Application of low-frequency sonophoresis and reduction of antibiotics in the aquatic systems.

A major concern in aquaculture is the use of chemical therapeutics, such as antibiotics, because of their impact on the environment as well as on the fish product. As a potential tool for reducing antibiotic use, we tested the application of low-frequency ultrasound as a method for enhancing antibiotic uptake. Rainbow trout juveniles (Oncorhynchus mykiss) were exposed to two different concentrations of oxytetracycline (OTC), flumequine (FLU) and florfenicol (FLO), administered by bath after the application of ultrasound. After exposure, concentrations of these substances were measured in the liver and blood of treated fish. Results showed that the ultrasound treatment can significantly increase the uptake for all three antibiotics. The uptake of OTC for example, in fish exposed to an OTC concentration of 20 mg L-1 after prior treatment with ultrasound, was similar to the OTC concentrations in their liver and blood to fish exposed to 100 mg L-1 without sonication. For FLU and FLO, the use of ultrasound caused significant differences of uptake in the liver at high antibiotic concentrations. This suggests that the use of ultrasound as a technique to deliver antibiotics to fish can ultimately reduce the amount of antibiotics discharged into the aquatic environment.

Nutritional status and gene expression along the somatotropic axis in roach (Rutilus rutilus) infected with the tapeworm Ligula intestinalis.

The tapeworm Ligula intestinalis inhibits gametogenesis of its fish host, the roach (Rutilus rutilus). We investigated whether L. intestinalis infection makes significant demands on nutritional resources and consequently manipulates the endocrine somatotropic axis of roach. Two groups of naturally infected and uninfected roach were studied: a field group (natural feeding) and a laboratory group (ad libitum food supply). In females, no significant impact of parasitization on storage substrates (glycogen, lipids, and protein) was detected, whereas in males, either lipid content of the liver (field group) or lipid of the muscle and glycogen of the liver (laboratory group) were slightly decreased. Except for the females of the field group, higher mRNA expression of growth hormone (gh) in the pituitary of infected fish was observed. Furthermore, the expression of hypophyseal somatolactin α and ß (slα, slß) was up-regulated in infected females of the field and laboratory group, respectively. In liver and muscle, mRNA expression of insulin-like growth factors (igf1, igf2) and igf receptor (igfr) remained either unchanged or were up-regulated with infection. Parasitization showed inconsistent effects on gh receptor 1 (ghr1) expression in liver and muscle, whereas ghr2 mRNA was mostly not influenced by infection. In general, the expression profile of genes involved in the somatotropic axis as well as the content of storage substances in infected roach did not resemble that of food-deprived fish either under natural or ad libitum feeding. In conclusion, the present study does not indicate starvation of L. intestinalis infected roach, and it is suggested that the inhibition of reproduction attenuated the nutritional demand of parasitization.

Influence of temperature on puberty and maturation of pikeperch, Sander lucioperca.

Among external factors, temperature is known to exhibit a prominent role in reproduction of temperate fish species. Here, temperature related induction of puberty in pikeperch Sander lucioperca was investigated. For the first time the key factors of the pikeperch brain-pituitary-gonad axis, targeting the mRNA expression of the luteinising hormone (LH) and the follicle stimulating hormone (FSH), as well as the plasma sex steroids estradiol (E2), testosterone (T), 11-ketotestosteron (11-KT) and 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) were addressed in the experiment. Concomitant the maturational stages were described histologically. After 3 months, female pikeperch kept at 12°C revealed significant increases in the GSI and plasma E2 concentration and 90% of the females were mid-vitellogenic. After 5 months, females kept between 9 and 15°C exhibited significant up-regulation of E2 and GSI as well as comparable histological outcome. At 6 and 23°C in nearly all females stagnation of oogenesis was recorded. Congruently, T was increased at 12 and 15°C. Expression analysis revealed a significant up-regulation of LHß and FSHß mRNA in females from early-vitellogenesis, and from mid-spermatogenesis in males, correlated to elevated plasma concentrations of steroids (except for E2 in males). In conclusion, moderate temperatures (12-15°C for) for at least 3 months were required to proceed with first maturation in juvenile pikeperch. The most efficient effect was observed at 12°C, while high (23°C) or low (6°C) temperatures prevented gonadal maturation. So temperature was identified as a prime factor in the induction of puberty in pikeperch, as revealed by histological as well as endocrine parameters.

In vivo treatment with progestogens causes immunosuppression of carp Cyprinus carpio leucocytes by affecting nitric oxide production and arginase activity.

In this study, carp Cyprinus carpio were injected with various steroid compounds, including synthetic and natural progestogens and the glucocorticoid cortisol, to investigate effects on leucocytes isolated from their kidneys. Injection of cortisol led to an increased spleeno-somatic index (I(S)) on day 21 post-injection (pi) and immunosuppressive effects measured as decreased nitric oxide (NO) production and increased arginase activity in isolated leucocytes on days 14 and 21 pi, respectively. Moreover, reduced NO production was also observed after injection of the synthetic progestogens, levonorgestrel (LEV) and medroxyprogesterone acetate. In addition, LEV influenced arginase activity in head kidney cells on day 14 and day 21 pi. This study is the first demonstration in fishes that the application of these steroid compounds in vivo affects NO production and arginase activity of isolated leucocytes.

Developmental regulation of gene expression in the thyroid gland of Xenopus laevis tadpoles.

Thyroid hormones (TH) are the primary morphogen regulating amphibian metamorphosis. However, knowledge about molecular mechanisms regulating thyroid gland activity in anuran tadpoles is very scarce. In this study, we characterized gene expression profiles in thyroids of Xenopus laevis tadpoles during spontaneous metamorphosis. Using real-time PCR, elevated expression of slc5a5, tpo, tshr, and sar1a mRNAs was detected at late prometamorphic and climax stages. For dio2 and dio3 but not dio1, developmental regulation of thyroidal expression was evident from a strong up-regulation at late stages. Conversely, expression of the DNA replication markers mcm2 and pcna declined at climax stages. The presence of functional feedback mechanisms at premetamorphic stages was examined in two experiments. Stage 52 tadpoles were exposed for 72 h to 1.0 microg/l thyroxine (T4). This treatment caused reduced mRNA expression of slc5a5, tpo, and dio2, whereas no significant changes were detectable for tshr expression in thyroids and tshb expression in the pituitary. In another experiment, stage 46 tadpoles were treated with 20 mg/l sodium perchlorate (PER) for 5 and 10 days. Within this period of time, control tadpoles developed to stages 50 and 52, respectively. PER treatment resulted in up-regulation of slc5a5, tpo, and tshr mRNAs at both time points and increased dio2 mRNA expression at day 10. Effects of PER on thyroid histology were only apparent on day 10. Together, our analyses of thyroidal gene expression demonstrate a marked developmental regulation for functional markers of thyroid activity, two deiodinases as well as for DNA replication markers. Expression patterns detected in PER- and T4-treated tadpoles indicate that functional feedback signaling controlling thyroid activity is already active during premetamorphosis.

Expression profiles of LHbeta, FSHbeta and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles.

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHbeta and FSHbeta mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHbeta and FSHbeta mRNA was observed at late premetamorphosis. A clear raise of LHbeta mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHbeta mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHbeta and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds.

Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 10(-8) M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects.

Perchlorate and ethylenethiourea induce different histological and molecular alterations in a non-mammalian vertebrate model of thyroid goitrogenesis.

Despite evidence for a conserved role of thyroid-stimulating hormone (TSH) in regulating vertebrate thyroid function, molecular data on thyroid responses to TSH are mainly limited to mammalian species. In this study, we examined histological and molecular changes in the thyroid of Xenopus laevis tadpoles during a 12-day treatment with 20mg/l perchlorate (PER) and 50mg/l ethylenethiourea (ETU). Inhibition of thyroid hormone (TH) synthesis by PER and ETU was evident from developmental retardation, reduced expression of TH-regulated genes and up-regulation of tshb-A mRNA. Thyroid histopathology revealed goiters with strikingly different follicular morphologies following PER and ETU treatment. Using real-time PCR, we analyzed thyroids sampled on day 12 for differential expression of 60 candidate genes. Further temporal analyses were performed for a subset of 14 genes. Relative to the control, PER and ETU treatment modulated the expression of 51 and 49 transcripts, respectively. Particularly genes related to TH synthesis and protein metabolism were similarly affected by PER and ETU. However, several genes were differentially expressed in PER- and ETU-treated tadpoles. Specifically, goiter formation in the PER treatment was associated with low expression of genes related to DNA replication but high expression of negative growth regulators. Results from this work provide for the first time a characterization of gene expression profiles during goitrogenesis in a non-mammalian vertebrate model. Overall, our data suggest that, in addition to TSH over-stimulation, further mechanisms related to the mode of goitrogen action contribute to the regulation of thyroid gene expression.

Effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings.

A 56-day feeding trial was conducted to access the effect of housefly maggot meal (magmeal) diets on the performance, concentration of plasma glucose, cortisol and blood characteristics of Oreochromis niloticus fingerlings. Seven feeds formulated to contain 36% protein and 20 kJ g(-1) gross energy (dry matter basis), were prepared by replacing fish meal with magmeal. Fifteen fingerlings (initial average weight 2.0 +/- 0.1 g) stocked per experimental tank were fed in triplicates at 5% body weight in two portions per day (a level previously established). Growth and food conversion ratio were adequate and comparable without any significant differences (p < 0.5) between feeding groups. Mean values for haematocrit and plasma glucose were not significantly different (p < 0.05) among the feeding groups. Fish group fed control diet (containing highest inclusion level of fish meal and without magmeal) gave the lowest haemoglobin concentration (5.96 +/- 0.22 g dl(-1)). This value was significantly different from other feeding groups. Stressful conditions in fish and in mammals are associated with decreased growth, haematocrit (packed cell volume) and haemoglobin values, increased whole blood glucose (hyperglycaemia) and plasma cortisol concentrations. No such physiological changes were observed in this study. Results suggest that feeding O. niloticus fingerling with magmeal diets did not cause any form of physiological stress. Magmeal can be used as a good alternative protein source in tilapia diets.

Influence of batch-specific biochemical egg characteristics on embryogenesis and hatching success in farmed pikeperch.

Low and variable egg quality remains a major issue in aquaculture impeding a reliable and continuous supply of larvae, particularly in emerging species, such as pikeperch, Sander lucioperca. We assessed the influence of batch-specific egg parameters (fatty acid (FA) profiles, cortisol content) on embryo life-stages until hatching (survival at 2, 24, 48, 72 h post fertilization (hpf), hatching rate) in an integrated study under commercial hatchery conditions (44 egg batches). Embryo mortality was elevated until 48 hpf (average 9.8% mortality between 2 and 48 hpf). Embryos surviving until 48 hpf were very likely (98.5%) to hatch successfully. The inherent egg FA composition was variable in-between batches. Total FA content ranged form 66.1 to 171.7 µg/mg (dry matter) total FA. Whereas specific FA ,18 : 0 and 20 : 5(n-3) (eicosapentaenoic acid) of the polar fraction and the ratio of 22 : 6(n-3) (docosahexaenoic acid) to 20 : 5(n-3) within the neutral fraction, were significantly correlated with early embryo development, contents of the respective FA did not differ between high (>90% hatching rate), mid (70% to 90% hatching rate) and low (<70% hatching rate) quality egg batches. Late embryo development and hatching were relatively independent of the FA profiles highlighting stage-dependent influences especially during early embryogenesis. Cortisol levels ranged from 22.7 to 293.2 ng/ml and did not directly explain for mortalities. However, high cortisol was associated with a lower content of specific FA, in particular highly unsaturated FA. These results demonstrate the magnitude of inter-individual differences in the batch-specific biochemical egg composition under stable hatchery conditions and suggest a stress-mediated lack of essential FA, which in turn affects early embryo survival. Surprisingly, embryos are able to cope well with a broad range of inherent egg parameters, which limits their predictive potential for egg quality in general. Still, specific FA profiles of high quality egg batches have potential for formulating species-specific broodstock diets and improving reproductive management in pikeperch.

Environmental agent susceptibility assessment using existing and novel biomarkers as rapid noninvasive testing methods.

This study is part of a project aimed at developing and validating novel noninvasive methods for the detection of biomarkers of endocrine disrupters (EDs) directly in the mucus of aquatic species, to identify novel functional biomarker(s) for EDs, and to verify their applicability for field studies. The multidisciplinary approach chosen aims at the development of an integrated testing strategy utilizing in vitro protocols to identify water and sediment fractions with potential endocrine-disrupting activity; the identification, characterization, and measurement of new biomarker(s) for EDs; the development and validation of a dipstick-based test method; and the development of (computer-assisted) predictive models. Some results of the first year of the project are presented here.

Effects of the non-steroidal anti-inflammatory drug(NSAID) naproxen on gene expression of antioxidant enzymes in zebrafish (Danio rerio).

The aim of this study was to investigate the effects of naproxen on the gene expression of antioxidant enzymes in adult zebrafish. Surprisingly, after 2 weeks exposure no significant effect on the mRNA expression of the target genes was found in the liver. However, mRNA levels of three genes were altered significantly in the intestine. The expression of Ucp-2 decreased at the environmental concentration of 1µg/L while mRNA expression of GST p2 increased at the concentration of 100µg/L. The mRNA level for the antioxidant enzyme CAT was up-regulated significantly at both the concentrations used. Exposure to naproxen caused only moderate effects on the expression of antioxidant genes in the intestine rather than in the liver, which demonstrates that the intestine is more sensitive to waterborne naproxen exposure than the liver. Interestingly, the adverse side effects of NSAIDs occur in the gastrointestinal tract of humans. To our knowledge, this is the first study that has focused on transcriptional effects of naproxen on zebrafish.

Characterization and localization of beta2-adrenergic receptors in the bovine oviduct: indication for progesterone-mediated expression.

Beta2-adrenergic receptors were detected in bovine oviductal epithelium by use of receptor binding studies and expression analysis. Complementary DNA cloning gave use to the first full-length bovine beta2-adrenoceptor messenger RNA sequence (2030 bases). Receptor bioactivity in oviduct epithelial cells was characterized by specific ligand interaction and consequent cAMP generation. Expression studies demonstrated an estrous cycle-dependent regulation, with higher transcript levels and significantly increased binding capacity during the luteal phase. After progesterone supplementation, oviduct epithelial cells showed elevated receptor expression in culture, supporting the hypothesis that progesterone up-regulates the beta2-adrenergic receptor within these cells. It seems likely that catecholamines from the circulation or from innervation might be able to influence reproductive success by regulating oviductal secretion.

Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver.

In spite of the importance of IGF-I for growth and development, knowledge about regulation of its production in submammalian species is rather limited. In order to create a tool for investigation of direct regulatory effects on the expression of IGF-I in bony fish liver, a primary cell culture of hepatocytes from Oreochromis mossambicus, the tilapia, was established. The cells were viable for up to 3 days and IGF-I mRNA synthesis was detected by northern blot and semiquantitative reverse transcriptase (RT)-PCR. Northern blot analysis of the primary cultured hepatocytes revealed four different IGF-I transcripts, 0.5, 1.9, 3.9 and 6.0 kb in size, which were identical to those in liver tissue. However, the expression rate was weaker than that in liver. The direct effects of recombinant tilapia (rt) growth hormone (GH) and salmon (s) IGF-I on the expression of IGF-I in primary cultured hepatocytes were investigated in time-course and dose-response experiments. In untreated cultures, IGF-I mRNA decreased with time. Hepatocytes treated with 100 nM rtGH resulted in a pronounced stimulation of IGF-I mRNA expression throughout the experiment. Treatment with rtGH in concentrations ranging from 0.1 nM to 1 microM caused a clear dose-dependent increase in the amount of IGF-I mRNA. Significant stimulation was obtained even with 0.1 nM, reaching a plateau with 10 nM. Neither significant inhibitory nor stimulatory effects were detected by adding sIGF-I from 0.1 nM to 1 microM to the hepataocytes. Our results indicate that the established primary cell culture of tilapia hepatocytes is a useful system in which to study direct effects of potential regulators of bony fish liver cell function.

Insulin-like growth factor-I and -II in the ovary of a bony fish, Oreochromis mossambicus, the tilapia: in situ hybridisation, immunohistochemical localisation, Northern blot and cDNA sequences.

There is accumulating evidence that insulin-like growth factor (IGF)-I and IGF-II are present in the mammalian ovary but comparable studies on bony fish remain scarce. Thus, the present study aims to analyse several parameters of the IGFs in the ovary of a bony fish, the tilapia, (Oreochromis mossambicus). Molecular biological and morphological techniques were applied. The IGF-I and IGF-II cDNA sequences established from the ovary indicate that the same molecules are present in ovary and liver. Northern blot analysis revealed four IGF-I mRNA transcripts (6.0, 3.9, 1.9, 0.5 kb) and three IGF-II mRNA transcripts (5.0, 4.0, 2.0 kb) in ovary and liver. The amounts of IGF-I and IGF-II mRNA in the ovary were considerably high when compared to those in liver (IGF-I: 80.7%; IGF-II: 63.7%). The expression of IGF-I mRNA and IGF-II mRNA in the ovary were studied by in situ hybridisation and the peptides located by immunohistochemistry. The expression of IGF-I varied between the different developmental stages. Both IGF-I mRNA and IGF-I immunoreactivity were present in small oocytes. Moderate IGF-I expression and immunoreactivity occurred in granulosa cells of follicles at the lipid stage. A high IGF-I expression was observed in the granulosa and theca cells surrounding oocytes at the yolk globule stages and mature oocytes but neither IGF-I mRNA nor IGF-I immunoreactivity occurred in oocytes of the later stages. Thus, the IGF-I production seems to change from the young oocyte to the surrounding follicle cells at the later stages. In contrast, IGF-II mRNA and IGF-II-immunoreactivity occurred only in granulosa cells of the late follicle stages. The results suggest that both IGF-I and IGF-II are involved in the maturation of bony fish oocytes and in follicle development in a paracrine/autocrine manner. IGF-I and IGF-II may exert their effects at different stages of development. Furthermore, the intraovarian IGF-I and IGF-II systems seem to have a long phylogenetic history indicating the importance of the IGFs in reproductive biology.

Insulin-like growth factor I in the anterior pituitary of the clawed frog Xenopus laevis: immunocytochemical and autoradiographic indication for a paracrine action and corelease with prolactin.

Insulin-like growth factor I (IGF-I) and its receptor are present in human and rat anterior pituitary. However, few data exist on the potential presence of IGF-I or its receptor in the non-mammalian pituitary and the cellular sites of IGF-I production have not been identified in any species. Thus, we investigated the anterior pituitary of the clawed frog Xenopus laevis which is widely used to study growth and differentiation. The study was performed with antisera against mammalian insulin-like growth factor I (IGF-I), prolactin (PRL) and growth hormone (GH) using immunohistochemical and immunocytochemical techniques. IGF-I binding was determined by in-vitro receptor autoradiography. The PRL-and GH-immunoreactive cells exhibited distinct distribution patterns. Neither at the light nor the electron microscopical level any colocalization of PRL-and GH-immunoreactivities was apparent. The PRL-immunoreactive cells exhibited round granules of medium electron density (mean diameter: 312 nm) and the GH-immunoreactive cells spherical granules of medium electron density (mean diameter: 165 nm). By the use of serial semithin sections IGF-I-immunoreactivity was exclusively located in PRL-immunoreactive cells. At the ultrastructural level, IGF-I-immunoreactivity was confined to the secretory granules in coexistence with PRL-immunoreactivity using the double labelling immunogold technique. Specific IGF-I binding sites were localized throughout the pituitary. The results provide evidence for a concomitant release of PRL and IGF-I and suggest autocrine/paracrine actions of IGF-I in the anterior pituitary.

Atrial natriuretic factor (ANF) binding sites in frog kidney and adrenal.

Atrial natriuretic factor (ANF) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. [125I]-rat ANF(99-126) binding was present in kidney glomeruli and in the outer layer of interrenal tissue in the adrenal gland. ANF binding exhibited positive cooperativity with a half-maximal binding concentration (EC50) of 102 +/- 16 pM in glomeruli and 93 +/- 19 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 1.33 +/- 0.16 and 1.21 +/- 0.36 fmol/mm2. [125I]-Rat ANF(99-126) binding was competitively displaced by unlabeled ANF analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. The presence of ANF binding in glomeruli and steroidogenic interrenal cells suggests physiological functions of ANF for the osmomineral regulation in the frog by influencing glomerular filtration rate and adrenal steroid secretion.

Angiotensin II binding sites in frog kidney and adrenal.

Angiotensin II (AII) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. AII binding was present in kidney glomeruli and in interrenal tissue of the outer zone of the adrenal gland. Saturation experiments showed that [125I]-[Val5]AII binds to a single class of binding sites with a dissociation constant (Kd) of 548 +/- 125 pM in glomeruli and 593 +/- 185 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 2.48 +/- 0.71 and 3.05 +/- 1.02 fmol/mm2, respectively. AII binding was displaced by unlabeled angiotensin analogues in the rank order: [Sar1]AII greater than human AII greater than [125I]-[Val5]AII = [Val5]AII = human AIII much greater than human AI. The AII binding sites in glomeruli and interrenal tissue suggest an influence of AII on glomerular filtration rate and adrenal steroid secretion to take part in osmomineral regulation of the frog.

Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding.

Many chemicals released into the environment without toxicological risks have the capacities to disrupt the function of endocrine systems. These endocrine disruptors disturb normal endocrine mechanisms and have been observed in nearly all classes of vertebrates. The aim of this research is to develop a comprehensive model to study endocrine disruption using the amphibian Xenopus laevis. The assessment of estrogenic potencies of endocrine disruptors includes several levels of investigation: (I) binding to liver estrogen receptor, (II) estrogenic activity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes, and (III) in vivo effects on sexual development caused by exposure of larvae. The present paper is focused on the first part by establishing a radioreceptorassay for [3H]17 beta-estradiol ([3H]E2) binding using liver cytosol fraction. In order to get optimum binding conditions we performed kinetic, saturation, and competitive displacement experiments. Association of [3H]E2 to estrogen receptor revealed that maximum specific binding is achieved between 18 and 48 h of incubation. Scatchard analyses of saturation experiments resulted in a homogenous saturable population of estrogen receptors having no significant differences of binding parameters between both sexes. The values of Kd (dissociation constant) in males and females were 22.4 +/- 6.0 and 15.0 +/- 2.8 nM (mean +/- S.E.M.; n = 5), respectively, while corresponding Bmax (maximum binding capacity) revealed 89 +/- 46 and 136 +/- 46 fmol [3H]E2/mg protein. The specificity of estrogen receptors as shown by competitive displacement experiments demonstrated receptors being highly specific just for estrogens, but not for other endogenous steroids having the following ranking of binding affinities: E2 > estrone > dehydroepiandrosterone > aldosterone > or = testosterone > or = corticosterone > or = progesterone. The affinity ranking of environmental chemicals compared to E2 was: E2 > tetrachlorbiphenyl > diethylphthalate > 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A) > or = 4-nonylphenol > or = 3-t-butyl-4-hydroxyanisole > or = 4-octylphenol > dichlor-diphenyl-trichlor-ethan (4,4'-DDT). Analyses of five sewage effluents for displacement of [3H]E2 binding resulted in three samples displacing more than 50% of specific binding at their original concentration. Taken together the established radioreceptorassay for [3H]E2 binding in Xenopus laevis liver cytosol is useful to screen estrogen receptor binding of pure compounds or complex mixtures of them, which is the prerequisite for causing either estrogenic or antiestrogenic effects.

Amphibians as a model to study endocrine disruptors: II. Estrogenic activity of environmental chemicals in vitro and in vivo.

Several environmental chemicals are known to have estrogenic activity by interacting with development and functions of endocrine systems in nearly all classes of vertebrates. In order to get a better insight of potential estrogenic effects on amphibians caused by environmental pollution this study aims to develop a model for investigating endocrine disruptors using the amphibian Xenopus laevis. In that model the potential estrogenic activity of endocrine disruptors is determined at several levels of investigation: (I) binding to liver estrogen receptor; (II) estrogenicity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes; and (III) in vivo effects on sexual development. Here we deal with establishing methods to assay estrogenic activity of environmental chemicals in vitro and in vivo. In vitro we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique to determine mRNA-induction of the estrogenic biomarker vitellogenin in primary cultured hepatocytes of male Xenopus laevis. Time courses of vitellogenin-mRNA in the presence and absence of 10(-6) M 17 beta-estradiol (E2) resulted in a marked loss of mRNA from controls after 2 days while E2 treatment kept vitellogenin-mRNA at a relatively stable level. After 36 h of incubation estrogenic activities of E2, 4-nonylphenol (NP), and 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A) at concentrations ranging from 10(-10) to 10(-5) M were assayed by RT-PCR of vitellogenin-mRNA and showed the following ranking of dose-dependent potency: E2 > NP > bisphenol A. These in vitro results were confirmed further by in vivo experiments determining sexual differentiation of Xenopus laevis after exposure to E2 and environmental chemicals during larval development. Concentrations of 10(-7) and 10(-8) M E2 as well as 10(-7) M of NP or bisphenol A caused a significant higher number of female phenotypes compared to controls indicating a similar ranking of estrogenic potencies in vivo as in vitro. In addition, butylhydroxyanisol and octylphenol, both showed feminization at 10(-7) M while octylphenol was also effective at 10(-8) M. In summary these results demonstrate for the first time the use of a semiquantitative RT-PCR technique for screening estrogenicity by assaying mRNA induction of the estrogenic biomarker vitellogenin in vitro. The combination of this newly developed method with classical exposure experiments is necessary for determination of the biological significance of estrogenic chemicals.