A critical assessment of the OECD collaborative study to validate the uterotrophic assay for the detection of oestrogenic and anti-oestrogenic chemicals

The design and execution of a recently completed validation study on the rat uterotrophic assay for detecting oestrogens and anti-oestrogens, managed by the OECD, are critically assessed with respect to internationally agreed criteria for the validation of new in vitro and in vivo toxicity test methods. It is concluded that, while the design of the study did not take account of several important criteria for validation, the uterotrophic assay appears to reliably detect the strong and weak oestrogenic substances used in the study, which act via binding to the oestrogen receptor in vivo. However, the reliability of the assay has not been substantiated for detecting anti-oestrogens that act as antagonists, due to the involvement of an insufficient number of experiments and test chemicals. Moreover, the data do not permit an assessment of the accuracy of the prediction of oestrogenicity, and the protocols have not been sufficiently optimised with regard to controlling variables. This problem has been exacerbated by a wish to introduce as much flexibility as possible into the protocols during the formal validation phase of the study, rather than during a separate prevalidation stage. In addition, the choice between surgically treated and/or immature animals, and details of housing and husbandry conditions that are necessary for increasing the sensitivity and efficiency of the assay, need to be clarified. The assay also lacks a well-defined prediction model by which the overall relevance of the data to toxicity, and especially to human hazard, can be assessed, and no performance criteria have been established. The results of this analysis of the study indicate that it would be premature to produce an OECD test guideline for the uterotrophic assay at this time, before some of the above issues have been satisfactorily resolved.     

Linking inter-individual variability to endocrine disruptors: insights for epigenetic inheritance

Endocrine disrupting chemicals (EDCs) can induce a myriad of adverse health effects. An area of active investigation is the multi- and transgenerational inheritance of EDC-induced adverse health effects referring to the transmission of phenotypes across multiple generations via the germline. The inheritance of EDC-induced adverse health effects across multiple generations can occur independent of genetics, spurring much research into the transmission of underlying epigenetic mechanisms. Epigenetic mechanisms play important roles in the development of an organism and are responsive to environmental exposures. To date, rodent studies have demonstrated that acquired epigenetic marks, particularly DNA methylation, that are inherited following parental EDC exposure can escape embryonic epigenome reprogramming. The acquired epimutations can lead to subsequent adult-onset diseases. Increasing studies have reported inter-individual variations that occur with epigenetic inheritance. Factors that underlie differences among individuals could reveal previously unidentified mechanisms of epigenetic transmission. In this review, we give an overview of DNA methylation and posttranslational histone modification as the potential mechanisms for disease transmission, and define the requirements for multi- and transgenerational epigenetic inheritance. We subsequently evaluate rodent studies investigating how acquired changes in epigenetic marks especially DNA methylation across multiple generations can vary among individuals following parental EDC exposure. We also discuss potential sources of inter-individual variations and the challenges in identifying these variations. We conclude our review discussing the challenges in applying rodent generational studies to humans.

     

Characterizing the effect of endocrine disruptors on human health: The role of epidemiological cohorts

Research on endocrine disruptors (EDs) developed from numerous disciplines. In this concert of disciplines, epidemiology is central to inform on the relevance for humans of mechanisms and dose-response functions identified in animals, to characterize the health impact (number of attributable disease cases), the cost associated with ED exposure, and the efficiency of the measures taken to limit exposure. Here, we present epidemiological tools to draw valid inference regarding effects of potential EDs. Epidemiology is generally observational, requiring care to control confounding bias. Many potential EDs have a short biological half-life; approaches relying on repeated biospecimens sampling allow limiting exposure misclassification and the resulting bias. For non-persistent compounds, couple–child cohorts are a central study design. Cohorts can now rely on molecular biology approaches to characterize exposures and intermediate pathways, which corresponds to the advent of molecular epidemiology and allows stronger interactions between epidemiology, toxicology, and molecular epidemiology to characterize the health effects of EDs.     

Application of the OECD 301F respirometric test for the biodegradability assessment of various potential endocrine disrupting chemicals

The biodegradability of several potential endocrine disrupting compounds, namely 4-n-nonylphenol (4-n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) was evaluated in this study, using OECD method 301F (manometric respirometry test) and activated sludge as inoculum. According to the results, 4-n-NP and BPA meet the strict definition of ready biodegradability and they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7+/-5.7%, n=3), TCS (52.1+/-8.5%, n=3) and NP1EO (25.9+/-8.1%, n=3), indicating their possible biodegradation in wastewater treatment systems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable compound showed the absence of co-metabolic phenomena during 4-n-NP, BPA and TCS biodegradation. Using first order kinetics to describe biodegradation of the target compounds, half-lives of 4.3+/-0.6, 1.3+/-0.2, 1.8+/-0.5, 6.9+/-2.6 days were calculated for 4-n-NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio fischeri showed that biodegradation of 4-n-NP, NP1EO, BPA and TCS is a simultaneous detoxification process, while possible abiotic or biotic transformations of NP2EO, DEHP, PFOA and PFNA during respirometric test resulted to significant increase of their toxicities.     

Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In na?ve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.     

The emerging role of endocrine disruptors in pathogenesis of insulin resistance: a concept implicating nonalcoholic fatty liver disease

Endocrine disruptors or endocrine-disrupting chemicals (EDCs) represent a highly heterogeneous group of molecules found in the environment or in consumer products. Toxicology and epidemiology studies have suggested the involvement of diverse EDCs in an increasing number of metabolic disorders, including insulin resistance (IR) and IR-related co morbidities, such as obesity, type 2 diabetes mellitus (T2DM) and polycystic ovary syndrome. Nonalcoholic fatty liver disease (NAFLD), another IR related condition, is emerging as a significant public health concern, affecting 30-45% of the general population in the Western world. To evaluate whether EDCs may also play a role in the pathogenesis of NAFLD, we reviewed the literature on well-studied EDCs, such as dioxins, bisphenol A, phthalates and other persistent organic pollutants, in relation to pathways that might contribute to the pathogenesis of fatty liver / NAFDL. Certain EDCs may be responsible for inducing alterations similar to those encountered in NAFLD either directly through a hepatotoxic effect and/or indirectly by triggering hepatic and systematic IR. Considering these effects, which act in concert with the effects of the epidemics of obesity and T2DM, EDCs may play a significant role in the pathogenesis of fatty liver, thereby increasing the prevalence of NAFLD worldwide. Translational studies and clinical trials investigating the association between EDCs and NAFLD are required to confirm and extent these studies.     

In vitro monitoring of i-NOS concentrations with an immunosensor: the inhibitory effect of endocrine disruptors on i-NOS release

The amperometric immunosensor has demonstrated the toxicity of endocrine disrupters (EDs) through monitoring the in vitro i-NOS concentration change, where the antibody of inducible nitric oxide synthase (i-NOS) was immobilized on the conducting polymer-gold nanoparticles composite. The performance of the sensor and the experimental parameters affecting the immunoreaction were optimized. Neuronal cells treated by EDs decreased in the in vitro i-NOS concentration. The effect of bisphenol A (BPA) on the i-NOS concentration released in the cells was investigated with different incubation times, and the interfering by nonspecific binding species present in a neuronal cell lysate was also examined. Of all the tested EDs, BPA showed the inhibitoriest effect and the minimum inhibitory concentration of BPA affecting the i-NOS concentration was 0.09 ± 0.005 μM. The result shows that monitoring of i-NOS in the neuronal cells treated by EDs will be a useful method to evaluate the toxic behavior of EDs.     

Development and validation of the spiral Salmonella assay: an automated approach to bacterial mutagenicity testing

Since its development by Dr. Bruce Ames and his colleagues more than a decade ago, the Salmonella/mammalian microsome mutagenicity assay has become a widely accepted tool to assist in the identification of chemicals with mutagenic and carcinogenic potential. Several automated approaches to Salmonella testing have been proposed in recent years but have failed to gain acceptance in the scientific community due to poor performance or lack of demonstrated usefulness. In this paper we report on an automated system that successfully generates dose-response data and, moreover, reduces the labor, materials, and sample mass required to obtain such information. In the standard plate-incorporation assay, dose-response relationships are defined by testing discrete doses of the test agent on a series of agar plates. In contrast, the spiral Salmonella assay generates dose-response data from a continuous concentration gradient on a single agar plate. Upon analysis, each spiral plate yields a dose-response curve consisting of 13 data points that span a concentration range of about 15:1, which is equivalent to 5 two-fold serial dilutions. The performance of the spiral Salmonella assay was compared to that of the conventional plate-incorporation assay using 13 mutagens and 7 nonmutagens selected from a variety of chemical classes. Concordant qualitative responses were obtained for all compounds tested, and comparable dose-response relationships were generated by all mutagens with the exception of sodium azide and cyclophosphamide, which are highly water-soluble and, thus, are unable to maintain a well-defined concentration gradient on a spiral plate due to rapid diffusion. In general, toxicity was expressed at a lower dose in the spiral assay, and the mutagenic potencies (slopes of the dose-response curves) were greater in the spiral assay relative to the plate-incorporation assay. These differences will be discussed, as will the applicability of the spiral plating technique to routine screening and its relevancy to future mutagenesis testing.     

Adding a radial dimension to the assessment of esophagogastric junction relaxation: validation studies of the 3D-eSleeve

High-resolution manometry (HRM) with esophageal pressure topography (EPT) allowed for the establishment of an objective quantitative measurement of esophagogastric junction (EGJ) relaxation, the integrated relaxation pressure (IRP). This study assessed whether or not a novel 3D-HRM assembly could improve on this measurement. Twenty-five normal subjects were studied with both a standard HRM assembly and a novel hybrid assembly (3D-HRM), including a 9.0 cm 3D-HRM segment composed of 96 radially dispersed independent pressure sensors. The standard IRP was computed using each assembly and compared with a novel paradigm, the 3D-IRP, an analysis premised on finding the axial maximum and radial minimum pressure at each sensor ring along the sleeve segment. Fourteen additional subjects underwent barium swallows with 3D-HRM and concurrent videofluoroscopy to compare the electronic sleeve (eSleeve) paradigm (circumferential average) to the 3D eSleeve paradigm (radial minimum) as a predictor of transphincteric flow. The 3D-IRP was significantly less than all other calculations of IRP with the upper limit of normal being 12 mmHg vs. 17 mmHg for the standard IRP. The sensitivity (0.78) and the specificity (0.88) of the 3D-eSleeve were also better than the standard eSleeve (0.55 and 0.85, respectively) for predicting flow permissive time verified fluoroscopically. The 3D-IRP and 3D-eSleeve calculated using the radial pressure minimum lowered the normative range of EGJ relaxation (upper limit of normal 12 mmHg) and yielded intraluminal pressure gradients that better correlated with bolus flow than did analysis paradigms based on circumferentially averaged pressure.     

Peer assessment in large undergraduate classes: an evaluation of a procedure for marking laboratory reports and a review of related practices

This study provides evidence that peer marking can be a reliable tool for assessing laboratory reports in large cohorts. It was conducted over a 4-yr period with first-year undergraduates (～180 students/cohort) taking a mammalian physiology course, but the procedure adopted would be applicable to any other laboratory-based discipline. The process was found to be efficient in staff time, enabling a summative practical report to be marked in <1 h (<5% of the time that had previously been required for staff marking), facilitating rapid feedback to students on their performance. When samples of the peer-assessed reports were marked by a single member of staff, there was excellent correlation between peer and staff marks (r = 0.96-0.98), although peer-awarded marks exceeded staff marks by an average of 2.5-3.0%. The validity of peer marking was independent of both the sex of the marker and the staff score awarded to the marker for the same piece of work. Feedback from students was largely positive; they reported that the procedure adopted was effective in increasing their understanding of the underlying physiology and contributed to their understanding of best practice in presenting a laboratory report. Seventy percent of students agreed that it was acceptable for peer assessment to contribute a small (up to 5%) component of the overall mark for the course. The results are discussed in relation to other reports of peer marking, particularly when used to assess an academic product or process in a scientific discipline.     

Welcome to the revolution: integrative biology and assessing the impact of endocrine disruptors on environmental and public health

Concern continues to grow over the negative impact of endocrine disrupting chemicals on environmental and public health. The number of identified endocrine disrupting chemicals is increasing, but biological endpoints, experimental design, and approaches for examining and assessing the impact of these chemicals are still debated. Although some workers consider endocrine disruption an "emerging science," I argue here that it is equally, a "merging science" developing in the tradition of integrative biology. Understanding the impact of endocrine disruptors on humans and wildlife is an examination of "context dependent development" and one that Scott Gilbert predicted would require a "new synthesis" or a "revolution" in the biological sciences. Here, I use atrazine as an example to demonstrate the importance of an integrative approach in understanding endocrine disruptors.Atrazine is a potent endocrine disruptor that chemically castrates and feminizes amphibians and other wildlife. These effects are the result of the induction of aromatase, the enzyme that converts androgens to estrogens, and this mechanism has been confirmed in all vertebrate classes examined (fish, amphibians, reptiles, birds, and mammals, including humans). To truly assess the impact of atrazine on amphibians in the wild, diverse fields of study including endocrinology, developmental biology, molecular biology, cellular biology, ecology, and evolutionary biology need to be invoked. To understand fully the long-term impacts on the environment, meteorology, geology, hydrology, chemistry, statistics, mathematics and other disciplines well outside of the biological sciences are required.     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.