Induction of micronuclei and other nuclear abnormalities in mussels exposed to bisphenol A, diallyl phthalate and tetrabromodiphenyl ether-47

Analysis of micronuclei, nuclear buds, bi-polynucleated and fragmented-apoptotic cells was performed in gills of blue mussels exposed for 3 weeks to sublethal concentrations of bisphenol A, diallyl phthalate (for the both nominal concentration 50 ppb) and to tetrabromodiphenyl ether-47 (nominal concentration 5 ppb). Fourteen specimens from each treatment and control group were used for the analysis. Our results demonstrated a significant increase in micronuclei frequency after the treatment with bisphenol A (P=0.0243), diallyl phthalate (P=0.0005) and tetrabromodiphenyl ether-47 (P<0.0001; Mann-Whitney U-test). Induction of bi-nucleated (P=0.0028), fragmented-apoptotic (P=0.0004) cells and nuclear buds (P=0.0101) was found in mussels exposed to tetrabromodiphenyl ether-47 while treatment with diallyl phthalate increased the level of fragmented-apoptotic cells (P=0.0283). Bisphenol A was the only agent that resulted only in induction of micronuclei but not any other kind of nuclear injuries.     

Immunological responses, histopathological finding and disease resistance of blue mussel (Mytilus edulis) exposed to treated and untreated municipal wastewater

This study provides new information on the response of the immune system of Mytilus edulis exposed to untreated and treated sewage, linking immune response to ecologically relevant endpoints, such as disease resistance. Our goal was to assess the potential effects of sewage on the immune system (phagocytic activity and production of cytotoxic metabolites, disease resistance) and gills (light microscope) of mussels through a bioassay and field study in an estuarine receiving environment (RE). A semi-static experiment was developed in a wastewater treatment plant in New Glasgow, NS Canada. Mussels were exposed for 21 days to 12.5%, 25%, 50% and 100% of untreated sewage influent and artificial seawater control. Sampling occurred after 7, 14 and 21 days of exposure. In the field study, eight sites were selected in East River and Pictou Harbour, NS, positioned upstream and downstream of sewage effluents outfalls. Caged mussels were exposed to the RE for 90 days (May-July 2005). Mussels were challenged to test their efficiency at eliminating the bacteria, Listonella anguillarium in the bioassay and field studies. The bioassay results showed that higher concentrations of untreated sewage could modulate the immune system of mussels through increased of phagocytic activity (PA), nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) production during 14 days of exposure, and decreased activity and production at 21 days, with the exception of H(2)O(2) production which was high even at 21 days. Mussels exposed to untreated sewage RE also presented a high PA, NO and H(2)O(2) production and lower number of haemocytes compared to mussels from reference sites. In the bacterial challenge, mussels pre-exposed to 100% sewage died 24h after being infected with L. anguillarium, while mussels pre-exposed to 50% eliminated bacteria had a mortality rate of 30%. Mussels from the control, 12.5% and 25% groups eliminated bacteria and no mortality was observed. No significant difference was observed in bacterial clearance in mussels exposed to effluents in the RE. The lesions observed in gills in both studies were: infiltration of haemocytes in the tissue, epithelium proliferation, lamellar fusion and dilated haemolymphatic sinus. In summary, untreated municipal wastewater affected the immune system of blue mussels during 21 days of exposure and the effects were reflected in their capability to resist pathogens. And an immune modulation was observed in mussels exposed to untreated sewage in a RE, but this modulation was not reflected in the mussel's capability in eliminating pathogens.     

Metabolomic analysis revealed that female mussel Mytilus galloprovincialis was sensitive to bisphenol A exposures

Bisphenol A (BPA) is a synthetic compound used in numerous chemicals, such as polycarbonate plastics and epoxy resins, and it can be released into aquatic environment and poses risk on aquatic organisms. In this work, metabolomics was applied to characterize the metabolic responses in mussel Mytilus galloprovincialis exposed to BPA. Our results indicated that the gonad of female mussel was sensitive to BPA exposures (1 and 10 μg/L) for one month. However, no significant metabolic responses were observed in male mussel gonads exposed to these two concentrations of BPA. Overall, this limited study suggested that the gender differences should be considered in marine ecotoxicology.     

Cytotoxic effects of BADGE (bisphenol A diglycidyl ether) and BFDGE (bisphenol F diglycidyl ether) on Caco-2 cells in vitro

Bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) are used as starting substances for the manufacturing of epoxy resins used in internal can coatings. They are obtained by a condensation reaction between epichlorohydrin with bisphenol A and bisphenol F, respectively. These potential endocrine disrupting chemicals are able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal cells. The aim of this study was to characterize BADGE and BFDGE toxicity in Caco-2 cells, in particular, at the cellular and molecular level. Using several approaches, we characterized BADGE- and BFDGE-induced cell toxicity in Caco-2 cells. The treatment was done using different concentrations up to cytotoxic doses and different times of exposure to the agents. We evaluated the effect of these compounds on cell morphology, cell detachment, cell proliferation, F-actin disruption and plasma membrane integrity. Both compounds are able to induce morphological changes, cell detachment from the substratum and to inhibit cell proliferation, being these effects time and dose-dependent. Moreover, BADGE and BFDGE induce F-actin depolymerization, this effect is very potent at 24 h of incubation with the agents and a complete F-actin disruption can be observed at 200 μM BADGE or BFDGE. In addition, cell integrity is not damaged, since neither propidium iodide uptake nor LDH release takes place in Caco-2 cells exposed to high doses of these agents for 24 h.     

Protein responses in blue mussels (Mytilus edulis) exposed to organic pollutants: a combined CYP-antibody/proteomic approach

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.     

Toxicity assessment and vitellogenin expression in zebrafish (Danio rerio) embryos and larvae acutely exposed to bisphenol A,endosulfan, heptachlor,methoxychlor and tetrabromobisphenol A

Organochlorine pesticides and brominated flame retardants, such as tetrabromobisphenol A and polybrominated diphenyl ethers, pose an environmental hazard owing to their persistence, low solubility and estrogenic effects, and concerns have been raised regarding their effects on aquatic biota. In the present study, zebrafish embryos and larvae were used as a model to investigate the sublethal and lethal effects of three different organochlorine pesticides, namely methoxychlor, endosulfan and heptachlor, as well as the flame retardant tetrabromobisphenol A, and its precursor compound bisphenol A. Preliminary data for chemical exposure tests were obtained by determining the 96 h median effective concentration EC₅₀ (hatching rate) and 96 h median lethal concentration LC₅₀. Quantitative polymerase chain reaction was used to investigate the gene expression levels of the biomarker vitellogenin (vtg1) after 96 h exposures to 10, 25, 50 and 75% of the 96 h EC₅₀ value for embryos and 96 h LC₅₀ value for larvae. The use of vtg1 mRNA induction in zebrafish embryos and larvae was found to be a sensitive biomarker of exposure to these organic compounds, and was helpful in elucidating their adverse effects and setting water quality guidelines. Copyright © 2012 John Wiley & Sons, Ltd.     

Human exposure to bisphenol A (BPA)

The plastic monomer and plasticizer bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. BPA is used in the production of polycarbonate plastics and epoxy resins used in many consumer products. Here, we have outlined studies that address the levels of BPA in human tissues and fluids. We have reviewed the few epidemiological studies available that explore biological markers of BPA exposure and human health outcomes. We have examined several studies of levels of BPA released from consumer products as well as the levels measured in wastewater, drinking water, air and dust. Lastly, we have reviewed acute metabolic studies and the information available about BPA metabolism in animal models. The reported levels of BPA in human fluids are higher than the BPA concentrations reported to stimulate molecular endpoints in vitro and appear to be within an order of magnitude of the levels needed to induce effects in animal models.     

Reproductive and behavioral effects of diisononyl phthalate (DINP) in perinatally exposed rats

Diisononyl phthalate (DINP) is a plasticizer abundantly used in consumer products as a substitute for other plasticizers prohibited in certain products due to reproductive toxicity. As anti-androgenic effects of DINP are suspected, DINP effects on reproduction and sexually dimorphic behavior were studied. Pregnant Wistar rats were gavaged from gestation day 7 to postnatal day (PND) 17 with vehicle, 300, 600, 750 or 900 mg DINP/kg bw/day. In fetal testes histopathological effects typical of phthalates were observed. In male offspring, DINP caused increased nipple retention, reduced anogenital distance, reduced sperm motility and increased sperm count. DINP affected spatial learning as female offspring performed better than controls and similarly to control males in the Morris Water Maze, indicating masculinization of behavior in DINP exposed females. These results show that DINP causes anti-androgenic effects on reproductive development, though less potent than DEHP, DBP and BBP, and further safety evaluation of DINP appears warranted.     

Relationship between domoic acid levels in the blue mussel (Mytilus edulis) and toxicity in mice

Monitoring of eastern blue mussels (Mytilus edulis), contaminated with domoic acid, involved mouse bioassays and quantitative analysis using HPLC. Mice undergo a typical scratching syndrome at sublethal as well as lethal doses of domoic acid. The onset of scratching behaviour and time of death in mice were inversely related to the dosage of domoic acid. An LD50 (i.p.) of 3.6 mg domoic acid/kg mouse was calculated. Toxic mussels held in tanks and flushed with uncontaminated sea water showed a decline in domoic acid concentration in mussel tissue with time. In addition, domoic acid concentrations in mussels from two infected rivers declined to negligible levels in 40-50 days under normal environmental conditions. The bulk of domoic acid and toxicity was located in the hepatopancreas which also contained large amounts of chlorophyll-A, an algae biomass indicator, relative to control mussels. These results support the conclusion that domoic acid was the primary causative factor in the shellfish poisonings from Prince Edward Island mussels in late 1987.     

Comparison of the short-term effects of di(2-ethylhexyl) phthalate, di(n-hexyl) phthalate, and di(n-octyl) phthalate in rats

This study compares changes in the livers of rats treated with di(2-ethylhexyl) phthalate (DEHP) and its straight-chain analogs di(n-hexyl) phthalate (DnHP) and di(n-octyl phthalate (DnOP). Groups of rats were fed diets containing 20,000 ppm of one of these compounds. Subgroups were killed after 3, 10, and 21 days, and the livers were examined by histological, cytological, and biochemical methods. The results show considerable differences between the effects of the branched-chain phthalate ester DEHP and its straight-chain analogs. The major effects on the liver following administration of diets containing DEHP were midzonal and periportal accumulation of small droplets of lipid, hepatomegaly accompanied by an initial burst of mitosis, proliferation of hepatic peroxisomes and of smooth endoplasmic reticulum accompanied by induction of peroxisomal fatty acid oxidation, damage to the peroxisomal membranes as evidenced by increased leakage of catalase to the cytosol, and centrilobular loss of glycogen and falls in glucose-6-phosphatase activity and in low-molecular-weight reducing agents. In contrast, diets containing DnHP or DnOP induced accumulation of large droplets of fat around central veins leading, by 10 days, to mild centrilobular necrosis and a very slight induction of one peroxisomal enzyme and an increase in liver weight, but no significant changes in any other parameters which were affected by DEHP.     

Effects of ocean acidification on early life stages of shrimp (Pandalus borealis) and mussel (Mytilus edulis)

Ocean acidification (OA) resulting from anthropogenic emissions of carbon dioxide (CO(2)) has already lowered and is predicted to further lower surface ocean pH. There is a particular need to study effects of OA on organisms living in cold-water environments due to the higher solubility of CO(2) at lower temperatures. Mussel larvae (Mytilus edulis) and shrimp larvae (Pandalus borealis) were kept under an ocean acidification scenario predicted for the year 2100 (pH 7.6) and compared against identical batches of organisms held under the current oceanic pH of 8.1, which acted as a control. The temperature was held at a constant 10°C in the mussel experiment and at 5°C in the shrimp experiment. There was no marked effect on fertilization success, development time, or abnormality to the D-shell stage, or on feeding of mussel larvae in the low-pH (pH 7.6) treatment. Mytilus edulis larvae were still able to develop a shell in seawater undersaturated with respect to aragonite (a mineral form of CaCO(3)), but the size of low-pH larvae was significantly smaller than in the control. After 2 mo of exposure the mussels were 28% smaller in the pH 7.6 treatment than in the control. The experiment with Pandalus borealis larvae ran from 1 through 35 days post hatch. Survival of shrimp larvae was not reduced after 5 wk of exposure to pH 7.6, but a significant delay in zoeal progression (development time) was observed.     

Reproductive function in rats exposed neonatally to bisphenol A and estradiol benzoate.

The reproductive function in rats treated subcutaneously (s.c.) with 300 microg/g bisphenol A or 2 microg/g estradiol benzoate from postnatal Day 1 to 5 was examined after puberty as well as histolopathogic changes in reproductive organs. All male and female rats treated postnatally with estradiol benzoate showed poor reproductive capability, including adverse effects on masculine sexual behavior, and marked histopathologic alterations of the reproductive organs. In addition, estradiol benzoate markedly reduced the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in males. On the other hand, all male and female rats treated postnatally with bisphenol A showed normal reproductive function and no histopathologic abnormalities of reproductive organs. Bisphenol A did not affect the volume of the SDN-POA. These results indicated that neonatal exposure to estradiol benzoate affects reproductive function in male and female rats, and treatment with bisphenol A at a fairly high dose was ineffective if given postnatally to male and female rats.     

Examination of the differential hepatotoxicity of diallyl phthalate in rats and mice

In this study we confirmed that diallyl phthalate (DAP) is more hepatotoxic to rats than to mice, and we demonstrated the same species difference in toxicity for allyl alcohol (AA). The data suggest that the toxicity of DAP probably results from AA cleaved from DAP. To determine if the species difference in susceptibility to hepatotoxicity resulted from differences in the disposition and metabolism of DAP, Fischer-344 rats and B6C3F1 mice were given [14C]DAP, 1, 10, or 100 mg/kg po or 10 mg/kg iv, and placed in metabolism cages for 24 hr. In rats, 25-30% of the DAP was excreted as CO2, and 50-70% appeared in the urine within 24 hr. In mice, 6-12% of the DAP was excreted as CO2, and 80-90% was excreted in the urine within 24 hr. Monoallyl phthalate (MAP), allyl alcohol, 3-hydroxypropylmercapturic acid (HPMA), and an unidentified polar metabolite (PM) were found in the urine of rats and mice dosed with DAP. The polar metabolite was present in the urine of rats dosed with DAP or AA, indicating that the compound is a metabolite of AA. There was no difference between the species in the quantity of AA excreted, but mice excreted more MAP (39 vs 33%), HPMA (28 vs 17%), and PM (20 vs 8%) than rats. Because DAP is metabolized to AA, a potent periportal hepatotoxicant, and because the mouse produced more HPMA than rats, we postulate that the differential hepatotoxicity of DAP is related to the extent of glutathione conjugation with allyl alcohol or acrolein (the active metabolite of AA).     

Study on anti-androgenic effects of bisphenol a diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE) and their derivatives using cells stably transfected with human androgen receptor, AR-EcoScreen.

We studied in vitro hormonal activity of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE), which are used as a material of interior coating for food cans. We also examined related compounds such as 2,2-bis[4-(3-chloro-2-hydroxypropoxy)phenyl]propane (BADGE.2HCl), and bis[4-(3-chloro-2-hydroxypropoxy)phenyl]methane (BFDGE.2HCl) etc. For this purpose, we constructed two stably transfected CHO-K1 cell lines (AR-EcoScreen for androgenic activity and c-luc for cell toxicity evaluation). One stably expresses luciferase with induction of androgen. The other stably expresses luciferase without androgen induction. Also, we have determined the androgenic and anti-androgenic effects of the test chemicals by reporter gene assay with these cell lines. None of the chemicals tested by this assay exhibited androgen agonistic activity. However, BADGE.2HCl and BFDGE.2HCl had the conspicuous antagonistic activity for androgen. These compounds had a high binding affinity for androgen receptor. Furthermore, these two compounds did not show the estrogenic activity in vitro assays. On the contrary, bisphenol A and bisphenol F exhibited anti-androgenic activity in vitro in addition to the estrogenic activity. These results suggest that these chlorohydroxy compounds of BADGE and BFDGE act as androgen antagonist through the process of binding to androgen receptor.     

Migration of BADGE (bisphenol A diglycidyl-ether) and BFDGE (bisphenol F diglycidyl-ether) in canned seafood

Migration of potentially toxic materials used for the lining of commercial can goods remains an important issue, especially with respect to certain types of processed foods. Seafood is one type where more information is needed with respect to other ingredients used for adding value to fishery products. Most cans are internally coated with starters of resins such as bisphenol A diglycidyl-ether (BADGE) and bisphenol F diglycidyl-ether (BFDGE), both considered as toxic compounds. Several seafood products, sardines, tuna fish, mackerel, mussels, cod and mackerel eggs, were manufactured in different conditions changing covering sauce, time and temperature of storage and heat-treated for sterilization in cans. Migration kinetics of BADGE and BFDGE from varnish into canned products were evaluated by HPLC in 70 samples after 6, 12 or 18 months of storage. Results showed that there is no migration of BADGE in tuna fish, sardines, mussels or cod. However, migration of BFDGE occurs in all species, in a storage time-dependent way and content of fat, although migration of these compounds is not affected by sterilization conditions. All samples analyzed presented values lower than 9 mg BADGE/kg net product without exceeding European limits. However, concerning BFDGE migration, European legislation does not allow the use and/or the presence of BFDGE. Main migration takes place in mackerel reaching the highest values, 0.74 mg BFDGE/kg and 0.34 mg BADGE/kg net product, in red pepper sauce.     

Preliminary inter-port study of the quality of environments using physiological responses of invertebrates exposed to chronic trace element and organic contamination in Corsica (Mediterranean Sea)

Ecotoxicology - Port areas are socio-ecosystems impacted by chronic mixture pollution. Some marine species benefit from living there and may be studied to define the ecological state of such...     

Natural radioactivity in the mussel Mytilus galloprovincialis derived from the central Adriatic Sea (Italy)

The aim of this study was to determine background levels of natural radionuclides such as uranium isotopes, (210)Pb, (210)Po, and (40)K in mussels Mytilus galloprovincialis, collected in the central Adriatic Sea along the Marche region as a mechanism to establish a biomonitoring model for human radiation exposure resulting from ingestion of this species. This mussel is an invasive warm-water species largely consumed by the local population and also exported to different countries. Among natural radionuclides, alpha emitters are considered responsible for a significant proportion of the radiation exposure of humans to background radiation, particularly through food consumption. The sampling was conducted in different seasons of the year in order to evaluate the spatial and temporal distribution of the natural radioactivity. Data was also compared to previous findings to corroborate our findings. The mean of activity concentration found was 2.34 +/- 0.61 and 149 +/- 58 Bq/kg dry for total uranium and (210)Po, respectively. In mussels the concentration trend of the studied radionuclides was (40)K > (210)Po > (210)Pb > uranium isotopes. The mean individual dose due to ingestion of mussels for (210)Po was in the range 1.65 yen 10(-2) to 9.20 yen 10(-2) mSv yr(-1). The dose derived from uranium isotopes, (40)K, and (210)Pb was negligible. Data show that mussels may be considered a reliable species model for human biomonitoring for radiation exposure.     

Accumulation of gliotoxin, a cytotoxic mycotoxin from Aspergillus fumigatus, in blue mussel (Mytilus edulis).

A strain of Aspergillus fumigatus has been isolated from sediments of a mussel bed. When cultured in hyper saline conditions (with sea-water), it produces a cytotoxic and immunosuppressive toxin, gliotoxin, which is excreted in an exudate. In order to know if this toxin could represent a risk for shellfish consumers, an experiment of bioaccumulation of gliotoxin in mussel has been carried out. After 6 days of contamination, toxin was accumulated in the meat of the mussels, at a level up to 2.9 microg/mg of extract weight, with a mode of contamination different to the classical digestive process described for a majority of marine toxins, but similar to the contamination mode of domoic acid.     

Acute myelotoxic responses in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Blood cells are derived from a multitiered system of progenitor stem cells that lose their capacity for proliferation and self-renewal as they continue along pathways of differentiation. Since these hematopoietic events can be readily monitored in vivo and in vitro in the mouse, we have utilized this system to examine altered cellular differentiation associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Progenitor cells were suppressed following acute exposure of mice to TCDD at doses as low as 1.0 micrograms/kg body wt. In vitro studies demonstrated that myelotoxicity occurs by a direct inhibition of proliferating stem cells. Genetic studies indicated that the myelotoxic responses to TCDD, both in vivo and in vitro, segregate with the Ah locus. In addition, the in vitro myelotoxicity of various polyhalogenated aromatic hydrocarbon congeners correlated with their previously reported ability to induce hepatic microsomal enzyme activity and to bind to an intracellular receptor for TCDD. TCDD was also found to bind specifically to bone marrow cells from Ah-responsive, but not nonresponsive mice, indicating that bone marrow cells possess a specific receptor for TCDD. These data indicate that the myelotoxic response to TCDD is regulated by the Ah receptor present in the target tissue and demonstrates the utility of this system for examining the cellular and molecular events associated with the toxicity of polyhalogenated aromatic hydrocarbons, the prototype for which is TCDD.