4-n-Nonylphenol and 17-beta estradiol may induce common DNA effects in developing barnacle larvae

There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17beta-estradiol (E2) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E2 induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E2 induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA levcl may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility.     

Juvenile sea bass biotransformation, genotoxic and endocrine responses to beta-naphthoflavone, 4-nonylphenol and 17 beta-estradiol individual and combined exposures

Juvenile sea bass, Dicentrarchus labrax L., were exposed during 2, 4, 8, and 24 h to 0.9 microM beta-naphthoflavone (BNF), 131 nM 17 beta-estradiol (E(2)), 4.05 microM 4-nonylphenol (NP), as well as to BNF combined either to E(2) or NP (maintaining the previous concentrations). Liver cytochrome P450 content (P450), ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST) activities were measured in order to evaluate biotransformation responses. Genotoxicity was assessed as erythrocytic nuclear abnormalities (ENA) frequency. The effects on endocrine function were evaluated as plasma cortisol and glucose. Cortisol was not affected by xeno/estrogens tested, either in single exposure or mixed with BNF. Nevertheless, the intermediary metabolism was affected since glucose concentration increased after 4 h exposure to E(2), and after all BNF+NP exposure lengths. Moreover, a synergism between BNF and NP was thoroughly demonstrated, whereas a sporadic antagonistic interaction was found at 4 h BNF + E(2) exposure. Liver EROD and GST activities were not significantly altered by single E(2) or NP exposure. However, both compounds were able to induce EROD activity in the presence of BNF. NP single exposure was able to significantly increase liver P450 content, while its mixture with BNF displayed an antagonistic interference. Considering the xeno/estrogens single exposures, only NP induced an ENA increase; however, both mixtures (BNF + E(2) and BNF + NP) displayed genotoxic effects. Fish responses to mixtures of xenobiotics are complex and the type of interaction (synergism/potentiation or antagonism) in a particular mixture can vary with the evaluated biological response.     

Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-n-nonylphenol

Chironomid mouthpart deformities have often been associated with sediment contamination and are, therefore, currently used to assess sediment quality. Deformities were only occasionally induced in laboratory bioassays. Mouthpart deformities results from a physiological disturbance during larval molting. In the past few years it has been shown that some chemicals can exert negative effects on both vertebrates and invertebrates at the level of endocrine regulation. As insect molting is hormonally regulated, we wanted to test the hypothesis that deformities are induced due to a hormonal disruption in the developmental process. The aim of the present study was to test whether the endocrine disrupter, 4-n-nonylphenol (4NP), induces mouthpart deformities in chironomids. A laboratory bioassay was performed exposing Chironomus riparius larvae to 10, 50 and 100 micrograms l-1 4NP. Survival of the larvae was not affected by the tested concentrations, but the frequency of mentum deformities increased significantly (P < 0.01) after exposure to 4NP.     

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.     

Developmental effects in Japanese medaka (Oryzias latipes) exposed to nonylphenol ethoxylates and their degradation products

The endocrine modulating potency of five alkylphenol compounds to fish, including nonylphenol (NP), three nonylphenol ethoxylate mixtures (NP1EO, NP4EO, NP9EO) and one nonylphenol ethoxycarboxylate (NP1EC) was assessed using in vivo tests conducted with Japanese medaka (Oryzias latipes). Medaka exposed to test materials from 1 day to 100 days post-hatch were monitored for alterations to sex ratios and secondary sex characteristics and development of gonadal intersex (i.e., testis-ova). The treatment with 100 microg l-1 NP (measured 29 microg l-1) induced gonadal intersex in over 80% of exposed males, mixed secondary sex characteristics in over 40% of exposed fish and suppression of the development of papillae on the anal fin of 100% of males. The 30 microg l-1 NP (measured 8.7 microg l-1) treatment induced gonadal intersex in only one of the 22 exposed males and mixed secondary sex characteristics in approximately 20% of the exposed fish. An elevated incidence of fish with mixed secondary sex characteristics and suppression of papillae development was also observed in the treatment with NP1EO at the highest test concentration of 300 microg l-1 (measured 105 microg l-1). There was no evidence of mixed secondary sex characteristics or gonadal intersex in treatments with the remaining test mixtures. This study confirms that NP is an estrogenic compound that could affect gonadal development in fish chronically exposed to concentrations in the range of 10 microg l-1. NP1EO is very weakly estrogenic at concentrations that are an order of magnitude higher than the lowest observed effect concentration for nonylphenol.     

Alteration of steroid hormone profile in juvenile turbot (Psetta maxima) as a consequence of short-term exposure to 17alpha-ethynylestradiol

The synthetic estrogen 17alpha-ethynylestradiol (EE2) is among the most potent xenoestrogens found in treated sewage. Adverse effects have been reported in fish exposed to this chemical, such as vitellogenin induction or alteration of gonad structure. In this study, the effects of EE2 on sex steroid profile in juvenile turbot (Psetta maxima) were investigated in a 15-day in vivo experiment. An analytical method was developed for the simultaneous gas chromatography/mass spectrometry determination of 14 steroids in gonads, plasma and bile. The impact of EE2 on the endocrine status of juvenile turbot was sex-dependent, since males were much more sensitive than females. Even at very low concentration (3.5 ng l(-1)), EE2 depressed androgen production as well as plasma levels in males. Androstenedione and 11-ketotestosterone were highly affected by EE2-exposure, since their production was suppressed in EE2-treated males. Furthermore, testis estrogen production and conjugation increased as a consequence of EE2 exposure. Overall, the ratio of androgens to estrogens in EE2-exposed males dropped in testis and plasma and reached values observed in females. These results indicate that juvenile male turbots are susceptible to hormonal imbalance as a consequence of short-term exposure to environmentally relevant EE2 levels.     

Whole-mount in situ TUNEL method revealed ectopic pattern of apoptosis in cadmium treated naupliar larvae of barnacle (Balanus amphitrite Darwin)

The effect of cadmium on stage II naupliar larvae of barnacle (Balanus amphitrite Darwin) was investigated. Barnacle larvae were exposed to 0-15 microM CdCl(2) for 24 h. Apoptotic cells were stained by the terminal deoxyribonucleotidyl transferase mediated dUTP nick end labelling (TUNEL) method. Incidence of apoptosis, as measured by numbers of animals with ectopic pattern of apoptosis as well as numbers of apoptotic cells per animal, was assessed using confocal microscopy. An increase in incidence of apoptosis was observed in the experimental animals with an increase in cadmium concentration. Mortality increased, and motility decreased, when barnacle larvae were exposed to an increasing concentration of cadmium. The relationship between the occurrence of apoptosis and swimming behaviour was investigated in larvae exposed to 10 microM CdCl(2). Significant increases in apoptosis were detected in the non-motile and dead nauplii. This study suggested that whole-mount in situ TUNEL method may be used to study increased occurrence of apoptotic cells in crustacean larvae.     

Impact assessment of cadmium contamination on rice (Oryza sativa L.) seedlings at molecular and population levels using multiple biomarkers

Assessment of environmental contamination on ecology (plant) at molecular and population levels is important in risk quantification and remediation study. Random amplified polymorphic DNA (RAPD) assay and related other fingerprinting techniques have been employed to detect the genotoxin-induced DNA damage and mutations. This research compared the effects occurring at molecular and population levels in rice seedlings exposed to cadmium (Cd) concentrations of 15-60 mg l(-1) for 8 days with quartz sand culture. Inhibition of root growth and increase of total soluble protein content in root tips of rice seedlings were observed with the increase of Cd concentration. For the RAPD analyses, 12 RAPD primers of 50-70% GC content were found to produce unique polymorphic band patterns and subsequently were used to produce a total of 180 bands of 179-3056 bp in molecular size in the control root tips of rice seedlings. Results produced by these RAPD primers indicate that changes in RAPD profiles of root tips after Cd treatment include modifications in band intensity and gain or loss of bands by comparison with control. The effect of changes was dose-dependent. Genomic template stability compares favourably with the traditional indices such as root growth and soluble protein content. The DNA polymorphisms detected by RAPD analysis can be applied as a suitable biomarker assay for the detection of genotoxic effects of Cd contamination on plants.     

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.     

Endocrine disruptors in bottled mineral water: total estrogenic burden and migration from plastic bottles

Background, aim, and scope  Food consumption is an important route of human exposure to endocrine-disrupting chemicals. So far, this has been demonstrated by exposure modeling or analytical identification of single substances in foodstuff (e.g., phthalates) and human body fluids (e.g., urine and blood). Since the research in this field is focused on few chemicals (and thus missing mixture effects), the overall contamination of edibles with xenohormones is largely unknown. The aim of this study was to assess the integrated estrogenic burden of bottled mineral water as model foodstuff and to characterize the potential sources of the estrogenic contamination. Materials, methods, and results  In the present study, we analyzed commercially available mineral water in an in vitro system with the human estrogen receptor alpha and detected estrogenic contamination in 60% of all samples with a maximum activity equivalent to 75.2 ng/l of the natural sex hormone 17β-estradiol. Furthermore, breeding of the molluskan model Potamopyrgus antipodarum in water bottles made of glass and plastic [polyethylene terephthalate (PET)] resulted in an increased reproductive output of snails cultured in PET bottles. This provides first evidence that substances leaching from plastic food packaging materials act as functional estrogens in vivo. Discussion and conclusions  Our results demonstrate a widespread contamination of mineral water with xenoestrogens that partly originates from compounds leaching from the plastic packaging material. These substances possess potent estrogenic activity in vivo in a molluskan sentinel. Overall, the results indicate that a broader range of foodstuff may be contaminated with endocrine disruptors when packed in plastics.     

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.     

Detection of estrogenic activity in municipal wastewater effluent using primary cell cultures from three-spined stickleback and chemical analysis

Environmental estrogens are substances that imitate the effects of endogenous estrogens. Effluents from municipal wastewater treatment plants are known to contain substances with estrogenic activity including steroidal estrogens and xenoestrogens. In the current study, a combination of biological and chemical analysis was applied to determine the estrogenic activity in municipal wastewater effluents in Finland. The male three-spined stickleback (Gasterosteus aculeatus) hepatocyte assay with vitellogenin induction as an endpoint was used for the detection of estrogenic activity in solid phase extracts of wastewater effluents, and 17beta-estradiol (E2) as a positive control. The wastewater extracts and E2 were found to induce vitellogenin production. The extracts were also subjected to chromatographic fractionation and the collected fractions were assayed. The only active fraction was the one in which E2, estrone and ethynylestradiol were eluted. Its activity corresponded to the activity of the original wastewater extract. The LC-MS/MS analyses of the wastewater extracts showed that the concentration of estrone was about 65 ng L(-1), the concentration of E2 was less than 1 ng L(-1), while estriol and 17alpha-ethynylestradiol could not be detected. These findings showed that the activity of the wastewater extracts and the chromatographic fraction was much higher than the activity which could have been expected on the base of the chemical analysis. This strongly indicates that other compounds, possibly acting by additivity or synergism, are playing a major role in the induced vitellogenin production by the hepatocytes.     

Acute toxicity of 353-nonylphenol and its metabolites for zebrafish embryos

Background, aim and scope  Nonylphenol (NP) can be detected in the aquatic environment all over the world. It is applied as a technical mixture of isomers of which 353-NP is the most relevant both in terms of abundance (about 20% of total mass) and endocrine potential. 353-NP is metabolised in sewage sludge. The aims of the present study were to determine and to compare the acute toxicity of t-NP, 353-NP and its metabolites as well as to discuss if the toxicity of 353-NP changes during degradation. Materials and methods  353-NP and two of its metabolites were synthesised. The zebrafish embryo test was performed according to standard protocols. Several lethal and non-lethal endpoints during embryonal development were reported. NOEL, LOEL and EC₅₀ were calculated. Results  All tested compounds caused lethal as well as non-lethal malformations during embryo development. 353-NP showed a higher toxicity (EC₅₀ for lethal endpoints 6.7 mg/L) compared to its metabolites 4-(3.5-dimethyl-3-heptyl)-2-nitrophenol (EC₅₀ 13.3 mg/L) and 4-(3,5-dimethyl-3-heptyl)-2-bromophenol (EC₅₀ 27.1 mg/L). Discussion  In surface water, concentrations of NP are far below the NOEC identified by the zebrafish embryo test. However, in soils and sewage sludge, concentrations may reach or even exceed these concentrations. Therefore, sludge-treated sites close to surface waters should be analysed for NP and its metabolites in order to detect an unduly high contamination due to runoff events. Conclusions  The results of the present study point out that the toxicity of 353-NP probably declines during metabolisation in water, sediment and soil, but does not vanish since the major metabolites exhibit a clear toxic potential for zebrafish embryos. Recommendations and perspectives  Metabolites of environmental pollutants should be included in the ecotoxicological test strategy for a proper risk assessment. An erratum to this article can be found at     

Testicular cancer and occupational exposures with a focus on xenoestrogens in polyvinyl chloride plastics

In a case-control study of 148 cases of testicular cancer and 314 healthy controls information was collected on lifetime working histories and specific exposures. The main finding was a six-fold increase in the risk for seminoma, one type of testicular cancer, among plastic workers exposed to polyvinyl chloride (PVC). No overrisks were observed for other plastics than PVC. There may be an aetiological link to xenoestrogens, i.e. chemicals in the environment with oestrogenic properties. Phthalates are used in PVC as plasticizer and have oestrogenic properties that could promote the growth of endocrine sensitive tumour cells. However, this "endocrine disruptors" hypothesis must be further evaluated in experimental models with regard to the causative mechanisms of the oestrogenic tumour promotion.     

DNA integrity of onion root cells under catechol influence

Catechol is a highly toxic organic pollutant, usually abundant in the waste effluents of industrial processes and agricultural activities. The environmental sources of catechol include pesticides, wood preservatives, tanning lotion, cosmetic creams, dyes, and synthetic intermediates. Genotoxicity of catechol at a concentration range 5?×?10^?1–5 mM was evaluated by applying random amplified polymorphic DNA (RAPD) and time-lapse DNA laddering tests using onion (Allium cepa) root cells as the assay system. RAPD analysis revealed polymorphisms in the nucleotidic sequence of DNA that reflected the genotoxic potential of catechol to provoke point mutations, or deletions, or chromosomal rearrangements. Time-lapse DNA laddering test provided evidence that catechol provoked DNA necrosis and apoptosis. Acridine orange/ethidium bromide staining could distinguish apoptotic from necrotic cells in root cells of A. cepa.     

Metal bioaccumulation and metallothionein concentrations in larvae of Crassostrea gigas

Larval stages of bivalve molluscs are highly sensitive to pollutants. Oysters from a hatchery from Normandy (English Channel) were induced to spawn, and fertilized eggs were exposed to copper or cadmium for 24 h. Metal accumulation (from 0.125 to 5 microg Cu L(-1) and from 25 to 200 microg Cd L(-1)) and MT concentrations were measured in larvae. Compared to controls, larvae accumulated copper and cadmium with an increase in MT concentrations particularly with cadmium (i.e. 130.96 ng Cu (mg protein)(-1) and 12.69 microg MT (mg protein)(-1) at 1 microg Cu L(-1) versus 23.19 ng Cu (mg protein)(-1) and 8.92 microg MT (mg protein)(-1) in control larvae; 334.3 ng Cd (mg protein)(-1) and 11.70 microg MT (mg protein)(-1) at 200 microg Cd L(-1) versus 0.87 ng Cd (mg protein)(-1) and 4.60 microg MT (mg protein)(-1) in control larvae). Larvae were also obtained from oysters of a clean area (Arcachon Bay) and a polluted zone (Bidassoa estuary) and exposed to copper in the laboratory, their MT concentration was measured as well as biomarkers of oxidative stress. Biomarker responses and sensitivity to copper for the larvae from Arcachon oysters were higher than for those from Bidassoa.     

Rapid-behaviour responses as a reliable indicator of estrogenic chemical toxicity in zebrafish juveniles

Whereas biochemical and molecular parameters have been well recognised as important “signposts” of individual disturbance to endocrine disrupting chemical’s (EDCs) exposure, behavioural endpoints are yet greatly overlooked as a routine tool in environmental risk assessment of EDCs. However, life histories are intimately associated with numerous inter- and intra-specific interactions, which invariably depend on the performance of effective behaviours. Within fish species, one of the most important factors influencing energy turnover earlier in the development is locomotor activity. This essential trait reflects the organism’s ability to generate and coordinate the metabolic energy required for both reproductive and non-reproductive behaviours. Inappropriate movement responses due to toxic effects of contaminants may ultimately impact important ecological variables.Therefore, in the present study, the swimming bursts of zebrafish juveniles exposed for 40 d to the synthetic estrogen ethinylestradiol (EE₂), tested at environmentally relevant concentrations (nominal concentrations of 0.5, 1 and 2 ng L⁻¹), were investigated in order to address the potential of rapid-behaviour patterns as an effective response indicator of estrogenic endocrine disrupting chemical’s exposure. This synthetic estrogen was selected due to its high prevalence in aquatic ecosystems, ability to mimic natural estrogens and proven record of causing negative effects in fish reproduction. The behavioural responses were compared with established endpoints used in the screening of EE₂ effects at adulthood. Results indicate that zebrafish juveniles’ swimming activity was significantly decreased upon EE₂ exposure. Since reduced locomotion of zebrafish may impact foraging, predator avoidance, drift and transport, and even interfere with social and reproductive behaviours, a fitness decline of wild fish populations can ultimately be hypothesized. Furthermore, behavioural endpoints were found to display higher sensitivity to EE₂ than either vitellogenin gene induction or reproductive parameters determined at adulthood.Overall, the findings of this work not only demonstrate the power of high-throughput behavioural responses, able to act as sensitive early warning signals of EDC exposure, but also highlight the potential of behavioural endpoints in providing a more comprehensive and non-invasive measure of EDC’s exposure.