Issues arising when interpreting results from an in vitro assay for estrogenic activity

Concern about possible adverse effects caused by the inadvertent exposure of humans and wildlife to endocrine-active chemicals, has led some countries to develop an in vivo–in vitro screening program for endocrine effects. In this paper, a previously described estrogen-inducible recombinant yeast strain (Saccharomyces cerevisiae) is used to investigate a number of issues that could potentially lead to the mislabeling of chemicals as endocrine disruptors. The chemicals studied were: 17-estradiol, dihydrotestosterone, testosterone, estradiol-3-sulfate, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, bisphenol-A, methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, butyl benzyl phthalate, 4-hydroxytamoxifen, and ICI 182,780. Alterations in assay methodology (for example, incubation time, initial yeast cell number, and the use of different solvents) did not affect the potency of bisphenol-A and 4-nonylphenol relative to 17-estradiol, but did alter the apparent potency of butyl benzyl phthalate. Other issues (including the metabolic activation of methoxychlor, the chemical purity of a steroid metabolite and unusual chemical artifacts observed with alkylphenolic chemicals) which affect data interpretation are described. Many of the issues raised will also affect other in vitro assays for endocrine activity, and some will be relevant to the interpretation of data from in vivo assays. These examples illustrate that considerable care and thought must be applied when interpreting results derived from any single assay. Only by using a suite of assays will we minimize the chances of wrongly labeling chemicals as endocrine disruptors

Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay

Copyright @ 2001 Environmental Health PerspectivesWe used a recombinant yeast estrogen assay to assess the activity of 73 phenolic additives that are used as sunscreens, preservatives, disinfectants, antioxidants, flavorings, or for perfumery. Thirty-two of these compounds displayed activity: 22 with potencies relative to 17 beta -estradiol, ranging from 1/3,000 to -estradiol. Forty-one compounds were inactive. The major criteria for activity appear to be the presence of an unhindered phenolic OH group in a para position and a molecular weight of 140-250 Da.This work was supported in part under contract with the U.K. Department of Trade and Industry as part of the Government Chemist Programme

Endocrine disruption in juvenile roach from English rivers: A preliminary study

Juvenile roach Rutilus rutilus from seven rivers of varying water quality were examined for evidence of endocrine disruption. The majority of roach from five of these rivers had femalelike reproductive ducts. The results suggest that juvenile, rather than adult, fish could be used in studies of endocrine disruption in wild fish populations

Preliminary data on the influence of rearing temperature on the growth and reproductive status of fathead minnows Pimephales promelas

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - Copyright @ 2011 Brian JV et al.An investigation into the influence of temperature on the growth and reproductive status of the fathead minnow Pimephales promelas revealed that, while there was no clear effect of treatment on sex differentiation, ovarian tissue from female fish reared under the highest temperature regime contained large amounts of undefined tissue containing no germ cells. Furthermore, both male and female fish exhibited differences in length mass, condition and somatic indices, and in the expression of secondary sexual characteristics. The patterns observed are discussed in the context of climate change

Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3,4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays

1-Keto-1,2,3,4-tetrahydrophenanthrene (THP-1) was reported by Cook et al in 1933 as the first synthetic estrogen. Estrogenic activity was assessed by the induction of vaginal cornification in ovariectomised rats. The corresponding 4-isomer (THP-4) was shown to be inactive. Both chemicals have been re-synthesised and assessed for hormonal activity. Each chemical bound weakly and to the same extent to isolated estrogen receptors, but only at high concentrations. However, they each lacked estrogenic or anti-estrogenic activity when evaluated in vitro using a yeast hER assay, and both failed to induce vaginal cornification or uterotrophic effects in ovariectomised rats. THP-1, and to a lesser extent THP-4, were shown to possess weak androgenic and anti-androgenic activity in vitro when evaluated using an hAR yeast assay. Estrogenic activity for bisphenol A (BPA) was subsequently demonstrated by Dodds and Lawson (1936) using the same ovariectomised rat protocol, and this activity has been confirmed and supplemented by positive uterotrophic effects for BPA in the same bioassays. The present results illustrate the complexity of deriving conclusions regarding the hormonal activities of chemicals. First, some activities observed in isolated hormonal receptor binding assays may not be expressed in functional hormonal assays. This indicates the need for functional hormonal assays in any screening programme. Second, that activities observed for a chemical in one hormonal assay may not be reflected in related hormonal assays. This indicates the need to define assay protocols with some precision when incorporating them into screening batteries. Finally, that some literature reports of hormonal activity for chemicals may not be capable of independent confirmation under apparently identical conditions of test. This illustrates the need to use lists of hormonally active chemicals with car

Selecting passive dosimetry technologies for measuring the external dose of terrestrial wildlife

Dosimeters attached to wild animals can be used to validate regulatory assessment approaches and models for estimating radiation exposure of wild animals. Such measurements are also necessary to ensure that robust dose-effect relationships can be developed from the results of field research programmes. This paper presents the first comprehensive evaluation of the different dosimetry technologies available for specifically measuring the external exposure of wildlife. Guidance is provided on the selection of appropriate passive dosimetry approaches for directly measuring external exposure of terrestrial wildlife under field conditions. The characteristics and performance of four available dosimetry technologies (thermoluminescent dosimeter (TLD), optically stimulated luminescent dosimeter (OSLD), radiophotoluminescent dosimeter (RPLD) and direct ion storage, (DIS)) are reviewed. Dosimeter properties, detection limit and dose range, study organisms and the intended application are variables that need to be considered when selecting a suitable dosimetry technology. Evaluated against these criteria, it is suggested that LiF based and Al2O3:C TLDs, OSLD and RPLD could all be used to estimate doses to wildlife. However, only LiF based TLDs have been used to directly measure wildlife doses in field studies to date. DIS is only suitable for comparatively large species (e.g. medium to large mammals), but has the advantage that temporal variation in dose can be recorded. In all cases, dosimeter calibration is required to ensure that the dose measurements reported can be interpreted appropriately for the organisms of interest

Radiocaesium transfer and radiation exposure of frogs in Fukushima Prefecture

The International Commission on Radiological Protection has proposed an environmental assessment framework. This includes ionising radiation exposure assessment for different frog life-stages, but radiocaesium transfer parameters are unavailable. We collate data from the Fukushima Prefecture (contaminated by the Fukushima accident) and estimate radiocaesium concentration ratio (CR ) values for tadpoles and adult frogs, presenting the largest available amphibian CR dataset. In total, 513 adult frogs and 2540 tadpoles were analysed in 62 and 59 composite samples respectively. Results suggest that equilibrium was reached between water and amphibian radiocaesium activity concentrations circa one-year after the accident. Radiocaesium transfer to tadpoles was higher than to adult frogs. Dose rates were estimated for different life-stages and species in both the aquatic and terrestrial environment. Estimated dose rates to adults and tadpoles were typically similar because external exposure dominated for both organisms; frogspawn dose rates were estimated to be orders of magnitude lower than other life-stages. For the two sites assessed, which were outside of the most contaminated areas of the Fukushima Prefecture, estimated dose rates were below those anticipated to present a risk to wildlife populations; it is likely that dose rates in more contaminated areas were in excess of some effects benchmark values

Hypoxia does not influence the response of fish to a mixture of estrogenic chemicals

The official published version can be obtained from the link below - Copyright @ 2009 American Chemical SocietyChemical risk assessment procedures assign a major role to standardized toxicity tests, in which the response of a particular organism to a single test substance is determined under otherwise constant and favorable conditions in the laboratory. This approach fails to consider the potential for chemical interactions, as well as failing to consider how the toxicological response varies, depending on the conditions of exposure. As yet, the issue of confounding factors on chemically mediated effects in wildlife has received little attention, despite the fact that a range of physicochemical parameters, including temperature, water quality, and pH, are known to modify chemical toxicity. Here, we consider how the estrogenic response of fish varies with regard to hypoxia. Fathead minnows (Pimephales promelas) were exposed to a mixture of estrogenic chemicals under hypoxic or normoxic conditions. Their estrogenic response was characterized using an in vivo assay, involving the analysis of the egg yolk protein, vitellogenin (VTG). The results revealed that there was no effect of hypoxia on the VTG response in either treatment group at the end of the exposure period. This suggests that this end point is robust and relatively insensitive to the effects of any physiological changes that arise as a result of hypoxia. The implications of these negative findings are discussed in terms of their relevance with regard to the development of risk assessment policy.This work was funded by a grant from the Natural Environment Research Council(NE/D00389X/1)

The rodent uterotrophic assay: Critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay

The major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used, and the specificity of the assay for estrogens. It is concluded that three daily oral administrations of test chemicals to 21- to 22-day-old rats, followed by determination of absolute uterus weights on the fourth day, provide a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favored over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is shown to be a useful, but nondefinitive, adjunct to the uterotrophic assay. The ability of test chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay for the purpose of detecting antiestrogens. The results of a series of studies on the environmental estrogen nonyl phenol (NP), and its linear isomer n -nonyl phenol, confirm that branching of the aliphatic side chain is important for activity. 17beta-Desoxyestradiol is shown to be of similar activity to estradiol in the uterotrophic assay and is suggested to represent the "parent" estrogen of NP. Benzoylation of NP and 17-desoxyestradiol did not affect their uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol. Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyze the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future. The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity

Radionuclide transfer to wildlife at a 'reference site' in the Chernobyl Exclusion Zone and resultant radiation exposures

This study addresses a significant data deficiency in the developing environmental protection framework of the International Commission on Radiological Protection, namely a lack of radionuclide transfer data for some of the Reference Animals and Plants (RAPs). It is also the first study that has sampled such a wide range of species (invertebrates, plants, amphibians and small mammals) from a single terrestrial site in the Chernobyl Exclusion Zone (CEZ). Samples were collected in 2014 from the 0.4 km2 sampling site, located 5 km west of the Chernobyl Nuclear Power complex. We report radionuclide (137Cs, 90Sr, 241Am and Pu-isotopes) and stable element concentrations in wildlife and soil samples and use these to determine whole organism-soil concentration ratios and absorbed dose rates. Increasingly, stable element analyses are used to provide transfer parameters for radiological models. The study described here found that for both Cs and Sr the transfer of the stable element tended to be lower than that of the radionuclide; this is the first time that this has been demonstrated for Sr, though it is in agreement with limited evidence previously reported for Cs. Studies reporting radiation effects on wildlife in the CEZ generally relate observations to ambient dose rates determined using handheld dose meters. For the first time, we demonstrate that ambient dose rates may underestimate the actual dose rate for some organisms by more than an order of magnitude. When reporting effects studies from the CEZ, it has previously been suggested that the area has comparatively low natural background dose rates. However, on the basis of data reported here, dose rates to wildlife from natural background radionuclides within the CEZ are similar to those in many areas of Europe