A型肉毒杆菌毒素

肉毒杆菌毒素是肉毒杆菌分泌的一种生物毒性蛋白质,其药理机制是与胆碱能神经末稍SNAP-25等蛋白产生作用,抑制乙酰胆碱释放,减少肌肉收缩和痉挛.目前,A型肉毒杆菌毒素获准用于治疗皱纹,其疗效平均持续4～6月,可以反复注射治疗.本文系统的回顾了A型肉毒杆菌毒的作用机制、临床试验、临床应用及不良反应.大量研究和病例分析已证明肉毒杆菌毒素注射术对于治疗面部各部位的皱纹是安全有效的.     

毒性中药蟾酥质量检测方法研究

目的:建立蟾蜍中多种药效及毒性成分蟾蜍毒素的定性定量检测方法。方法:采用液相色谱-质谱/质谱联用方法检测蟾酥中多种蟾蜍毒素成分。结果:建立了华蟾毒它灵、蟾蜍灵、华蟾毒配基及脂蟾毒配基的LC-MS/MS检测方法。比较了蟾酥样本的液相色谱-质谱/质谱联用分析数据。对4个不同来源中华大蟾蜍及黑眶蟾蜍的蟾酥样本进行检测,检出华蟾毒它灵、蟾蜍灵、华蟾毒配基及脂蟾毒配基的含量范围为0.05%~5.12%(w/w)。结论:液相色谱-质谱/质谱联用方法可用于蟾酥定性定量检测,并为质量评价提供参考。     

银环蛇粗毒若干有效组分对SWO细胞的作用

目的　测定银环蛇毒素组分是否抑制人神经胶质瘤细胞SWO的生长 ,以及确定抑制的原因是诱导凋亡或是杀伤。方法　用MTT和流式细胞术等方法探讨银环蛇粗毒及其有效组分对人神经胶质瘤细胞SWO的作用。结果　MTT试验结果显示 ,SWO细胞对银环蛇粗毒、第Ⅲ峰毒素、α 银环蛇毒素 (α BTX)的作用比较敏感 ,同时SWO细胞比 3T3细胞更敏感。SWO对其他组分的作用不敏感。 3种毒素作用于SWO细胞 ,流式细胞术检测未见凋亡峰。结论银环蛇粗毒、第Ⅲ峰毒素、α BTX对SWO细胞有杀伤作用 ,无凋亡作用     

蚯蚓毒素及其去除方法的初步研究

目的　研究蚯蚓毒素的来源及其成分 ,探索其去除方法。方法对蚯蚓应激分泌液进行透析、可见及紫外分光光度法检测透析袋内外液组分、电泳分析蛋白组分、动物体内试验检测毒性。结果蚯蚓应激分泌液的蛋白组分显示剧毒性 (最小致死量为 0 .2 42mg/kg)。PAGE分离主要有两条带 ;蚯蚓对不同刺激产生的应激分泌液成分相同。结论蚯蚓应激分泌液的蛋白组分有毒 ,用加盐或微电流刺激方法可去除毒素。     

眼镜蛇毒细胞毒素的分离、纯化及其抗癌活性

目的：研究眼镜蛇毒细胞毒素的抗癌活性。方法：应用Ｓｅｐｈａｄｅｘ　 Ｇ１００和 ＣＭ－Ｓｅｐｈａｒｏｓｅ ＦＦ柱层析,从眼镜蛇毒中分离、纯化细胞毒素组分,用ＭＴＴ法测定该组分对体外培养的人癌细胞的细胞毒性作用。结果：眼镜蛇毒细胞毒素对人癌细胞ＳＧＣ－７９０１、Ｂｅｌ－７４０２、Ｋ５６２和Ｕ９３７的抑制作用呈良好的量效关系,半数抑制浓度分别为 ４．１０、 ２． ０８、 ０． ２９和 ０．１７ μｇ／ｍｌ。结论：眼镜蛇毒细胞毒素对体外培养的人癌细胞有很强的杀伤作用。     

成纤维细胞生长因子安全性研究进展

成纤维细胞生长因（Fibroblast growth factor,FGF）是广泛存在于多种组织中的一类多肽因子,能影响多种细胞的生长、分化及功能,具有广泛的临床研究价值。为确保研制的新药的安全性,并提供新药对人类健康危害程度的科学依据,需对新药进行临床前安全性评价,其主要有急性毒性试验、长期毒性试验（反复给药毒性试验）、特殊毒性试验、皮肤用药及腔道用药毒性试验、药物依赖性试验及抗生育药及细胞毒性抗肿瘤药的毒性评价。本文综述了成纤维细胞生长因子的毒性试验的研究。     

NAC拮抗As_2O_3对外周血淋巴细胞的毒性作用

目的探讨N-乙酰-L-半胱氨酸(N acetylcysteine,NAC)拮抗As2O3对人淋巴细胞的毒性作用及机制。方法从正常人外周血中分离淋巴细胞,台盼蓝染色检测细胞活性;FITC-Annexin-V/PI染色检测细胞凋亡;电镜观察形态学改变;流式细胞术观察细胞内ROS水平及Bcl-2表达水平。结果 As2O3抑制人外周血淋巴细胞的增殖,细胞呈现典型的凋亡细胞特征性形态学改变,FITC-Annexin-V/PI染色发现凋亡细胞增多,同时,细胞内ROS水平增高和Bcl-2蛋白的表达水平下降。NAC与As2O3联合作用于淋巴细胞,细胞增殖抑制和凋亡均明显降低,细胞内ROS水平明显降低,Bcl-2蛋白表达水平回升。结论 As2O3对人淋巴细胞有明显的毒性效应,NAC可通过清除细胞内ROS和促进Bcl-2蛋白的表达而拮抗As2O3对淋巴细胞的毒性。     

神经毒素β-N-甲氨基-L-丙氨酸的毒理学与检测方法研究进展

β-N-甲氨基-L-丙氨酸(BMAA)是一种具有神经毒性的非蛋白氨基酸。研究表明,BMAA能够损伤运动神经元。在自然环境中,BMAA可以沿食物链传递,具有生物放大现象,可能参与肌萎缩侧索硬化-帕金森痴呆综合征的发病过程。大部分浮游蓝细菌(蓝藻)能够产生BMAA,环境中广泛存在的蓝藻可能会对人类健康构成威胁。本文对BMAA的理化性质、毒性、致毒机制、检测方法及其沿食物链的传递行为等进行了综述,以期为蓝藻毒素对人体健康的风险评估提供参考依据。     

氯化镉、对乙酰氨基酚对HepGⅡ、HEK293的细胞毒性

与传统方法相比，体外试验研究外来物质毒作用的靶器官具有简便、快捷、试验条件可控和节省实验动物等优点，因此成为目前毒理学技术的主要发展方向之一。但单个体外试验系统与机体内的实际情况有较大差距，得到的毒性数据比较有限，为了从体外毒理学试验体系中获得较全面的毒性资料，可以通过对多个体外试验系统获得的数据进行综合分析。本研究拟应用分别来源于肝脏和肾脏的2个细胞株对2种化学物进行细胞毒性试验，探讨将细胞毒性试验用于预测外源性化学物靶器官的可能性。     

蓖麻中毒与检测方法的研究进展

目的为进一步研究蓖麻中毒性成分的快检方法及在医药领域中的应用提供参考。方法查阅近年来公开发表的论文、专著等资料,对蓖麻碱和蓖麻毒蛋白的提取检测进行概述。结果明确了蓖麻中毒性成分蓖麻碱和蓖麻毒蛋白的理化性质及提取、纯化方法,总结了这2种毒性物质的提取、分离和检测方法。结论建立快速有效的检测鉴定方法对蓖麻中毒的解救是十分必要的。     

Effects of ochratoxins A and B on prechondrogenic mesenchymal cells from chick embryo limb buds

Prechondrogenic mesenchymal cells from the limb buds of 4-day chick embryos were cultured together with either ochratoxin A (OA) or ochratoxin B (OB) for 6 days. Both toxins inhibited the accumulation of cartilage proteoglycans and general protein synthesis in a concentration-related manner. The IC50 was 1.9 microM for OA and 6.2 microM for OB. Incubating the micromass with OA for periods ranging from 2 h up to 6 days did not produce any metabolites, indicating that OA was the proximal toxic compound in these studies. Neither preincubation with OB nor simultaneous exposure to non-toxic concentrations of OB together with various concentrations of OA influenced the toxicity of OA. The data indicate that interference of OA with general protein synthesis, both in vivo and in vitro, is an important mechanism underlying OA-induced embryotoxicity.     

Studies of the comparative in vitro toxicology of the cyanobacterial metabolite deoxycylindrospermopsin

Cyanobacteria are capable of producing metabolites that are in some cases toxic to humans and other animals. Of these metabolites, the toxin cylindrospermopsin (CYN) is produced by a number of species of cyanobacteria including Cylindrospermopsis raciborskii, and its toxicity has been documented. The CYN analog deoxycylindrospermopsin (deoxyCYN) is commonly produced in varying proportions by the cyanobacteria that produce CYN. The toxicological profile of CYN suggests that it is primarily a hepatotoxin, but with the capacity to damage other organs and tissues. Limited in vivo information is available on the toxicity of deoxyCYN and suggests it to be of low potency. The aim of this research was to determine the comparative toxicology of deoxyCYN using in vitro systems. Using cell viability assays, it was shown that deoxyCYN had inhibitory effects on cell viability and proliferation of a similar magnitude to that of CYN. Morphological changes in deoxyCYN-treated cells were similar to those of CYN. Investigation of protein synthesis inhibition demonstrated that deoxyCYN was of similar potency to CYN. Inhibition of protein synthesis is an acknowledged mechanism of toxicity for CYN, and the results produced here suggest that deoxyCYN operates by similar toxicological mechanisms to CYN and that in vivo animal testing should be undertaken to clarify the potential for risk to humans from this toxin.     

The total activity of a mixture of okadaic acid-group compounds can be calculated by those of individual analogues in a phosphoprotein phosphatase 2A assay

Monitoring of okadaic acid (OA)-group toxins in seafood is of paramount importance for the protection of consumer health from diarrheic shellfish poisoning. The property of OA-group compounds to inhibit type 2A serine/threonine phosphoprotein phosphatase (PP2A) has been exploited for the detection of OA in several experimental settings, but the performance of PP2A inhibition assays in the quantification of mixtures of OA-group compounds has not been reported yet. We have used a PP2A inhibition assay to analyze the total effect of mixtures including OA and one of its analogues, okadaol (OOH), by measuring the activity of individual compounds and of toxin mixtures through the inhibition they exert on the PP2A enzyme. We found that both OA and OOH inhibit PP2A under our experimental conditions, with IC₅₀ values of 0.37 ± 0.04 nM and 4.3 ± 0.8 nM, respectively, confirming that OOH is about ten-fold less potent than OA. PP2A assays were also carried out with predefined mixtures of OA and OOH, covering the full dose-response of one compound in the presence of increasing concentrations of the other toxin. The experimental data we obtained were used to analyze their correlation with those that could be calculated by adding the relative effects exerted by individual analogues, and we found that a good correlation exists between the observed and the expected data, when the predicted effect was calculated on the basis of toxicity equivalence factors. Our findings show that an additive model based on the use of toxicity equivalence factors of individual toxins is appropriate for the calculation of the total activity of multi-component mixtures of OA-group compounds in unknown samples.     

Ochratoxin binding to phenylalanyl-tRNA synthetase: computational approach to the mechanism of ochratoxicosis and its antagonism.

Ochratoxin A (OA) is a toxic isocoumarin derivative released by various species of mold which grow on grain, coffee, and nuts, representing a serious worldwide health problem. Among other mechanisms of toxicity, it has been suggested that OA inhibits phenylalanyl-tRNA synthetase (PheRS), thereby reducing protein synthesis. Using the crystal structure of PheRS from Thermusthermophilus, we have modeled its interactions with OA as well as with phenylalanyl adenylate (FAMP), the high-affinity intermediate substrate of PheRS. Our results indicate that while OA may be capable of weakly inhibiting PheRS, the OA-PheRS complex cannot adopt the same conformation as does FAMP-PheRS, contrary to previous assumptions. Relative to FAMP, the phenylalanyl moiety is found to bind more shallowly and in a different overall conformation. Free-energy perturbation calculations of the relative free energies of binding of OA with the phenolic moiety protonated versus deprotonated suggest that the protonated form binds significantly more strongly. Two alternative binding modes were also identified which cannot be discounted on the basis of these calculations. Our results, however, do not suggest binding stronger than millimolar for any of the binding modes, a conclusion which is in agreement with more recent experimental findings. This, in turn, suggests that the previously observed antagonistic effects of aspartame and piroxicam are more likely due to their prevention of OA binding to human serum albumin than to PheRS, which is in agreement with binding studies as well as with preliminary simulations performed in our laboratory.     

The use of okadaic acid to elucidate the intracellular role(s) of protein phosphatase 2A: lessons from the mast cell model system

In recent years a heightened appreciation has emerged for the role(s) that phosphatases play in regulating signal transduction pathways and other cellular processes. The tumor-promoting agent okadaic acid (OA) has been an invaluable tool in efforts aimed at delineating the contributions of the most abundant mammalian serine/threonine phosphatase, protein phosphatase 2A (PP2A), to intracellular signaling and cell function. PP2A, which is ubiquitous and vital in virtually every cell system studied, continues to be the focus of much research on phosphorylation control machinery. Mast cells represent an excellent in vitro model for the study of protein phosphorylation events because they possess a number of distinct signaling pathways that lead to the production and/or release of discreet mediators in response to different stimuli. The utility of OA in analyzing PP2A function has been demonstrated in mast cells across several species. Results of these studies have contributed to the current recognition that PP2A plays a crucial role in the biology of mast cells and other cell types.     

Influence of amino acids on okadaic acid production.

Okadaic acid (OA) (1)) was the first example of a group of polyether toxins known to be produced by marine microalgae, which are responsible for the natural phenomena known as Diarrhetic Shellfish Poisoning (DSP) red tides. It is also a highly selective inhibitor of protein phosphatases type 1 (PP1) and 2A (PP2A), as well as being a potent tumour promoter. For these reasons, OA is an extremely useful tool for studying cellular processes and an important standard for polluted shellfish control. In this paper, we report on a double objective: to improve the production of toxins and verify the apparent participation of amino acids in the formation of these polyethers by monitoring their influence on the promotion of growth, total cell yield and increased in toxicity in Prorocentrum lima of the PL2V strain in batch cultures, in a modified K medium.     

Oxygen reactive radicals production in cell culture by okadaic acid and their implication in protein synthesis inhibition

Okadaic acid (OA), a diarrhetic shellfish toxin is a potent promoter of tumours in mouse skin and a specific inhibitor of protein phosphatases 1 and 2A. Recently it has been shown that OA inhibited protein synthesis in a cell-free system, with 50% inhibitory concentration of 6.3x10(-12) M but the mechanism whereby this inhibition is mediated was still unclear. In the present study, the effect of OA on protein synthesis in Vero cell cultures was investigated. Protein synthesis was inhibited by OA alone in Vero cells in a concentration-dependent manner (IC50=27 ng/ml i.e. 3. 3x10(-8) M). Since OA also induced lipid peroxidation and likely oxygen reactive radicals, it was interesting to know whether these radicals impair the protein synthesis process. Therefore, SOD+catalase known as scavenger of active oxygen radicals were added in the culture medium in the presence of OA and labelled leucine. These enzymes partially prevented the inhibition of protein synthesis induced by OA, indicating that the formation of high reactive oxygen free radicals could be one of the pathways this marine toxin induces its toxicity. Since the prevention by SOD+catalase was only partial (the IC50 increased from 27 ng/ml to 48 ng/ml i.e. 3.3x10(-8) M to 5.9x10(-8) M) it was speculated that the production of oxygen reactive radical scavengered by SOD+catalase is not the main mechanism whereby OA induces its cytotoxicity. Vitamins E and C completely prevent the lipid peroxidation induced by OA in cells, but failed to reduce the inhibition of protein synthesis to the same level, indicating that a more specific mechanism might be responsible for protein synthesis inhibition. That is the hyperphosphorylation of elongation factor EF-2 in the protein synthesis machinery. However our results pointed to lipid peroxidation being a precocious phenomenon following the OA exposure, since a concentration with enhanced MDA production was lower than that inducing significant cellular protein synthesis inhibition.     

Understanding mechanisms of toxicity: insights from drug discovery research

Toxicology continues to rely heavily on use of animal testing for prediction of potential for toxicity in humans. Where mechanisms of toxicity have been elucidated, for example endocrine disruption by xenoestrogens binding to the estrogen receptor, in vitro assays have been developed as surrogate assays for toxicity prediction. This mechanistic information can be combined with other data such as exposure levels to inform a risk assessment for the chemical. However, there remains a paucity of such mechanistic assays due at least in part to lack of methods to determine specific mechanisms of toxicity for many toxicants. A means to address this deficiency lies in utilization of a vast repertoire of tools developed by the drug discovery industry for interrogating the bioactivity of chemicals. This review describes the application of high-throughput screening assays as experimental tools for profiling chemicals for potential for toxicity and understanding underlying mechanisms. The accessibility of broad panels of assays covering an array of protein families permits evaluation of chemicals for their ability to directly modulate many potential targets of toxicity. In addition, advances in cell-based screening have yielded tools capable of reporting the effects of chemicals on numerous critical cell signaling pathways and cell health parameters. Novel, more complex cellular systems are being used to model mammalian tissues and the consequences of compound treatment. Finally, high-throughput technology is being applied to model organism screens to understand mechanisms of toxicity. However, a number of formidable challenges to these methods remain to be overcome before they are widely applicable. Integration of successful approaches will contribute towards building a systems approach to toxicology that will provide mechanistic understanding of the effects of chemicals on biological systems and aid in rationale risk assessments.     

Review on the use of zebrafish embryos to study the effects of anesthetics during early development

Abstract

Over the years, the potential toxicity of anesthetics has raised serious concerns about its safe use during pregnancy. As evidence emerged from research in animal models, showing that some anesthetic drugs are potential teratogenic, the determination of the risk of exposures to anesthetic drugs at early life stages became mandatory. However, due to inaccessibility and ethical constrains related to experimental conditions, the use of early life stages in mammalian models is limited. In this regard, some animal and nonanimal models have been suggested to surpass mammalian use in experimentation. Among them, the zebrafish embryo test has been recognized as a promising alternative in toxicology research, as well as an inexpensive and practical test. Substantial information collected from developmental research following compounds exposure, has contributed to the application of zebrafish assays in research, although only a few studies have focused on the use of early life stages of zebrafish to evaluate the developmental effects of anesthetics. Based on the recent advances of science and technology, there is a clear potential for zebrafish early life stages to provide new insights into anesthetics teratogenicity. This review provides an overview of recent anesthesia research using zebrafish embryos, demonstrating its usefulness to the anesthesia field, discussing the recent findings on various aspects related to the effects of anesthetics during early life development and the strengths and limitations of this model system.     

Toward in vitro biomarkers for developmental toxicity and their extrapolation to the in vivo situation

INTRODUCTION: Reliable in vitro and in silico assays as alternatives for in vivo developmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivo developmental toxicity in in vitro assays are needed. AREAS COVERED: The present review gives an overview of alternative assays, as described in literature, for in vivo developmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivo developmental toxicity, and how in vitro effect data can be translated to the in vivo situation using physiologically based kinetic (PBK) modeling. EXPERT OPINION: Relevance of readouts in in vitro developmental toxicity assays as predictive biomarkers for in vivo developmental toxicity should be evaluated by comparing the obtained in vitro effect concentrations with in vivo internal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitro effect concentrations to in vivo dose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitro toxicity data in the safety assessment of compounds.