Helmut Segner

Screening and Testing for Endocrine Disruption in Fish—Biomarkers As “Signposts,” Not “Traffic Lights,” in Risk Assessment

Biomarkers are currently best used as mechanistic “signposts” rather than as “traffic lights” in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect oncentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38–55% (comparable to published CVs of 19–58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the “biomarkers as signposts” approach is a pragmatic way forward in the current risk assessment of EDCs.

AN ENVIRONMENTALLY RELEVANT CONCENTRATION OF ESTROGEN INDUCES ARREST OF MALE GONAD DEVELOPMENT IN ZEBRAFISH,DANIO RERIO

The aim of the present study was to elucidate how full life-cycle exposure to estrogens impacts zebrafish development and reproduction, compared to partial life-cycle exposure only, and whether the estrogen-induced effects in zebrafish are reversible or irreversible. Zebrafish were exposed in a flow-through system to an environmentally relevant concentration (3 ng/L) of the synthetic estrogen 17alpha-ethinylestradiol (EE2) either from fertilization until the all-ovary stage of gonad development (i.e., 42 d postfertilization [DPF] in our experiment) or from fertilization until the reproductive stage (i.e., 118 DPF). Reversibility of the estrogen-induced effects was assessed after 58 d of depuration in EE2-free water until 176 DPE Early life exposure led to a lasting induction of plasma vitellogenin (VTG) in adult females but altered neither the sex ratio nor the reproductive capabilities. Full life-cycle exposure resulted in elevated VTG concentrations and caused gonadal feminization in 100% of exposed fish and thus inhibited reproduction. Two types of ovaries were observed in continuously exposed adult fish, immature ovaries with primary growth stage oocytes only and mature ovaries containing the full range of all oocyte maturation stages. Fish with immature ovaries had plasma VTG levels like control males, while fish with mature ovaries had female-like VTG levels. The effects of full life cycle exposure were at least partly reversible, and 26% of fish of the previous all-female cohort developed fully differentiated testes. These findings suggest that continuous estrogen exposure had arrested the developmental transition of the gonads of genetic males from the early all-ovary stage to functional testes. After the exposure had ceased, however, these males apparently were able to accomplish testicular differentiation.

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption.

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.

Antiestrogenicity of β-naphthoflavone and PAHs in cultured rainbow trout hepatocytes : evidence for a role of the arylhydrocarbon receptor

The aims of the present study were to assess, (1) if polyaromatic hydrocarbons (PAHs) are able to inhibit estradiol-regulated vitellogenin synthesis in fish; and (2) if this antiestrogenic activity is mediated through the binding of PAHs to the arylhydrocarbon receptor (AhR). Cultured liver cells of rainbow trout, Oncorhynchus mykiss, were co-exposed to PAHs and 17beta-estradiol (E2), and the resulting effects on induction of AhR-regulated 7-ethoxyresorufin-O-deethylase (EROD) activity and on E2-regulated vitellogenesis were investigated. The following test compounds were compared: the PAH 3-methylcholanthrene (3MC), which is a strong EROD inducer, the PAH anthracene (ANT), which is not an inducer of EROD activity, and the model EROD inducer, beta-naphthoflavone (betaNF). 3MC and betaNF led to significant decreases of E2-triggered hepatocellular VTG synthesis, whereas ANT exerted no antiestrogenic activity. The rank order of the antiestrogenic activity of the test substances agreed with their EROD-inducing potency suggesting that their antiestrogenicity might be mediated through the AhR. Further evidence for this assumption comes from the observation that inhibitors such as alpha-naphthoflavone which interferes with ligand-AhR binding, and 8-methoxypsoralen (8MP), which prevents binding of the occupied AhR to responsive DNA elements, clearly reduced the antiestrogenic effects of the xenobiotics. Furthermore, from the comparison of estradiol concentrations in media of liver cells exposed to the CYP 1A-inducing agents and in media of control cells it is unlikely that the observed antiestrogenic effects were caused by an enhanced E2 catabolism. In conclusion, the results from this study indicate that, (1) AhR-binding PAHs possess an antiestrogenic activity; and (2) that the antiestrogenic activity is mediated through the AhR.

Potencies of estrogenic compounds in in vitro screening assays and in life cycle tests with zebrafish in vivo.

The objective of this study was to compare the estrogenic potency of environmental estrogens at two testing tiers: at the initial level of in vitro screening assays, and at the level of definitive fish reproduction tests in vivo. The in vitro tests included a recombinant yeast estrogen receptor (ER) assay, a competitive radioreceptor assay using the hepatic ER of carp (Cyprinus carpio), and assays on vitellogenin induction in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss) and carp. In vivo, full life cycle tests with zebrafish (Danio rerio) were performed, using fertilization success as estrogen-sensitive reproductive endpoint. The test compounds included the natural estrogen 17beta-estradiol (E2) (only applied in the in vitro assays); the synthetic estrogen ethynylestradiol (EE2); and two xenoestrogens, 4-tert-octylphenol (OP) and bisphenol A (BPA). Among the in vitro assays, differences were observed in the relative ranking of the test substances, and in the absolute sensitivity (EC50 values), although the interassay differences of EC50 values were within one order of magnitude. The in vivo activity of the test compounds was not accurately predicted by the in vitro assays, with respect to neither sensitivity nor ranking. The in vitro assays tended to overestimate the relative potency of the xenoestrogens; i.e. the ratio between the activity of the reference compound, EE2, and that of the test compound. The best prediction of the in vivo fish test results was obtained from the recombinant yeast assay.

Cytochrome P4501A (CYP1A) in teleostean fishes. A review of immunohistochemical studies

Cytochrome P4501A monooxygenase has an important function in the biotransformation of many xenobiotics, including polynuclear aromatic hydrocarbons, and planar organochlorine compounds. The metabolism can lead to detoxification or activation to reactive intermediates. Exposure of fish leads to a receptor-mediated induction of CYP1A gene expression. The induction response can be quantitatively analysed by means of molecular techniques (RT-PCR, Northern Blotting), immunochemical approaches (ELISA, Western Blotting), and enzymatic methods (7-ethoxyresorufin-O-deethylase, EROD) at the catalytical level. Immunohistochemical studies have provided qualitative information on cell and tissue distribution of CYP1A in teleost fish. The liver is the major organ of CYP1A activity in fish, but the enzyme is additionally expressed in numerous extrahepatic organs, including kidney, alimentary canal, heart, gills, olfactory system, gonads, brain and endocrine tissues. In many tissues, the vascular endothelia show a strong CYP1A immunoreactivity. As indicated from immunohistochemical studies with fish embryos and larvae, the typical cell and tissue distribution of CYP1A is established early during fish ontogeny.

Estrogen-mediated suppression of cytochrome P4501A (CYP1A) expression in rainbow trout hepatocytes: role of estrogen receptor

Hepatic CYP1A expression in fish can be modulated by the female sex hormone, 17beta-estradiol (E2), however neither the mechanism of E2 suppression of CYP1A nor the capacity for hormonal regulation to overcome CYP1A induction by xenobiotics are known. The present study investigates for the first time in fish if the estrogen receptor (ER) is involved in the suppressive action of E2 on CYP1A gene expression. The study further examines, if the E2 effect is able to overcome xenobiotic induction of CYP1A. As experimental model, in vitro cultures of rainbow trout, Oncorhynchus mykiss, hepatocytes were used. The effect of E2 on CYP1A was assessed by measuring the CYP1A-associated 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity, and CYP1A mRNA contents. E2 at non-cytotoxic concentrations caused a significant time- and concentration-dependent decline of basal but not of induced hepatic EROD activities. The inhibitory action of E2 on basal CYP1A was also evident at the mRNA level. The presence of the ER antagonist tamoxifen abolished the inhibitory action of E2 on CYP1A expression. The results from these in vitro experiments provide evidence (a) that the ER is involved in the suppressive action of E2 on CYP1A, and (b) that E2 inhibitory action does not overcome xenobiotic induction of CYP1A.

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc.

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.

Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio

Partial or full life-cycle tests are needed to assess the potential of endocrine-disrupting compounds (EDCs) to adversely affect development and reproduction of fish. Small fish species such as zebrafish, Danio rerio, are under consideration as model organisms for appropriate test protocols. The present study examines how reproductive effects resulting from exposure of zebrafish to the synthetic estrogen 17α-ethinylestradiol (EE2) vary with concentration (0.05 to 10 ng EE2 L−1, nominal), and with timing/duration of exposure (partial life-cycle, full life-cycle, and two-generation exposure). Partial life-cycle exposure of the parental (F1) generation until completion of gonad differentiation (0–75 d postfertilization, dpf) impaired juvenile growth, time to sexual maturity, adult fecundity (egg production/female/day), and adult fertilization success at 1.1 ng EE2 L−1 and higher. Lifelong exposure of the F1 generation until 177 dpf resulted in lowest observed effect concentrations (LOECs) for time to sexual maturity, fecundity, and fertilization success identical to those of the developmental test (0–75 dpf), but the slope of the concentration-response curve was steeper. Reproduction of zebrafish was completely inhibited at 9.3 ng EE2 L−1, and this was essentially irreversible as a 3-mo depuration restored fertilization success to only a very low rate. Accordingly, elevated endogenous vitellogenin (VTG) synthesis and degenerative changes in gonad morphology persisted in depurated zebrafish. Full life-cycle exposure of the filial (F2) generation until 162 dpf impaired growth, delayed onset of spawning and reduced fecundity and fertilization success at 2.0 ng EE2 L−1. In conclusion, results show that the impact of estrogenic agents on zebrafish sexual development and reproductive functions as well as the reversibility of effects, varies with exposure concentration (reversibility at ≤ 1.1 ng EE2 L−1 and irreversibility at 9.3 ng EE2 L−1), and between partial and full life-cycle exposure (exposure to 10 ng EE2 L−1 during critical period exerted no permanent effect on sexual differentiation, but life-cycle exposure did).

Future water quality monitoring - Adapting tools to deal with mixtures of pollutants in water resource management

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.