a model to study MMP-13 expression both during normal embryogenesis and following acute exposure to two glucocorticoids, dexamethasone, and hydrocortisone. MMP-13 is one of three collagenases identified in vertebrates that catalyzes the degradation of type I collagens at neutral pH. MMP-13 expression varied during zebra fish development, with peak expression at 48 h post-fertilization (hpf). Morpholino knockdown studies showed that MMP-13 expression is necessary for normal zebra fish embryogenesis. Acute exposure to dexamethasone and hydrocortisone resulted in abnormal zebra fish development including craniofacial abnormalities, altered somitogenesis, blood pooling and pericardial and yolk sac edema as well as increased MMP-13 mRNA and activity at 72 hpf. In situ hybridization experiments were used to confirm the increase in MMP-13 expression following glucocorticoid treatment and showed elevated MMP-13 expression in the rostral trunk, brain, eye, heart, and anterior kidney of treated embryos. These data demonstrate that normal zebra fish embryogenesis requires MMP-13 and that dexamethasone and hydrocortisone modulate the expression of this gene, leading to increased activity and potentially contributing to subsequent dysmorphogenesis.