Effects of the model androgen methyltestosterone on vitellogenin in male and female eelpout, Zoarces viviparus (L).

In the present experiment male and female eelpout, Zoarces viviparus were exposed to different doses of the model androgen 17alpha-methyltestosterone (MT) and the effects on the plasma level of vitellogenin and the gonadosomatic index were investigated. In females exposed to different doses of MT (nominal concentrations 10, 25, 50, 100 and 500 ng/L) in the ambient seawater, the concentrations of the circulating yolk-precursor protein vitellogenin (vtg) were shown to decrease in all groups but only significantly in the MT-100 group when compared to controls. No significant effects could be observed on the GSI during early vitellogenesis (April/May). Males exposed to E2 under flow through conditions during 10 days showed an induced synthesis of vtg as depicted by a high level of circulating vtg. When the estrogen-exposed males were subsequently exposed to different doses of MT only, during another 10 days, the levels of vtg decreased significantly in three of the MT-treated groups when compared with the level of vtg in plasma of the estrogen-treated group after the first 10 days. The gonadosomatic index was observed to decrease by the estradiol-treatment but to increase in a dose-dependent manner in the MT-exposed groups, indicating that MT had the capacity to override the effect of the preceding exposure to estradiol-17beta.

Effects of an environmental estrogen, 17alpha-ethinyl-estradiol, on the maternal-fetal trophic relationship in the eelpout Zoarces viviparus (L).

In the present experiment pregnant eelpout Zoarces viviparus females were exposed to 0, 5, 10, 50, 500 ng/l of the synthetic estrogen ethinylestradiol (EE2) and to 500 ng/l estradiol-17beta (E2) under flow-through conditions during 3 weeks and the maternal-fetal trophic response was investigated. The circulating yolk-precursor protein vitellogenin, measured by ELISA, increased from a mean control value of 0.017-36 mg/ml in the plasma of the motherfish exposed to the highest concentration of EE2 This increase in vitellogenin was also depicted by a 288% increase in circulating calcium levels. During pregnancy the ovary represents a new route of calcium loss from the maternal blood for the growth of the embryos. However, a significant decrease (120% in the group exposed to the highest concentration of EE2) in the calcium level in the ambient medium of the embryos, the ovarian fluid, was observed concomitant with the increase in maternal plasma calcium in the EE2-exposed females. In contrast, the level of circulating amino acids decreased in the maternal blood, with a slight concomitant increase in the ovarian fluid of the exposed fish. These and other metabolic observations indicate that exposure to different doses of EE2 and to estradiol-17beta affects the maternal-fetal trophic relationship.

Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio).

The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg x ml(-1) and the practical detection limit was 40 ng Vtg x g(-1) fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng x l(-1) of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 x l(-1) induced a 200-fold increase in Vtg levels.

Concentration-dependent effects of nonylphenol on testis structure in adult platyfish Xiphophorus maculatus.

Nonylphenol has been found to exert estrogenic effects in fish and may affect the fertility of male fish. The objective of this study was to use the platyfish, Xiphophorus maculatus, as a model to evaluate the concentration-dependent effects of nonylphenol on the testicular structure. A tendency of a concentration-dependent decrease in the gonadosomatic index was observed after 28 days of treatment with nonylphenol. Histological examination revealed marked effects of nonylphenol on the testis structure. The effects were more pronounced at the higher concentrations of nonylphenol. This study strongly suggests that exposure to nonylphenol can result in decreased fertility in X. maculatus males.

gamma-Glutamyl transpeptidase as a possible marker of Sertoli cells in fish testes for studies of xenoestrogens.

Estrogenic chemicals are known to have marked effects on the reproductive system of male fish. Finding useful markers of reproductive effects are therefore of great importance and interest. gamma-Glutamyl transpeptidase (gamma-GTP) is a possible marker of Sertoli cells in testes of fish. In the present study we have examined the relationship between the activity of gamma-GTP and the histological structure of the Sertoli cells in testes of five fish species (guppy, Poecilia reticulata; platyfish, Xiphophorus maculatus; eelpout, Zoarces viviparus; rainbow trout, Oncorhynchus mykiss; flounder, Platichthys flesus). In general we found that the more distinct the Sertoli cells the higher the activity of gamma-GTP.

Characterization of a cytosolic estrogen receptor and its up-regulation by 17 beta-estradiol and the xenoestrogen 4-tert-octylphenol in the liver of eelpout (Zoarces viviparus).

Estrogen binding activity was revealed in the cytosolic fraction of hepatic extracts from adult male and female eelpout (Zoarces viviparus). The binding moiety was characterized by a single class of high affinity binding sites (K(d)=0.59+/-0.05 nM in males and 1.06+/-0.10 nM in females). The affinity was significantly higher in males. Binding sites were satiable and binding capacity was significantly elevated in vitellogenic females (2.92+/-0.28 pmol/g) compared to males (1.67+/-0.11 pmol/g). The binding was specific to known estrogens but not to other tested steroids. The binding moiety was able to bind to DNA-cellulose and was extractable by high salt concentrations. A time-course study of estrogen binding activity in liver cytosol and of vitellogenin (Vtg) in plasma, after intraperitoneal (i.p.) injections of 17 beta-estradiol (E(2)) in male eelpout, was carried out. It was shown that both are inducible by E(2). Estrogen binding activity was significantly elevated 48 h and Vtg 72 h after E(2) treatment. The binding moiety was hereafter designated as a cytosolic estrogen receptor (ER). The estrogenicity of 4-tert-octylphenol (OP) was evaluated by measuring ER and Vtg after i.p. treatment. OP-treatment increased both receptor levels and Vtg concentrations in male fish, indicating that OP acts as an estrogen in male eelpout.

Effects of nonylphenol and 17beta-estradiol on vitellogenin synthesis and testis morphology in male platyfish Xiphophorus maculatus.

Nonylphenol has been found to exert estrogenic effects in fish and may influence the fertility of male fish. In the present study, the effects of nonylphenol and 17beta-estradiol on vitellogenin synthesis and testis morphology in platyfish Xiphophorus maculatus were investigated. Vitellogenin was observed in the plasma of all fish exposed to nonylphenol or 17beta-estradiol. Exposure to 17beta-estradiol resulted in a significant reduction in the gonadosomatic index. A tendency for a dose-dependent reduction in the gonadosomatic index in the nonylphenol exposed groups was observed. Histological examination revealed dose-dependent effects of nonylphenol on the testis structure. The testes of control fish contained numerous cysts with spermatogenetic cells. The testes of fish exposed to nonylphenol or 17beta-estradiol showed a decrease in the number of cysts concomitant with an increase in the amount of hypertrophied Sertoli cells present. Formation of spermatozeugmata is compulsory for this species, but free spermatozoa were observed in the efferent ducts of the treated fish. The study indicates that nonylphenol has estrogenic potency, and that both nonylphenol and 17beta-estradiol have marked effects on the testis morphology of X. maculatus. The ambient concentration of nonylphenol was measured by high pressure liquid chromatography during the experiment. The measurements revealed that the actual concentrations of nonylphenol in the water were about 30-40 % of the nominal concentrations.

The preservatives ethyl-, propyl- and butylparaben are oestrogenic in an in vivo fish assay.

The widely used phenolic preservatives ethylparaben, propylparaben, butylparaben and their common metabolite p-hydroxybenzoic acid were tested for their ability to evoke an oestrogenic response in vivo. Yolk protein induction in sexually immature rainbow trout was used as an oestrogen-specific endpoint after repeated injections of the compounds. All tested parabens were oestrogenic in doses between 100 and 300 mg/kg, while the metabolite showed no activity. Ethylparaben was found to be approximately sixty times weaker than propyl- and butylparaben which had oestrogenic potencies comparable to those previously found for bisphenol A.

In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncorhynchus mykiss).

The in vivo estrogenic activity of the two branched alkylphenols, tert-octylphenol and technical nonylphenol, and the two linear isoforms, n-octylphenol and n-nonylphenol, was compared. The compounds were administered to juvenile rainbow trout (Oncorhynchus mykiss) by either intraperitoneal injection or water exposure and their estrogenic potential was evaluated by ELISA measurements of induced plasma vitellogenin. Intraperitoneal injections (50 mg/kg) of the two branched alkylphenols resulted in a significant vitellogenic response after 12 days whereas no significant induction was seen with the two linear isomers. Water exposure for 9 days to a nominal concentration of 150 micrograms/l of the alkylphenols elicited the same response pattern as seen for the injection experiment. Furthermore, in the present vitellogenin assay tert-octylphenol was giving a higher estrogenic response compared to technical nonylphenol using either of the two exposure routes.

Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals.

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

Vitellogenin in Zoarces viviparus: purification, quantification by ELISA and induction by estradiol-17 beta and 4-nonylphenol.

Vitellogenin was isolated by gel filtration and ion-exchange chromatography from plasma of estradiol-treated male Zoarces viviparus. The purification of vitellogenin was followed through each step by native polyacrylamide gelelectrophoresis (PAGE). The purified vitellogenin was used to raise anti-vitellogenin antibodies in rabbits. The specificity of the affinity purified antibody raised against vitellogenin was assessed by Western blot analysis. No crossreactivity was observed with plasma from non-induced control males. A direct enzyme-linked immunosorbent assay (ELISA) was developed by use of the raised anti-vitellogenin. The detection limit for the purified standard vitellogenin by the ELISA method was 5 ng ml-1 Vitellogenin levels were quantified in plasma of pregnant female Zoarces viviparus, which were held in aquaria with different concentrations of the estrogenic compound 4-nonylphenol dissolved in the ambient seawater. A marked effect of exposure to ambient 4-nonylphenol was observed by significant dose-dependent increases in the level of plasma vitellogenin of the pregnant fish as well as of embryos exposed in vitro.

Effects of nonylphenol and 17 beta-oestradiol on vitellogenin synthesis, testicular structure and cytology in male eelpout Zoarces viviparus.

Nonylphenol has been found to be oestrogenic in fish and may influence the reproductive system of male fish. In the present study, the effects of low (10 microg g-1 week-1) and high (100 microg g-1 week-1) doses of nonylphenol and of 17 beta-oestradiol on the synthesis of vitellogenin and on testicular structure and cytology were investigated in male eelpout Zoarces viviparus during active spermatogenesis (May) and late spermatogenesis (June). Twenty-five days after injection, a significant dose-dependent increase in the plasma vitellogenin concentration, measured by enzyme-linked immunosorbent assay, was observed in the treated groups. A highly significant reduction in the gonadosomatic index was observed concomitant with the increase in the plasma vitellogenin concentration. Macroscopically, milt was observed to be present in the control fish, but was sparse or absent in the treated fish. Histological examination using light microscopy revealed severe effects of nonylphenol as well as of oestradiol treatment on testicular structure. Control fish had seminiferous lobules containing spermatogenic cysts and only a few spermatozoa (May) or had the walls of their seminiferous lobules lined with cuboidal Sertoli cells (June). In the treated fish, the seminiferous lobules were degenerated (May) or were filled with numerous spermatozoa and the Sertoli cells appeared very squamous (June). Electron microscopy revealed greater numbers of phagocytosed spermatozoa in these Sertoli cells. In rats, -glutamyl transpeptidase (-GTP) has been used as a specific marker of Sertoli cell function. In the present study, both nonylphenol and 17 beta-oestradiol treatment resulted in a reduction in the activity of this enzyme. The study provides evidence that nonylphenol is oestrogenic, as indicated by the large increase in vitellogenin synthesis, and that both nonylphenol and oestradiol have marked effects on the testicular structure and cytology of germ cells and Sertoli cells of male Z. viviparus.

Ammonia and urea in the maternal-fetal trophic relationship of the viviparous blenny (eelpout) Zoarces viviparus.

A high correlation was observed between the concentration of urea in the maternal plasma and the concentration of urea in the ovarian fluid during post-yolk sac growth of the embryos in the ovary of the viviparous blenny (eelpout) Zoarces viviparus. A high correlation was observed between maternal plasma and ovarian fluid ammonia as well. Ammonia but not urea was excreted to the external medium by the mother fish during pregnancy. Intraovarian loading with either urea or ammonia resulted in a steady decrease from initial high concentrations in the ovarian fluid and a concomitant increase in the maternal plasma levels of both nitrogenous compounds. Injected ammonia was eliminated much faster from the ovarian fluid than urea. No significant effect of any ammonia could be observed on urea excretion rates by embryos in vitro. Patterns of accumulation for urea and ammonia in the external medium were investigated by exposure of embryos in vitro to concentrations of urea and ammonia similar to those normally found in the ovarian fluid. No significant changes could be observed in the external urea or ammonia levels during the experiment. The results indicate that during post-yolk sac development of embryos in vivo, net catabolism of nitrogen-containing organics and formation of urea may be reduced by high ambient concentrations of urea in the ovarian fluid.

Gluconeogenesis in hepatocytes of immature rainbow trout (Oncorhynchus mykiss): control by estradiol.

Estradiol treatment of starving immature rainbow trout dramatically alters the metabolic performance of isolated hepatocytes. One and two weeks postimplantation with estradiol, the rate of de novo glucose synthesis from [14C]alanine is reduced fourfold from 0.4 mumol/g/hr to 0.1 mumol/g/hr, compared with vehicle-injected control fish. After 6 weeks, the rate of glucose production on a gram wet weight basis is identical in both treatment groups, but significantly larger (by 80%) in the estradiol-treated group than in the controls, if expressed normalized to the hepatosomatic index (HSI). Estradiol treatment caused preferential partitioning of alanine carbon into oxidative pathways away from gluconeogenesis, indicated by a significantly lower ratio of glucose production over CO2 production in hepatocytes isolated from estradiol-treated animals. Incorporation of [14C]alanine into acid-precipitable protein is significantly larger in the estradiol-treated group after 2 weeks, and also after 6 weeks, when normalized to the HSI, indicating that part of the protein synthesized in the estradiol-treated groups is vitellogenin. No differences were detected between estradiol-treated animals and control animals in the activities of enzymes associated with gluconeogenesis [phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase (FBPase)] and amino acid metabolism (alanine and aspartate aminotransferases) in the time course investigated (expressed on a wet weight basis). Activities normalized to the HSI are higher in fish implanted with estradiol compared with controls at 2 and 6 weeks. In keeping with the increased potential of hepatocytes for CO2 production from alanine, estradiol treatment doubled and tripled the maximum activity of pyruvate kinase 1 and 2 weeks postimplantation, respectively. Fish were fasted to avoid erratic feeding due to treatments. Superimposed on estradiol actions are effects of starvation: a fourfold increase in the rate of gluconeogenesis, a threefold increase in oxidative flux, and a fivefold increase in the activity of FBPase--all normalized to hepatocyte weight.

Incidence of lung cancer by histological type among asbestos cement workers in Denmark.

OBJECTIVE: A significant twofold increased risk of lung cancer was found among 8000 men employed in the Danish asbestos cement industry between 1928 and 1984. The histological pattern of 104 lung cancer cases was studied with the aim of evaluating a relation between specific morphological types, duration of employment, and time since first employment. METHODS: Age, sex, and calendar time specific incidence of morphological subtypes of lung cancer (adenocarcinoma, squamous cell carcinoma, anaplastic carcinoma, and unspecified malignant tumour) for all Danish men were computed from 1943 to 1984, from data routinely collected by the Danish Cancer Registry. Person-years of observation were counted from 15 years after the date of first employment until date of diagnosis of cancer, death, emigration, or the end of follow up on 31 December 1984. Expected numbers of cases were computed by applying person-years at risk to the appropriate incidence rates. Observed numbers were distributed accordingly and the relative risk calculated. RESULTS: The relative risk for adenocarcinoma was 3.31 (observed (O) 24, expected (E) 7.26), for squamous cell carcinoma 1.67 (O, 37, E, 22.12), for anaplastic carcinoma 1.58 (O, 23, E, 14.53), and for unspecified malignant tumour 1.57 (O, 18, E, 11.46). An increased risk by duration of employment and time since first employment was most pronounced for adenocarcinoma. CONCLUSION: The link between adenocarcinoma and asbestos was confirmed in this, the first study of risk of lung cancer by histological category based on incident cancer cases for a whole population during a 50 year period.

Exposure to cement dust at a Portland cement factory and the risk of cancer.

The relation between exposure to cement dust and cancer was examined in a population of 546 cement workers and a reference population of 858 randomly sampled men of similar age and area of residence. In 1974 all men gave lifelong occupational and smoking histories; information on incidence of cancer in the period 1974-85 was obtained from the Danish Cancer Registry. No increased risk of overall cancer was found among cement workers. Among men with more than 20 years exposure to cement dust, 14 cases of respiratory cancer were observed (observed/expected (O/E) 1.52, 95% confidence interval (95% CI) 0.90-2.57) when compared with all Danish men. Men with 1-20 years exposure had O/E 1.14 (95% CI 0.59-2.19) based on nine cases of cancer. After excluding all men with documented exposure to asbestos during employment in an asbestos cement factory no increased risk of overall cancer or respiratory cancer was found among cement workers compared with white collar workers from the local reference population, using a Cox regression model controlling for age and smoking habits. Relative risks were 0.5 (95% CI 0.1-1.5) and 1.0 (95% CI 0.4-2.6) for men with 1-20 and more than 20 years of exposure to cement dust respectively compared with white collar workers.