Oxidative stress and genotoxicity of 1-methyl-3-octylimidazolium chloride on loach (Misgurnus anguillicaudatus)

This study was a preliminary step to evaluate the acute toxicity of 1-methyl-3-octylimidazolium chloride ([C₈mim]Cl) on loach (Misgurnus anguillicaudatus) by determining the effects on hepatic antioxidant enzyme activities and by the comet assay. The results showed that [C₈mim]Cl had acute toxicity at concentrations above 20 mg L^?1, inducing oxidative stress and genotoxicity on fish liver cells. In respect to enzyme activities, [C₈mim]Cl induced changes in the activities of superoxide dismutase, catalase, and glutathione content the livers of fish exposed at 20–80 mg L^?1. [C₈mim]Cl at the same exposure level caused a remarkable increase in malondialdehyde level. The comet assay indicated that [C₈mim]Cl at 20–80 mg L^?1 induced genotoxicity in liver cells. With increased exposure concentration and time, the two comet parameters trailing rate and tail moment were significantly increased, with significant differences (P < 0.05) observed between control group and each treatment group. The present study shows that ionic liquids can be a threat to the health of aquatic organism when accidentally released to aquatic ecosystems.     

In vitro genotoxicity of fly ash leachate in earthworm coelomocytes

Fly ash is the major coal combustion byproduct from thermal power plants. Considering its plant–nourishing constituents, its soil amendment in farmland is one of its promoted disposal methods. A substantial amount of heavy metals present in fly ash, which may leach out due to rainwater or irrigation water, may cause serious problem with long term use, especially to soil organisms. These metals may cause DNA damage through Reactive Oxygen Species (ROS) generation. In the present study, single cell gel electrophoresis [(SCGE) i.e., comet assay] was used to detect DNA damage in earthworm (Dichogaster curgensis) coelomocytes, following an in vitro exposure. Significant DNA damage was observed at the lowest concentration of fly ash leachate (6.25%) examined. DNA damage by all the tested concentrations (6.25%, 12.5%, 25%, 50%) differed significantly (p?<?0.001) from that of the negative control. Hence, long-term application of fly ash might prove harmful for earthworm populations.     

人血淋巴细胞检测浊漳河地表水的遗传毒性

为评价浊漳河水体中有机污染物的致突变性,采用单细胞凝胶电泳(SCGE)技术,研究了浊漳河水体3个不同样点的有机提取物对人外周血淋巴细胞DNA损伤效应。结果显示,各水样中的有机提取物均能引起DNA损伤,且随剂量的增高,有机物对DNA的损伤加重;细胞DNA损伤与对照组相比均有显著性差异(p<0.01)。这表明人外周血淋巴细胞对水体有机污染物的毒作用非常敏感。细胞彗星尾部DNA含量(%DNAT)和染毒剂量呈极显著正相关(R2>0.8547,p<0.01)。以上结果表明,浊漳河水体受到一定程度的有机物污染,这些有机污染物可诱导淋巴细胞DNA损伤,具有遗传毒性。SCGE技术作为一种简便、快速和灵敏的检测方法在水环境遗传毒性监测方面具有较大的应用价值。     

Lysosomal,genetic and chromosomal damage in haemocytes of the freshwater bivalve (Unio pictorum) exposed to polluted sediments from the River Cecina (Italy)

The response to chemical disturbance of an Italian river (Cecina River) characterised by high levels of trace metals was assessed in the sentinel species freshwater painter’s mussel (Unio pictorum). We exposed U. pictorum in the laboratory to metal, metalloid and other trace element polluted river sediments in order to evaluate genetic and cellular biomarkers. Bivalves exposed to sediments taken from all the impacted sites of the river basin exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. The lysosomal membrane stability of circulating haemocytes, assessed by the Neutral Red Retention Time, was found to be higher in specimens from Lake Maggiore and Berignone, while exhibited a significant decrease in specimens exposed to Ponteginori sediments. An increase of DNA migration in specimens exposed to the two contaminated sites in comparison with controls was found. Moreover, higher micronucleated haemocytes frequencies were observed in bivalves exposed to sediments collected at Possera 1 site. This study suggested that biomarkers evaluating cellular impairment, genetic and chromosomal damages, are highly sensitive and suitably applied to laboratory experiments. The painter’s mussel was confirmed to be an appropriate sentinel species for quality assessment in metal-contaminated freshwater environments.     

彗星实验技术及其应用

彗星实验是瑞典科学家Ostling和Johanson于1984年发明的检测毒物DNA损伤效应的方法。它经历了从最初的微电泳技术、中性彗星实验、碱性彗星实验、酶切彗星实验和双向垂直彗尾彗星实验等不断完善的发展过程。在毒理学、遗传学和环境生态科学等领域有着重要的应用,是经济合作与发展组织(OECD)和欧洲食品安全局等国际组织推荐的测定遗传毒性的方法之一。彗星实验的关键点包括单细胞悬液的制备、细胞裂解液的成分与比例,低熔点琼脂糖凝胶的浓度,电泳条件等。在典型应用领域,如蚯蚓、鱼、两栖动物、鼠和人的彗星实验很难找到标准实验方案。成功的彗星实验还需关注,实验设计时必须包括阳性对照,结果表述时必须有图为证,实验方案可能因物种或细胞而异。     

Toxicity of endosulfan on growth,photosynthesis, and nitrogenase activity in two species of Nostoc (Nostoc muscorum and Nostoc calcicola)

Treatments of the cyanobacteria Nostoc muscorum and Nostoc calcicola with the insecticide endosulfan (5, 10, and 20?µg?mL^?1) inhibited growth, photosynthetic pigments, photosynthetic, and nitrogenase activities. The sensitivity of N. muscorum to endosulfan was higher than that of N. calcicola. The toxic effect of endosulfan was more pronounced on phycocyanin; however, a considerable reduction in chlorophyll a and carotenoids was also noticed. ¹⁴C-fixation appeared to be more sensitive to the insecticide than photosynthetic oxygen evolution. Endosulfan caused strong inhibition of photosystem (PS) II activity whereas PS I was least affected. The inhibition of PS II activity was partially restored by electron donors (DPC, NH₂OH, and MnCl₂) at low dose of endosulfan. Nitrogenase activity was significantly suppressed in both species by the endosulfan at high dose (20?µg?mL^?1). On the basis of our comparative analysis, N. calcicola was found to be endosulfan resistant and can be used in paddy fields for better productivity under pesticide stress.     

泥鳅在污染物毒性评价中的应用研究

泥鳅营底栖生活,生存能力强,易于饲养,便于采集和处理,有作为模式生物的潜力。受到污染物胁迫时,泥鳅在分子、细胞、组织、器官和个体水平上会发生明显的变化,其存活、生长和繁殖从而受到影响。泥鳅的这些特性可以被用来进行污染物毒性评价。从急性毒性、蓄积毒性、生殖力毒性、遗传毒性等方面,综述和分析了泥鳅在污染物毒性评价中的应用研究进展,并对未来研究重点进行了展望。     

蚯蚓体腔细胞彗星试验检测阿散酸在泥浆体系中降解前后的遗传毒性变化

配制不同浓度的阿散酸标准液,与某养猪场附近的表层土壤混合形成泥浆系统,放在摇床上连续培养30d.在第0、1、2、3、4周取出适量降解液,分别用分光光度法测定阿散酸的降解率;用彗星试验检测降解液对赤子爱胜蚓体腔细胞DNA的损伤.结果表明,阿散酸低浓度时在泥浆体系中能被较快降解,500mg/L的降解率达到了25%;蚯蚓体腔细胞彗星试验表明,所有降解液均具有遗传毒性,可导致蚯蚓体腔细胞DNA明显损伤,与对照相比具有显著性差异(P<0.05).图1表1参17     

敌百虫致小鼠外周血淋巴细胞DNA-蛋白质交联作用研究

为探讨敌百虫致DNA-蛋白质交联作用,以昆明小鼠为受试动物,敌百虫按0、20、40、60 mg·kg-14个剂量水平,灌胃染毒小鼠2周。第5组以提取的正常小鼠外周血淋巴细胞经50μmol·L-1的H2O2处理为阳性对照组。采用经改进的彗星试验方法来检测染毒后外周血淋巴细胞DNA-蛋白质交联效应。结果表明,与空白组相比,各敌百虫染毒组都能引起DNA损伤作用(P0.01)并引起一定程度的DNA-蛋白质交联效应,在较高浓度(40、60 mg·kg-1)时DNA-蛋白质交联尤为明显,存在一定的潜在突变风险。     

三氯生和三氯卡班对人体肝细胞DNA损伤的研究

三氯生（TCS）和三氯卡班（TCC）作为优良的抗菌和消毒剂,在药物和个人护理用品中广泛使用。近几年来,TCS和TCC在各种环境介质和生物体中被频繁检出,并引起国内外环境科学工作者的重点关注。目前,有关TCS和TCC对水生生物的毒性研究已有大量的报道,其对人体健康危害的报道仍较少。为了评估TCS和TCC对人体的遗传毒性,利用彗星实验研究两种污染物对正常人肝细胞（L02）DNA的损伤效应。结果发现：三氯生和三氯卡班皆能引起人体肝细胞的DNA损伤,并呈现显著的剂量-效应关系和时间效应关系。相比于TCS而言,L02细胞对TCC更为敏感,在低暴露剂量（2.38μmol.L-1）下就可以引起DNA断裂损伤。该结果有助于进一步揭示TCS和TCC对人体毒害的机制,更全面的评估两种污染物对人体健康的风险。     

Effect of fomesafen on glutathione S-transferase and cellulase activity and DNA damage in the earthworm (Eisenia fetida)

Biochemical responses and DNA damage of earthworm (Eisenia fetida) exposed to various concentrations of fomesafen (0, 10, 100, and 500 μg kg^?1) for 3, 7, 14, 21, and 28 days were investigated in vivo. Glutathione S-transferase (GST) activity was significantly stimulated upon treatment with 500 μg kg^?1 fomesafen for 21 days, while the cellulase activity was markedly inhibited after 14 days of treatment. On day 28, the activities of GST and cellulase had recovered to the level similar to that of controls. Low or mild DNA damage in earthworm was induced within 14 days of exposure, and the damage was reduced or disappeared with the extension of exposure.     

全氟辛烷磺酸暴露对赤子爱胜蚓的急性毒性效应

采用人工土壤培养法,通过14 d急性暴露试验,研究了全氟辛烷磺酸(PFOS)对赤子爱胜蚓(Eisenia fetida)的死亡率、谷胱甘肽硫转移酶(GST)和过氧化氢酶(CAT)活性以及DNA损伤的影响。研究结果表明：PFOS对蚯蚓14 d-LC₅₀为478.0 mg·kg^-1,显示为低毒;急性暴露期间,PFOS显著抑制蚯蚓生长,生长抑制率随着PFOS浓度增加而增大,且有良好的剂量效应关系(r=0.951,P<0.01);PFOS对蚯蚓GST和CAT活性均没有显著性影响,没有观察到剂量效应关系;PFOS可致蚯蚓体腔细胞DNA损伤,不同浓度的PFOS对蚯蚓体腔细胞尾长、尾部DNA含量和尾矩的影响具有一定的剂量效应关系(P<0.01),与对照组之间存在显著性差异(P<0.05)。上述结果表明,PFOS对蚯蚓的存活影响显示为低毒,但是对生长有一定影响,可致蚯蚓体腔细胞DNA损伤。     

基于彗星实验的洛克沙胂对秀丽隐杆线虫胚胎细胞DNA损伤的研究

为评价洛克沙胂的遗传毒性,采用单细胞凝胶电泳(彗星实验),研究了不同浓度的洛克沙胂对秀丽隐杆线虫胚胎细胞脱氧核醣核酸(DNA)的损伤作用。提取秀丽隐杆线虫的胚胎细胞,分别暴露于0(空白对照)、50、250、500μg·L~(-1)含洛克沙胂的溶液染毒1 h。用彗尾DNA百分比含量(TDNA%)、彗星尾长(TL)和Olive尾矩(OTM)作为DNA损伤的指标。实验结果表明,与空白对照组比较,处理组中彗尾DNA百分比含量、彗星尾长以及Olive尾矩显著增加(P0.01)。随着洛克沙胂浓度的增加,彗尾DNA百分比含量、彗星尾长以及Olive尾矩逐渐增加,其相关系数r0.99,说明在实验浓度范围内,存在极显著的浓度-效应关系。洛克沙胂对秀丽隐杆线虫胚胎细胞DNA具有损伤作用,彗星实验操作简便、快速、灵敏度高,能够反映出洛克沙胂的遗传毒性。因此,通过彗星实验建立实验室检测洛克沙胂遗传毒性的方法具有可行性。     

彗星试验技术在水生与土壤环境中的毒理学应用

彗星试验是一种检测真核细胞脱氧核糖核酸(DNA)损伤的技术,快速、低成本、适用于几乎所有真核细胞,因此常应用于遗传毒理学、辐射生物学、肿瘤细胞学、毒性检测等方面。尤其以灵敏度高、检测联合毒性的特点大量运用于环境研究中。主要介绍彗星试验方法学发展历程,彗星试验的原理及其在环境领域中的应用,并指出应用中的受限因素以及未来的发展前景,为今后进一步推广该试验技术提供理论上的指导与参考。     

Protective effect of Kappaphycus alvarezii (Rhodophyta) extract against DNA damage induced by mercury chloride in marine fish

Marine organisms are continuously exposed to agents, both exogenous and endogenous, that damage DNA. Consequently, it is important to determine the ability of compounds to provide protection against damaging chemicals. The aim of this study was to evaluate the anti-genotoxic activity of crude aqueous extracts of Kappaphycus alvarezii (Rhodophyceae), collected from the Southeast coast of India. This study focused on possible anti-genotoxic potential of aqueous extract of K. alverazii to interfere with clastogenicity induced by mercury chloride (HgCl₂) in marine fish, Therapon jarbua as measured by cytogenetic endpoints such as cell viability and comet assay. In the first set of experiments, fish were exposed to a single treatment of Hg at 0.125, 0.25, 0.5, 1, or 2?ppm along with controls. Mercury exposure produced significant DNA damage in all comet classes, maximum as >79% (Class 4) at 0.5, 1, and 2?ppm exposure in a time dependent manner. Algal extract did not induce genotoxicity when given alone and prevented Hg-induced genotoxicity. The algal extract reduction in genotoxicity was significant but not time- and concentration-dependent. Results suggested that under present experimental conditions, K. alvarezii extract exhibit potent anti-genotoxicity effects in this fish model; and thus these extracts may be recommended as a supplement in fish meal and may benefit humans ingesting Hg-contaminated fish.     

干燥和复吸水条件下发菜过氧化物氧还蛋白差异表达与基因克隆

发菜(Nostoc flagelliforme)是一种陆生固氮蓝藻,为探讨其耐旱的分子机理及对极端干旱环境的适应和保护机制,运用双向电泳技术(2-DE)、凝胶图像分析、MALDI-TOF-TOF/MS质谱鉴定和数据库检索,分析发菜过氧化物氧还蛋白(Peroxiredoxin)在持续干燥48 h和复吸水4 h后的差异表达水平,根据鉴定的过氧化物氧还蛋白已知氨基酸序列设计简并性引物克隆该基因,分析基因和氨基酸序列的同源性及对蛋白质二级结构进行预测,并研究其原核表达.结果表明,发菜过氧化物氧还蛋白在复吸水后的表达量明显高于干燥状态下的表达量。根据简并性引物克隆获得长度为639 bp的过氧化物氧还蛋白基因,GenBank登陆号为HM854286.序列比较分析显示该基因具有较高的保守性,蛋白质二级结构主要由α螺旋、β折叠和随机卷曲构成.将过氧化物氧还蛋白基因在大肠杆菌中表达,获得符合预期的外源重组蛋白(26.5×103),经Western blotting验证,该外源蛋白为过氧化物氧还蛋白.图10表1参25     

利用大型蚤和斑马鱼评价腈纶废水好氧-厌氧处理过程的急性毒性和遗传毒性变化

腈纶废水毒性较强且难以去除,对腈纶废水毒性及污水处理过程毒性削减能力进行评价,可为开发该类废水毒性减排技术提供科学依据。利用大型蚤及斑马鱼评价某腈纶废水的急性毒性和遗传毒性,及厌氧-好氧工艺对该废水毒性的削减能力。采用大型蚤活动抑制率和斑马鱼致死率表征废水急性毒性,采用斑马鱼肝细胞彗星尾矩表征废水遗传毒性。腈纶废水处理前对大型蚤和斑马鱼的急性毒性单位(TU)分别为1.2和2.9,经厌氧-好氧工艺处理后分别降至0.4和0.5。遗传毒性结果表明腈纶废水对斑马鱼肝细胞造成DNA损伤作用,经处理后遗传毒性仍显著高于阴性对照组。理化指标与毒性指标相关性分析表明,该废水氨氮与毒性显著相关,推测氨氮可能是该废水中的重要致毒因子之一。研究结果表明该腈纶废水采用现有厌氧-好氧工艺无法有效削减毒性,对受纳水体水生态环境造成潜在危害。     

Effect of calcium supplementation on lead-induced genotoxicity and bioaccumulation in suckling Wistar rats

The study investigated the effect of calcium (Ca) supplementation on lead-induced toxicity in suckling male Wistar rats. The sucklings were exposed to lead acetate (5 mg/kg b wt./week) orally for a period of six weeks. The unexposed group of sucklings was the negative control. Out of the lead-exposed groups, two groups were supplemented with Ca at 3% and 6% as CaHPO₄ × 2H₂O in suspension in cow's milk. The artificial feeding regimen was followed for 7 h each day for 6 weeks. Sucklings exposed to lead with Ca supplementation recorded a significantly higher increase in body weight and a significant reduction of lead concentration in all tissues. The lead-induced histopathological alterations in the liver and kidney were also reversed with Ca supplementation. Sodium rhodizonate staining of liver sections showed distinct presence of lead granules in the group exposed to lead while the Ca-supplemented groups were comparable to the control. Lead exposure reduced the expression of rad 51 in the testis and liver which was markedly enhanced with Ca supplementation and was restored to the level in the control. Thus, Ca supplementation during the suckling period was effective in ameliorating the lead-induced toxicity.     

Fine atmospheric particles emitted by industrial,traffic and urban sources in France: characterization and genotoxicity

Health risks associated with inhalation of fine particulate matter of 2.5 µm in diameter or smaller depend on their atmospheric levels and physicochemical properties. The relationships between chemical compositions and genotoxic activities of particles emitted by mineral industries, traffic and urban sources during summer and winter in the region of Provence-Alpes-Côte d'Azur (France) were investigated.

The fine particles were separated in respect to water-soluble (13 minerals and metals) and organic-extractable (16 polycyclic aromatic hydrocarbons) components that were quantified. The chromosome damaging properties of the hydrophilic and lipophilic extracts were assessed using the centromeric micronucleus assay on a human lung fibroblast cell line.

The composition of the fine particulate matter was variable and depended upon the sources and seasons. Both the hydrophilic and lipophilic extracts induced chromosome damage: (1) in hydrophilic extracts, Ca and Zn affected chromosome losses induction; (2) acenapthylene affected chromosome damage (breakages and losses) induction and naphthalene affected chromosome damage and losses induction in lipophilic extracts without metabolic activation; and (3) benzo[a]pyrene affected chromosome losses induction in lipophilic extracts with metabolic activation. Fine particulate matter arising from coal-fired power station, road traffic, and other urban sources were the most efficient to induce chromosome breakage.     

检测细胞DNA断裂损伤效应的彗星实验法的改良

为了解决彗星实验过程中常出现的脱胶、细胞核分离操作繁琐、重复性低等问题,对彗星实验方法进行了改良,初步建立了彗星实验的快速操作流程。结果显示,通过对载玻片进行预处理,可确保凝胶悬挂均匀;采用改良机械法分离的细胞核浓度适中;以0.5%(w/v)涂层琼脂糖作为基层、以1.5%(w/v)低熔点包埋琼脂糖作为叠加层的"双层凝胶法",辅以"推片法"铺胶,操作便捷且不发生脱胶现象;细胞核膜经裂解处理后再进行电泳和荧光观察,彗星图像清晰,杂质少。应用改良后的彗星实验方法,操作简便,耗时更短,实验效果良好,可快速检测出细胞DNA损伤效应。