Characterization of cholinesterase activity from different tissues of Nile tilapia (Oreochromis niloticus)

Cholinesterases (ChE) from brain, muscle and liver in Nile tilapia (Oreochromis niloticus) were characterized using three substrates: acetylthiocholine iodide, propionylthiocholine iodide, and butyrylthiocholine iodide. Eserine was used as a total ChE inhibitor; BW284c51 and iso-OMPA were used as selective inhibitors for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), respectively. The results indicate that AChE is the enzyme present in brain, whereas in both liver and muscle, the presence of atypical ChEs are suggested. These findings indicate that characterization of ChE is necessary prior to use in monitoring programs.     

Acetylthiocholine (ATC)--cleaving cholinesterase (ChE) activity as a potential biomarker of pesticide exposure in the Manila clam, Ruditapes philippinarum, of Korea

The acetylthiocholine (ATC) - cleaving cholinesterase (ChE) activity in Manila clam, which is widely distributed throughout the coastal environment of Korea, was assayed as a potentially useful biomarker of organophosphorous pesticides (OPs). A clear dose-response relationship was determined between inhibited ChE in adductor muscle of clams and four OPs (methidathion, chlorpyrifos, diazinon, IBP) which are heavily used OPs in Korea. The measured EC(50)-24?h values of methidathion, chlorpyrifos, diazinon, and IBP for Ruditapes philippinarum were 7.16?μg?l(-1), 0.34?mg?l(-1), 3.01?mg?l(-1), and 3.41?mg?l(-1), respectively. In field studies, ChE activity in Manila clams collected from 23 stations in the mid-western coastal region demonstrated spatial variation with statistical differences. These results suggest that ChE activity in R. philippinarum is a potential biomarker for assessing organophosphorous pesticide contamination in coastal environments.     

久效磷对单环刺螠体内几种酶活性影响的初步研究

运用冰冻切片技术和酶组织化学的方法研究了长期暴露于不同质量浓度久效磷(0,50,100,150ng/L)下的单环刺螠(Urechisunicinctus)体内的乙酰胆碱酯酶(AChE)、细胞色素C氧化酶(CCO)、碱性磷酸酶(AKP)的活性变化。实验表明,AChE在单环刺螠的体壁上呈现出较强的酶活性,活性位点处为红棕色的颗粒沉淀、并随久效磷胁迫浓度的提高显色越来越深;消化道切片上基本上没有AChE的活性。CCO在单环刺螠的体壁及肠道上都呈现出较强的酶活性,活性位点显示为棕色;肠道上,不同质量浓度组由低到高其CCO活性位点依次增多、显色也依次加深。低质量浓度久效磷(<150ng/L)胁迫对单环刺螠体内的AKP活性没有明显的影响。     

Cholinesterase activities in the Antarctic scallop Adamussium colbecki: Tissue expression and effect of ZnCl2 exposure

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl₂ was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25–29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10⁻⁴–10⁻¹⁰ M (IC₅₀ 10⁻⁶) while in vivo exposure to ZnCl₂ did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.     

Cholinesterase activities in the Antarctic scallop Adamussium colbecki: tissue expression and effect of ZnCl2 exposure

Biochemical characterization of cholinesterase activity (ChE) was carried out on the Antarctic scallop Adamussium colbecki collected in winter 2000 from Campo Icaro (Ross Sea, Antarctica) in order to increase its suitability as a sentinel organism for monitoring the Antarctic environment. The digestive gland, gills and adductor muscle were investigated for substrate specificity and inhibitors sensitivity using acetylthiocholine iodide (ASCh) and butyrylthiocholine iodide (BSCh) as substrates and tetra (monoisopropyl)pyrophosphor-tetramide (Iso-OMPA), 1,5-bis(4-allyldimethylammoniumphenyl)-penthan-3-one dibromide (BW284c51) and the insecticide chlorpyrifos as inhibitors. Effect of in vivo exposure to ZnCl(2) was also investigated. All the tissues expressed ChE activity (gill > adductor muscle > digestive gland) and low substrates specificity throughout the hydrolysis of both ASCh and BSCh substrates. Partial (25-29%) and total inhibition (100%) of ChE activity in gills was demonstrated following in vitro incubation with Iso-OMPA and BW284c51 (3 mM), respectively. Concentration-dependent inhibition was also evident with chlorpyrifos in the range 10(-4)-10(-10) M (IC(50) 10(-6)) while in vivo exposure to ZnCl(2) did not seem to affect ChE activity in the scallop. The potential use of ChE in the A. colbecki as biomarker for monitoring water contamination in the marine Antarctic environment is discussed.     

Environmental monitoring using acetylcholinesterase inhibition in vitro. A case study in two Mexican lagoons.

Cholinesterase inhibition is considered a specific biomarker of exposure and effect for organophosphorous pesticides. Its use for monitoring has been hindered, particularly in tropical countries where organophosphates are widely used for malaria and dengue control, because of the frequent lack of suitable controls. An in vitro technique is proposed as a biochemical method for monitoring pollutant mixtures in sediment toxicity tests. Brain homogenate from the fish Oreochromis niloticus is used as the enzyme source. Optimum incubation time, extraction solvent and effect of crude oil on acetylcholinesterase (AChE) are reported. The method described was used in sediments from two Mexican lagoons, located in an oil extraction area where pesticides are used in agriculture and vector control campaigns. AChE inhibitions from 3 to 21% were found in these lagoons, even in the presence of high concentrations of petroleum.     

海鱼AChE监测海水有机磷农药的可行性研究

有机磷农药(OPs)对乙酰胆碱酯酶(AChE EC 3.1.1.7)具有专一性抑制作用,因此AChE常作为淡水和土壤OPs污染的特异性标识物(biomarker).本文在海水背景下,通过测定两种有机磷农药(马拉硫磷和甲基对硫磷)对海洋鱼类-鲈鱼[Lateolabrax japonicus(cuvier)]脑组织AChE的抑制效应,分析了海鱼AChE监测海水OPs的可行性.结果显示,抑制时间为20min时,抑制程度达到相对稳定;在低浓度OPs抑制下,纯酶液比粗酶液表现出更好的敏感性,对马拉硫磷和甲基对硫磷的灵敏度分别达到0.28μg*L-1和0.2μg*L-1;在一定浓度范围内,AChE活力对OPs具有显著的线性响应.研究表明,利用鲈鱼AChE指示和监测海水OPs的微污染是完全可行的.     

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides.     

Organic and trace metal contaminants in sediments and English sole tissues from Vancouver Harbour,Canada

As part of a multinational workshop on marine environmental quality, sediments were collected from seven sites in Vancouver Harbour and analyzed for polynuclear aromatic hydrocarbons (PAHs) and organochlorines (OCs), and for trace metals. English sole were collected from five sites, and muscle was analyzed for trace metals and liver for OCs. As expected, sediment PAH and OC concentrations and tissue OC concentrations were higher at sites east of the First Narrows, compared to the outer harbor and reference sites. Sediment PAH concentrations east of the First Narrows were similar to concentrations at moderately contaminated sites in Puget Sound, south of Vancouver Harbour. In contrast, concentrations of OCs in sediments and tissue were low to moderate, even at relatively contaminated sites within Vancouver Harbour. Although several trace metals in sediments were higher than in contaminated sediments from Puget Sound, trace metals measured in fish muscle were lower.     

Biomarker responses to pollution in two invertebrate species: Scrobicularia plana and Nereis diversicolor from the Cádiz bay (SW Spain)

The clam Scrobicularia plana and the polychaete worm Nereis diversicolor were collected in several sites from a littoral enclosure in SW Spain. The aim of our study was to relate various biomarker responses in these species to a pollution gradient caused by untreated domestic discharges and to verify the adequacy of the selected species as sentinels in this habitat. The biomarkers selected were the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX) and DT-diaphorase (DT-D). In addition, the activities of cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity, the phase II detoxifying enzyme glutathione S-transferase (GST) and the neurotoxicity marker acetylcholinesterase (AChE) were measured. Metallothionein levels were selected as biomarkers of heavy metals exposure in both species. The results suggest a different response in the water filtering organism (clam) and the sediment eater (polychaete), probably as a consequent of different pollution exposure and that samples from the “Caño Sancti-Petri” were exposed to biologically active compounds that altered some of their biochemical responses. AChE was the most sensitive biomarker in both species and N. diversicolor proved to be a more robust sentinel in this ecosystem.     

枝管藻多糖对久效磷暴露水体中鱼类保护作用的研究

以金鱼(Carassius auratus)体内超氧化物歧化酶(Superoxide dismutase，SOD)活力、乙酰胆碱酯酶(Acetylcholinesteras，AChE)活力变化为指标，观察了枝管藻多糖对久效磷暴露的水体中鱼类生理功能的影响。结果表明：用枝管藻多糖灌胃鲫鱼15d，枝管藻多糖能显著降低久效磷对鱼类体内SOD活力的诱导和有效阻止久效磷对鱼类中枢神经系统AChE活性的抑制，具有拮抗久效磷农药对鱼类毒害的作用。     

栉孔扇贝不同组织SOD、CAT和MPO活力的比较分析

对2龄和3龄栉孔扇贝外套膜、鳃、消化盲囊、肾、闭壳肌、性腺组织超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和髓过氧化物酶(MPO)活力分析表明,检测组织均存在这三种酶,且在不同组织中活力表现存在明显差别,其中肾组织SOD、CAT活力最高,而MPO在消化盲囊中表现最高活力,与其它组织相比均呈显著性差异(P＜0.05).不同贝龄的栉孔扇贝酶活力比较表明,三龄贝各组织SOD、CAT和MPO活力均高于二龄贝.     

荣成天鹅湖越冬大天鹅粪便的重金属水平

为对大天鹅(Cygnus cygnus)保护及湖区管理提供基础数据,作者于2011年11月~2012年3月研究了山东荣成天鹅湖越冬大天鹅食物来源及其粪便的重金属水平。结果表明:所选择的食物来源中,Zn、As和Cr在硬毛藻(Chaetomorpha linum)中含量最高,Cd在大叶藻(Zostera marina L.)叶片中含量最高,Pb在大叶藻根茎中含量最高,Cu在石莼(Ulva pertusa)中含量最高,Hg在小麦(Triticum aestivum)中含量最高;水生植物来源的粪便中Cd含量极显著高于陆地植物来源的粪便,陆地植物来源的粪便中Cr,Pb和Hg含量显著高于水生植物来源的粪便;摄食小麦苗可能造成大天鹅对Cd和Hg的积累。     

Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius

Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment.     

大港原油对毛蚶部分抗氧化酶和代谢酶的影响研究

为研究港原油对毛蚶部分抗氧化酶和代谢酶的影响,设置0.01、0.1、1、3mg/L大港原油水溶液性成分(WSF),采用暴露法研究毛蚶天津群体的鳃、斧足中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)抗氧化酶等抗氧化酶及酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、钠/钾泵(Na^+/K^+ATPase)、钙泵(Ga²⁺/Mg²⁺ATPase)等代谢相关酶的活性变化,测定丙二醛(MDA)含量,采用整合生物标志物(Integratedbiomarkerresponse,IBR)进行分析。结果表明,毛蚶鳃和斧足中SOD、CAT、GPX表现出一定的剂量-效应关系, MDA含量呈先升高后降低的趋势, 3 mg/L组表现出MDA累积;ACP、AKP、Na^+/K^+ATPase、Ga²⁺/Mg²⁺ATPase表现出一定剂量-效应关系,酸性磷酸酶较碱性磷酸酶响应更迅速, Na^+/K^+ATPase较Ga²⁺/Mg²⁺ATPase更易受WSF影响;鳃中酶类活性受WSF影响更明显。鳃中SOD、GPX、MDA较斧足高,而CAT则相反。斧足、鳃组织RIB值呈现先下降后上升趋势,与WSF浓度及暴露时间存在剂量-效应关系和时间-效应关系,两组之间存在差异性,斧足累积RIB值高于鳃组织。     

10 个半滑舌鳎家系 MHC IIB 基因多态性初步研究

为检测10个半滑舌鳎(Cynoglossus semilaevis)家系MHCⅡB(Cyse-DAB)基因的多态性水平、MHCⅡ类B位点数目以及平衡选择的作用,作者利用多聚酶链式反应(PCR)和直接测序的方法对10个半滑舌鳎家系MHCⅡB基因位点遗传变异和平衡选择进行了研究。用特异性引物和PCR扩增的半滑舌鳎MHCⅡB基因片段大约397 bp,包含一部分第一外显子,全部第一内含子和全部第二外显子。10个半滑舌鳎家系中,每家系选取5个体,每个体5个克隆序列分析发现60个不同序列,代表60个等位基因,其中有28个是新发现的,已提交到Gen Bank。同源分析表明60个等位基因相似性为89.36%。共50个个体中,有6个存在5个不同等位基因,表明在半滑舌鳎至少存在3个座位。有9个家系的MHCⅡB序列多肽结合区(PBR)的非同义替换(dN)显著高于同义替换(dS)。     

Brain acetylcholinesterase as a marine pesticide biomarker using Brazilian fishes

Brain acetylcholinesterase (AChE) of some fishes from the coast of Rio de Janeiro State was studied as a possible pesticide biomarker in marine environmental monitoring. AChE specific activity in brain varied from 145 to 530 U/g of proteins and the Michaelis-Menten constant (K(M)) for acetylthiocholine varied from 104 to 291 microM among the 20 species studied. The enzyme sensitivity to methyl paraoxon, evaluated by the inhibition kinetic constants, shows that some species (Paralonchurus brasiliensis and Genidens genidens) are more sensitive (IC50-30 min=455 and 468 nM, respectively). The less sensitive Merluccius hubbsi and Percophis brasiliensis (IC50-30 min=3339 and 3259 nM, respectively) belong to the super-order Paracanthopterygii, which includes the more ancient species. On the other hand, more susceptible species belong to the super-order Acanthopterygii, which includes more recent species. These results suggest a possible evolutionary linkage for AChE sensitivity to methyl paraoxon. The application of inhibition kinetic constants for fish brain AChE in phylogenetic studies is still being investigated. The results have shown that a fish sentinel species should have the highest brain AChE level among the more sensitive ones.     

有机磷农药对海洋动物乙酰胆碱酯酶(AChE)的毒性效应研究进展

近几十年来，农业和海水养殖业所使用的有机磷农药(OPs)造成沿岸海水的严重污染，威胁到海洋动物的生存。有机磷农药的海洋生态毒理学研究已经成为海洋环境科学中的热门领域之一。作为动物神经传导过程中的关键酶，乙酰胆碱酯酶(AChE)能够被有机磷农药专一性抑制，因此，有机磷农药对海洋动物AChE的影响受到更多的关注。本文以近年来该领域的研究成果为基础，综述了有机磷农药对海洋动物乙酰胆碱酯酶的毒性效应。