Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT)

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.     

Effects of prochloraz and ethinylestradiol on sexual development in Rana temporaria

A wide range of environmental xenobiotics that mimic hormones (endocrine-disrupting chemicals) may cause alterations in sexual development or reproductive function in aquatic organisms such as amphibians when exposed during early sensitive stages. We exposed tadpoles of the Common frog, Rana temporaria, from hatch to metamorphosis, to two different endocrine disruptors, the synthetic estrogen 17 alpha-ethinylestradiol and the fungicide prochloraz. The object of the study was to assess the effects of these two compounds on the sexual development of the tadpoles by investigating sex ratio, gonadal development, sex steroid concentrations and vitellogenin induction. Histology revealed that a large percentage of all groups were juvenile hermaphrodites at metamorphosis. Tadpoles exposed to 115 and 251 microg/L prochloraz showed a significant increased proportion of males. However, the testosterone concentrations were depressed in those groups. Ethinylestradiol in concentrations of 77 and 159 ng/L EE(2) increased whole-body calcium levels in a dose-dependent manner indicating induction of the egg yolk protein vitellogenin, verified also by gel electrophoresis. The study shows that ethinylestradiol may induce vitellogenesis and prochloraz may affect the sexual development in Common frogs.     

Reproductive effects of prenatal exposure to nonylphenol on zebrafish (Danio rerio)

Mature female and male zebrafish were separated and exposed to nonylphenol (NP) at 0.1, 1, 10, 50, 100 and 500 microg/L, respectively, for 3 weeks. Gonadosomatic index (GSI) in both sexes and vitellogenin (VTG) induction in males was measured as the bioindicators for the impairment to the parents. The results indicated that 50 microg/L of NP was the non-observed effect concentration (NOEC) for GSI and VTG induction. Afterwards, the 50 microg/L NP exposed females and males, and the control females and males were cross-wise pair-bred in the control water for one week to examine the reproductive effects. The embryonic cathepsin D (CAT D) activity, eggshell thickness, fecundity, hatching rate and malformation (vertebral column flexure) rate of offspring were determined in the four pair-bred groups. While endpoints remained unchanged in the groups with exposed males, prenatal exposure of females to 50 microg/L of NP resulted in the impairment of reproduction in groups with exposed females including inhibition of CAT D activity (P < 0.05), decrease of eggshell thickness (by 23.6%) and elevation of malformation rate (P < 0.001). These results suggested NP could induce reproductive damage to zebrafish at NOEC for parents. The results also imply that alterations of CAT D activity and eggshell thickness may be more sensitive biomarkers to indicate the reproductive effects caused by endocrine disrupting chemicals.     

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus)

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.     

Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish (Danio rerio)

In this study, a phytosterol preparation ("ultrasitosterol"; 80% beta-sitosterol) and an oxidized ultrasitosterol preparation were evaluated for reproductive effects in zebrafish. Adults were exposed in a continuous flow to 10 microg/L and 100 microg/L ultrasitosterol and oxidized ultrasitosterol, and to 0.27 microg/L 17beta-estradiol and 0.28 microg/L testosterone for 3 weeks. Biomarkers analysed included plasma vitellogenin, testosterone, 11-ketotestosterone, 17beta-estradiol, and gonadal histopathology. Ovarian steroid production of testosterone and 17beta-estradiol was examined in isolated zebrafish follicles exposed in vitro to the compounds at the same concentrations as in vivo. Vtg was induced in males exposed to ultrasitosterol, and in males and females exposed to 17beta-estradiol. Males exposed to oxidized phytosterols showed increased levels of testosterone and 11-ketotestosterone, and accelerated spermatogenesis. Increased follicular atresia was observed in females exposed to oxidized phytosterols and 17beta-estradiol. Correlation analyses between biomarkers revealed more intercorrelated values for females than for males, and the strongest associations were found in females exposed to oxidized phytosterols. Testosterone production was significantly increased in follicles exposed to the oxidized phytosterol preparations. These findings indicate that the phytosterol mixture is weakly estrogenic in male fish, and that the oxidized phytosterol mixture contains substances that may interfere with spermatogenesis, oogenesis and gonadal steroidogenesis in zebrafish.     

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.     

Effects of handling on endocrinology and reproductive performance of the fathead minnow

Anaesthesia with MS-222 followed by intra-peritoneal (ip) injection (with a 10% ethanol in corn oil carrier) of fathead minnow either as one or three (weekly) treatments did not affect survival, behaviour or secondary sexual characteristics of the fish. Fecundity of the fish, as indicated by fertility and hatching success, was also unaffected. Gonadal condition (relative gonad mass, histopathology) was not altered in either sex. Male and female plasma sex steroids (β;-oestradiol, testosterone, 11-ketotestosterone) and male vitellogenin concentrations were not significantly affected by the treatments. Females subjected to either ip treatment regime had significantly higher plasma vitellogenin concentrations than control females. However, based on previous data, this difference did not appear to be treatment-related. Overall, exposure of fathead minnows to chemicals via the ip route should not confound the interpretation of toxicity tests with potential endocrine disrupting chemicals.     

The effects of 4-nonylphenol contamination on livers of Tilapia fish (Oreochromus spilurs) in Jeddah

Alkylphenol polyethoxylates is a group of estrogenic compounds. Natural or synthetic types of these compounds react with the endocrine system by binding hormone receptors, resulting in interference with their action, which is why they are called endocrine disrupting chemicals. Among their hydrolytic products are nonylphenols (NP), which are considered pollutants of aquatic environments. The objective of this study was to evaluate the pathological alterations on liver tissue of fish exposed to these compounds for long durations, starting from beginning of life and during the period of sexual maturity. Tilapia fish were obtained from Abhur fish farms, reared in the laboratory in special basins, and divided into two groups. The first maternal group was untreated and their larvae were divided into three sub-groups: control; exposed to 15μg/L; and exposed to 30 μg/L. The second maternal group was divided into 2 sub-groups: with larvae exposed to 15μg/L; and with their larvae exposed to 30 μg/L. Larvae and mother exposed to different concentrations of NP (15 and 30 μg/L) showed an increased accumulation of NP in both livers and muscles compared to the control group due to bioaccumulation. Tissue section examinations of the treated group (15 μg NP /L) showed disruption of liver architecture, with lyses, loss of nuclei, necrosis, and fatty infiltration. The changes were more marked in tissues exposed to (30 μg NP /L). Although this pollution was not lethal, its effect may be reflected in vital activities and in the economy.     

Toxicity of the aromatase inhibitor letrozole to Japanese medaka (Oryzias latipes) eggs, larvae and breeding adults

Letrozole is a synthetic aromatase inhibitor and interferes in the committed step in the synthesis of endogenous estrogens from androgens. To evaluate potential effects on the early life stages of Japanese medaka, larvae and fertilized eggs were exposed to letrozole for 96 h and 14 days, respectively. No larvae died and no adverse effects were found on embryonic development at concentrations up to 3125 microg/L. Reproductive effects were assessed by exposing adults to 1, 5, 25, 125 and 625 microg/L letrozole for 21 days. A dose-dependent decrease in fecundity (>25 microg/L) and fertility (>5 microg/L) accompanied by histological changes suggested the inhibition of oocyte growth and possibly maturation. At 625 microg/L, the fish ceased spawning during the last week of exposure. Letrozole (>5 microg/L) reduced plasma vitellogenin levels in females in a dose-dependent manner. Transgenerational effects were evaluated by removing freshly-laid F1 eggs from letrozole-contaminated water and raising them to 15 days post-hatching in control water. Hatchability and time to hatching were detrimentally affected (>5 microg/L), but no morphological deformities were observed. Furthermore, a dose-dependent increase in the proportion of genotypic F1 males was found (>5 microg/L).     

Exposure to Paper Mill Effluent at a Site in North Central Florida Elicits Molecular-Level Changes in Gene Expression Indicative of Progesterone and Androgen Exposure

Endocrine disrupting compounds (EDCs) are chemicals that negatively impact endocrine system function, with effluent from paper mills one example of this class of chemicals. In Florida, female Eastern mosquitofish (Gambusia holbrooki) have been observed with male secondary sexual characteristics at three paper mill-impacted sites, indicative of EDC exposure, and are still found at one site on the Fenholloway River. The potential impacts that paper mill effluent exposure has on the G. holbrooki endocrine system and the stream ecosystem are unknown. The objective of this study was to use gene expression analysis to determine if exposure to an androgen receptor agonist was occurring and to couple this analysis with in vitro assays to evaluate the presence of androgen and progesterone receptor active chemicals in the Fenholloway River. Focused gene expression analyses of masculinized G. holbrooki from downstream of the Fenholloway River paper mill were indicative of androgen exposure, while genes related to reproduction indicated potential progesterone exposure. Hepatic microarray analysis revealed an increase in the expression of metabolic genes in Fenholloway River fish, with similarities in genes and biological processes compared to G. holbrooki exposed to androgens. Water samples collected downstream of the paper mill and at a reference site indicated that progesterone and androgen receptor active chemicals were present at both sites, which corroborates previous chemical analyses. Results indicate that G. holbrooki downstream of the Fenholloway River paper mill are impacted by a mixture of both androgens and progesterones. This research provides data on the mechanisms of how paper mill effluents in Florida are acting as endocrine disruptors.     

In vivo genotoxicity of the pyrethroid pesticide beta-cyfluthrin using the comet assay in the fish Bryconamericus iheringii

Environmental pollution by pesticide residues is a major environmental concern due to the extensive use of these substances in agriculture. The insecticide beta-cyfluthrin is a synthetic pyrethroid widely used in agricultural and other domestic activities. The aim of the present study was to assess the genotoxic effects of a sublethal exposure of the fish Bryconamericus iheringii (Characidae) to a commercial formulation of beta-cyfluthrin using the comet assay. Fish were exposed to sublethal concentrations (4.2 and 5.6 microg/L) of beta-cyfluthrin under static conditions during 24- and 48-h exposure periods. Fish in tap water were used as negative controls. Results obtained by the comet assay revealed genotoxic effects of the pyrethroid in the higher concentration and at the longer exposure period. The mean DNA damage index of fish exposed to 5.6 microg/L beta-cyfluthrin for 48 h was significantly higher (145.9 +/- 51.8) than in the control group (69.3 +/- 39.5). These findings indicate that native fish species might be at risk for genotoxic damage in waters contaminated with beta-cyfluthrin.     

Hepatic Protein Expression Networks Associated with Masculinization in the Female Fathead Minnow (Pimephales promelas)

Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen 5-alpha dihydrotestosterone (DHT) (100 μg/L), the ureic-based herbicide linuron (LIN) (100 μg/L), and a mixture of DHT and LIN (100 μg/L each) to better characterize androgen action in females. LIN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT + LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, ～2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and LIN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts.     

Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback (Gasterosteus aculeatus)

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.     

Endocrine Toxicants and Reproductive Success in Fish

There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.     

Ligula intestinalis infection as a potential source of bias in the bioindication of endocrine disruption in the European chub Leuciscus cephalus

European chub Leuciscus cephalus collected from five localities in the lowland and subalpine regions of Austria were analysed for oestrogenic effects of endocrine-disrupting chemicals and the presence of the plerocercoid of the tapeworm Ligula intestinalis. Of 1494 chub analysed, only seven (six males, one female) were found to be infected with single, but large plerocercoids up to 15 cm in length. Ligula-infected fish showed comparatively immature gonads, as demonstrated by the gonadosomatic index and gamete developmental stages. Plasma levels of the egg precursor protein vitellogenin also showed concentrations ranging below the detection limit. The present results indicate that chub infected with L. intestinalis and exposed to exogenous oestrogenic compounds can result in reduced gonadal maturation and produce false oestrogen-positive diagnoses in male fish. For plasma vitellogenin levels, L. intestinalis infections can result in false oestrogen-negative diagnoses in male and female fish.     

Effect of androgen mediated by the estrogen receptor of fish liver: Vitellogenin accumulation

We have investigated the action of high doses of androgens in $\text{Gobius niger L.}$, a marine teleostean fish, by characterizing specific steroid receptors in liver and by assaying the plasma vitellogenin concentration under different hormonal treatments. Estrogen and androgen receptors were characterized in the liver nuclear extracts according to their binding specificity. The maximum binding capacity was 25 fmoles/mg protein for the estrogen and androgen receptors. $\text{In vivo}$, high doses of DHT(·) increased the concentration of plasmatic vitellogenin as assayed by immunodiffusion while low doses were inefficient. In spite of a similar number of estrogen and androgen nuclear receptor sites (25 fmoles/mg protein), DHT was at least 70 fold less active than et on yolk protein and vitellogenin induction both in male and female $\text{Gobius niger}$. In addition, the antiestrogen tamoxifen, which was inactive by itself, inhibited the e₂ and the DHT induced accumulation of vitellogenin. Progesterone (2 mg/fish) was also totally inactive in inducing vitellogenin. We conclude that the induction of vitellogenin by DHT is mediated by the estrogen receptor rather than by the androgen receptor.In addition to the estradiol induced protein in rat uterus and to other estrogenic responses obtained by androgens in mammary cancer, fish vitellogenin is another estrogen regulated protein which can be induced by high doses of androgens. (·) 17β-hydroxy-5α-androstan-3-one.     

Pathogenesis of Mycobacterium spp. in zebrafish (Danio rerio) from research facilities

One of the most common diseases that we have diagnosed in zebrafish is mycobacteriosis, caused by several Mycobacterium spp. The severity of the disease ranged from severe outbreaks to incidental infections. We conducted an in vivo study to evaluate the pathogenesis of six isolates of Mycobacterium from zebrafish with mycobacteriosis from four research facilities and one wholesale supplier of zebrafish in the United States: Mycobacterium abscessus, Mycobacterium peregrinum, Mycobacterium chelonae (2 isolates), and Mycobacterium marinum. We also included two isolates of M. marinum from other fishes. Fish were exposed by intraperitoneal injection at a target does of 5 x 10(4) bacteria/fish, and were held in static aquaria at 28 degrees C for 8 weeks. Fish were examined by histology and culture, and mortalities were recorded. The M. marinum isolates caused 100% infection and mortality between 30% and 100%. None of the other Mycobacterium species caused significant mortalities, but several of these fish had granulomatous lesions in visceral organs. Mycobacteria were consistently recovered in culture from fish exposed to M. marinum, and from only 9% of fish exposed to the other species. This study suggests that, of the isolates tested, only M. marinum is highly pathogenic and virulent to healthy zebrafish.