A Potential Neurotoxic Mechanism: Bisphenol S-Induced Inhibition of Glucose Transporter 1 Leads to ATP Excitotoxicity in the Zebrafish Brain.

Many environmental pollutants have neurotoxic effects, but the initial molecular events involved in these effects are unclear. Here, zebrafish were exposed to the neurotoxicant bisphenol S (BPS, 1, 10, or 100 µg/L) from the embryonic stage to the larval stage to explore the ability of BPS to interfere with energy metabolism in the brain. BPS, which is similar to a glucose transporter 1 (GLUT1) inhibitor, inhibited GLUT1 function but increased mitochondrial activity in the brains of larval zebrafish. Interestingly, GLUT1 inhibitor treatment and BPS exposure did not reduce energy production in the brain; instead, they increased ATP production by inducing the preferential use of ketone bodies. Moreover, BPS promoted the protein expression of the purinergic 2X receptor but inhibited the purinergic 2Y-mediated phosphatidylinositol signaling pathway, indicating that excess ATP acts as a neurotransmitter to activate the purinergic 2X receptor under the BPS-induced restriction of GLUT1 function. BPS-induced inhibition of GLUT1 increased the number of neurons but promoted apoptosis by activating ATP-purinergic 2X receptors in the brain, causing ATP excitatory neurotoxicity. Our data reveal a potential neurotoxic mechanism induced by BPS that may represent a new adverse outcome pathway.

Bisphenol S causes excessive estrogen synthesis by activating FSHR and the downstream cAMP/PKA signaling pathway.

Estrogen excess in females has been linked to a diverse array of chronic and acute diseases. Emerging research shows that exposure to estrogen-like compounds such as bisphenol S leads to increases in 17ß-estradiol levels, but the mechanism of action is unclear. The aim of this study was to reveal the underlying signaling pathway-mediated mechanisms, target site and target molecule of action of bisphenol S causing excessive estrogen synthesis. Human ovarian granulosa cells SVOG were exposed to bisphenol S at environmentally relevant concentrations (1 µg/L, 10 µg/L, and 100 µg/L) for 48 h. The results confirms that bisphenol S accumulates mainly on the cell membrane, binds to follicle stimulating hormone receptor (FSHR) located on the cell membrane, and subsequently activates the downstream cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway, leading to enhanced conversion of testosterone to 17ß-estradiol. This study deepens our knowledge of the mechanisms of environmental factors in pathogenesis of hyperestrogenism.

Highly sensitive lateral flow immunoassays based on Ag@Au nanoflowers with marine medaka (Oryzias melastigm) vitellogenin as a target analyte.

In order to improve the sensitivity of lateral flow immunoassays (LFIAs) for the detection of piscine vitellogenin (Vtg), a well-established biomarker for environmental estrogens, Au coated Ag nanoflowers (Ag@Au NFs) were used as labeling probes to develop a LFIA for marine medaka Vtg. The synthesized Ag@Au NFs with good monodispersity had an average diameter of 44.1 nm and absorbance peak of 524 nm. When the concentration of goat anti-mouse IgG and anti-Vtg polyclonal antibody (anti-Vtg PAbs) were 1.3 and 0.4 mg/mL, respectively, the detection range of the LFIA was 0.19-25 ng/mL, and the visual detection limit was 0.1 ng/mL, which was approximately 80 times lower than that of LFIAs based on other nanoparticles (Au NPs, Ag NPs, Au NFs, and FM). After evaluation of its specificity and robustness, the usefulness of Ag@Au NFs labeled LFIA was validated by measuring Vtg induction in the plasma of marine medaka exposed to bisphenol A, a weak estrogenic chemical. This highly sensitive lateral flow immunoassay could detect Vtg biomarker within 15 min without the need of expensive and complicated instruments, and thus offered an ultrasensitive and robust on-site detection method for estrogenic activity in field environment.

Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein.

Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17ß-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens. Chg genes (ChgL and ChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9-250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE2). The results showed that 2 ng/L EE2 notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE2 induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments.

Polystyrene microplastics increase estrogenic effects of 17α-ethynylestradiol on male marine medaka (Oryzias melastigma).

Microplastics (MPs) and endocrine disrupting chemicals are ubiquitous pollutants in marine environments, but their combined ecological risk is unclear. This study exposed male marine medaka (Oryzias melastigma) to 10 ng/L 17α-ethynylestradiol (EE2) alone or EE2 plus 2, 20, and 200 µg/L polystyrene MPs for 28 days to investigate the impacts of MPs on the reproductive disruption of EE2. The results showed that 10 ng/L EE2 alone did not affect biometric parameters, while co-exposure to EE2 and 20, 200 µg/L MPs suppressed the growth and decreased gonadosomatic and hepatosomatic indices. Compared to EE2 alone, EE2 plus MPs exposure significantly increased plasma 17ß-estradiol (E2) levels in a dose-dependent manner, and co-exposure to EE2 and 20, 200 µg/L MPs significantly increased the ratios of E2/testosterone (T). Moreover, EE2 plus MPs exposure elevated the transcription levels of estrogen biomarker genes vitellogenin and choriogenin, and estrogen receptor (ERα and ERß). Morphological analysis also showed that co-exposure to EE2 and MPs induced more severe damage to the testes and livers, indicating that MPs increased the toxicity of EE2. The actual EE2 concentrations in the solution increased with the exposure concentrations of MPs, suggesting that MPs changed the fate and behavior of EE2 in the seawater. These findings demonstrate that MPs could increase the estrogenic effects of EE2 on marine fish, suggesting that the combined health risk of MPs and endocrine disrupting chemicals on marine organisms should be paid great attention.

Improving the Activity of Antimicrobial Peptides Against Aquatic Pathogen Bacteria by Amino Acid Substitutions and Changing the Ratio of Hydrophobic Residues.

With the increasing number of drug-resistant bacteria, there is an urgent need for new antimicrobial agents, and antimicrobial peptides (AMPs), which exist in the human non-specific immune system, are one of the most promising candidates. It is an effective optimization strategy to modify antimicrobial peptides (AMPs) according to the distribution of amino acids and hydrophobic characteristics. The addition of bacterial pheromones to the N short peptide can increase the ability to recognize bacteria. In this study, we designed and synthesized AMP1-6 by amino acid substitution of mBjAMP1. Additionally, P-6, S-6, and L-6 were designed and synthesized by adding bacterial pheromones based on 1-6. Functional tests showed that the four AMPs had the ability to kill Gram-negative Vibrio anguillarum, Pseudomonas mendocina, and Vibrio parahaemolyticus, and Gram-positive Micrococcus luteus and Listeria monocytogenes. Additionally, all four AMPs induced permeabilization and depolarization of bacterial cell membranes and increased intracellular reactive oxygen species (ROS) levels. Importantly, they had little or no mammalian cytotoxicity. At the same time, 1-6 and L-6 protected the stability of intestinal flora in Sebastes schlegelii and increased the relative abundance of Lactobacillaceae. In summary, our results indicate that the designed AMPs have broad application prospects as a new type of polypeptide antimicrobial agent.

Distribution of vitellogenin in Japanese flounder (Paralichthys olivaceus) for biomarker analysis of marine environmental estrogens.

Estrogen pollution in marine environments has become a research hotspot due to its adverse effects on the reproduction of wild organisms. To early detection of estrogen pollution, this study developed two methods for detecting Japanese flounder vitellogenin (Vtg), a sensitive biomarker for environmental estrogens. Firstly, monoclonal antibodies (mAb) specific to Vtg were prepared using purified lipovitellin (Lv), a main Vtg-derived yolk protein. Anti-Lv mAb (C1F1) had the highest titer (1:256,000) and was labeled with fluorescein isothiocyanate to establish a direct immunofluorescence (DIF) method for histological detection of Vtg in tissues. Additionally, using the purified Lv and mAb, an enzyme-linked immunosorbent assay (ELISA) was developed and this assay had a detection limit of 0.75 ng/mL and a working range of 1.95-250 ng/mL. Furthermore, Vtg induction in the plasma of Japanese flounder exposed to 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), and bisphenol A (BPA) were quantified by ELISA, and Vtg induction in the liver of EE2-exposed Japanese flounder were measured by DIF. Finally, the distribution of Vtg in Japanese flounder was detected using these two methods. The results revealed that Vtg mainly appeared in the terminal tail fin, liver, kidney, intestine, and spleen. Considering the high concentration of Vtg and easy sample collection, the terminal tail fin could be a new alternative to plasma for Vtg quantification, while kidney and liver are suitable for histological detection of Vtg.

Polystyrene microplastics cause tissue damages, sex-specific reproductive disruption and transgenerational effects in marine medaka (Oryzias melastigma).

The ubiquity of microplastics in the world's ocean has aroused great concern. However, the ecological effects of microplastics at environmentally realistic concentrations are unclear. Here we showed that exposure of marine medaka (Oryzias melastigma) to environmentally relevant concentrations of 10 µm polystyrene microplastics for 60 days not only led to microplastic accumulation in the gill, intestine, and liver, but also caused oxidative stress and histological changes. Moreover, 2, 20, and 200 µg/L microplastics delayed gonad maturation and decreased the fecundity of female fish. Alterations of the hypothalamus-pituitary-gonadal (HPG) axis were investigated to reveal the underlying mechanisms, and gene transcription analysis showed that microplastic exposure had significantly negative regulatory effects in female HPG axis. Transcription of genes involved in the steroidogenesis pathway in females were also downregulated. This disruption resulted in decreased concentrations of 17ß-estradiol (E2) and testosterone (T) in female plasma. Furthermore, parental exposure to 20 µg/L microplastics postponed the incubation time and decreased the hatching rate, heart rate, and body length of the offspring. Overall, the present study demonstrated for the first time that environmentally relevant concentrations of microplastics had adverse effects on the reproduction of marine medaka and might pose a potential threat to marine fish populations.

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.

Typhoons increase the abundance of microplastics in the marine environment and cultured organisms: A case study in Sanggou Bay, China.

Microplastic contamination in the ocean has emerged as an environmental issue of global importance. The most effective strategy to control microplastic pollution is to reduce the terrestrial input, but severe weather conditions make it difficult. This study investigated microplastic abundance and characteristics in the seawater, sediments, and cultured oysters (Crassostrea gigas) of Sanggou Bay (China) before and after two typhoons with an average rainfall of 19.2 mm/d over 8 days. Prior to the typhoons, microplastic levels in the seawater, sediment, and oysters were 63.6 ±â€¯37.4 items/L, 2178 ±â€¯369 items/kg, and 41.0 ±â€¯15.5 items/individual, with fibers being the predominant shape. Typhoons increased the average concentrations of microplastics in the seawater and sediments by approximately 40%, and the proportions of fragments, spherules, and granules in the sediments increased by 9.6%, 4.0%, and 4.3%, respectively. The majority of microplastics in seawater, sediments, and oysters collected before the typhoons could be grouped into sizes of 0.1-0.5 mm (36.7%), 0.05-0.1 mm (42.6%), and 0.1-0.5 mm (47.1%), respectively. After the typhoons, the most abundant size classes of microplastics in the three environmental compartments were 0.05-0.1 mm (39.2%) for seawater, 0.1-0.5 mm (37.1%) for sediments, and 0.05-0.1 mm (29.9%) for oysters. The typhoons also altered color distribution of microplastics and increased the proportions of polypropylene, polystyrene, and polyethylene terephthalate in seawater. Scanning electron microscopy/energy dispersive spectroscopy showed that organic matter and heavy metals were present on the microplastics collected from oysters. Our results suggest that weather conditions should be considered when investigating marine microplastics.

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.

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.

Oxidative damage induced by copper in testis of the red swamp crayfish Procambarus clarkii and its underlying mechanisms.

Copper (Cu) is one of the most widespread environmental pollutants and is known to exert multiple toxic effects including reproductive toxicity. In this study, we investigated the toxic effect of Cu on reproduction of the red swamp crayfish (Procambarus clarkii), an economic crustacean species, by exposing adult male crayfish to 0.03 and 3.00 mg/L Cu2+ for 7 days. The results showed that Cu2+ exposure induced oxidative stress accompanied by elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels in testes, and resulted in decreased sperm quality and abnormal testicular structures with apoptotic germ cells and vacuolisation in Sertoli cells. To reveal the molecular mechanism of Cu2+-induced oxidative damage in crayfish testes, we sequenced, assembled and annotated the transcriptome for crayfish testes, using the Illumina sequencing approach. After the 3.00 mg/L Cu2+ treatment, 6745 genes with differentially expressed profile were identified, of which many genes were involved in cellular response to ROS based on Gene Ontology enrichment analysis. Further, KEGG analysis demonstrated that genes with up-regulated expression levels significantly enriched in mitochondria oxidative phosphorylation pathway, suggesting disturbed mitochondrial electron transport chain was probably a main source of Cu2+-induced ROS production in testes. This study represented the first use of transcriptome to investigate the toxic effect of Cu2+ on male crayfish reproduction, and the pathways identified underlying Cu2+ toxicity at molecular level provide a novel insight into the reproductive toxicity of Cu in crustaceans.

High levels of microplastic pollution in the sediments and benthic organisms of the South Yellow Sea, China.

Microplastics, emerging contaminants in the ocean, are thought to sink and accumulate in sediments, and thus may pose a potential ecological risk to benthic communities. In this study, abundances and characteristics of microplastics in sediments and benthic organisms from the South Yellow Sea were investigated. First, we optimized the sediment sampling for microplastic analysis and found that the top layer (0-5 cm) had the highest abundance, and microplastic abundances decreased significantly with increase in sediment depth. The abundance of microplastics was 560-4205 n/kg dry weight in the surface sediments (the topmost 3 cm) of 14 sites and 1.7-47.0 n/g wet weight in the tissues of benthic organisms. Moreover, microplastic abundances in sediments and benthic organisms were both positively correlated with water depth. Fibers, transparent microplastics, and small microplastics (<0.5 mm) were the most dominant types in sediments and organisms. FTIR analysis showed that polypropylene (PP, 31%), polyester (PE, 24%), nylon (19%), and polystyrene (PS, 15%) were the most abundant polymers in sediments. The results of SEM showed rough surfaces and obvious cracks on the microplastics isolated from sediments. In addition, characteristics of microplastics in Ophiura sarsii, Crangon affinis, and Acila mirabilis were compared. Our results demonstrate that a comprehensive investigation of microplastics in sediments and benthic communities will help to fully understand the ecological risk of microplastic pollution.

Development of ELISAs for the detection of vitellogenin in three marine fish from coastal areas of China.

Estrogenic pollution has aroused great concern for its adverse effects on marine organisms. This study aimed to establish biomarker-based methods for detecting environmental estrogens using vitellogenin (Vtg) of teleost fishes inhabiting coastal areas of China. Firstly, Vtgs in marbled flounder (Pseudopleuronectes yokohamae), black rockfish (Sebastes schlegelii) and fat greenling (Hexagrammos otakii) were purified, characterized and used to prepare antibodies. Then, Vtg ELISA for each species was developed using purified Vtg and its antibody. Marbled flounder Vtg ELISA had a working range of 3.9-500 ng/mL and a detection limit of 2.1 ng/mL, and black rockfish Vtg ELISA had strong cross-reactivity with marbled flounder Vtg. Furthermore, Vtg induction in male marbled flounder exposed to pentadecafluorooctanoic acid (PFOA) was measured by developed ELISA. Plasma Vtg concentrations were significantly increased with PFOA concentrations in seawater and fish muscle. Therefore, Vtg ELISAs for these species might be useful tools for monitoring marine environmental estrogens.

Development of homologous enzyme-linked immunosorbent assays to quantify two forms of vitellogenin in guppy (Poecilia reticulata).

Guppy (Poecilia reticulata) is a promising model organism in toxicological studies, and vitellogenin (Vtg) is a commonly used biomarker for environmental estrogens. Although an ELISA for guppy Vtg has been developed previously, we found that guppy had two forms of Vtgs. In this study, two Vtgs were characterized and enzyme-linked immunosorbent assays (ELISAs) for each Vtg were developed. Two Vtgs purified from 17ß-estradiol (E2)-exposed guppy were characterized as phospholipoglycoproteins with molecular weights of ~ 520 and ~ 480 kDa, respectively. In SDS-PAGE, one purified Vtg appeared as three major bands of ~ 210, ~ 126, and ~ 102 kDa, and the other revealed a clear band of ~ 68 kDa. Matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry analysis showed that they were VtgAb and VtgC. Using purified Vtgs and their corresponding antibodies, two sandwich ELISAs with working ranges of 7.8~1000 and 15.6~500 ng/mL were developed. Precision tests showed that intra- and inter-assay coefficients of variations of both ELISAs were below 10%. Parallelism between Vtg standard curves and serial dilutions of whole body homogenate from E2-exposed guppy confirmed that two ELISAs could quantify guppy Vtgs. Furthermore, two ELISAs were used to measure Vtg inductions in liver, caudal fin and whole body of male guppy exposed to 17a-ethinylestradiol to validate their use for detecting estrogenic effects of exogenous chemicals. These homologous Vtg ELISAs will promote the use of guppy as a model organism to study estrogenic chemicals.

Quantitative analysis of in-vivo responses of reproductive and thyroid endpoints in male goldfish exposed to monocrotophos pesticide.

Cross-regulation occurs at many points between the hypothalamic-pituitary-gonad (HPG) and hypothalamic-pituitary-thyroid (HPT) axes. Monocrotophos (MCP) pesticide could disrupt HPG and HPT axes, but its direct target within the endocrine system is still unclear. In the present study, hormone concentrations and transcriptional profiles of HPG and HPT genes were examined in male goldfish (Carassius auratus) exposed to 0, 4, 40, and 400 µg/L MCP for 2, 4, 8, and 12 d. In vivo data were analyzed by multiple linear regression and correlation analysis, quantitatively indicating that MCP-induced plasma 17ß-estradiol (E2) levels were most associated with alteration of cyp19a transcription, which was also a potential point indirectly modulated by the MCP-altered thyroid hormones (THs) status; disturbance of THs pathways was most related with effect of MCP on regulation of the hypothalamic-pituitary hormones involved in the thyroid system, and the increased E2 levels might enhance the impact of MCP on HPT axis by modulating hepatic deiodinase expression. Our finding, based on these correlational data, gave a whole view of the regulations, especially on the cross-talk between sex hormone and thyroid hormone pathways upon exposure to chemicals with unknown direct target in vivo, and cautions should be exercised when developing adverse outcome pathway networks for reproductive and thyroidal endocrine disruption.

The anti-androgenic effect of chronic exposure to semicarbazide on male Japanese flounder (Paralichthys olivaceus) and its potential mechanisms.

Semicarbazide (SMC), a new marine pollutant, has anti-estrogenic effects on female Japanese flounder (Paralichthys olivaceus). However, whether SMC also affects the reproductive endocrine system of male marine organisms is currently unclear. In this study, Japanese flounder embryos were exposed to 1, 10, and 100 µg/L SMC for 130 days. Plasma testosterone (T) and 17ß-estradiol (E2) concentrations were significantly decreased in male flounders after SMC exposure. The expression of genes involved in T and E2 synthesis, including steroidogenic acute regulatory protein, cytochrome P450 11A1, 17α-hydroxylase, 17ß-hydroxysteroid dehydrogenase and cytochrome P450 19A, was down-regulated in the gonads, which may explain the decrease in plasma sex hormones levels. Moreover, SMC-mediated changes in the transcription of these steroidogenic genes were associated with reduced levels of follicle-stimulating hormone beta subunit (fshß), luteinizing hormone beta subunit (lhß), follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr) mRNA. In addition, down-regulated transcription of fshß and lhß in the SMC exposure groups was affected by reduced mRNA levels of seabream gonadotropin-releasing hormone (sbgnrh), g-protein-coupled receptor 54 (gpr54) in the kisspeptin/gpr54 system, as well as the gamma-aminobutyric acid (GABA) synthesis enzyme glutamic acid decarboxylase (gad). Overall, our results showed that environmentally relevant concentrations of SMC exerted anti-androgenic effects in male flounders via impacting HPG axis, kiss/gpr54 system and GABA synthesis, providing theoretical support for investigating reproductive toxicity of environmental pollutants that interfere with the neuroendocrine system.

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.

The anti-estrogenicity of chronic exposure to semicarbazide in female Japanese flounders (Paralichthys olivaceus), and its potential mechanisms.

This study investigated the anti-estrogenic effects of chronic exposure to a new marine pollutant, semicarbazide (SMC; 1, 10, and 100µg/L), in female Paralichthys olivaceus, as well as the associated mechanism. After 130days of exposure, plasma 17ß-estradiol and testosterone concentrations, and hepatic estrogen receptors, vitellogenin, and choriogenin mRNA levels decreased significantly in SMC-exposed groups. Moreover, down-regulation of genes in the hypothalamic-pituitary-gonadal axis, including gonadotropin-releasing hormone, gonadotropic hormones and their receptors, the steroidogenic acute regulatory protein, 17α-hydroxylase, 17ß-hydroxysteroid dehydrogenase, and cytochrome P450 19A, was observed after SMC exposure. Furthermore, the kisspeptin/g protein-coupled receptor 54 (kiss/gpr54) system and gamma-aminobutyric acid-ergic (GABAergic) system were also affected by SMC: SMC significantly down-regulated mRNA expression of kiss2, gpr54, and the GABA synthesis enzyme gad67. Our results demonstrated for the first time that environmentally relevant concentrations of SMC exerted anti-estrogenicity in female flounders, providing theoretical support for ecological risk assessments of SMC in marine environments.