Vitellogenin synthesis in primary cultures of fish liver cells as endpoint for in vitro screening of the (anti)estrogenic activity of chemical substances

Concern over possible adverse effects of endocrine-disrupting compounds on fish has caused the development of appropriate testing methods. In vitro screening assays may provide initial information on endocrine activities of a test compound and thereby may direct and optimize subsequent testing. Induction of vitellogenin (VTG) is used as a biomarker of exposure of fish to estrogen-active substances. Since VTG induction can be measured not only in vivo but also in fish hepatocytes in vitro, the use of VTG induction response in isolated fish liver cells has been suggested as in vitro screen for identifying estrogenic-active substances. The main advantages of the hepatocyte VTG assay are considered its ability to detect effects of estrogenic metabolites, since hepatocytes in vitro remain metabolically competent, and its ability to detect both estrogenic and anti-estrogenic effects. In this article, we critically review the current knowledge on the VTG response of cultured fish hepatocytes to (anti)estrogenic substances. In particular, we discuss the sensitivity, specificity, and variability of the VTG hepatocyte assay. In addition, we review the available data on culture factors influencing basal and induced VTG production, the response to natural and synthetic estrogens as well as to xenoestrogens, the detection of indirect estrogens, and the sources of assay variability. The VTG induction in cultured fish hepatocytes is clearly influenced by culture conditions (medium composition, temperature, etc.) and culture system (hepatocyte monolayers, aggregates, liver slices, etc.). The currently available database on estrogen-mediated VTG induction in cultured teleost hepatocytes is too small to support conclusive statements on whether there exist systematic differences of the VTG response between in vitro culture systems, VTG analytical methods or fish species. The VTG hepatocyte assay detects sensitively natural and synthetic estrogens, whereas the response to xenoestrogens appears to be more variable. The detection of weak estrogens can be critical due to the overshadow with cytotoxic concentrations. Moreover, the VTG hepatocyte assay is able to detect antiestrogens as well as indirect estrogens, i.e substances which require metabolic activation to induce an estrogenic response. Nevertheless, more chemicals need to be analysed to corroborate this statement. It will be necessary to establish standardized protocols to minimize assay variability, and to develop a set of pass-fail criteria as well as cut-offs for designating positive and negative responses.

Assessment of estrogenic exposure in brown trout (Salmo trutta) in a Swiss midland river: integrated analysis of passive samplers, wild and caged fish, and vitellogenin mRNA and protein

This field study examined the vitellogenin (VTG) biomarker response under conditions of low and fluctuating activities of environmental estrogenicity. The present study was performed on immature brown trout (Salmo trutta) exposed to the small river Luetzelmurg, which is located in the prealpine Swiss midland region and receives effluents from a single sewage treatment plant (STP). To understand better factors influencing the relationship between estrogenic exposure and VTG induction, we compared VTG levels in caged (stationary) and feral (free-ranging) fish, VTG levels in fish from up- and downstream of the STP, and two different methods for quantifying VTG (enzyme-linked immunosorbent assay vs real-time reverse transcription-polymerase chain reaction), and we used passive samplers (polar organic chemical integrative sampler [POCIS]) to integrate the variable, bioaccumulative estrogenic load in the river water over time. The POCIS from the downstream site contained approximately 20-fold higher levels of bioassay-derived estrogen equivalents than the POCIS from the upstream site. In feral fish, this site difference in estrogenic exposure was reflected in VTG protein levels but not in VTG mRNA. In contrast, in caged fish, the site difference was evident only for VTG mRNA but not for VTG protein. Thus, the outcome of VTG biomarker measurements varied with the analytical detection method (protein vs mRNA) and with the exposure modus (caged vs feral). Our findings suggest that for environmental situations with low and variable estrogenic contamination, a multiple-assessment approach may be necessary for the assessment of estrogenic exposure in fish.

Pathogenic infection confounds induction of the estrogenic biomarker vitellogenin in rainbow trout

To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17beta-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA levels than healthy fish. Transcriptome data suggest that this was due to energetic constraints. Reduced responsiveness of the VTG biomarker in parasitized fish might obscure detection of low-level field exposure.

17Beta-estradiol affects the response of complement components and survival of rainbow trout (Oncorhynchus mykiss) challenged by bacterial infection

Research on the endocrine role of estrogens has focused on the reproductive system, while other potential target systems have been less studied. Here, we investigated the possible immunomodulating role of 17beta-estradiol (E2) using rainbow trout (Oncorhynchus mykiss) as a model. The aims of the study were to examine a) whether estrogens can modulate immune gene transcription levels, and b) whether this has functional implications for the resistance of trout towards pathogens. Trout were reared from fertilization until 6 months of age under (1) control conditions, (2) short-term E2-treatment (6-month-old juveniles were fed a diet containing 20 mg E2/kg for 2 weeks), or c) long-term E2-treatment (twice a 2-h-bath-exposure of trout embryos to 400 mug 17beta-estradiol (E2)/L, followed by rearing on the E2-spiked diet from start-feeding until 6 months of age). Analysis of plasma estrogen levels indicated that the internal estrogen concentrations of E2-exposed fish were within the physiological range and analysis of hepatic vitellogenin mRNA levels indicated that the E2 administration was effective in activating the endogenous estrogen receptor pathway. However, expression levels of the hepatic complement components C3-1, C3-3, and Factor H were not affected by E2-treatment. In a next step, 6-month-old juveniles were challenged with pathogenic bacteria (Yersinia ruckeri). In control fish, this bacterial infection resulted in significant up-regulation of the mRNA levels of hepatic complement genes (C3-1, C3-3, Factor B, Factor H), while E2-treated fish showed no or significantly lower up-regulation of the complement gene transcription levels. Apparently, the E2-treated trout had a lower capacity to activate their immune system to defend against the bacterial infection. This interpretation is corroborated by the finding that survival of E2-treated fish under bacterial challenge was significantly lower than in the control group. In conclusion, the results from this study suggest that estrogens are able to modulate immune parameters of trout with functional consequences on their ability to cope with pathogens.

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 concentration) 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.

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 17alpha-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 < or = 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).

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Estrogen Modulates Hepatic Gene Expression and Survival of Rainbow Trout Infected with Pathogenic Bacteria Yersinia ruckeri

In the aquatic environment, fish are exposed to various stimuli at once and have developed different response mechanisms to deal with these multiple stimuli. The current study assessed the combined impacts of estrogens and bacterial infection on the physiological status of fish. Juvenile rainbow trout were exposed to two different concentrations of 17 beta-estradiol (E2) (2 or 20 mg/kg feed) and then infected with three concentrations of Yersinia ruckeri, a bacterial pathogen causing massive losses in wild and farmed salmonid populations. Organism-level endpoints to assess the impact of the single and combined treatments included hepatic vitellogenin transcript expression to evaluate the E2 exposure efficiency and survival rate of pathogen-challenged fish. The two E2 doses increased vitellogenin levels within the physiological range. Infection with Y. ruckeri caused mortality of trout, and this effect was significantly enhanced by a simultaneous exposure to high E2 dose. The hormone reduced survival at intermediate and high (10(4) and 10(6) colony forming units, cfu) bacterial concentrations, but not for a low one (10(2) cfu). Analysis of hepatic gene expression profiles by a salmonid 2 k cDNA microarray chip revealed complex regulations of pathways involved in immune responses, stress responses, and detoxicification pathways. E2 markedly reduced the expression of several genes implicated in xenobiotic metabolism. The results suggest that the interaction between pathogen and E2 interfered with the fish's capability of clearing toxic compounds. The findings of the current study add to our understanding of multiple exposure responses in fish.

Persistence of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the androgen 17β-trenbolone.

The aim of the present study was to investigate the effects of the androgenic endocrine disruptor 17β-trenbolone on the sexual development of zebrafish (Danio rerio) with special emphasis on the question of whether adverse outcomes of developmental exposure are reversible or persistent. An exposure scenario including a recovery phase was chosen to assess the potential reversibility of androgenic effects. Zebrafish were exposed to environmentally relevant concentrations of 17β-trenbolone (1 ng/L-30 ng/L) from fertilization until completion of gonad sexual differentiation (60 d posthatch). Thereafter, exposure was either followed by 40 d of recovery in clean water or continued until 100 d posthatch, the age when zebrafish start being able to reproduce. Fish exposed for 100 d to 10 ng/L or 30 ng/L 17β-trenbolone were masculinized at different biological effect levels, as evidenced from a concentration-dependent shift of the sex ratio toward males as well as a significantly increased maturity of testes. Gonad morphological masculinization occurred in parallel with decreased vitellogenin concentrations in both sexes. Changes of brain aromatase (cyp19b) mRNA expression showed no consistent trend with respect to either exposure duration or concentration. Gonad morphological masculinization as well as the decrease of vitellogenin persisted after depuration over 40 d in clean water. This lack of recovery suggests that androgenic effects on sexual development of zebrafish are irreversible.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.