Streamlined beta-galactosidase assay for analysis of recombinant yeast response to estrogens

Here, we describe a rapid, convenient, and quantitative beta-galactosidase assay in liquid culture of recombinant yeast that expresses the estrogen receptor. This assay allows large-scale screening of chemicals (more than 600 samples/day) for the evaluation of their direct estrogenic potency and accurate determination of their EC50 with minimal manipulations. The assay, which is based on digestion of the yeast cell wall by lyticase (zymolase), a beta-glucanase isolated from Arthrobacter luteus, followed by a hypoosmotic shock lysis, is performed completely in 96-well plates. This protocol for using recombinant yeast with the two-hybrid technology significantly advances recombinant yeast manipulation.     

Monitoring Endocrine Disrupter Compounds in the Tunisian Hamdoun River using In Vitro Bioassays

Anthropogenic chemicals occurring in the environment, namely endocrine-disrupting chemicals (EDCs), have generated growing concern over their potential adverse effects on human wildlife health and ecosystem processes. This interest resulted particularly from their abilities to mimic the effect of endogenous hormones. In this study, we used stable transfected reporter cell lines to investigate the endocrine-disrupting profile of water as well as sediment samples. Samples are collected from up- and downstream of an industrial wastewater discharge point at the Hamdoun River in the vicinity of an industrial zone located at the center of Tunisia. The analysis of estrogen, androgen, and xenobiotic (pregnane X and dioxin) ligands receptors expressed by chimeric cell lines indicated that while the water and sediment samples from upstream sites have lower levels of estrogenic activity, those from downstream exhibited stronger estrogenic, aryl hydrocarbon receptor (AhR), and Pregnane X Receptor (PXR) activities. Moreover, collected samples have shown hormonal activity in terms of all tested receptors except the androgenic ones. In vitro recombinant estrogen receptor competitive binding assays revealed that while the estrogenic activities of the downstream water sample compounds had a strong affinity for estrogen receptor α (ERα), those present in the sediment samples showed a weaker one. These findings were consolidated by subsequent chemical analysis (high-performance liquid chromatography with UV detectors). Our results indicate that the water and sediment discharges at the Hamdoun River represent a major sink for EDCs from natural and industrial effluents, particularly those of the textile industry, with pernicious potential to disrupt normal endocrine functions.     

Determining Indicators of Exposure and Effects for Endocrine Disrupting Chemicals (EDCs): An Introduction

Endocrine disruptors are characterized by their influence on animal endocrine systems resulting in reproductive, developmental, neurological, and immune dysfunction. The purpose of this overview is to provide the reader with a sense of the activities within the U.S. Environmental Protection Agency (USEPA), in particular NHEERL, that address the many facets of research on endocrine disrupting chemicals (EDCs) and to highlight the approach being taken at the different organizational levels within the USEPA, including screening, testing and evaluating endocrine disrupting chemicals. As a part of this endeavor, the USEPA continues to evaluate the current research activities in order to better understand and refine the process of risk characterization of EDCs. Thus, the participants in this session were asked to review their research within the framework of a better identification of EDC effects, better characterization of those compounds that have endocrine disrupting activity and how to incorporate this information into the risk assessment paradigm. Specifically, the goals of the ensuing papers were to compare individual vs. population indicators of endocrine disrupting effects, examine comparable and multiple mechanisms of toxicity, and describe the use of effects as indicators to identify toxicants and their sources. Mammalian and fish reproductive endpoints served as models to emphasize commonalities between human and wildlife risks.     

Studies on the endocrine properties of a new progestational steroid 4,6-dichloro-16-methylene-17-hydroxy-4,6-pregnadiene-3,20-dione 17-acetate (MDAP)

MDAP (4,6-dichloro-16-methylene-17-hydroxy-4,6-pregnadiene-3,20-dione 17-acetate) was found to be approximately 100 times more potent than progesterone and 5 to 10 times more potent than chlormadinone acetate (6-chloro-17-hydroxy-4,6-pregnadiene-3, 20-dione 17-acetate) in the Clauberg-McPhail assay. MDAP manifested significant antigonadotropic and anti-androgenic activities and caused adrenal atrophy. The compound was also weakly uterotropic, thymolytic, and anti-estrogenic.     

Estrogenic, anti-estrogenic and cytotoxic effects elicited by water from the type localities of the endangered goodeid fish Girardinichthys viviparus

Girardinichthys viviparus is a Mexican endangered endemic fish species living only in Lake Texcoco (LTX), one of two extant type localities for this species. The other type locality is Lake Zumpango (LZ). LTX and LZ are fed by wastewater treated at secondary level that contains several endocrine disrupting chemicals. Our goal was to assess the estrogenic and anti-estrogenic effects elicited in G. viviparus by water from the two type localities and by these same matrices enriched with PCBs in order to understand potential damage due to increased xenobiotic levels. Estrogenic and anti-estrogenic effects were evaluated in vitro by E-screen assay in MCF-7 cells and cytotoxicity by MTT assay. PCBs were quantified in type localities. In vivo vitellogenin (VTG) induction was determined by a hybrid ELISA in adult laboratory-born fish exposed during 1, 2, 4, 8 and 16 days to LTX or LZ water in static exposure systems, and by the same matrices enriched with PCBs. We found PCBs only in LTX, but the water from both type localities elicit estrogenic and anti-estrogenic effects in vitro. Cytotoxicity was not observed in MCF-7 cells exposed to LTX or LZ water. VTG induction was higher with LTX water than with LZ water; also the response of males was greater than in females. In the PCB-enriched matrices, VTG induction in both sexes exposed to LTX water was reduced compared to un-enriched matrices. Thus, the sublethal increases in PCB levels may be hazardous to both sexes since they are linked probably to hepatotoxicity.     

Establishment of a transgenic yeast screening system for estrogenicity and identification of the anti-estrogenic activity of malachite green

Endocrine disruptors refer to chemical compounds in the environment which interfere with the endocrine systems of organisms. Among them, environmental estrogens pose serious problems to aquatic organisms, in particular fish. It is therefore important and necessary to have a fast and low-cost system to screen the large number of different chemical compounds in the aquatic environment for their potential endocrine disrupting actions. In this study, a screening platform was developed to detect xenoestrogens in the aquatic environment using the fission yeast Schizosaccharomyces pombe, and applied for compound screening. The aim was to demonstrate any significant potential differences between the fish screening system and the human screening system. To this end, a yeast expression vector harboring a fish estrogen receptor alpha and a reporter vector containing the estrogen responsive element fused with the Escherichia coli LacZ gene were constructed. After transformation with these two vectors, the transformed yeast clones were confirmed by Western blotting and selected on the basis of the beta-galactosidase activity. In this transgenic yeast system, the natural estrogen (estradiol) and other known xenoestrogens such as diethylstilbestrol, bisphenol A, genistein and dichloro-diphenyl-trichloroethane exhibited dose-dependent activities. Using this system, more than 40 putative endocrine disruptors including phytoestrogens, pesticides, herbicides, industrial dyes and other industrial chemicals were screened. Ten of them were demonstrated to exhibit estrogenic actions. Industrial dyes such as malachite green (MG) that disrupt thyroid hormone synthesis are extensively used and are widely distributed in the aquatic environment. Using this system, MG did not show any estrogenic action, but was demonstrated to exhibit anti-estrogenic activity.     

Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.     

New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment

Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Trends in Risk Assessment of Chemicals in the European Union

Current legislation in the European Union (EU) requires a risk assessment for industrial chemicals. The underlying procedures and paradigms of such EU risk assessment for new and existing chemicals are explained. The risk assessment is performed according to a harmonised methodology, laid down in the Technical Guidance Documents (TGD). Important new, technical risk assessment aspects covered in a recent revision round of the TGD are highlighted. The most prominent change in the environmental TGD part is the addition of the marine risk assessment, including a Persistent Bioaccumulation and Toxicity (PBT) assessment. In the human health part a significant change is the new data requirement for reproductive toxicity. The performance of both the risk assessment and the risk reduction phase of EU existing chemicals have been evaluated. An important conclusion was that our a priori knowledge on possible risks of chemicals is poor. The European Commission has recently launched a proposal (REACH) for drastically changing the risk management process of industrial chemicals in the EU. Major changes are a shift in responsibility from authorities to industry (including downstream users) for the safe use of chemicals, an acceleration of data collection for ‘non-assessed’ chemicals, and an authorization step for substances of very high concern.     

Relationship between estrogen receptor-binding and estrogenic activities of environmental estrogens and suppression by flavonoids

In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERbeta) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERbeta than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17alpha-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17beta-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.     

Health Risk Assessment of Workers' Exposure to Organic Compounds in a Tire Factory

This study focuses on a health risk assessment related to chemical exposure via inhalation for workers in a tire factory. Specifically, several volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs) were measured in the four different points of the vulcanization unit. A chemical transport model was developed in order to better represent the workers' exposure to the chemicals. Then, a risk assessment methodology was employed to evaluate the potential adverse health effects of the chemicals according to their carcinogenicities. Concentrations measured near the milling machine and press in the vulcanization unit were generally higher than the respective occupational exposure limit values. The corresponding estimated cumulative cancer risks for the carcinogens at the each sampling point were higher than the designated acceptable risk level of 1 × 10^{? 4}. With respect to non-carcinogenic risks, the hazard indexes, both individually and cumulatively, were lower than the specified level of one. The high cancer risk estimated in this study suggests that the VOCs and SVOCs exposure for workers in the vulcanization unit should not be neglected. The results obtained in this study are valuable to plant managers, government officials, and regulators in the risk evaluation process.     

The effect of apigenin,an aryl hydrocarbon receptor antagonist,in Phthalate-Exacerbated eosinophilic asthma model

Endocrine disrupting chemicals have been known to contribute to the aggravation of inflammatory diseases including asthma. We aimed to investigate the effects of mono-n-butyl phthalate (MnBP) which is one of the representing phthalates, and its antagonist in an eosinophilic asthma mouse model. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA) with alum and followed by three nebulized OVA challenges. MnBP was administered through drinking water administration throughout the study period, and its antagonist, apigenin, was orally treated for 14 days before OVA challenges. Mice were assessed for airway hyperresponsiveness (AHR), differential cell count and type 2 cytokines in bronchoalveolar lavage fluid were measured in vivo. The expression of the aryl hydrocarbon receptor was markedly increased when MnBP was administered. MnBP treatment increased AHR, airway inflammatory cells (including eosinophils), and type 2 cytokines following OVA challenge compared to vehicle-treated mice. However, apigenin treatment reduced all asthma features, such as AHR, airway inflammation, type 2 cytokines, and the expression of the aryl hydrocarbon receptor in MnBP-augmented eosinophilic asthma. Our study suggests that MnBP exposure may increase the risk of eosinophilic inflammation, and apigenin treatment may be a potential therapy for asthma exacerbated by endocrine-disrupting chemicals.     

Hydrogeochemical evolution and risk assessment of human health in a riverbank filtration site,northeastern China

Intensive agriculture and industrial activities have resulted in contamination in rivers and groundwater quality, which threatens human health. In this study, we used comprehensive physiochemical indicators to assess the quality of groundwater used for drinking and irrigation in addition to the potential risks to local residents in a riverbank filtration site. Human health risks through drinking water intake and dermal contact were also estimated. Moreover, we analyzed the spatial distribution regularities of health risk values in a riverbank filtration site. The assessment results revealed that NH₄–N, NO₂–N, F^?, Mn, and As are main contaminants affecting groundwater quality and that 62% of the total samples is suitable for a variety of purposes. All groundwater in the study area is suitable for irrigation based on the sodium adsorption ratio (SAR), residual sodium carbonate (RSC), Na percentage (%Na), and U.S. Salinity Laboratory (USSL) and Wilcox diagrams. The health risk assessment suggests that residents in the study area are at high health risk, and women and children face higher risk than men in both non-carcinogenic and carcinogenic risks. The spatial distribution regularities of health risk values suggest that the human health risk value of each groundwater sample is different in the study area and has certain regularity. Therefore, effective measurements must be taken to address the groundwater contamination and to reduce the human health risks.     

三种转基因酵母法筛选类雌激素活性物质方法的比较

利用转基因酵母菌株,分别采用摇瓶法、96孔板法和单板法筛选类雌激素活性化学品,并对实验参数进行了优化。通过对工作光密度及β半乳糖苷酶反应时间的优化,使改进后的转基因酵母筛选法筛选一批样品的时间从原来需要的几天缩短为半天。并且在时间缩短的同时,仍保留原来检测的高灵敏度和准确性,也节省了大量的试验耗材,达到了能应用于环境样品中类雌激素活性物质的快速测定的目的。       

Reduction,refinement and replacement: putting the immune system to work

Many chemicals are known to be, or have been implicated as, contact allergens, and allergic contact dermatitis is an important occupational and environmental health issue. It is the responsibility of toxicologists to identify those chemicals that have the potential to induce skin sensitisation, and to assess the conditions under which there will exist a risk to human health. This article describes progress that has been made in the development of new approaches to the toxicological evaluation of skin sensitisation, and the benefits to animal welfare that such developments have already produced, and are likely to produce in the future. In this context, the local lymph node assay is described with regard to hazard identification and risk assessment, and possible strategies for the development of in vitro approaches to safety assessment are discussed.     

Risk Assessment of the Abandoned Jukjeon Metal Mine in South Korea Following the Korean Guidelines

Toxic metal contamination in the vicinity of Korean abandoned metal mines has been reported. A risk assessment for these metals was performed for the inhabitants in the area of the abandoned Jukjeon metal mine. Soil, groundwater, and crop samples were collected around the mine. After pretreatment of these samples, metal concentrations were measured and then a risk assessment was performed using the Korean soil-contamination risk assessment guidelines. Phytoaccumulation of metals in crops was observed in soybeans (As and Zn), red peppers (Zn), sweet potatoes (As and Zn), and cabbage (Cu), which had higher metal concentrations than soils in the area. The metal intake rate was highest for inhalation of soil. Cancer risk was highest from ingestion of As-contaminated crops. The sum of carcinogenic risks was 6.29 × 10^–3. The non-carcinogenic risk was highest for ingestion of As-contaminated crops (8.17). Most of the risks were attributable to As, Pb, and Hg contamination, therefore these three metals must be considered as the principal metals toxic to human health in the sampled area. In particular, the inhalation of metal-contaminated soil should be considered for risk assessment along with ingestion of water and crops in abandoned mine areas.