Modulation of steroidogenesis by coastal waters and sewage effluents of Hong Kong, China, using the H295R assay

BACKGROUND, AIM, AND SCOPE: The presence of a variety of pollutants in the aquatic environment that can potentially interfere with the production of sex steroid hormones in wildlife and humans has been of increasing concern. The aim of the present study was to investigate the effects of extracts from Hong Kong marine waters, and influents and effluents from wastewater treatment plants on steroidogenesis using the H295R cell bioassay. After exposing H295R cells to extracts of water, the expression of four steroidogenic genes and the production of three steroid hormones were measured. MATERIALS AND METHODS: Water samples were collected during the summer of 2005 from 24 coastal marine areas and from the influents and effluents of two major waste water treatment plants (WWTPs) in Hong Kong, China. Samples were extracted by solid phase extraction (SPE). H295R cells were exposed for 48 h to dilutions of these extracts. Modulations of the expression of the steroidogenic genes CYP19, CYP17, 3betaHSD2, and CYP11beta2 were determined by measuring mRNA concentrations by real-time polymerase chain reaction (Q-RT-PCR). Production of the hormones progesterone (P), estradiol (E2), and testosterone (T) was quantified using enzyme linked immunosorbent assays (ELISA). RESULTS: Extracts from samples collected in two fish culture areas inhibited growth and proliferation of H295R cells at concentrations greater or equal to 10(5) L equivalents. The cells were exposed to the equivalent concentration of active substances in 10,000 L of water. Thus, to observe the same level of effect as observed in vitro on aquatic organisms would require a bioaccumulation factor of this same magnitude. None of the other 22 marine samples affected growth of the cells at any dilution tested. Twelve of the marine water samples completely inhibited the expression of CYP19 without affecting E2 production; inhibition of CYP17 expression was observed only in one of the samples while expression of CYP11beta2 was induced as much as five- and ninefold after exposure of cells to extracts from two locations. The expression of the progesterone gene 3betaHSD2 was not affected by any of the samples; only one sample induced approximately fourfold the production of E2. Although more than twofold inductions were observed for P and T production, none of these values were statistically significant to conclude effects on the production of these two hormones. While influents from WWTPs did not affect gene expression, an approximately 30% inhibition in the production of E2 and a 40% increase in P occurred for the exposure with influents from the Sha Tin and Stonecutters WWTPs, respectively. Effluents from WWTPs did not affect the production of any of the studied hormones, but a decrement in the expression of the aldosterone gene CYP11beta2 was observed for the Sha Tin WWTP exposure. No direct correlation could be established between gene expression and hormone production. DISCUSSION: Observed cytotoxicity in the two samples from fish culture areas suggest the presence of toxic compounds; chemical analysis is required for their full identification. Although effluents from WWTPs did not affect hormone production, other types of endocrine activity such as receptor-mediated effects cannot be ruled out. Interactions due to the complexity of the samples and alternative steroidogenic pathways might explain the lack of correlation between gene expression and hormone production results. CONCLUSIONS: Changes observed in gene expression and hormone production suggest the presence in Hong Kong coastal waters of pollutants with endocrine disruption potential and others of significant toxic effects. The aromatase and aldosterone genes seem to be the most affected by the exposures, while E2 and P are the hormones with more significant changes observed. Results also suggest effectiveness in the removing of compounds with endocrine activity by the WWTPs studied, as effluent samples did not significantly affect hormone production. The H295R cell showed to be a valuable toll in the battery required for the analysis of endocrine disrupting activities of complex environmental samples. RECOMMENDATIONS AND PERSPECTIVES: Due to the intrinsic complexity of environmental samples, a combination of analytical tools is required to realistically assess environmental conditions, especially in aquatic systems. In the evaluation of endocrine disrupting activities, the H295R cell bioassay should be used in combination with other genomic, biological, chemical, and hydrological tests to establish viable modes for endocrine disruption and identify compounds responsible for the observed effects.     

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Background, Aim and Scope Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented for long range transport of these conjugates in plants, rather than storage in the vacuole. To our knowledge this is the first report about the unidirectional long range transport of xenobiotic conjugates in plants and the exudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds which give them similar advantages as animals. Materials and Methods: Barley plants (Hordeum vulgare L. cv. Cherie) were grown in Petri dishes soaked with tap water in the greenhouse. - Fluorescence Microscopy. Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers for our experiments. Their transport in the root could be followed sensitively with very good temporal and spatial resolution. Roots of barley seedlings were cut under water and the end at which xenobiotics were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). - Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. - Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results: The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min-1. The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. Discussion: It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. - On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Conclusions: Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. Recommendations and Perspectives: The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exuded metabolites on the biology of rhizosphere bacteria and fungi.     

优质稻的穗肥施用技术研究

优质稻田间穗肥试验表明,在叶色转赤的基础上,施穗肥一般以复合肥112.5~150.0 kg/hm2加氯化钾37.5 kg/km2为宜; 施穗肥比不施穗肥增产10.7%~15.5%。施穗肥的效果还与肥料中的氮钾比例有关,N∶K2O以1∶0.7~1.0较适宜。     

几种水稻涂层专用肥一次性施用的肥效试验

一次性施用水稻涂层专用肥,水稻在整个生育期内均能正常生长。几种水稻涂层专用肥的肥效试验表明,水稻专用涂层复混肥1号成本低、增产稳定,值得推广应用。     

论可持续发展视野中的农村社会支持

本文阐述了如何可持续发展观来指导农村社会支持,并提出在农村社会支持活动中,引进社会保险、社会服务、生态保护和心理培育等新的机制,运用综合性评价指标体系对各种农村社会支持项目及其结果进行经济效益、社会效益和心理效益三个方面的分析。     

薄膜扩散梯度技术在重金属生物有效态监测中的应用

重金属生物有效性是评估重金属元素迁移性、生物可利用性和生态影响的关键参数。薄膜扩散梯度技术(DGT)是一种原位被动采样技术,因其具有原位富集性、形态选择性,可提供被监测物质在监测时间段内的平均浓度等优点,可作为生物对重金属摄取的模拟替代物对环境介质中重金属的生物可利用度进行预测,已被广泛应用于环境介质中重金属生物有效性的测定。研究主要介绍了DGT技术的原理、组成和特点,评述了其近年来在水体、土壤、沉积物中重金属生物有效态应用方面的新进展,提出了DGT技术未来要提高抗生物污染能力及寻找可与DGT技术联用的相关技术的观点。     

典型地区农用地污染调查及风险管控标准探讨

针对《土壤污染风险管控标准——农用地土壤污染风险管控标准》(GB 15618—2018),提出以土壤中全量浓度筛选值和管控值作为衡量农用地土壤污染风险管控的标准,对湖南省部分稻田农用地土壤及点对点稻米样品中镉、铅、砷、汞的总量和有效态浓度及稻米中含量进行监测,根据重金属总量浓度分为低风险、中风险、高风险3组。结果显示:(1)土壤及稻米中镉含量基本为随着风险级别的升高而增加,铅、砷在土壤和稻米中含量无规律性结果,汞监测结果均为未检出。(2)低风险组稻米镉超标率为12. 0%,高风险组稻米镉达标率为33. 3%,表明利用总量浓度对农用地土壤潜在风险进行分组存在一定的局限性。(3)依据4种重金属在土壤中总量及稻米(早稻)中含量情况,对风险级别进行调整并综合判断:有58个样品为低风险组,占样品总数的68. 2%,超标率为零;有15个样品为中风险组,占样品总数的17. 7%,超标率为80. 0%;有12个样品为高风险组,占样品总数的14. 1%,超标率为100. 0%。调整后评价结果与上述标准的划分目标更接近,能够提高上述标准的准确性和实用性。     

复杂气井套管力学分析与可靠度评价

为建立考虑地层和套管参数随机性的套管可靠度评价理论方法,以便得到复杂井况下套管传统设计安全系数与可靠指标之间的关系,提出了非均匀地应力和内压联合作用下,沿套管最大外挤力方向管壁任意位置发生屈服失效时外壁等效均匀外挤力的计算方法;建立了套管抗挤和抗内压三轴强度计算公式以及有效内压计算方法;根据套管载荷和强度影响因素统计参数以及评价过程中参数测试标准值,利用蒙特卡洛法（MC）建立了完整的套管可靠度计算和评价方法;通过实例对传统安全系数与可靠指标的对应关系进行了研究。研究结果表明:指定条件下,套管安全系数与可靠指标之间存在对应关系;利用建立的方法编制计算程序可以为传统设计法中安全系数代表的安全程度进行量化;可靠度评价方法能够为安全系数的选取提供指导。     

Optimizing mixture properties of biodiesel production using genetic algorithm-based evolutionary support vector machine

Nowadays, biodiesel is used as one of the alternative renewable energy due to the increasing energy demand. However, optimum production of biodiesel still requires a huge number of expensive and time-consuming laboratory tests. To address the problem, this research develops a novel Genetic Algorithm-based Evolutionary Support Vector Machine (GA-ESIM). The GA-ESIM is an Artificial Intelligence (AI)-based tool that combines K-means Chaotic Genetic Algorithm (KCGA) and Evolutionary Support Vector Machine Inference Model (ESIM). The ESIM is utilized as a supervised learning technique to establish a highly accurate prediction model between the input--output of biodiesel mixture properties; and the KCGA is used to perform the simulation to obtain the optimum mixture properties based on the prediction model. A real biodiesel experimental data is provided to validate the GA-ESIM performance. Our simulation results demonstrate that the GA-ESIM establishes a prediction model with better accuracy than other AI-based tool and thus obtains the mixture properties with the biodiesel yield of 99.9%, higher than the best experimental data record, 97.4%.     

The in vitro effect of selected essential oils on the growth and mycotoxin production of Aspergillus species

The aim of the present study was to assess the antifungal and anti-toxinogenic activity of 15 essential oils (EOs) against three fungi of the genus Aspergillus (A. parasiticus KMi-227-LR, A. parasiticus KMi-220-LR and A. flavus KMi-202-LR). The minimum inhibitory doses (MIDs) of the tested essential oils and their antifungal activity were determined using the micro-atmosphere method. The original commercial essential oil samples of Jasminum officinale L., Thymus vulgaris L., Syzygium aromaticum (L.) Merrill &; Perry, Rosmarinus officinalis L., Ocimum basilicum L., Eucalyptus globulus Labill., Salvia officinalis L., Citrus limon (L.) Burm, Origanum vulgare L., Lavandula angustifolia Mill., Carum carvi L., Citrus sinensis (L.) Osbeck., Zingiber officinalis Rosc., Mentha piperita L. and Cinnamomum zeylanicum Nees. (C. verum J.S.Presl.) were produced in Slovakia (Calendula a.s., Nová ?ubovňa, Slovakia). All essential oils exhibited activity against all tested strains of fungi. After 14 days of incubation, A. flavus (KMi-202-LR) showed the highest susceptibility with a growth inhibition percentage (GIP) of 18.70% to C. limon and 5.92% to C. sinensis, while A. parasiticus (KMi-220-LR) exhibited a GIP of 20.56% to J. officinale. The minimum inhibitory doses (MIDs) of EOs with the most significant activity were recorded. The best antifungal activity, using the micro-atmosphere method was found in S. aromaticum with an MID of 62.5 μL L^?1 air, T. vulgaris (MID of 62.5 μL L^?1 air) and O. vulgare (MID of 31.5 μL L^?1 air) against all tested strains. Mycotoxin production of the tested strains was evaluated by the thin layer chromatography (TLC) method. Mycotoxin production of AFB₁ and AFG₁ was inhibited following all treatments with C. carvi, R. officinale and S. officinale, Eucalyptus globulus L. and O. basilicum L. Essential oils exhibited a potential inhibition activity against toxic fungi, although, these affected only the production of AFB₁.