An investigation into the biological effects of indirect potable reuse water using zebrafish embryos.

Advanced treatment technologies are being assessed as a proactive measure to assist with the transformation of treated wastewater into a source of water for potable water production. We investigated the biological effects along an advanced water treatment pilot plant, using zebrafish embryos throughout early development. The study compared phenotypic observations with global transcriptome responses, enabling us to keep an open mind about the chemicals that might influence the biological activity. There was no evidence of acute toxicity at any treatment stage, but skeletal, cardiovascular and pigmentation changes occurred in a small proportion of embryos along the treatment process, and in a tap water; not detected in the aquarium water control. Reverse osmosis (RO) reduced the concentration of measured chemical contaminants in the water the most, while eliminating the occurrence of abnormalities detected in fish embryos. Conversely, advanced oxidation reversed the benefits of RO treatment by increasing the frequency of teratogenic and sub-lethal abnormalities seen. Using the molecular responses of zebrafish embryos to different IPR water, we report the bioactivity within the water at different stages of advanced treatment and associate these to perturbed biological functions. Transcriptomic analysis revealed alterations to the retinoid system, which was consistent with the observed teratogenic effects. Changes to tryptophan metabolism (associated with the production of melatonin required for the control of normal circadian rhythms) and somatolactin-beta (associated with normal pigmentation in fish) were also found. We show that underexplored forms of biological activity occur in treated wastewater effluent, and/or may be created depending on the type of advanced treatment process used. By integrating the available analytical chemistry we highlight chemical groups associated to this response. Our study shows that more detailed and in-depth characterisation of chemicals and biological pathways associated with advanced treatment water systems are needed to mitigate possible risks to downstream organisms.

Rapid loss of estrogenicity of steroid estrogens by UVA photolysis and photocatalysis over an immobilised titanium dioxide catalyst.

The presence of low levels of natural and synthetic steroid estrogens in the aquatic environment, and their biological effects on aquatic organisms, are presently issues of concern. In this study, we investigated the temporal removal of estrogenic activity of several potent and environmentally relevant steroid estrogens by photocatalysis over an immobilised titanium dioxide (TiO2) catalyst. We used a recombinant yeast assay to measure estrogenic activity, which provided detection limits within the reactor of 53 ng/l for 17beta-estradiol and 17alpha-ethinylestradiol, and 100 ng/l for estrone. Pseudo-first-order kinetic data showed that photocatalysis over titanium dioxide was equally effective at removing the estrogenic activity of all three steroid substrates in aqueous solutions (initial concentrations of 10 microg/l) with a 50% reduction in estrogenicity within 10 min. In control experiments without TiO2 catalyst, the rate of UVA photolysis of the steroid substrates varied, but was most effective with 17alpha-ethinylestradiol followed by estrone, and was least effective with 17beta-estradiol (0.42, 0.2 and < 0.1 times the rate achieved with photocatalysis, respectively). The application of photocatalysis for the removal of steroid compounds within STW effluent released into the aquatic environment is discussed.

Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta.

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.

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: 17beta-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 17beta-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.

Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic.

The inadvertent estrogenicity of certain synthetic chemicals, and their subsequent effects on the endocrine system of humans and wildlife, is of concern. In this paper we report findings from in vitro and in vivo (uterotrophic) studies which confirm that a range of alkyl hydroxy benzoate preservatives (parabens) are weakly estrogenic. In a receptor-binding assay, butylparaben was able to compete with 3H-estradiol for binding to the rat estrogen receptor with an affinity approximately 5 orders of magnitude lower than that of diethylstilboestrol, and between 1 and 2 orders of magnitude less than nonylphenol. In an in vitro yeast-based estrogen assay, the four most widely used parabens (namely methyl-, ethyl-, propyl-, and butylparaben) were all found to be weakly estrogenic with the most potent (butylparaben) being 10,000-fold less potent than 17 beta-estradiol. The estrogenic activity of parabens was inhibited by 4-hydroxy tamoxifen in vitro, illustrating the requirement of these chemicals to interact with the estrogen receptor in order to activate the yeast. When administered orally to immature rats, the parabens were inactive. However, subcutaneous administration of butylparaben produced a positive uterotrophic response in vivo, although it was approximately 100,000 times less potent than 17 beta-estradiol. Given their use in a wide range of commercially available topical preparations, it is suggested that the safety in use of these chemicals should be reassessed, with particular attention being paid to estimation of the actual levels of systemic exposure of humans exposed to these chemicals. The acquisition of such data is a prerequisite to the derivation of reliable estimates of the possible human risk of exposure to parabens.

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.

Structural features of alkylphenolic chemicals associated with estrogenic activity.

The ability of certain man-made chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals are of increasing concern. The growing list of reported hormone-mimics includes the alkylphenolic (AP) compounds, a small number of which have been reported to be weakly estrogenic. In their most basic form, APs are composed of an alkyl group, which can vary in size, branching, and position, joined to a phenolic ring. The aim of this project was to identify the important structural features responsible for the estrogenic activity of AP chemicals. This was achieved by incubating APs with different structural features in a medium containing a previously described estrogen-inducible strain of yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor and comparing their activity spectrophotometrically by the resulting color change of the medium. The results were compared to the effects of the main natural estrogen 17beta-estradiol. The data indicate that both the position (para > meta > ortho) and branching (tertiary > secondary = normal) of the alkyl group affect estrogenicity. Optimal estrogenic activity requires a single tertiary branched alkyl group composed of between 6 and 8 carbons located at the para position on an otherwise unhindered phenol ring. The results are discussed in relation to the purity and composition of the chemicals tested.

Impact of an environmentally-realistic intake of cadmium on calcium, magnesium, and phosphate metabolism in bank voles, Clethrionomys glareolus.

Oral intake of cadmium can disrupt calcium metabolism in laboratory rodents. If this occurs in free-living wild rodents, reproductive potential, growth, and survival could be adversely affected. This study investigated whether an environmentally-realistic intake of cadmium (1.5-1.7 mg/kg BW/24h) by bank voles, Clethrionomys glareolus, affected uptake and assimilation of calcium and also that of magnesium and phosphorus, two elements metabolized in close association with calcium. Mineral metabolism was monitored in bank voles fed four different diets; two diets were relatively low (0.12%) in calcium and also contained either no cadmium (HC) or cadmium (LCCd), while the remainder were relatively high (0.74%) in calcium and also contained either no cadmium (HC) or cadmium (HCCd). Dietary calcium levels were varied because calcium can affect gut absorption of cadmium, and calcium levels in natural forage vary with habitat soil type. Cadmium had a significant effect on voles fed a low calcium diet. Animals given LCCd diet had significantly poorer calcium net gut absorption efficiency than animals fed the corresponding control (LC) diet and were in negative calcium balance, losing approximately 0.5-1% of body calcium reserves each day. In contrast, calcium inputs matched total outputs in animals fed the LC diet. Cadmium had no effect on net gut absorption of magnesium and phosphorus, but renal reabsorption and subsequent assimilation of these elements was greater in animals fed the LCCd diet than in those given the LC diet. Cadmium had no clear effect on macroelement metabolism in bank voles fed a diet relatively high in calcium.(ABSTRACT TRUNCATED AT 250 WORDS)