Screening breeding sites of the common toad (Bufo bufo) in England and Wales for evidence of endocrine disrupting activity.

Anuran amphibians are often present in agricultural landscapes and may therefore be exposed to chemicals in surface waters used for breeding. We used passive accumulation devices (SPMD and POCIS) to sample contaminants from nine breeding sites of the Common toad (Bufo bufo) across England and Wales, measuring endocrine activity of the extracts in a recombinant yeast androgen screen (YAS) and yeast estrogen screen (YES) and an in vitro vitellogenin induction screen in primary culture of Xenopus laevis hepatocytes. We also assessed hatching, growth, survival, and development in caged larvae in situ, and sampled metamorphs for gonadal histopathology. None of the SPMD extracts exhibited estrogen receptor or androgen receptor agonist activity, while POCIS extracts from two sites in west-central England exhibited concentration-dependent androgenic activity in the YAS. Three sites exhibited significant estrogenic activity in both the YES and the Xenopus hepatocyte. Hatching rates varied widely among sites, but there was no consistent correlation between hatching rate and intensity of agricultural activity, predicted concentrations of agrochemicals, or endocrine activity measured in YES/YAS assays. While a small number of intersex individuals were observed, their incidence could not be associated with predicted pesticide exposure or endocrine activitity measured in the in vitro screens. There were no significant differences in sex ratio, as determined by gonadal histomorphology among the study sites, and no significant correlation was observed between proportion of males and predicted exposure to agrochemicals. However, a negative correlation did become apparent in later sampling periods between proportion of males and estrogenic activity of the POCIS sample, as measured in the YES. Our results suggest that larval and adult amphibians may be exposed to endocrine disrupting chemicals in breeding ponds, albeit at low concentrations, and that chemical contaminants other than plant protection products may contribute to endocrine activity of surface waters in the agricultural landscape.

Molecular cloning and characterization of ligand- and species-specificity of amphibian estrogen receptors.

Estrogens are essential for normal reproductive activity in both males and females as well as for ovarian differentiation during a critical developmental stage in most vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in amphibians, we isolated cDNAs encoding the estrogen receptors (ERalpha and ERbeta) from the Japanese firebelly newt (Cynops pyrrhogaster), Tokyo salamander (Hynobius tokyoensis), axolotl (Ambystoma mexicanum), and Raucous toad (Bufo rangeri). Full-length amphibian ER cDNAs were obtained using 5' and 3' rapid amplification of cDNA ends. The predicted amino acid sequences of these amphibian ERs showed a high degree of amino acid sequence identity (over 70%) to each other. We analyzed the relationships of these amphibian ER sequences to other vertebrate ER sequences by constructing a phylogenetic tree. We verified that these were bona fide estrogen receptors using receptor dependent reporter gene assays. We analyzed the effects of natural estrogens, ethinylestradiol, and DDT and its metabolites on the transactivation of the four amphibian species listed above, and Xenopus tropicalis ERs and found that there were species-specific differences in the sensitivity of these ERs to hormones and environmental chemicals. These findings will expand our knowledge of endocrine-disrupting events in amphibians.

Thyroid histopathology assessments for the amphibian metamorphosis assay to detect thyroid-active substances.

In support of an Organization for Economic Cooperation and Development (OECD) Amphibian Metamorphosis Assay (AMA) Test Guideline for the detection of substances that interact with the hypothalamic-pituitary-thyroid axis, a document was developed that provides a standardized approach for evaluating the histology/histopathology of thyroid glands in metamorphosing Xenopus laevis tadpoles. Here, a consolidated description of histology evaluation practices, core diagnostic criteria and severity grading schemes for the AMA, an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis, and the core diagnostic criteria with examples of severity grades is provided. Core diagnostic criteria include thyroid gland hypertrophy/atrophy, follicular cell hypertrophy, and follicular cell hyperplasia. The severity grading scheme is semiquantitative and employs a four-grade approach describing ranges of variation within assigned ordinal classes: not remarkable, mild, moderate, and severe. The purpose of this severity grading approach is to provide an efficient, semi-objective tool for comparing changes (compound-related effects) among animals, treatment groups, and studies. Proposed descriptions of lesions for scoring the four core criteria are also given.

Application of metamorphosis assay to a native Japanese amphibian species, Rana rugosa, for assessing effects of thyroid system affecting chemicals.

The aims of this study were to assess the utility of a metamorphosis assay for detecting thyroid hormone-disrupting chemicals using Rana rugosa, a domestic frog species in Japan, and to compare species differences in sensitivity to thyroxine (T(4)) and propylthiouracil (PTU) among R. rugosa, Xenopus laevis and Xenopus (Silurana) tropicalis. Tadpoles of R. rugosa (TK stages III/IV) were exposed to standard test chemicals for acceleration (T(4)) and inhibition (PTU) of metamorphosis for 28 days in semi-static condition and total body length and developmental stage (TK stage) were recorded every week. T(4) (0.61 and 2.24 microg/L in actual concentrations) and PTU (19.73, 76.83, and 155.67 mg/L in actual concentrations) induced significant acceleration and inhibition of metamorphosis, respectively. The present results indicate that the metamorphosis assay is successfully applied to the domestic frog species, R. rugosa, suggesting this assay can be used for the assessment of chemicals on ecological impacts in wild frog species.

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis.

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.

Development of biomarkers of endocrine disrupting activity in emerging amphibian model, Silurana (Xenopus) tropicalis.

Because amphibians show peculiar ecological features and interesting responses to some hormones, it is conceivable that amphibians are very useful animals for assessing the toxic effects of environmental contaminants, including endocrine disrupters. To develop methods of detecting endocrine toxicity of environmental chemicals in amphibians, we have started to assemble a biomarker tool kit for an emerging amphibian model, Silurana (Xenopus) tropicalis. We isolated full-length cDNAs encoding estrogen receptor alpha (ERalpha), ERbeta, thyroid hormone receptor alpha (TRalpha), and TRbeta of S. (X.) tropicalis to develop a reporter gene assay system, as an estimation tool for environmental chemicals. The amino acid sequences inferred from the four full-length cDNAs were highly homologous to those of ERalpha, TRalpha and TRbeta of X. laevis, and ERbeta of the Japanese quail. In particular, the S. (X.) tropicalis ERalpha shared a higher similarity of amino acid sequence with X. laevis ERalpha than the previously reported S. (X.) tropicalis ERalpha, as determined by Wu et al. RT-PCR analysis showed that the two ERalpha and ERbeta transcripts were expressed relatively abundantly in the brain, liver, and gonad/kidney complex of the S. (X.) tropicalis tadpole after gonadal sex differentiation occurring at developmental stages 54-59, suggesting that they are susceptible to estrogenic substances. A similar result was obtained in the two TR transcripts, although their expression levels were lower in the gonad/kidney complex than in the other tissues. Moreover, we identified vitellogenin A (Vtg A) and Vtg B as estrogen-responsive genes expressed in the female S. (X.) tropicalis liver using macroarray analysis and RT-PCR. In addition, Rana japonica Vtg was purified from serum using anion-exchange chromatography to produce anti-Vtg antibody as a protein marker. In the future, we are going to construct reporter gene assay systems using the full-length ER and TR cDNAs, analyze histologically the differentiation of gonads and thyroid glands in the S. (X.) tropicalis tadpole exposed to estrogenic chemicals, and produce sex-reversed male S. (X.) tropicalis to obtain all-male tadpoles. Using these tools, we hope to be able to identify endocrine disrupting compounds in laboratory experiments for hazard assessment purposes, and also detect endocrine toxicity in environmental samples as part of an integrated approach to assessing the impact of environmental contaminants on wild amphibian populations in Japan and the UK.

Investigating potential for effects of environmental endocrine disrupters on wild populations of amphibians in UK and Japan: status of historical databases and review of methods.

Concern over global declines among amphibians has resulted in increased interest in the effects of environmental contaminants on amphibian populations, and more recently, this has stimulated research on the potential adverse effects of environmental endocrine disrupters in amphibians. Laboratory studies of the effects of single chemicals on endocrine-relevant endpoints in amphibian, mainly anuran, models are valuable in characterizing sensitivity at the individual level and may yield useful bioassays for screening chemicals for endocrine toxicity (for example, thyroid disrupting activity). Nevertheless, in the UK and Japan as in many other countries, it has yet to be demonstrated unequivocally that the exposure of native amphibians to endocrine disrupting environmental contaminants results in adverse effects at the population level. Assessing the potential of such effects is likely to require an ecoepidemiological approach to investigate associations between predicted or actual exposure of amphibians to (endocrine disrupting) environmental contaminants and biologically meaningful responses at the population level. In turn, this demands recent but relatively long-term population trend data. We review two potential sources of such data for widespread UK anurans that could be used in such investigations: records for common frogs and common toads in several databases maintained by the Biological Records Centre (UK Government Centre for Ecology and Hydrology), and adult toad count data from 'Toads on Roads' schemes registered with the UK wildlife charity 'Froglife'. There were little abundance data in the BRC databases that could be used for this purpose, while count data from the Toads on Roads schemes is potentially confounded by the effects of local topology on the detection probabilities and operation of nonchemical anthropogenic stressors. For Japan, local and regional surveys of amphibians and national ecological censuses gathering amphibian data were reviewed to compile survey methodologies and these were compared with methods used in the UK and other countries. Substantial consensus exists in amphibian survey methodologies and this should be exploited in the initiation of coordinated monitoring programs for widespread and common anuran amphibians in Japan and the UK to generate long-term robust and standardized population trend data. Such data would support comparative ecoepidemiological assessments of the impact of environmental endocrine disrupters in these two cooperating countries.

Medaka (Oryzias latipes) for use in evaluating developmental effects of endocrine active chemicals with special reference to gonadal intersex (testis-ova).

Japanese medaka (Oryzias latipes) has been widely used for the evaluation of the toxicity of endocrine active chemicals (EACs) and other chemicals as well as for monitoring the adverse effects of effluent discharges in relation to sexual development and function. It is useful for these evaluations for many reasons including the following: 1) it has a short life cycle facilitating studies extending over long phases of development and over multigenerations, 2) it is easy to rear, 3) male and female phenotypes can easily be distinguished on the basis of secondary sex characteristics, and 4) a genetic marker (DMY) is available for identifying the true genotypic sex. Several biomarkers have been found to be useful for identifying the effects of exposure to estrogenic and androgenic chemicals in medaka and they include increased levels of hepatic vitellogenin (VTG) and testis-ova induction in males for exposure to estrogenic chemicals, and decreased levels of hepatic VTG in females and an altered morphology of dorsal and anal fins and formation of papillae for androgenic chemicals. In this paper, we present a critical analysis of the use of medaka as a test species for studies of endocrine disruption and report on the use of sex-related genetic markers and alterations in gonadal development, including the induction of testis-ova formation, for assessing the disruptive effects of EACs. In this paper, we focus on some of the more recent studies and findings.

Molecular cloning of estrogen receptor alpha (ERalpha; ESR1) of the Japanese giant salamander, Andrias japonicus.

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERalpha (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERalpha mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

Estrogenic activity of phthalate esters by in vitro VTG assay using primary-cultured Xenopus hepatocytes.

Estrogenic activity of phthalate esters in dental soft resins was evaluated with an amphibian system consisting of a vitellogenin (VTG)-detecting Enzyme-Linked Immunosorbent Assay and a primary-cultured hepatocyte assay using adult male Xenopus laevis. In particular, phthalate esters--Di-n-butyl phthalate (DBP), Butyl phthalyl butyl glycolate (BPBG), Benzyl butyl phthalate (BBP), and Benzyl benzoate (BB)--were investigated. Bisphenol A (BPA) was prepared for comparison with these chemicals, and 17beta-estradiol (E2) was used as a positive control. The chemicals were diluted in dimethyl sulfoxide (DMSO) to obtain final concentrations ranging from 10(-11) to 10(-4) mol/l. BPA induced estrogenic activity at a concentration of 1.1x10(-6) mol/l, while E2 showed at 4.1x10(-11) mol/l. DBP, BBP, BB, and BPBG showed no estrogenic activity at concentrations between 4x10(-7) mol/l and 1x10(-4) mol/l. The latter result indicated that these phthalate esters might be metabolically transformed into non-estrogenic substances in Xenopus hepatocytes. Furthermore, this study demonstrated that through in vitro metabolism assessment, the estrogenic activity of chemical substances could be directly detected in terms of VTG secretion in primary-cultured Xenopus hepatocytes.

Availability of in vitro vitellogenin assay for screening of estrogenic and anti-estrogenic activities of environmental chemicals.

Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelope proteins and their mRNAs are produced in the liver of oviparous species by stimulation of endogenous estrogen and exogenous estrogenic chemicals. The VTG assay based on enzyme-linked immunosorbent assay (ELISA) has been widely used for many fish species to screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents using immature fish and/or male fish. In order to reduce the number of fish for screening of estrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can be used. In fact, primary cultured hepatocytes have been successfully used for the detection of estrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fish species, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream (Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish (Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these include frogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallus domesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantitative reverse transcription-polymerase chain reaction has also been successfully applied in the primary cultured hepatocytes of various species.

Description and initial evaluation of a Xenopus metamorphosis assay for detection of thyroid system-disrupting activities of environmental compounds.

A need is recognized for the development and evaluation of bioassays for detection of thyroid system-disrupting compounds. The issue of testing for thyroid disruption can be addressed by exploiting amphibian metamorphosis as a biological model. In the present study, a test protocol for a Xenopus metamorphosis assay (XEMA) was developed and its interlaboratory transferability was evaluated in an informal ring test with six laboratories participating. In the XEMA test, exposure of Xenopus laevis tadpoles was initiated at stages 48 to 50 and continued for 28 d. Development and growth of tadpoles were assessed by means of developmental stage and whole body length determinations, respectively. For initial test protocol evaluation, thyroxine (T4), and propylthiouracil (PTU) were used as positive controls for thyroid system-modulating activity, and ethylenethiourea (ETU) was used as a test compound. Exposure of tadpoles to 1 microg/L T4 produced a significant acceleration of metamorphosis whereas PTU concentrations of 75 and 100 mg/L completely inhibited metamorphosis. Five different ETU concentrations (5, 10, 25, 50, and 100 mg/L) were tested and a concentration-dependent inhibition of metamorphosis was observed. None of the compounds affected tadpole survival, and only PTU caused a slight retardation in tadpole growth. This study demonstrates that the XEMA test provides a sensitive, robust, and practical testing approach for detection of compounds with both agonistic and antagonistic effects on the thyroid system in Xenopus tadpoles.

Metal ion-responsive transgenic Xenopus laevis as an environmental monitoring animal.

We generated germ line-transgenic Xenopus laevis that monitors environmental heavy metal ions. Sperm nuclei were transduced with cDNA of enhanced green fluorescent protein (EGFP) driven by murine metallothionein-1 gene promoters and were microinjected into unfertilized eggs. The eggs developed to sexually matured adults. The transgenic tadpoles at the premetamorphic stage were reared in water containing Zn(2+) and Cd(2+) separately at the concentrations of 0.38-1.52 and 0.09-0.44 µM, respectively. These animals responded to Zn(2+) at as low as 0.38 µM and Cd(2+) at as low as 0.44 µM. The level of EGFP fluorescence emitted by tadpoles increased as the concentration increased up to 1.52 µM and the exposure time prolonged up to 120 h. The fluorescent response was much more sensitive to Cd(2+) than to Zn(2+). We concluded that these transgenic tadpoles are useful as an animal indicator of environmental heavy metal ions.

Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental contaminant. TCDD binds and activates the transcription factor aryl hydrocarbon receptor (AHR), leading to adverse biological responses via the alteration of the expression of various AHR target genes. Although small amounts of TCDD are consumed via contaminated daily foodstuffs and environmental exposures, the effects of low-dose TCDD on gene expression in animal tissues have not been clarified, while a number of genes affected by high-dose TCDD were reported. In this study, we comprehensively analyzed gene expression profiles in livers of C57BL/6N mice that were orally administered relatively low doses of TCDD (5, 50, or 500 ng/kg body weight (bw) day(-1)) for 18 days. The hepatic TCDD concentrations, measured by gas chromatography-mass spectrometry, were 1.2, 17, and 1063 pg toxicity equivalent quantity (TEQ)/g, respectively. The mRNA level of the cytochrome P450 CYP1A1 was significantly increased by treatment with only TCDD 500 ng/kg bw day(-1). DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver with at all doses of TCDD employed. However, repression of expression of genes involved in energy metabolism was not observed in the livers of Ahr-null mice that were administered the same dose of TCDD. These results indicate that changes in gene expression by TCDD are mediated by AHR and that exposure to low-dose TCDD could affect energy metabolism via alterations of gene expression.

Vitellogenin-inducing activities of natural, synthetic, and environmental estrogens in primary cultured Xenopus laevis hepatocytes.

Vitellogenin (VTG)-inducing activities of natural estrogens (E1: estrone, E2:17beta-estradiol, E3: estriol, alpha-E2: 17alpha-estradiol), synthetic estrogens (EE2: 17alpha-ethynyl estradiol, DES: diethylstilbestrol,), phytoestrogen (GEN: genistein), and xeno-estrogens (BPA: bisphenol A, NP: nonylphenol, OP: octylphenol) were investigated by an assay system using primary-cultured hepatocytes of Xenopus laevis. An enzyme-linked immunoabsorbent assay (ELISA) was able to detect VTG at a minimum detection limit of 0.06 ng/mL. Relative estrogenic activities of the compounds were determined from their dose-response curves. The activities relative to E2 activity were 138% for DES, 121% for EE2, 6.1% for E3, 0.33% for E1, 0.29% for alpha-E2, 0.037% for GEN, 0.008% for BPA, 0.005% for NP, and 0.002% for OP. Comparison with data reported for other bioassay systems revealed that there were significant interspecies-and cell-type-differences in the activities of DES, E3, E1 and alpha-E2. BPA was found to have a substantial antagonistic activity (approximately 0.8% of tamoxifen activity) under the influence of physiological concentrations of E2. Complex-effects of endocrine disrupters on aquatic animals will be discussed.

Detection of Legionella pneumophila serogroup 7 strain from bathwater samples in a Japanese hospital.

Hospital-acquired legionellosis is one of the serious problems in nosocomial infection. For risk assessment of nosocomial Legionella infection, we surveyed samples from bathrooms for public use in three hospitals and two nursing homes to determine whether Legionella pneumophila was present. A total of 70 hot bathwater samples and samples wiped from bathtubs were collected at 1-h intervals. Fifteen shower-water and 15 inner-head samples were obtained at the start of a bath. Water samples were cultured using the Legionella spp. selective medium, and discrimination between L. pneumophila and other Legionella spp. was performed by PCR analysis. L. pneumophila serogroup 7 was detected in 1 bathwater and 1 wiped sample, both of which were collected 1 h after daily use from the same bathtub in a hospital. However, L. pneumophila SG7 was not detected in any other samples. Furthermore, the concentrations of free residual chlorine in most bath- and shower-water samples were lower than 0.1 mg/l. These results suggest that L. pneumophila has become a potential pathogen for nosocomial infections in public-type hospital baths. From the point of view of an infection-control program, it might be advisable to hold the concentration of free residual chlorine at 0.2-0.4 mg/l, which is generally required for public baths in Japan.

Development of metamorphosis assay using Silurana tropicalis for the detection of thyroid system-disrupting chemicals.

The West African clawed frog (Silurana tropicalis), which resembles the South African clawed frog (Xenopus laevis), but is somewhat smaller, has a diploid genome and a shorter generation time. Therefore, S. tropicalis has the potential for use as a new model in ecotoxicology. We demonstrated a S. tropicalis metamorphosis assay based on Xenopus Metamorphosis Assay (XEMA) using 1 microg/L thyroxine (T4) and 75 mg/L propylthiouracil (PTU). Tadpoles at developmental stages 48-50 were exposed to chemicals for 28 days and total body length, developmental stage, and hind limb length were recorded every 7 days. Significant differences in developmental stage and total body length were found for both T4 and PTU after 7-day exposure, which were similar to the results of the XEMA ring-test using the same chemicals. Moreover, in the present study, we measured hind limb length as a new endpoint of thyroid axis. Significant differences in the hind limb length were encountered in both T4 and PTU treatments after 7 days of exposure. These results suggest that S. tropicalis can be used in a XEMA-like protocol to detect agonist and antagonist effects of chemicals on the thyroid system. Hind limb length is also a suitable endpoint in such protocols. A new test protocol detecting both thyroid disruption and reproductive effects of chemicals using S. tropicalis should be established in the near future.

All ZZ male Xenopus laevis provides a clear sex-reversal test for feminizing endocrine disruptors.

We investigated the possibility of using all ZZ male Xenopus laevis tadpoles produced by mating normal ZZ males with feminized ZZ males to detect estrogenic chemical activity. We examined the effects of 17beta-estradiol (E2) on sex differentiation by treating NF stage 49/50 to stage 57 tadpoles with 0.1, 1, 10, and 20 nM E2 for 4 weeks. Following this, the tadpoles were allowed to develop in clean water until the animals reached stage 66. Increased developmental abnormalities and mortality were not observed in all E2-exposed groups during metamorphosis. Feminization of gonads was detected at all E2 concentrations, whereas nonexposed controls developed testes. Morphological and histological analyses showed that feminized gonads were ovaries. Five and one hermaphroditic frogs were found in the 0.1 and 1 nM E2 groups, respectively, showing testicular as well as ovarian regions within one gonad. These results indicate that phenotypically normal females can be produced from genetic males and demonstrate the utility of a sex-reversal test based on all ZZ males for examining in vivo effects of chemicals with estrogenic activity. The testing of all ZZ male tadpoles might be a useful tool for assessment of feminizing compounds not only estrogenic substance.

Effects of an androgenic growth promoter 17beta-trenbolone on masculinization of Mosquitofish (Gambusia affinis affinis).

Endocrine disrupting chemicals can affect normal hormone dependent processes through numerous mechanisms, including ligand mimicky. 17beta-Trenbolone (TB), a pharmaceutical, androgenic, anabolic steroid, is a potent agonist of androgen receptors, and has been extensively used as a growth promoter for beef cattle in the US. The effects of TB on adult and newborn mosquitofish (Gambusia affinis affinis) were examined. Two forms of mosquitofish androgen receptor (AR), ARalpha and ARbeta, were cloned. The mRNA expression levels of ARalpha and ARbeta were transiently increased in the anal fin of adult females at day 3 following exposure to TB (1-10 microg/L) or methyltestosterone (MT) (0.1-10 microg/L), a pharmaceutical androgen used as a positive control. Gonopodium differentiation from the adult female anal fin was induced after 28 days of exposure to TB (1-10 microg/L) or MT (0.1-10 microg/L). Gonopodium differentiation also was induced in all mosquitofish fry exposed for 28 days to 0.3, 1 or 10 microg/L TB. Furthermore, spermatozoa were observed histologically in the testes of male fry exposed for 28 days to 1 or 10 microg/L TB; spermatozoa are normally observed only in the testes of mature males. Surprisingly, all female fry exposed for 28 days to 1 or 10 microg/L TB displayed the formation of an ovotestis, as spermatozoa were found in the ovary. Thus, TB, like MT, induced masculinization of the anal fin accompanied by a transient up-regulation of ARalpha and ARbeta in adult females. TB also induced differentiation of the anal fin into a gonopodium in fry of both sexes, stimulated precocious spermatogenesis in the testes of males and the formation of ovotestes in females.

Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 beta effects on whole animals and primary-cultured hepatocytes.

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.