Development of a sandwich enzyme-linked immunosorbant assay for the quantification of vitellogeinin in Bullfrog (Rana catesbeiana)

A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p<0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.     

Effects of waterborne exposure to 4-nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp (Cyprinus carpio)

4-Nonylphenol (NP) has been shown to elicit estrogenic responses both in vivo and in vitro. The mechanism by which NP exerts estrogenic and other endocrine-modulating effects in vivo remains unclear, however. The goal of this study was to evaluate the ability of NP to elicit estrogenic responses through indirect mechanisms of action involving the modulation of endogenous steroid hormone concentrations. Sexually mature male common carp (Cyprinus carpio) were exposed to aqueous NP concentrations ranging from <0.05 to 5.4 microg NP/l for 28-31 d. Approximately 0.5-3.5 ppm of NP was detected in pooled plasma samples or tissue samples from the carp studied. NP exposure did not significantly increase plasma concentrations of 17beta-estradiol (E2), testosterone (T) or vitellogenin (VTG). Excluding outliers, plasma E2 concentrations ranged from <175 to 700 pg E2/ml. T concentrations ranged from 940 to 24,700 pg T/ml plasma. The greatest VTG concentration detected was 52 microg/ml. One-third of the plasma samples tested contained <1 microg VTG/ml. Overall, the results of this study did not support the hypothesis that exposure to waterborne NP can modulate concentrations of steroid hormones in the plasma of sexually mature male carp. The results did, however, raise a number of questions regarding the utility of estradiol equivalent (EEQ) estimates as a means of predicting in vivo effects of estrogenic substances. Furthermore, they provide information regarding the concentrations and variability of E2, T, and VTG in the plasma of sexually mature male carp, which may aid in design and interpretation of future studies.     

A sensitive and reliable quantification method for Bisphenol A based on modified competitive ELISA method

Bisphenol A (BPA), generally known as bisphenols, has been identified as a potential estrogenic substance. BPA must be conjugated to carrier protein and BSA was commonly used. 4,4-Bis(4-hydroxyphenyl) valeric acid (BHPVA) has a bisphenolic structure and a long carbon chain with a reactive carboxyl group on the end. In this study, BHPVA-BSA was used to produce polyclonal antibody against bisphenolic structure, and a modified competitive ELISA method for quantification of BPA was developed. This system was based on BHPVA-BSA for polyclonal antibody production against bisphenolic structure, and BHPVA-HRP for determination of BPA substituting detection antibody in competitive reaction. Recovery was assessed at 10 different concentrations (2-1000 ng/ml) of BHPVA, and the recovery range was from 96.3% to 107.2%. The variation was from 6.2% to 9.8% for intra assay and from 10.1% to 12.6% for inter assay. The quadratic was used to establish the curve regression. The range was found to be between 2 and 1000 ng/ml. This modified competitive ELISA method has proven to be a very useful tool for quantification of BPA without the unexpected interaction of BSA and anti-BSA polyclonal antibody.     

Vitellogenin content in European eel (Anguilla anguilla) in Flanders, Belgium

As part of a large-scale monitoring program of bioaccumulating contaminants in the European eel (Anguilla anguilla) in Flanders (Belgium), we investigated potential effects of xenoestrogens in these fish. The present paper describes the results of the plasma vitellogenin (VTG) content, measured in 142 eels sampled at 20 different locations, in relation to the internal pollution levels. To validate the blood VTG assays, a small number of eels (n=8) was exposed to 10 microg ethinylestradiol/l (EE2) for 9 days. In this experiment, VTG was detected as a protein with a molecular weight of 214 kDa and confirmed by Western blotting. Compared with the solvent controls, significantly higher concentrations of VTG were measured in EE2 exposed eel. However, the VTG content was relatively low compared with other fish species exposed to high concentrations of estrogens. The plasma VTG content of eels from the field study was very low, despite a very high internal load of endocrine disrupters. These results, together with previously published studies, suggest that immature yellow European eel might not be the best sentinel species to study the effects of estrogenic compounds on VTG levels of wild fish populations.     

Measurement of bisphenol A concentrations in human colostrum

Bisphenol A (BPA), an estrogenic endocrine disrupting chemical, has been reported to affect embryos and alter their postnatal development. In the present study, we measured the concentrations of BPA in human colostrum by a competitive enzyme-linked immunosorbent assay (ELISA) with the aim of understanding the present status of BPA burden in human breast milk in Shizuoka, Japan. Human colostral samples were collected from 101 healthy mothers within three days after delivery. The BPA concentrations of colostral samples were estimated by ELISA after the acetonitrile extraction and solid phase extraction column purification. BPA in 101 samples was detected in the concentration range of 1-7 ng ml(-1). The mean concentration of BPA was 3.41+/-0.13 (mean+/-SD) ng ml(-1). This is the first demonstration as to what BPA concentrations are in human colostrum. The BPA concentrations in colostrum were higher than those in blood sera samples obtained from healthy women in a previous study. In our study, there was no significant correlation between the concentrations of BPA in colostrum and the age and parity of mothers.     

Minimal transmission of zearalenone to milk of dairy cows

Milk and plasma levels of zearalenone (ZEN), alpha-zearalenol (alpha-ZEL), beta-zearalenol (beta-ZEL) and conjugated metabolites were determined after feeding lactating cows with ZEN. In those instances where ZEN and alpha- and beta-ZEL were detected in milk or plasma, they occurred only as conjugates hydrolysable by treatment with a mixture of beta-glucuronidase and aryl sulfatase. With studies where 50 or 165 mg was fed daily to three cows for 21 day periods, neither dosage showed the presence of ZEN or metabolites in either milk or plasma (detection limits: milk, 0.5 ng/ml, ZEN, alpha-ZEL; 1.5 ng/ml, beta-ZEL; plasma, 2-3 times higher). A dose of 544.5 mg zearalenone per day given to a single cow for 21 days yielded maximum concentrations of only 2.5 ng ZEN/ml and 3.0 ng alpha-ZEL/ml in the milk. In plasma, up to 3 ng ZEN/ml could be detected during the initial 4 days of treatment. At a dose of 1.8 g of zearalenone given over a one day feeding period, maximum milk levels of 4.0 ng ZEN/ml, 1.5 ng alpha-ZEL/ml, and 4.1 ng beta-ZEL/ml were observed during the initial 2 days; corresponding maximum levels after a one day dose of 6.0 g zearalenone were 6.1, 4.0 and 6.6 ng/ml milk on days 2-3. In plasma, peak ZEN concentrations (9 and 13 ng/ml at the lower and higher one-day doses, respectively) occurred 12 hr after initial dosing, and declined to negligible levels by days 5-7. Neither alpha- nor beta-ZEL were detected in plasma. Since measurable levels required very high oral doses of ZEN, milk would not normally pose a human health hazard as a result of feeding rations containing ZEN to lactating dairy cows.     

Toxic effects of chlorpromazine on Carassius auratus and its oxidative stress

Under laboratory conditions, ecotoxicological effects of chlorpromazine (CPZ) on freshwater goldfish (Carassius auratus) were examined using the toxic culture experiment. The results showed that the median lethal concentration (LC(50)) of CPZ toxic to Carassius auratus in 24, 48 and 96 h was 1.11, 0.43 and 0.32 mg/L, respectively. Thus, CPZ is an extreme toxicant to goldfish. Furthermore, there were significantly positive correlations between the ecotoxicological effects of CPZ and its concentrations, and the toxicity became higher as the exposure time increased. The activity of superoxide dismutase (SOD) and catalase (CAT) in goldfish livers was significantly influenced by CPZ. At the same exposure time, the activity of SOD reduced first, and increased then, whereas the activity of CAT enhanced first and decreased then. At the same exposure levels of CPZ, the activity of SOD and CAT changed similarly, decreased first, then increased and decreased at last. Within the range of exposure concentrations, the changes in the activity of CAT can more easily reflect the oxidation stress in Carassius auratus by CPZ than those of SOD.     

Plasma vitellogenin in male teleost fish from 43 rivers worldwide is correlated with upstream human population size

It has been previously demonstrated that vitellogenin (VTG) - a precursor egg yolk protein - is produced in male fish exposed to estrogenic compounds in wastewater treatment plant (WWTP) effluent. However, little attention has been given to examine whether any patterns of male VTG production exists across fish species on a global scale. We hypothesized that a composite measure of human population size over river discharge would best explain variations of protein levels in male fish. We compiled VTG data in 13 fish species from 43 rivers receiving municipal WWTP effluent on 3 continents. We found that human population size explained 28% of the variation in male VTG concentrations, whereas population/flow rate failed to significantly correlate with VTG. We suggest this result may be explained by the low solubility of estrogenic compounds, resulting in localized contamination near WWTP outfalls, rather than dilution by river water.     

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.     

Biological response of high-back crucian carp (Carassius auratus) during different life stages to wastewater treatment plant effluent

This study presents the adverse effects of endocrine disrupting chemicals (EDCs) in effluent of wastewater treatment plants (WwTPs) on fish health. A study of chronic exposure to WwTPs effluent for 10 months was undertaken in high-back crucian carp (Carassius auratus) during different life stages, covering early-life-stage (ELS), prespawning period, and postspawning period. Condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) levels were employed as indicators to assess biological effects of effluent on this gynogenesis species. Meanwhile, some high-back crucian carp were caged in Demonstration Base of Biological Purification for Filter-feeding Fish (hereinafter, Demonstration Base), as WwTPs effluent exposure controls. In the meantime, a depuration study was carried out to determine whether or not the estrogenic effects caused by effluent exposure could be reduced after moving fish into EDCs-free water. CF, HSI, GSI, and plasma Vtg levels of high-back crucian carp caged in Demonstration Base were generally in accordance with seasonal change. Effluent exposure inhibited gonadal growth, reducing GSI in ELS while increasing it around spawning, sharpened liver burdens, increasing HSI, and induced abnormal Vtg expression in juvenile high-back crucian carp, augmenting Vtg concentrations in plasma. Around spawning period, Vtg in high-back crucian carp were mainly induced by endogenous estrogens, and EDCs in effluent had less influence on them. Staying in EDCs-free water for 30 days made high-back crucian carp recover from effects of previous effluent exposure, relieving inhibition of gonadal development and hypertrophy of liver as well as reducing Vtg induced by EDCs in effluent. The results revealed that high-back crucian carp in ELS are more sensitive to WwTPs effluent exposure. Additionally, the depuration study showed a clearance of the estrogenic effects caused by effluent.     

Monoclonal antibody produced by heterologous indirect enzyme-linked immunosorbent assay and its application for parathion residue determination in agricultural and environmental samples

A heterologous indirect enzyme-linked immunosorbent assay (ELISA) was developed, which was based on monoclonal antibody (Mab) to determine parathion residue in agricultural and environmental samples. Eight Mabs were produced by introducing the heterologous indirect ELISA into the screening procedure. It was shown that these Mabs had more broad-reactivity with twenty competitors than that of 5H7 (Mab produced by homologous screening). So it became much easier using these new Mabs to develop heterologous immunoassays. In addition, all the developed heterologous ELISAs could be used to determine parathion residue in semiquantitative or quantitative level, and their detection limitation (LOD) was around 2 ng/mL. Compared with the previous heterologous ELISA (hapten 11/5H7) with IC₅₀ of 13.3 ng/mL, a better heterologous ELISA (hapten 11/1E1) was obtained with IC₅₀ of 3.8 ng/mL, which improved the sensitivity 3.5 times. Finally, the latter was applied to parathion residue determination in spiked agricultural and environmental samples without extraction or cleanup. The average recoveries of parathion added to water, soil, cucumber, Chinese cabbage and carrot were between 80.4 % and 111.8 %. The LOD for water and soil samples was 5 ng/mL, and the LOD for cucumber, rice and corn samples was 10 ng/mL.     

Development of enzyme-linked immunosorbent assays for plasma vitellogenin in Chinese rare minnow (Gobiocypris rarus)

Due to the wide occurrence of endocrine disrupting chemicals in the environment, it is much of importance to develop high throughput screening method for the analysis of this kind of pollutants. Using anion-exchange membrane chromatography, vitellogenin (VTG) from the plasma of 17β-estradiol (E₂) treated Chinese rare minnow was rapidly purified within 15 min. Both polyclonal antibody (PcAb) and monoclonal antibody (McAb) against rare minnow VTG (R-VTG) were prepared in rabbit and Balb/c mice, respectively. The competitive enzyme-linked immunosorbent assays (ELISA) based on either PcAb or McAb were developed to identify and quantify R-VTG in the plasma, and these two methods showed similar characteristics. The detection limits of both assays were lower than 3 ng mL⁻¹ with the working ranges covering three magnitudes. The recovery efficiencies of PcAb and McAb based ELISA were 104.2% and 102.6%, respectively; and the intra-assay and inter-assay of these two assays were 6.2% and 9.2%, 8.6% and 12.8%, respectively. These results indicated that the described competitive ELISA methods were sensitive and valuable tools for quantifying vitellogenin in rare minnow plasma. These methods were then applied to measure R-VTG concentrations in plasma of male fish exposed to a series of E₂ concentrations. When E₂ levels were less than 10 ng L⁻¹, R-VTG levels in plasma were comparable to that in solvent control, while R-VTG levels significantly increased 15-folds and 350-folds, respectively when E₂ exposure concentrations were controlled at 10 and 50 ng L⁻¹. The high sensitivity of Chinese rare minnow to E₂ was demonstrated, making it a valuable model species to study environmental estrogens.     

Input/output balance of estrogenic active compounds in a major municipal sewage plant in Germany

24 h samples of untreated and treated wastewater were taken in parallel from a modern municipal sewage plant in southern Germany in March and June 1998. After solid phase extraction, total estrogenic activity was quantitatively measured with a miniaturized E-screen assay and the levels of nine estrogenic phenolic chemicals analyzed by HRGC/LRMS. 17Beta-estradiol equivalent concentrations (EEQ) were 58 and 70 ng/l in the influent and 6 ng/l in the effluent, indicating that the load of estrogenic activity of the wastewater was reduced by about 90% in the sewage plant. Less than 3% of the estrogenic activity was found in the sludge. 4-t-octylphenol, 4-nonylphenol, bisphenol A, 2-hydroxybiphenyl, and 4-chloro-3-methylphenol were detected in the untreated wastewater at levels from 0.13 to 3.6 microg/l. 4-t-octylphenol, 4-nonylphenol, and bisphenol A were present in the effluent at concentrations from 0.16 to 0.36 microg/l, 2-hydroxybiphenyl and 4-chloro-3-methylphenol were not detectable. The contribution of the quantified levels of phenolic xenoestrogens to total estrogenic activity in the sewage was 0.7-4.3%.     

Histological alternation and vitellogenin induction in adult rare minnow (Gobiocypris rarus) after exposure to ethynylestradiol and nonylphenol

Adult rare minnow (Gobiocypris rarus) were exposed to 0, 1, 5, and 25 ng/l (nominal concentrations) of 17alpha-ethynylestradiol (EE2) and 3, 10, and 30 microg/l (nominal concentrations) of 4-nonylphenol (NP) under flow-through conditions for a period of 28 d. Low mortality was observed at 5 and 25 ng/l EE2 and the growth of fish reduced significantly at 25 ng/l EE2 compared to controls. However, the gonadosomatic indices (GSI) of male fish were significantly higher in 1 ng/l EE(2) treatments and in 10 and 30 microg/l NP treatments (p<0.05). Renal somatic indices (RSI) of male fish in EE2 treatments were significantly higher than those in controls (p<0.05). In contrast, significantly decreased GSI and RSI of female fish could only be observed in 5 and 25 ng/l EE(2) treatments (p<0.05). Hepatosomatic indices (HSI) of male fish were significantly higher in 25 ng/l EE(2) treatments. However, significantly increased of HSI of female fish could only be observed in 1 ng/l EE(2) treatments. Plasma vitellogenin (VTG) induction could be observed in males after exposed to different concentrations of EE2 and NP, and plasma VTG concentrations in females exposed to 5 and 25 ng/l EE(2) were also significantly higher than in controls (p<0.05). At level higher than 5 ng/l EE2 or 30 microg/l NP, hepatic tissue and renal tissue impairment of males could be observed. The pathological male liver was associated with a hypertrophy of hepatocytes and damages to cellar structure and accumulated eosinophilic material. Renal tissue showed different pathological effects which was reflected by accumulated eosinophilic material, hemorrhages within the kidney tubules and hypertrophy of the tubular epithelia. Also at these levels of exposure, feminization of male fish could be noticed and parts of males manifested the testis-ova phenomenon. Ovaries of female rare minnow in 25 ng/l EE2 treatment group were degenerated. Therefore when exposed to EE2 and NP even at environmental observed concentrations, adverse effects could occur in the reproductive system of adult fishes. The observed hepatic tissue and renal tissue impairment should be due to the induction and accumulation of VTG in organs, especially in males.     

Vitellogenin gene expression in males of the Antarctic fish Trematomus bernacchii from Terra Nova Bay (Ross Sea): a role for environmental cadmium?

Synthesis of vitellogenin (VTG) in male fish is a widely recognized effect for estrogenic pollutants in temperate environments, while similar investigations are still lacking for Antarctic organisms. In this study, a preliminary characterization of vitellogenin gene expression was performed by RT-PCR in the key species Trematomus bernacchii sampled in different phases of reproductive cycle and food availability. Females exhibited the highest gene expression during the spawning period, but VTG mRNA was always detected also in males; a significant increase of gene expression was observed both in males and females at the end of the feeding season. These results were not fully supported by a differential exposure to phyto- or anthropogenic estrogens during the planctonic cycle; on the other side, the endocrine properties of cadmium, naturally elevated in Terra Nova Bay and increasing during algal bloom, would explain both the presence of VTG mRNA in males and the seasonal changes of gene induction. Laboratory exposures did not reveal short-term estrogenic effects of cadmium while an elevated responsiveness of T. bernacchii was observed toward a classical estrogenic receptor agonist (17beta-estradiol). Different hypotheses were considered to suggest delayed endocrine effects of cadmium, including the early interaction with other cellular detoxification systems or alterations at multiple levels of the hypothalamus-pituitary-gonad-liver axis. Although molecular mechanisms of VTG gene expression in males of T. bernacchii remain unclear, obtained results provide interesting insights on this species which should stimulate future research activities.     

Occurrence of estrogenic effects in sewage and industrial wastewaters in Beijing, China

Estrogenic potencies of the effluents or water samples from wastewater treatment plants (WWTPs), industries and hospitals and some receiving rivers in Beijing city were estimated by using a human estrogen receptor recombinant yeast assay. Estrogenic activity of industrial wastewaters was found to range from 0.1 to 13.3 ng EEQ/L and decreased to the range of 0.03-1.6 ng EEQ/L after treatment. Estrogenic activity in WWTP influent ranged from 0.3 to 1.7 ng EEQ/L and decreased to the range of 0.05-0.5 ng EEQ/L after treatment. In the receiving river waters, the estrogenic effect range was 0.1-4.7 ng EEQ/L. These data suggest that treated industrial effluents and WWTP effluents of concern are not the only source of estrogenic pollution in surface waters in Beijing city. EEQ levels in Beijing river water are likely attributable to untreated municipal and industrial wastewaters discharged directly into the river.     

Perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in Flanders (Belgium): relationships with biochemical and organismal effects

A perfluorooctane sulfonic acid (PFOS) assessment was conducted on gibel carp (Carassius auratus gibelio), carp (Cyprinus carpio), and eel (Anguilla anguilla) in Flanders (Belgium). The liver PFOS concentrations in fish from the Ieperlee canal (Boezinge, 250-9031 ng/g wet weight, respectively) and the Blokkersdijk pond (Antwerp, 633-1822 ng/g wet weight) were higher than at the Zuun basin (Sint-Pieters-Leeuw, 11.2-162 ng/g wet weight) and among the highest in feral fish worldwide. Eel from the Oude Maas pond (Dilsen-Stokkem) and Watersportbaan basin (Ghent) had PFOS concentrations ranging between 212 and 857 ng/g wet weight. The hepatic PFOS concentration was significantly and positively related with the serum alanine aminotransferase activity, and negatively with the serum protein content in eel and carp. The hepatic PFOS concentration in carp correlated significantly and negatively with the serum electrolyte concentrations whereas a significant positive relation was found with the hematocrit in eel. Although 13 organochlorine pesticides, 22 polychlorinated biphenyl (PCB) congeners and 7 polybrominated diphenyl ethers (PBDEs) were also measured in the liver tissue, only PCB 28, PCB 74, gamma-hexachlorocyclohexane (gamma-HCH) and hexachlorobenzene (HCB) were suggested to contribute to the observed serological alterations in eel.