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。     

全氟辛烷磺酸(PFOS)和多壁碳纳米管(MWCNTs)复合对斑马鱼外周血红细胞DNA的损伤

为了评价全氟辛烷磺酸(PFOS)和多壁碳纳米管(MWCNTs)对水环境及鱼类的影响,以斑马鱼为模式生物,研究了PFOS和MWCNTs复合对斑马鱼外周血红细胞的DNA损伤。将成年斑马鱼暴露于PFOS(0.2、0.4、0.8、1.6 mg·L-1)、MWCNTs(50 mg·L-1)、PFOS+MWCNTs(0.2+50、0.4+50、0.8+50、1.6+50 mg·L-1)和对照溶液中30 d后,断尾取血进行微核试验和彗星试验。结果表明:PFOS和MWCNTs均可造成斑马鱼外周血红细胞的DNA损伤。1.6 mg·L-1PFOS处理组的微核率、Olive尾矩及尾长分别为(36.3±0.25)‰、(87.91±14.90)μm和(250.49±34.71)μm。PFOS与MWCNTs复合后,斑马鱼外周血红细胞的DNA损伤效应明显降低。复合处理组斑马鱼外周血红细胞的微核率、Olive尾矩及尾长均低于PFOS单独处理相。1.6 mg·L-1复合处理组的微核率、Olive尾矩及尾长比PFOS单独处理组分别降低了24.7%、68.9%、52.4%。因此,在实验浓度范围内,MWCNTs可以降低PFOS对斑马鱼外周血红细胞的DNA损伤。     

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.     

Heterogeneous oxidation of SO2 by O3-aged black carbon and its dithiothreitol oxidative potential

Ozone (O₃) is an important atmospheric oxidant. Black carbon (BC) particles released into the atmosphere undergo an aging process via O₃ oxidation. O₃-aged BC particles may change their uptake ability toward trace reducing gases such as SO₂ in the atmosphere, leading to different environmental and health effects. In this paper, the heterogeneous reaction process between O₃-aged BC and SO₂ was explored via in-situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). Combined with ion chromatography (IC), DRIFTS was used to qualitatively and quantitatively analyze the sulfate product. The results showed that O₃-aged BC had stronger SO₂ oxidation ability than fresh BC, and the reactive species/sites generated on the surface had an important role in the oxidation of SO₂. Relative humidity or 254 nm UV (ultraviolet) light illumination enhanced the oxidation uptake of SO₂ on O₃-aged BC. The oxidation potentials of the BC particles were detected via dithiothreitol (DTT) assay. The DTT activity over BC was decreased in the process of SO₂ reduction, with the consumption of oxidative active sites.     

河豚毒素检测技术研究进展

对检测河豚毒素的主要方法包括生物检测方法、理化检测方法、免疫学方法、传感器方法进行了介绍,并对目前检测方法的优缺点进行比较分析。小鼠测定法是河豚毒素检测的标准方法;理化检测法方法准确,但是对设备要求高,前处理严格;免疫学方法费用较高;传感器方法是新方法,还有待进一步规范。各种检测方法都有其优缺点,河豚毒素检测时应根据要求选择方法。     

Monoclonal antibody produced by heterologous indirect enzyme-linked immunosorbent assay and its application for parathion residue determination in agricultural and environmental samples

A heterologous indirect enzyme-linked immunosorbent assay (ELISA) was developed, which was based on monoclonal antibody (Mab) to determine parathion residue in agricultural and environmental samples. Eight Mabs were produced by introducing the heterologous indirect ELISA into the screening procedure. It was shown that these Mabs had more broad-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening). So it became much easier using these new Mabs to develop heterologous immunoassays. In addition, all the developed heterologous ELISAs could be used to determine parathion residue in semiquantitative or quantitative level, and their detection limitation (LOD) was around 2 ng/mL. Compared with the previous heterologous ELISA (hapten 11/5H7) with IC₅₀ of 13.3 ng/mL, a better heterologous ELISA (hapten 11/1E1) was obtained with IC₅₀ of 3.8 ng/mL, which improved the sensitivity 3.5 times. Finally, the latter was applied to parathion residue determination in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, Chinese cabbage and carrot were between 80.4 % and 111.8 %. The LOD for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.     

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.     

甲基营养型酵母表达载体系统研究进展

酵母作为生产真核异源蛋白的宿主菌 ,具备了易于分子遗传操作 ,原核微生物的生长性质及真核蛋白的翻译后加工修饰等特点 .其中Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ (酿酒酵母 )是第一个作为异源蛋白基因表达系统并加以应用的酵母 .人们认为Ｓ .ｃｅｒｅｖｉｓｉａｅ并非表达外源基因的最理想的酵母宿主菌 .在一群优于Ｓ .ｃｅｒｅｖｉｓｉａｅ的表达菌中 ,甲基营养型酵母Ｐｉｃｈｉａｐａｓｔｏｒｉｓ研究得最多 ,最具应用潜力 .在 2 0世纪 70年代期间 ,Ｐ .ｐａｓｔｏｒｉｓ是单细胞蛋白 (Ｓｉｎｇｌｅ ｃｅｌｌｐｒｏｔｅｉｎ…     

Spatial distribution of ciguateric fish in the Republic of Kiribati

Ciguatera is food poisoning caused by human consumption of reef fish contaminated with ciguatoxins (CTXs). The expanding international trade of tropical fish species from ciguatera-endemic regions has resulted in increased global incidence of ciguatera, and more than 50 000 people are estimated to suffer from ciguatera each year worldwide. The Republic of Kiribati is located in the Pacific Ocean; two of its islands, Marakei and Tarawa, have been suggested as high-risk areas for ciguatera. The toxicities of coral reef fish collected from these islands, including herbivorous, omnivorous and carnivorous fish (24% [n = 41], 8% [n = 13] and 68% [n = 117], respectively), were analyzed using the mouse neuroblastoma assay (MNA) after CTX extraction. The MNA results indicated that 156 fish specimens, or 91% of the fish samples, were ciguatoxic (CTX levels >0.01 ng g⁻¹). Groupers and moray eels were generally more toxic by an order of magnitude than other fish species. All of the collected individuals of eight species (n = 3-19) were toxic. Toxicity varied within species and among locations by up to 10 000-fold. Cephalapholis argus and Gymnothorax spp. collected from Tarawa Island were significantly less toxic than those from Marakei Island, although all individuals were toxic based on the 0.01 ng g⁻¹ threshold. CTX concentrations in the livers of individuals of two moray eel species (Gymnothorax spp., n = 6) were nine times greater than those in muscle, and toxicity in liver and muscle showed a strong positive correlation with body weight. The present study provides quantitative information on the ciguatoxicity and distribution of toxicity in fish for use in fisheries management and public health.     

Effects of organic contaminants in reactive oxygen species, protein carbonylation and DNA damage on digestive gland and haemolymph of land snails

The present study focused on early responses of land snails Eobania vermiculata to organic environmental contaminants, by investigating the use of a newly-established method for the measurement of protein carbonylation as a new biomarker of terrestrial pollution, as well as by measuring the ROS production and the DNA damage. Land snails were exposed to different concentrations of chlorpyrifos, parathion-methyl or PAHs in vivo or in vitro in the laboratory. The susceptibility of exposed snails was increased in relation to oxidative stress induced by contaminants tested. A statistically significant increase in ROS production, protein carbonylation and DNA damage was revealed in the snails treated with pollutants, compared to the untreated ones. The results indicated the effectiveness of measuring ROS production and DNA damage and reinforce the application of the present ELISA method in organic terrestrial pollution biomonitoring studies.