Nitrate-N-mediated toxicological responses of Scenedesmus acutus and Daphnia pulex to cadmium, arsenic and their binary mixture (Cd/Asmix) at environmentally relevant concentrations.

Several biomarkers used for ecological risk assessment have been established for single contaminant toxicity, many of which are less predictive of the influence of media and/or dietary nutrients on toxicity outcomes of contaminant mixtures. In this study, we investigate toxicological responses and life traits of Scenedesmus acutus and Daphnia pulex to heavy metals (cadmium-Cd, arsenic-As, binary mixture-Cd/Asmix) in media and diets with varied nutrient (nitrate-N) conditions (low-LN, median-MN, optimum-COMBO). Results showed that nitrate-N-mediated metal inhibitory effects on growth and productivity of primary producer (S. acutus) were significantly interactive (p < 0.05; effect size, ƞ2≤56 %). Cadmium toxicities (Cd-IC50s) in S. acutus were 1.2×, 5.3×, and 4.3× As-IC50s in LN, MN and COMBO media, respectively, while mixture (Cd/Asmix) toxicities were synergistic in MN medium and partial additivity in COMBO and LN media. Nitrate-N and metal exposure effects on S. acutus nutrient stoichiometry, metal uptake and bioaccumulation were significantly interactive (p < 0.05, ƞ2≤100 %). Moreover, survival of primary consumer (D. pulex) was significantly impaired by single and mixed dietary-metal exposures with greater effect under LN condition coupled with significant interactive effects on reproductive capacity (p < 0.05, ƞ2≤21.2 %) but not on swimming activity. We recommend that nitrate-N-mediated metal exposure effects/toxicity in bioindicator species should be considered during ecological risk assessments.

Trophic-Level Interactive Effects of Phosphorus Availability on the Toxicities of Cadmium, Arsenic, and Their Binary Mixture in Media-Exposed Scenedesmus acutus and Media and Dietary-Exposed Daphnia pulex.

Various anthropogenic activities simultaneously alter essential mineral nutrients and contaminant content in the environment. Depending on essential nutrient conditions, the uptake and effects of contaminants in exposed organisms may be altered. The addressing of ecological risk assessment (ERA) of contaminant mixtures has proven difficult. Furthermore, most assessments involving single contaminant exposures do not consider the interaction of essential nutrients on toxicological end points. Hypotheses for toxicological effects of cadmium (Cd), arsenic (As), and their binary mixture (Cd/Asmix) include alteration under varying dietary and media phosphorus (P) conditions. However, interactive effects and effect size (η2) are largely unknown. Here, we investigated the toxicities of Cd-, As-, and Cd/Asmix-treated media and diets on Scenedesmus acutus (a primary producer) and Daphnia pulex (a primary consumer), under varied media and dietary P conditions [low (LP), median (MP), and optimum (COMBO)]. Our results showed significant (p < 0.05) interactive effects and concentration dependent growth inhibition of S. acutus. The toxicity (at day 7) of Cd against S. acutus was 2×, 11×, and 4× that of As in LP, MP, and COMBO conditions, respectively, while the joint toxicity effects of Cd/Asmix were partially additive in LP and COMBO, and synergistic in MP media. Furthermore, acute lethal toxicity (96 h) of Cd in D. pulex was ∼60× that of As, while Cd/Asmix joint toxicity was synergistic. Chronic toxicity (14 d) in D. pulex showed significant (p < 0.05) interaction of As and P-availability on survival, reproduction, and behavior (distance moved, velocity, acceleration and mobility), while Cd and P availability showed significant interactive effect on rotational behavior. Dose response effects of Cd, As, and Cd/Asmix in S. acutus and D. pulex were either monophasic or biphasic under varying nutrient conditions. This study provides empirical evidence of the interactive effects of media/dietary P and toxic metals (Cd, As, and Cd/Asmix) at environmentally relevant concentrations, emphasizing the need for consideration of such interactions during ERA.

Persistence of elevated concentrations of PM, affiliated pharmaceuticals, and tetracycline resistance genes downwind of feedyards.

Beef cattle feedyards have been identified as sources of large amounts of particulate matter (PM) which may transport affiliated chemicals including steroids, beta agonists, and antibiotics from feedyards into the environment. This study is the first to examine persistence of PM-affiliated pharmaceuticals downwind of feedyards using multiple downwind samples collected at increasing distances from feedyard boundaries (n = 5). Concentrations of antibiotics and ractopamine per gram of PM remained consistent at all downwind locations (out to 4.8 km) whereas concentrations per m3 air decreased significantly at distances between 0.1 and 0.7 km downwind, corresponding to significant decreases in mass of PM. Monensin was present in the highest concentrations of any measured pharmaceutical, with concentrations of 37 µg/g PM (376 ng/m3) air in samples collected within 0.1 km downwind of feedyards. Total copy count of tetracycline resistance genes (tetW, tetQ, tetO, tetM, tetL, and tetB) were also significantly increased in samples collected within 0.1 km downwind of feedyards (106 copies) as compared to samples collected upwind (103 copies) and farther downwind (104 copies) of feedyard boundaries. These results suggest that transport of pharmaceutical-laden PM into the terrestrial environment is occurring primarily via PM deposition within 0.7 km of the feedyard, while aerial transport persists over longer distances (>4.8 km).

Crude oil impairs immune function and increases susceptibility to pathogenic bacteria in southern flounder.

Exposure to crude oil or its individual constituents can have detrimental impacts on fish species, including impairment of the immune response. Increased observations of skin lesions in northern Gulf of Mexico fish during the 2010 Deepwater Horizon oil spill indicated the possibility of oil-induced immunocompromisation resulting in bacterial or viral infection. This study used a full factorial design of oil exposure and bacterial challenge to examine how oil exposure impairs southern flounder (Paralichthys lethostigma) immune function and increases susceptibility to the bacteria Vibrio anguillarum, a causative agent of vibriosis. Fish exposed to oil prior to bacterial challenge exhibited 94.4% mortality within 48 hours of bacterial exposure. Flounder challenged with V. anguillarum without prior oil exposure had <10% mortality. Exposure resulted in taxonomically distinct gill and intestine bacterial communities. Mortality strongly correlated with V. anguillarum levels, where it comprised a significantly higher percentage of the microbiome in Oil/Pathogen challenged fish and was nearly non-existent in the No Oil/Pathogen challenged fish bacterial community. Elevated V. anguillarum levels were a direct result of oil exposure-induced immunosuppression. Oil-exposure reduced expression of immunoglobulin M, the major systemic fish antibody, and resulted in an overall downregulation in transcriptome response, particularly in genes related to immune function, response to stimulus and hemostasis. Ultimately, sediment-borne oil exposure impairs immune function, leading to increased incidences of bacterial infections. This type of sediment-borne exposure may result in long-term marine ecosystem effects, as oil-bound sediment in the northern Gulf of Mexico will likely remain a contamination source for years to come.

Effects of thyroid endocrine manipulation on sex-related gene expression and population sex ratios in Zebrafish.

Thyroid hormone reportedly induces masculinization of genetic females and goitrogen treatment delays testicular differentiation (ovary-to-testis transformation) in genetic males of Zebrafish. This study explored potential molecular mechanisms of these phenomena. Zebrafish were treated with thyroxine (T4, 2nM), goitrogen [methimazole (MZ), 0.15mM], MZ (0.15mM) and T4 (2nM) (rescue treatment), or reconstituted water (control) from 3 to 33days postfertilization (dpf) and maintained in control water until 45dpf. Whole fish were collected during early (25dpf) and late (45dpf) testicular differentiation for transcript abundance analysis of selected male (dmrt1, amh, ar) and female (cyp19a1a, esr1, esr2a, esr2b) sex-related genes by quantitative RT-PCR, and fold-changes relative to control values were determined. Additional fish were sampled at 45dpf for histological assessment of gonadal sex. The T4 and rescue treatments caused male-biased populations, and T4 alone induced precocious puberty in ∼50% of males. Male-biased sex ratios were accompanied by increased expression of amh and ar and reduced expression of cyp19a1a, esr1, esr2a, and esr2b at 25 and 45dpf and, unexpectedly, reduced expression of dmrt1 at 45dpf. Goitrogen exposure increased the proportion of individuals with ovaries (per previous studies interpreted as delay in testicular differentiation of genetic males), and at 25 and 45dpf reduced the expression of amh and ar and increased the expression of esr1 (only at 25dpf), esr2a, and esr2b. Notably, cyp19a1a transcript was reduced but via non-thyroidal pathways (not restored by rescue treatment). In conclusion, the masculinizing activity of T4 at the population level may be due to its ability to inhibit female and stimulate male sex-related genes in larvae, while the inability of MZ to induce cyp19a1a, which is necessary for ovarian differentiation, may explain why its "feminizing" activity on gonadal sex is not permanent.

Health implications of engineered nanoparticles in infants and children.

BACKGROUND: The nanotechnology boom and the ability to manufacture novel nanomaterials have led to increased production and use of engineered nanoparticles (ENPs). However, the increased use of various ENPs inevitably results in their release in or the contamination of the environment, which poses significant threats to human health. In recent years, extraordinary economic and societal benefits of nanoproducts as well as their potential risks have been observed and widely debated. To estimate whether ENPs are safe from the onset of their manufacturing to their disposal, evaluation of the toxicological effects of ENPs on human exposure, especially on more sensitive and vulnerable sectors of the population (infants and children) is essential. DATA SOURCES: Papers were obtained from PubMed, Web of Science, and Google Scholar. Literature search words included: "nanoparticles", "infants", "children", "exposure", "toxicity", and all relevant cross-references. RESULTS: A brief overview was conducted to 1) characterize potential exposure routes of ENPs for infants and children; 2) describe the vulnerability and particular needs of infants and children about ENPs exposure; 3) investigate the current knowledge about the potential health hazards of ENPs; and 4) provide suggestions for future research and regulations in ENP applications. CONCLUSIONS: As the manufacturing and use of ENPs become more widespread, directed and focused studies are necessary to measure actual exposure levels and to determine adverse health consequences in infants and children.

A multiple endpoint analysis of the effects of chronic exposure to sediment contaminated with Deepwater Horizon oil on juvenile Southern flounder and their associated microbiomes.

Exposure to oiled sediments can negatively impact the health of fish species. Here, we examine the effects of chronic exposure of juvenile southern flounder, Paralichthys lethostigma, to a sediment-oil mixture. Oil:sediment mixtures are persistent over time and can become bioavailable following sediment perturbation or resuspension. Juvenile flounder were exposed for 32 days under controlled laboratory conditions to five concentrations of naturally weathered Macondo MC252 oil mixed into uncontaminated, field-collected sediments. The percent composition of individual polycyclic aromatic hydrocarbons (PAHs) of the weathered oil did not change after mixing with the sediment. Spiked exposure sediments contained 0.04-395mg/kg tPAH50 (sum of 50 individual PAH concentration measurements). Mortality increased with both exposure duration and concentration of sediment-associated PAHs, and flounder exposed to concentrations above 8mg/kg tPAH50 showed significantly reduced growth over the course of the experiment. Evident histopathologic changes were observed in liver and gill tissues of fish exposed to more than 8mg/kg tPAH50. All fish at these concentrations showed hepatic intravascular congestion, macrovesicular hepatic vacoulation, telangiectasia of secondary lamellae, and lamellar epithelial proliferation in gill tissues. Dose-dependent upregulation of Cyp1a expression in liver tissues was observed. Taxonomic analysis of gill and intestinal commensal bacterial assemblages showed that exposure to oiled sediments led to distinct shifts in commensal bacterial population structures. These data show that chronic exposure to environmentally-relevant concentrations of oiled sediments produces adverse effects in flounder at multiple biological levels.

Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 µg/L endosulfan or 0, 2.5, and 5.0 µg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even though expression of XPC and CSA were induced by exposure to UVB or pesticides, XPA was the limiting factor in the NER pathway. Our results suggest that pesticides may increase the accumulation of UVB-induced DNA photo-adducts and one likely mechanism is the alteration of critical NER gene expression. The present study provides important implications for evaluating the combined risks of pesticide usage and potentially increasing UVB radiation in aquatic ecosystems.

Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle feed yards via particulate matter.

BACKGROUND: Emergence and spread of antibiotic resistance has become a global health threat and is often linked with overuse and misuse of clinical and veterinary chemotherapeutic agents. Modern industrial-scale animal feeding operations rely extensively on veterinary pharmaceuticals, including antibiotics, to augment animal growth. Following excretion, antibiotics are transported through the environment via runoff, leaching, and land application of manure; however, airborne transport from feed yards has not been characterized. OBJECTIVES: The goal of this study was to determine the extent to which antibiotics, antibiotic resistance genes (ARG), and ruminant-associated microbes are aerially dispersed via particulate matter (PM) derived from large-scale beef cattle feed yards. METHODS: PM was collected downwind and upwind of 10 beef cattle feed yards. After extraction from PM, five veterinary antibiotics were quantified via high-performance liquid chromatography with tandem mass spectrometry, ARG were quantified via targeted quantitative polymerase chain reaction, and microbial community diversity was analyzed via 16S rRNA amplification and sequencing. RESULTS: Airborne PM derived from feed yards facilitated dispersal of several veterinary antibiotics, as well as microbial communities containing ARG. Concentrations of several antibiotics in airborne PM immediately downwind of feed yards ranged from 0.5 to 4.6 µg/g of PM. Microbial communities of PM collected downwind of feed yards were enriched with ruminant-associated taxa and were distinct when compared to upwind PM assemblages. Furthermore, genes encoding resistance to tetracycline antibiotics were significantly more abundant in PM collected downwind of feed yards as compared to upwind. CONCLUSIONS: Wind-dispersed PM from feed yards harbors antibiotics, bacteria, and ARGs.

Distinct expression profiles of stress defense and DNA repair genes in Daphnia pulex exposed to cadmium, zinc, and quantum dots.

The ever-increasing production and use of nanocrystaline semiconductors (Quantum dots; QDs) will inevitably result in increased appearance of these nanomaterials in the aquatic environment. However, the behavior and potential toxicity of heavy metal constituted nanoparticulates in aquatic invertebrates is largely unknown, especially with regard to molecular responses. The freshwater crustacean Daphnia pulex is a well-suited toxicological and ecological model to study molecular responses to environmental stressors. In this study, D. pulex were exposed for 48 h to sublethal doses of QDs (25% and 50% of LC50) with differing spectral properties (CdTe and CdSe/ZnS QDs) and Cd and Zn salts. Our data suggest that acute exposure to both CdSO4 and Cd-based QDs leads to Cd uptake in vivo, which was biologically supported by the observation of increased expression of metallothionein (MT-1). Furthermore, Cd, Zn, and CdSe/ZnS QDs induced different patterns of gene expression regarding stress defense and DNA repair, which furthers our knowledge regarding which response pathways are affected by nanoparticulate forms of metals versus ionic forms in aquatic crustaceans.

Comparison of cytotoxicity and expression of metal regulatory genes in zebrafish (Danio rerio) liver cells exposed to cadmium sulfate, zinc sulfate and quantum dots.

Recent advances in the ability to manufacture and manipulate materials at the nanometer scale have led to increased production and use of many types of nanoparticles. Quantum dots (QDs) are small, fluorescent nanoparticles composed of a core of semiconductor material (e.g. cadmium selenide, zinc sulfide) and shells or dopants of other elements. Particle core composition, size, shell, and surface chemistry have all been found to influence toxicity in cells. The aim of this study was to compare the toxicities of ionic cadmium (Cd) and zinc (Zn) and Cd- and Zn-containing QDs in zebrafish liver cells (ZFL). As expected, Cd(2+) was more toxic than Zn(2+), and the general trend of IC50-24 h values of QDs was determined to be CdTe < CdSe/ZnS or InP/ZnS, suggesting that ZnS-shelled CdSe/ZnS QDs were more cytocompatible than bare core CdTe crystals. Smaller QDs showed greater toxicity than larger QDs. Isolated mRNA from these exposures was used to measure the expression of metal response genes including metallothionein (MT), metal response element-binding transcription factor (MTF-1), divalent metal transporter (DMT-1), zrt and irt like protein (ZIP-1) and the zinc transporter, ZnT-1. CdTe exposure induced expression of these genes in a dose dependent manner similar to that of CdSO4 exposure. However, CdSe/ZnS and InP/ZnS altered gene expression of metal homeostasis genes in a manner different from that of the corresponding Cd or Zn salts. This implies that ZnS shells reduce QD toxicity attributed to the release of Cd(2+), but do not eliminate toxic effects caused by the nanoparticles themselves.

Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells.

Increasing use of quantum dots (QDs) makes it necessary to evaluate their toxicological impacts on aquatic organisms, since their contamination of surface water is inevitable. This study compares the genotoxic effects of ionic Cd versus CdTe nanocrystals in zebrafish hepatocytes. After 24h of CdSO4 or CdTe QD exposure, zebrafish liver (ZFL) cells showed a decreased number of viable cells, an accumulation of Cd, an increased formation of reactive oxygen species (ROS), and an induction of DNA strand breaks. Measured levels of stress defense and DNA repair genes were elevated in both cases. However, removal of bulky DNA adducts by nucleotide excision repair (NER) was inhibited with CdSO4 but not with CdTe QDs. The adverse effects caused by acute exposure of CdTe QDs might be mediated through differing mechanisms than those resulting from ionic cadmium toxicity, and studying the effects of metallic components may be not enough to explain QD toxicities in aquatic organisms.

Impact of environmental estrogens on nucleotide excision repair gene expression in embryonic zebrafish.

Estrogens and estrogen mimics are aquatic contaminants that can elicit a variety of deleterious effects in exposed fauna. One of the most potent xenoestrogens found in the aquatic environment is 17α-ethinylestradiol (EE(2)), the pharmaceutically derived semi-synthetic hormone found in oral contraceptives and hormone replacement therapies. Exposure to 100 ng/L EE(2) has previously been shown to profoundly decrease functional hepatic nucleotide excision repair (NER) processes in adult zebrafish in correlation with dramatic decreases in the abundance of hepatic XPC and XPA transcripts; however, its effects on these processes in embryos are currently unknown. Because developing organisms are known to have increased sensitivities to endocrine disrupting compounds such as EE(2), the goal of this study was to examine the impacts of estrogen exposure on mRNA expression of these two key NER genes in zebrafish embryos during the first 4 days of development. Embryos were exposed from 0 h post fertilization (hpf) to waterborne EE(2), its major metabolite, estrone (E(1)), or combinations of the two compounds and sampled at 12, 24, 48, 72 and 96 hpf. Increased abundance of vitellogenin-1 (VTG1) mRNA, a bioindicator of estrogen exposure, was evident as early as 24 hpf in embryos that were co-exposed to EE(2) and E(1) and this effect was sustained throughout 96 hpf. Embryos exposed to EE(2) alone exhibited elevated VTG1 beginning at 72 hpf. In contrast to observations from adult zebrafish exposed to EE(2), embryos did not show any change in mRNA abundance of the excision repair gene, XPC, during the first 4 days of development. However, co-exposure to EE(2) and E(1) elicited an increase in XPA mRNA abundance at 48 and 72 hpf, which was the opposite response as that observed in exposed adults where hepatic XPA mRNA abundance decreased after EE(2) exposure. These differences between embryos and adults suggest that alteration of NER gene transcription by EE(2) is operating under different stimuli during development.

Zinc hyperaccumulation in squirrelfish (Holocentrus adscenscionis) and its role in embryo viability.

Female squirrelfish (Fam. Holocentridae) can accumulate and temporarily sequester copious amounts of zinc (Zn) in their livers. There, it is initially compartmentalized before a subsequent, estrogen-triggered redistribution to the ovaries. Here we show that cellular uptake of Zn is also influenced by estrogen signaling, and that estrogen increases concentrations of the plasma Zn-binding protein vitellogenin (VTG). However, estrogen-mediated increases in VTG are not sufficient to accommodate the magnitude of hepato-ovarian Zn transfer in female squirrelfish (Holocentrus adscensionis). These findings suggest that holocentrids have acquired the ability to use hormonal cues to drive hepatic uptake and storage of Zn, signal for its physiological redistribution, and influence the capacity for systemic transport of Zn beyond the mediation of increased plasma VTG concentrations. Such specific adaptations suggest an advantage for the oocyte, which is corroborated in further studies where we determined that oocyte Zn concentrations are positively correlated with egg viability in captive-spawned squirrelfish. The novel nature of these findings underlies the importance of Zn in squirrelfish reproductive biology.

Effects of cyanobacterial lipopolysaccharides from microcystis on glutathione-based detoxification pathways in the zebrafish (Danio rerio) embryo.

Cyanobacteria ("blue-green algae") are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems.

Efficacy of pharmacological estrogen receptor antagonists in blocking activation of zebrafish estrogen receptors.

A variety of pharmacological agonists, antagonists and selective estrogen receptor modulators (SERM) have been used to better understand the role of specific receptors in various physiological processes. Despite similar structure and function, less is known about the effect of agonists and antagonists on teleost estrogen receptors and the results of these studies have indicated wide variation among species. The goal of this study was to determine the ability of two human SERMs to modulate activation of three zebrafish estrogen receptor isoforms. Full length cDNA of zebrafish estrogen receptor 1 (esr1), estrogen receptor 2a (esr2a) and estrogen receptor 2b (esr2b) were cloned into expression vectors and transfected into cells that do not endogenously express any estrogen receptor along with an estrogen responsive luciferase vector. Cells transfected with any of the zebrafish estrogen receptors individually and then exposed to 17ß-estradiol (E2) or 17α-ethinylestradiol (EE2) exhibited a dose dependent increase in luciferase activity. None of the pharmacological antagonists, ICI 182, 780, methyl-piperidino-pyrazole (MPP) or pyrazolo [1,5-a] pyrimidine (PHTPP), were able to independently transactivate luciferase expression with any of the zebrafish estrogen receptors. Of the three ER antagonists, only ICI 182, 780 was able to block EE2 induced luciferase activity, although a 10 to 100-fold excess of ICI 182, 780 was necessary with all receptors. Neither MPP nor PHTPP were able to block EE2 induced luciferase activity with any isoform of zebrafish estrogen receptor. These results indicate that the difference between human ER and zebrafish ER ligand binding is not conserved enough for the SERMs MPP or PHTPP to elicit similar effects in zebrafish as those manifested in humans.

Cyanobacterial LPS potentiates cadmium toxicity in zebrafish (Danio rerio) embryos.

Cyanobacteria are prevalent in the freshwater environment, reaching critical mass in harmful algal blooms. These organisms produce a variety of toxins including endotoxins such as lipopolysaccharides (LPS), which have been previously shown to decrease glutathione-S-transferase (GST) activity in zebrafish (Danio rerio) embryos. GST plays a vital role in detoxification response during oxidative stress and provides a first line of defense after toxic heavy metal insult, before increased metallothionein expression. Although some attention has focused on cyanobacterial LPS, little research has focused on effects of concurrent exposures with other toxicants. Because cyanobacterial LPS can alter detoxification enzymes including GST, we hypothesized that cyanobacterial LPS could potentiate metal toxicity. This study investigated the effects of LPS from two cyanobacterial species, Lyngbya spp. and Microcystis aeruginosa, on cadmium toxicity in zebrafish embryos. Forty-eight-hour CdCl(2) LC(50) values showed that coexposure of cadmium and Lyngbya LPS or Microcystis LPS resulted in significantly increased cadmium toxicity in comparison with cadmium alone. However, increased cadmium toxicity was not due to decreased GST activity as initially hypothesized. In concurrent Microcystis LPS-cadmium exposures, GST activity was significantly increased in comparison with control embryos at all time points and cadmium concentrations sampled. Concurrent Lyngbya LPS-cadmium exposures also resulted in increased GST activity at most exposure concentrations. These results indicate that regardless of mechanism, cyanobacterial LPS can potentiate the toxic effects of heavy metals. This represents a significant risk for aquatic organisms exposed to combinations of LPS and metals in the environment.

Activity of Phase I and Phase II enzymes of the benzo[a]pyrene transformation pathway in zebrafish (Danio rerio) following waterborne exposure to arsenite.

The environmental pollutants inorganic arsenic (iAs) and benzo[a]pyrene (B[a]P) are carcinogens often found together in groundwater. The hepatic metabolism of B[a]P is a multi-step process requiring several Phase I and Phase II enzymes, notably cytochrome p450 1A (CYP1A), epoxide hydrolase (EH), and glutathione S-transferase (GST). The purpose of this study was to examine the effect of arsenite (As(III)) on the activity of these enzymes in vivo utilizing adult zebrafish (Danio rerio). Zebrafish were exposed to either 0.4 microM B[a]P, 0.4 microM B[a]P+0.4 microM As(III), 0.4 microM B[a]P+8 microM As(III), 0.4 microM As(III), or 8 microM As(III) for 7 days. Co-exposures to As(III) and B[a]P led to significant decreases in CYP1A enzyme activity (approximately 3-fold) when compared to exposure to B[a]P alone. No similar effects occurred with EH or GST, although B[a]P exposure did significantly increase EH activity. Furthermore As(III) and B[a]P co-exposures significantly decreased CYP1A transcript levels (up to 35-fold) when compared to B[a]P. However, B[a]P-induced CYP1A protein levels remained elevated following co-exposures to As(III). This evidence suggests that As(III) has the potential to modify components of the B[a]P biotransformation pathway in vivo via a disruption of CYP1A activity by way of both pre- and post-translational mechanisms.

Wastewater treatment effluent alters nucleotide excision repair in zebrafish (Danio rerio).

Wastewater treatment effluent is a complex mixture of anthropogenic pollutants including estrogenic substances and chemicals, such as polyaromatic hydrocarbons, that cause bulky DNA adducts. Significant research focuses on reproductive effects of aquatic estrogens from wastewater treatment plants. However, other studies suggest genotoxic and immunological effects occur at lower concentrations of wastewater treatment effluent than reproductive endpoints. Recently, effects of estrogen on DNA repair processes in zebrafish have been suggested as a possible mechanism by which estrogen can modulate incidence of DNA mutations. Because wastewater treatment facilities are a significant source of estrogenic compounds, we hypothesized that exposure to whole effluents would also affect DNA repair in zebrafish (Danio rerio). Exposure to effluent from multiple treatment facilities in northern Maine decreased repair of DNA adducts in zebrafish liver cells. Expression of two nucleotide excision repair genes, XPC and XPA, were quantified and showed varied response after exposure in adult male zebrafish. Evidence of estrogen and aryl hydrocarbon receptor activation after exposure varied between treatment plants and temporally within treatment plants when evaluated using a traditional biomarker, vitellogenin-1 (VTG) and, cytochrome p450 1A1 (CYP1A1) mRNA abundance. This research highlights the continuing importance of examining non-reproductive effects of wastewater treatment effluent.

17alpha-Ethinylestradiol hinders nucleotide excision repair in zebrafish liver cells.

Nucleotide excision repair (NER) is the primary mechanism that removes bulky DNA adducts such as those caused by ubiquitous environmental mutagens including benzo(a)pyrene and other polycyclic aromatic hydrocarbons. Recent data suggest that exposure to environmentally relevant concentrations of estrogen decreases hepatic mRNA abundance of several key NER genes in adult zebrafish (Danio rerio). However, the impact of decreased hepatic NER expression on NER function was not investigated in the previous study. The goal of this study was to examine the effect of the potent estrogen receptor agonist 17alpha-ethinylestradiol (EE(2)) on rate and magnitude of bulky DNA adduct repair. Here we show that exposure of zebrafish liver (ZFL) cells to physiologically relevant concentrations of EE(2) resulted in reduced ability of ZFL cells to repair damaged DNA in comparison to control cells. Co-exposure to EE(2) and a complete estrogen receptor antagonist (ICI 182,780) also resulted in reduced NER capacity, whereas ICI 182,780 alone did not affect the ability of ZFL cells to repair UV damage. These results indicate that estrogen exposure can decrease cellular NER capacity and that this effect can occur in the presence of an estrogen receptor antagonist, suggesting that EE(2) can affect NER processes through mechanisms other than nuclear estrogen receptor activation.