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.     

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.     

Suppression of estrogenic activity of 17-beta-estradiol by beta-cyclodextrin

The suppressive effects of cyclodextrins (CDs) on the strong estrogenic activity of 17β-estradiol (E2) in water environments were investigated in this study. Cyclodextrins are doughnut-shaped molecules that possess a hydrophobic cavity and a hydrophilic exterior. The cavity can incorporate nonpolar molecules as guests to form inclusion complexes. β-CD and 2-hydroxypropyl-β-CD (HP-β-CD) were the most successful in forming a complex with E2 and improving its low aqueous solubility. The E2/CDs complexes bound to the estrogen receptor in a cell-free system as determined by ELISA and suppressed the hormone activities as measured by a yeast two-hybrid assay. These results indicate that hydrophobic E2 is easily transported through the lipid zone of the plasma membrane into the target cell and can bind to the nuclear receptor. However, the hydrophilic E2/β-CD and E2/HP-β-CD complexes do not penetrate the membrane. Therefore, these CDs are able to suppress the hormone activity of E2 through complex formation.     

Denaturing high-performance liquid chromatography (DHPLC)-based prenatal diagnosis for tuberous sclerosis

Tuberous sclerosis (TSC) is a frequent autosomal-dominant condition (affecting 1 in 6000 individuals) caused by various mutations in either the hamartin (TSC1) or the tuberin gene (TSC2). This allelic and non-allelic heterogeneity makes genetic counseling and prenatal diagnosis difficult, especially as a significant proportion of TSC cases are due to de novo mutations. For this reason the identification of the disease causing mutation is mandatory for accurate counseling, yet current mutation detection methods such as single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) are labor intensive with limited detection efficiency. Denaturing high-performance liquid chromatography (DHPLC) is a high-throughput, semi-automated mutation detection system with a reported mutation detection rate close to 100% for PCR fragments of up to 800 bp. We used a recently described DHPLC assay allowing the efficient detection of mutations in TSC1 to analyze the DNA extracted from a chorion villus sample in order to perform a prenatal diagnosis for TSC. The fetus was found not to have inherited the deleterious mutation and the DHPLC diagnosis was confirmed by haplotype analysis. This represents the first DHPLC-based prenatal diagnosis of a genetic disease. Copyright © 2001 John Wiley & Sons, Ltd.     

镉引起蚕豆(Vicia faba)叶片DNA损伤和细胞凋亡研究

以蚕豆为研究对象,采用碱性、半碱性和中性彗星实验方法研究Cd对植物DNA的损伤,同时还采用DAPI染色法从细胞形态学入手研究Cd诱导的蚕豆叶片的细胞凋亡.用3种彗星实验研究Cd处理蚕豆叶片DNA损伤,结果表明Cd能够引起蚕豆叶片DNA损伤,但是不同Cd浓度引起DNA损伤的种类不同:5mg·L^-1Cd处理主要引起单链DNA损伤和碱性不稳定位点的形成;10mg·L^-1Cd处理时开始检测到DNA双链断裂;20mg·L^-1Cd处理下,各种类型的DNA损伤均明显增加,且DNA双链断裂尤其明显.DAPI染色法通过细胞形态学变化来检测蚕豆叶片细胞凋亡的结果表明,蚕豆叶片细胞在Cd处理下发生凋亡的过程与DNA损伤过程有很大的相关性.结果表明,Cd是一种基因毒性物质,同时证明Cd引起的DNA损伤是引起细胞发生凋亡的机制之一.