Ecological risk assessment of heavy metals in desulfurized seawater discharged from a coal-fired power plant in Qingdao.

Despite the prevalence of discharge of large volumes of heavy-metal-bearing seawater from coal-fired power plants into adjacent seas, studies on the associated ecological risks remain limited. This study continuously monitored concentrations of seven heavy metals (i.e. As, Cd, Cr, Cu, Hg, Pb, and Zn) in surface seawater near the outfall of a coal-fired power plant in Qingdao, China over three years. The results showed average concentrations of As, Cd, Cr, Cu, Hg, Pb, and Zn of 2.63, 0.33, 2.97, 4.63, 0.008, 0.85, and 25.00 µg/L, respectively. Given the lack of data on metal toxicity to local species, this study investigated species composition and biomass near discharge outfalls and constructed species sensitivity distribution (SSD) curves with biological flora characteristics. Hazardous concentrations for 5% of species (HC5) for As, Cd, Cr, Cu, Hg, Pb, and Zn derived from SSDs constructed from chronic toxicity data for native species were 3.23, 2.22, 0.06, 2.83, 0.66, 4.70, and 11.07 µg/L, respectively. This study further assessed ecological risk of heavy metals by applying the Hazard Quotient (HQ) and Joint Probability Curve (JPC) based on long-term heavy metal exposure data and chronic toxicity data for local species. The results revealed acceptable levels of ecological risk for As, Cd, Hg, and Pb, but unacceptable levels for Cr, Cu, and Zn. The order of studied heavy metals in terms of ecological risk was Cr > Cu ≈ Zn > As > Cd ≈ Pb > Hg. The results of this study can guide the assessment of ecological risk at heavy metal contaminated sites characterized by relatively low heavy metal concentrations and high discharge volumes, such as receiving waters of coal-fired power plant effluents.

Bisphenol S Impairs Behaviors through Disturbing Endoplasmic Reticulum Function and Reducing Lipid Levels in the Brain of Zebrafish.

The number of neurotoxic pollutants is increasing, but their mechanism of action is unclear. Here, zebrafish were exposed to 0, 1, 10, and 100 µg/L bisphenol S (BPS) for different durations beginning at 2 h postfertilization (hpf) to explore the neurotoxic mechanisms of BPS. Zebrafish larvae exposed to BPS displayed abnormal neurobehaviors. At 48 and 120 hpf, BPS inhibited yolk lipid consumption and reduced the lipid distribution in the zebrafish brain. Moreover, BPS downregulated the mRNA levels of genes involved in fatty acid elongation in the endoplasmic reticulum (ER) and activated ER stress pathways at 48 and 120 hpf, and KEGG analysis after RNA-seq showed that the protein processing pathway in the ER was significantly enriched after BPS exposure. Exposure to ER toxicants (thapsigargin and tunicamycin), two positive controls, induced neurotoxic effects on zebrafish embryos and larvae similar to those of BPS exposure. These data suggested that BPS and ER toxicants disturbed ER function and reduced brain lipid levels. Continued exposure to BPS into adulthood not only inhibited brain fatty acid elongation and ER function but also caused abnormal swelling of the ER in zebrafish. Our data provide new insights into the neurotoxic mechanism of BPS.

Brightened body coloration in female guppies (Poecilia reticulata) serves as an in vivo biomarker for environmental androgens: The example of 17ß-trenbolone.

In vivo testing systems for environmental androgens are scarce. The aim of this study was to evaluate the potential of male-specific brightened body coloration in female guppies (Poecilia reticulata) to serve as an in vivo biomarker of environmental androgens using 17ß-trenbolone as an example. The high bioaccumulation of 17ß-trenbolone in the skin of female guppies suggests that it is a potential target tissue of environmental androgens. The coloration index, pigment cell ultrastructure, pigment levels, sexual attractiveness, and reproductive capability of female guppies were analyzed following 28 days of exposure to 20 ng/L, 200 ng/L, and 2000 ng/L 17ß-trenbolone. Increases in the coloration index caused by 17ß-trenbolone exposure were attributable to increased pteridine and melanin levels. Decreases in the sexual attractiveness, number of offspring, and survival rate of offspring suggested that the changes in body coloration translated into adverse outcomes. Finally, mRNA sequencing indicated that 17ß-trenbolone increased pteridine levels by activating genomic effects of androgen receptor on xanthine dehydrogenase and increased melanin levels by exerting non-genomic effects targeting microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 1 that were mediated by mitogen-activated protein kinase and calcium signaling pathways. We have derived a robust adverse outcome pathway of environmental androgens, and our findings suggest that indicators at different biological levels related to brightened body coloration in female guppies can serve as less-invasive or noninvasive in vivo biomarkers of short-term exposure to environmental androgens.

Bisphenol S Induces Ectopic Angiogenesis in Embryos via VEGFR2 Signaling, Leading to Lipid Deposition in Blood Vessels of Larval Zebrafish.

Bisphenol S (BPS), used as a bisphenol A substitute, has been detected in various environments. However, the safety of BPS is still unclear. Here, zebrafish embryos were exposed to BPS (0, 1, 10, and 100 µg/L) for 24, 48, 72, 96 h, and 15 days. BPS induced ectopic sprouting of budding blood vessels in embryos, but the blood flow velocity within these lesions was unchanged at 48 h. At 72 h postfertilization (hpf), by observing the subintestinal venous plexus responsible for yolk absorption, we found that VEGFR2 transduced an angiogenic signal and that the subsequent reduction in blood flow velocity inhibited yolk absorption. At 96 hpf, yolk consumption was still delayed because of the disturbed transportation route, resulting in transient extensive lipid retention in the blood vessels. After feeding, obvious atherogenic lipids were discovered in the blood vessels, especially in bends, bifurcations, and stenoses. This dynamic visualization of the pathogenesis demonstrates a plausible mechanistic link between BPS exposure-induced embryonic vessel overgrowth and an increased atherosclerosis risk.

Bisphenol S promotes the cell cycle progression and cell proliferation through ERα-cyclin D-CDK4/6-pRb pathway in MCF-7 breast cancer cells.

Bisphenol S (BPS), exhibiting estrogenic activity, has been reported to promote cell proliferation in MCF-7 breast cancer cells; however, the underlying mechanism remains unclear. In this study, BPS (1-100 µM) significantly promoted cell proliferation in ERα positive MCF-7 cells, but not in ERα negative MDA-MB-231 or SK-BR-3 cells, confirming the important role of ERα in BPS-induced cell proliferation. Results of the flow cytometry analysis indicated that 10 µM BPS promoted MCF-7 proliferation by accelerating G1 to S phase transition of the cell cycle. BPS increased cyclin D1 expression and phospho-retinoblastoma (p-Rb) levels, resulting in the release of E2F transcription factors and the increased expression of downstream cyclin E2 and cyclin A2 genes to promote the cell cycle progression. Moreover, BPS-induced Rb phosphorylation and cell cycle progression were prevented by the ERα inhibitor ICI 182,780 and methylpiperidino pyrazole, as well as cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitor PD 0332991, indicating that the underlying mechanisms involve ERα-dependent pathways but also mediated by cyclin D-CDK4/6. Overall, our result showed, for the first time, that BPS promoted cell cycle progression and cell proliferation through the ERα-cyclin D-CDK4/6-pRb pathway in MCF-7 breast cancer cells. This study provides a novel insight regarding the potential role of cyclin D-CDK4/6-pRb pathway in mediating the proliferative effects of BPS in breast cancer cells.

Cu accumulation, detoxification and tolerance in the red swamp crayfish Procambarus clarkii.

Copper is an essential metal but potentially toxic to aquatic animals at high levels. The present study investigated physiologically adaptive responses to waterborne Cu2+ exposure (0, 0.03, 0.30, 3.00 mg/L) in a representative species of crustaceans, the red swamp crayfish (Procambarus clarkii) for 7 d, followed by a 7-d depuration period. The tissue-specific distribution of Cu showed that crayfish hepatopancreas was the primary accumulating site among internal tissues. During Cu2+ exposure, crayfish repressed the expression level of Cu homeostasis genes (Ctr1, Atox1, copper-transporting ATPase 2, MTF-1/2, and MT) in hepatopancreas to inhibit intracellular Cu transporting. Cu2+-exposed crayfish increased activities of GPx and GST, GSH contents, and mRNA expression of antioxidative enzyme genes (Cu/Zn-sod, cat, gpx, gst) to cope with the Cu2+-induced oxidative stress which accompanied by an increased MDA content. Additionally, after a 7-d depuration, crayfish effectively eliminated excess Cu from hepatopancreas by up-regulating expression level of Cu homeostasis genes, and recovered from oxidative damage by enhancing antioxidative enzyme gene expression (Cu/Zn-sod, cat, gpx, gst) and consuming more GSH, which thereby caused a return of the MDA level to the control value. Overall, our study provided new insights into the regulatory mechanisms of cellular Cu homeostasis system and antioxidative system, contributing to Cu detoxification and tolerance ability exhibited by crayfish under Cu2+ stress and after withdrawal of Cu2+ stress.

2,2'-Dithiobis-pyridine induced reproductive toxicity in male guppy (Poecilia reticulata).

Metal pyrithiones (MePTs) are frequently used antifouling biocides in marine coatings. Their main degradation product, 2,2'-dithiobis-pyridine ((PS)2), has been widely detected in seawater and may pose potential ecological risks. In the present study, sexually mature guppies (Poecilia reticulata) were exposed to (PS)2 at concentrations of 0, 20, 200, and 2000 ng/L for 28 days to investigate its reproductive toxicity. The results showed that (PS)2 significantly reduced testosterone (T) levels, spermatogenic cyst number and sperm motility, impeded spermatogenic cell differentiation in male guppies and delayed embryo development in females. These results indicated that (PS)2 could cause reproductive toxicity in guppies. We also examined mRNA expression of indices involved in the hypothalamic-pituitary-gonadal axis and reproductive behaviors. We found that 200 and 2000 ng/L (PS)2 decreased T synthesis by downregulating 17ßHSD and CYP17 mRNA levels, and upregulating the mRNA level of CYP19a1a, which converted T to 17ß-estradiol. (PS)2 also upregulated GnRH1, FSHß, LHß, and LHR mRNA levels, a positive feedback regulation due to the decrease of T levels in male guppies. Furthermore, (PS)2 significantly decreased CYP19a1b mRNA levels in all three exposure groups and thus reduced the display frequency of male guppies. This study was the first to report that (PS)2 could induce reproductive toxicity, which would provide a basis for future assessment of its ecological risk.

New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin.

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17ß-ethinylestradiol (EE2) was measured using these three methods. Exposure to 10 and 50 ng/L EE2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments.

An effervescence-assisted switchable fatty acid-based microextraction with solidification of floating organic droplet for determination of fluoroquinolones and tetracyclines in seawater, sediment, and seafood.

This study developed a new effervescence-assisted switchable fatty acid-based microextraction combined with solidification of a floating organic-droplet (EA-SFAM-SFO) for simple and rapid determination of fluoroquinolones and tetracyclines in seawater, sediment, and seafood. Five medium-chain fatty acids (pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, and nonanoic acid) were tested as an extraction solvent, given their ability to change between hydrophobic and hydrophilic forms by pH adjustment. As nonanoic acid had the highest extraction recovery (>92%) for the six antibiotics and the ability to transform from liquid to a solidified floating state at low temperature, it was selected as the optimum extraction solvent. The prominent advantages of the newly developed method are: (1) reaction between the procedures salt and fatty acid changed extraction solvent from the hydrophobic to hydrophilic state; (2) bubbling with CO2 greatly increased the contact area between fatty acid and analytes resulting in improved extraction recovery; and (3) solidification of the fatty acid at a low temperature provided good separation and avoided the use of specialized equipment. Single-factor screening and optimization of the main factors were conducted using Plackett-Burman design and central composite design, respectively. The main parameters were optimized as follows: 258 µL fatty acid, 406 µL H2SO4 (98%), 3.9 min vortex time, and 354 µL Na2CO3 (2 mol L-1). Under optimized conditions, limits of detection were 0.007-0.113 µg L-1 or µg kg-1 and extraction recoveries were 82.2%-116.7% for six fluoroquinolone and tetracycline antibiotics in seawater, sediments, and seafood. The newly developed method combines the advantages of effervescence-assisted dispersion, hydrophobic/hydrophilic switchable solvent, and liquid/solid transition induced by low temperature. Overall, the new method is simple, quick, and environment-friendly with low detection limits and high recoveries. Thus, the newly developed method has excellent prospects for sample pretreatment and analysis of antibiotics in marine environmental and food samples. Graphical Abstract ᅟ.

Long-term exposure to bisphenol S damages the visual system and reduces the tracking capability of male zebrafish (Danio rerio).

Bisphenol S (BPS) is widely detected in aquatic environments and in human bodies. BPS has reproductive and thyroid disrupting effects, but its effect on the visual system remains unknown. In the present study, zebrafish embryos were exposed to BPS at concentrations of 1, 10, 100 and 1000 µg l-1 until 120 days post-fertilization in a semistatic system, and the effect of BPS on the visual behavior was examined using the optokinetic response and the optomotor response tests in male zebrafish. The retinal histology, mRNA expression of photoreceptor opsin genes (zfrho, zfblue, zfgr1, zfred and zfuv) and apoptosis-related genes (bax and bcl-2) were also assessed. Long-term BPS exposure decreased the tracking capability of male zebrafish, consistent with structural damage to the retina. BPS induced different amounts of vacuoles in the retinal pigment epithelium, and 1000 µg l-1 BPS exposure decreased the length of the inner plexiform layer, ganglion cell layer and retina, and induced an irregular arrangement of photoreceptor cells. The expression levels of the opsin genes (zfred, zfgr1 and zfrho) were significantly elevated, indicating an enhanced spectral sensitivity to red, green and dim light to compensate for the reduction of the optomotor response. Together, the results showed for the first time that long-term exposure to BPS damaged the structure of male zebrafish retina and reduced their tracking capability.

Bisphenol S exposure impairs glucose homeostasis in male zebrafish (Danio rerio).

Bisphenol S (BPS) is a substitute of the plastic additive bisphenol A (BPA). Its concentrations detected in surface waters and urine samples are on the same order of magnitude as BPA. Human exposure to BPA has been implicated in the development of diabetes mellitus; however, whether BPS can disrupt glucose homeostasis and increase blood glucose concentration remains unclear. We extensively investigated the effects of environmentally relevant concentrations of BPS on glucose metabolism in male zebrafish (Danio rerio) and the underlying mechanisms of these effects. Male zebrafish were exposed to 1, 10, or 100µg/L of BPS for 28 d. Fasting blood glucose (FBG) levels, glycogen levels in the liver and muscle, and mRNA levels of key glucose metabolic enzymes and the activities of the encoded proteins in tissues were evaluated to assess the effect of BPS on glucose metabolism. Plasma insulin levels and expression of preproinsulin and glucagon genes in the visceral tissue were also evaluated. Compared with the control group, exposure to 1 and 10µg/L of BPS significantly increased FBG levels but decreased insulin levels. Gluconeogenesis and glycogenolysis in the liver were promoted, and glycogen synthesis in the liver and muscle and glycolysis in the muscle were inhibited. Exposure to 100µg/L of BPS did not significantly alter plasma insulin and blood glucose levels, but nonetheless pronouncedly interfered with gluconeogenesis, glycogenolysis, glycolysis, and glycogen synthesis. Our data indicates that BPS at environmentally relevant concentrations impairs glucose homeostasis of male zebrafish possibly by hampering the physiological effect of insulin; higher BPS doses also pronouncedly interfered with glucose metabolism.

Impairment of bisphenol F on the glucose metabolism of zebrafish larvae.

Bisphenol F (BPF) is a substitute of bisphenol A in the production of epoxy resin and polycarbonate. Its extensive use in consumer products leads to a wide human exposure at high levels. Although the adverse effects of BPF on animal health are of increasing public concern, its risks on systematic glucose metabolism and blood glucose concentrations still remain largely unknown. Using zebrafish larvae as the model animal, we investigated the disturbance of BPF exposure on glucose metabolism and the underlying mechanisms. Zebrafish larvae at 96 h post fertilization were exposed to 0.1, 1, 10, and 100 µg/L of BPF for 48 h. Compared with the control group, glucose levels of larvae increased significantly in the 10 and 100 µg/L exposure groups, which are associated with enhancement of gluconeogenesis and suppression of glycolysis induced by high doses of BPF. Additionally, both mRNA expressions and protein levels of insulin increased significantly in the 10 and 100 µg/L exposure groups, while transcription levels of genes encoding insulin receptor substrates decreased significantly in these groups, indicating a possibly decreased insulin sensitivity due to impairment of insulin signaling transduction downstream of insulin receptor. Further, compared with BPF alone, co-exposure of larvae to BPF and rosiglitazone, an insulin sensitizer, significantly attenuates increases in both glucose levels and mRNA expressions of a key gluconeogenesis enzyme. Our data therefore indicate impairing insulin signaling transduction may be the main mechanism through which BPF disrupts glucose metabolism and induces hyperglycemia. Results of the present study inform the health risk assessment of BPF and also suggest the use of zebrafish larvae in large-scale screening of chemicals with possible glucose metabolism disturbing effect.

Investigation of the molecular mechanisms of hepatic injury upon naphthalene exposure in zebrafish (Danio rerio).

Naphthalene has been used worldwide as a commercial insecticide for decades, which when detected in the environment can have various negative effects on non-target organism, such as hepatotoxicity. However, the molecular mechanisms of how naphthalene acts to affect the liver in zebrafish (Danio rerio) remains unknown. In this study, we evaluated the potential toxic effects of naphthalene on livers in female adult zebrafish over a 21-day subacute exposure. Global hepatic gene expression was examined by microarrays and the results indicated the regulated genes were associated significantly with vital hepatic injury pathways and GO categories upon naphthalene exposure, such as disruptions in lipid metabolism, inflammatory response, and the carcinogenic processes. According to our observations of liver histology, nuclear enlargement as a potential indicator of cancers and hepatic lipometabolic disorder precisely were supported. The 96 h acute naphthalene tests on Tg(lysC:DsRed) and LiPan lines larvae revealed recruitment of neutrophils by the liver, as well as decreased liver size, which further confirmed hepatic inflammation response to naphthalene exposure. Thus, these findings advance the field of ecotoxicology by unveiling a new role of naphthalene as a leading cause of liver damage and provide potential biomarker-genes for environmental naphthalene monitoring.

Effects of monocrotophos pesticide on cholinergic and dopaminergic neurotransmitter systems during early development in the sea urchin Hemicentrotus pulcherrimus.

During early development in sea urchins, classical neurotransmitters, including acetylcholine (ACh), dopamine (DA), and serotonin (5-HT), play important roles in the regulation of morphogenesis and swimming behavior. However, the underlying mechanisms of how organophosphate pesticides cause developmental neurotoxicity by interfering with different neurotransmitter systems are unclear. In this study, we investigated the effects of 0.01, 0.10, and 1.00mg/L monocrotophos (MCP) pesticide on the activity of acetyltransferase (ChAT), acetylcholinesterase (AChE), monoamine oxidase, the concentration of DA, dopamine transporter, and the transcription activity of DA receptor D1 and tyrosine hydroxylase, during critical periods in cholinergic and dopaminergic nervous system development in sea urchin (Hemicentrotus pulcherrimus) embryos and larvae. At the blastula stages, MCP disrupted DA metabolism but not 5-HT metabolism, resulting in abnormal development. High ChAT and AChE activity were observed at the gastrulation-completed stage and the two-armed pluteus stage, respectively, MCP inhibited ChAT activity and AChE activity/distribution and resulted in developmental defects of the plutei. From the gastrula stage to the two-armed pluteus stage, we found ubiquitous disrupting effects of MCP on ACh, DA, and 5-HT metabolism, particularly at critical periods during the development of these neurotransmitter systems. Therefore, we propose that this disruption is one of the main mechanisms of MCP-related developmental neurotoxicity, which would contribute better understanding insight into the mechanism of MCP pesticide's toxic effects.

Development of an immunosensor for quantifying zebrafish vitellogenin based on the Octet system.

Vitellogenin (Vtg) is a sensitive biomarker for environmental estrogens. In this study, an immunosensor for quantifying zebrafish Vtg was developed using the Octet system. First, Protein A sensors were immobilized with purified anti-lipovitellin (Lv) antibody that demonstrated specificity to Vtg. Then, antibody-coated biosensors were immersed into zebrafish Lv standards and diluted samples. The Octet system measured and recorded kinetic parameters between antigens and captured antibody within 5 min. Sample Vtg concentrations were automatically calculated by interpolating relative binding rates observed with each sample and the immobilized anti-Lv antibody into the developed standard curve. The sensor arrays exhibited a wide linear range from 78 to 5000 ng/mL, and the inter-assay coefficient of variation was 0.66-1.97%. Furthermore, the performance of the immunosensor in detecting Vtg was evaluated by quantifying Vtg induction in juvenile zebrafish exposed to 17ß-estradiol (E2). Compared with conventional immunoassay techniques, the Vtg immunosensor developed based on the Octet system was much simpler and less time-consuming, allowing rapid Vtg quantification within 15 min. Moreover, Protein A sensors could be reused many times to ensure that the assays have high reproducibility. Therefore, we suggest that immunosensors based on the Octet system are an easily operated detection method for ecotoxicological research.

Estrogenic effects associated with bisphenol a exposure in male zebrafish (Danio rerio) is associated with changes of endogenous 17ß-estradiol and gene specific DNA methylation levels.

The binding affinity of bisphenol A (BPA) to estrogen receptors (ERs) is much lower than that of 17ß-estradiol (E2), and whether there are other molecular mechanisms responsible for the estrogenic action of BPA in vivo currently remains unknown. The objective of this study was to explore the potential association between the estrogenic effect induced by bisphenol A in vivo and changes of endogenous E2 and gene specific DNA methylation levels. After a waterborne exposure of male zebrafish to 500, 1000, or 1500µg/L of BPA for 21d, vitellogenin (VTG) concentration in whole body homogenate, plasma E2 and testosterone levels, hepatic ERs mRNA expressions, gonadal cyp19a1a and cyp17a1 mRNA expressions, and methylation levels of hepatic esr1 and gonadal cyp19a1a's promoters were determined. Our results indicated that for the 500 and 1500µg/L treatment groups, VTG might be induced mainly by the elevated E2 levels; increases of E2 levels could be partly explained by the up-regulated expression of gonadal aromatase, mRNA levels of which were found to be negatively related to the methylation levels of both its promoter and one CpG site. In addition, upon BPA exposure, hepatic esr1 mRNA levels were also negatively related to the methylation levels of both its promoter and one CpG site. These observations provide evidence for the non-ERs mediated mechanisms underlying the estrogenic action of BPA on male zebrafish.

A novel enzyme-linked immunosorbent assay based on anti-lipovitellin monoclonal antibodies for quantification of zebrafish (Danio rerio) vitellogenin.

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95-250ng/mL, with a detection limit of 0.78ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E2-treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17ß-estradiol.

Semicarbazide-induced thyroid disruption in Japanese flounder (Paralichthys olivaceus) and its potential mechanisms.

Semicarbazide (SMC) is a carcinogenic and genotoxic substance that has been found in aquatic systems. SMC may also cause thyroid follicular epithelial cell injury in rats, but the thyroid-disrupting properties of SMC and its potential mechanisms remain unclear. In this study, we exposed fertilized eggs of Japanese flounder (Paralichthys olivaceus) to 1, 10, 100, and 1000µg/L SMC for 55 d to assess the impact of SMC exposure on the thyroid system. The number of larvae in each metamorphic stage was counted, the concentrations of whole-body thyroid hormones (THs) 3,5,3'-triiodothyronine (T3) and thyroxine (T4) were measured, and the transcription levels of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis and gamma-aminobutyric acid (GABA) synthesis were quantified. The results showed that 10µg/L SMC significantly increased whole-body T3 levels, and 100 and 1000µg/L SMC markedly enhanced whole-body T4 and T3 levels. Furthermore, 100µg/L SMC exposure shortened the time it took for flounder larvae to complete metamorphosis by 2 d as compared to the control group. Thus, this study demonstrated that SMC exerted thyroid-disrupting effects on Japanese flounder. SMC-mediated stimulation of TH levels was primarily related to transcriptional alterations of pituitary-derived thyroid stimulating hormone ß-subunit (tshß) and hepatic deiodinase (id). In the 10 and 100µg/L SMC exposure groups, the increased TH levels may have resulted from inhibition of TH metabolism caused by down-regulation of id3 mRNA expression, while at 1000µg/L SMC-exposed group, up-regulation of tshß and id1 transcripts was expected to enhance the synthesis of T4 and the conversion of T4 to T3 and, consequently, result in higher T4 and T3 levels. In addition, 1000µg/L SMC-induced down-regulation in glutamic acid decarboxylase gad65 and gad67 transcription may have also contributed to the increased TH levels. The thyroid-disrupting effects of 10 and 100µg/L SMC indicated that environmentally relevant concentrations of SMC posed potential environmental risks to aquatic organisms. Overall, our results demonstrated for the first time that SMC exhibited thyroid-disrupting properties by affecting the HPT axis and GABA synthesis, providing theoretical support for environmental risk assessment.

An in vivo assay performed using multiple biomarkers related to testosterone synthesis and conversion for assessing the androgenic potency of refuse leachate.

Refuse leachate is likely an important source of androgens. However, common in vitro bioassays underestimate the potential androgenic activity of leachate, owing to non-receptor-mediated mechanisms that modify the balance of sex hormones and promote the accumulation of endogenous androgens. This study aimed to develop an in vivo assay by using multiple biomarkers related to testosterone synthesis and conversion for assessing the potential androgenic activity of refuse leachate sampled from a municipal solid waste treatment plant in Qingdao, China. The results indicated that exposure to leachate increased the levels of testosterone and luteinizing hormone, but decreased those of 17ß-estradiol in both male and female goldfish (Carassius auratus), suggesting a potential androgenic activity. Further, Leydig cell hyperplasia and decreased gonadal P450 aromatase mRNA levels were observed; these alterations might promote the biosynthesis of testosterone and hinder the conversion of testosterone to 17ß-estradiol, which in turn enhance testosterone accumulation. Exposure to leachate also resulted in reproductive impairments, including decreased gonadosomatic index and plasma vitellogenin levels of female goldfish, as well as decreased testicular enzyme activities in male goldfish. The integrated use of biochemical, molecular, and histological markers not only improved our understanding of the androgenic effects of leachate but also verified the reliability and validity of the results. Therefore, the in vivo bioassay described in this study might allow the investigation of the androgenic effects of other complex contaminant mixtures in the future.

Preparation of a polyclonal antibody against goldfish (Carassius auratus) vitellogenin and its application to detect the estrogenic effects of monocrotophos pesticide.

Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17ß-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish.