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Se, an essential biological trace element, is required for fish growth. However, the underlying mechanisms remain unclear. Protein deposition in muscle is an important determinant for fish growth. This study was conducted on juvenile rainbow trout (Oncorhynchus mykiss) to explore the nutritional effects of Se on protein deposition in fish muscle by analysing the postprandial dynamics of both protein synthesis and protein degradation. Trout were fed a basal diet supplemented with or without 4 mg/kg Se (as Se yeast), which has been previously demonstrated as the optimal supplemental level for rainbow trout growth. After 6 weeks of feeding, dietary Se supplementation exerted no influence on fish feed intake, whereas it increased fish growth rate, feed efficiency, protein retention rate and muscle protein content. Results of postprandial dynamics (within 24 h after feeding) of protein synthesis and degradation in trout muscle showed that dietary Se supplementation led to a persistently hyperactivated target of rapamycin complex 1 pathway and the suppressive expression of numerous genes related to the ubiquitin–proteasome system and the autophagy–lysosome system after the feeding. However, the ubiquitinated proteins and microtubule-associated light chain 3B (LC3)-II:LC3-I ratio, biomarkers for ubiquitination and autophagy activities, respectively, exhibited no significant differences among the fish fed different experimental diets throughout the whole postprandial period. Overall, this study demonstrated a promoting effect of nutritional level of dietary Se on protein deposition in fish muscle by accelerating postprandial protein synthesis. These results provide important insights about the regulatory role of dietary Se in fish growth.

In this study, bighead carp (Aristichthys nobilis) were exposed to waterborne Cr6+ of 0.01, 0.1, 1 and 5 mg/L for 14 days and subsequently transferred to clean water for another 14 days. The Cr6+ contents in some edible parts, such as dorsal muscle, ventral muscle and head were detected. The Cr6+ concentrations in the three parts were in the order of: head > ventral muscle > dorsal muscle with significant increase during exposure period and remarkable decrease when kept at clean water during depuration stage. The head contained higher fat than that of muscle and the Cr6+ levels of these parts showed significantly positive correlation with fat content, however, the Cr6+ contents in the separated fat were extremely low. The Cr6+ levels determined in tissues can tell a real story rather than the correlation coefficient. Fish head poses a higher potential health risk than muscle due to heavy metals pollution.

BACKGROUND Selenium (Se) status is closely related to skeletal muscle physiological status. However, its influence on skeletal muscle growth has not been well studied. OBJECTIVES This study aimed to analyze the impacts of overall Se status (deficient, adequate, and high) on skeletal muscle growth using a growing zebrafish model. METHODS Zebrafish (1.5-mo-old) were fed graded levels of Se (deficient: 0.10 mg Se/kg; marginally deficient: 0.22 mg Se/kg; adequate: 0.34 mg Se/kg; high: 0.44, 0.57, and 0.69 mg Se/kg) as Se-enriched yeast for 30 d. Zebrafish growth, and Se accumulation, selenoenzyme activity, selenotranscriptome profiles, and oxidative status in the whole body, and selenotranscriptome profiles, histological characteristics, biochemicals, and gene and protein expression profiles related to muscle growth in the skeletal muscle were analyzed by model fitting and/or 1-factor ANOVA. RESULTS Se status biomarkers within the whole body and skeletal muscle indicated that 0.34 mg Se/kg was adequate for growing zebrafish. For biomarkers related to skeletal muscle growth, compared with 0.34 mg Se/kg, 0.10 mg Se/kg decreased the white muscle cross-sectional area (WMCSA) and the mean diameter of white muscle fibers (MDWMF) by 14.4%-15.1%, inhibited protein kinase B-target of rapamycin complex 1 signaling by 63.7%-68.5%, and stimulated the autophagy-lysosome pathway by 1.07 times and the ubiquitin-proteasome pathway (UPP) by 96.0% (P

Microcystin-LR (MC-LR) is regarded as one of the most toxic microcystins (MCs) isoforms. Microcystins could cause multiple organs dysfunction, and more attention has been drawn to the toxic effects on the gastrointestinal disorder. By using ex vivo everted gut sac model in 6 fish (Carassius auratus, Megalobrama amblycephala, Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idellus and Cyprinus carpio) and determining the accumulation of MC-LR in zebrafish intestine, we found a dose-dependent manner in the absorption and accumulation of MC-LR. Until now, little studies have been reported concerning the gut microbiota composition caused by different MC-LR exposure. The present study is the first time characterized the phylogenetic composition and taxonomic of the bacterial communities growth in the intestines of zebrafish treated with MC-LR using 16S rRNA pyrosequencing. After 30 days of treatment with 0, 1, 5 or 20 μg/L MC-LR, the alpha and beta diversity did not generate significant differences, indicating the existence of a core microbiota. However, db-RDA analysis showed that treatment with 20 μg/L MC-LR changed the characteristics of high abundances microbiota. The expression of Oatp2b1, stress related enzyme activities in gut and their associations with gut microbiota were also determined. The identified phylotypes including Actinobacteria, Lactobacillus and some opportunistic pathogens highlight the increasing risks of pathogen invasion and recovery tendency via potential probiotics resistance in zebrafish exposed to MC-LR.

As a nutritionally essential trace element, selenium (Se) is crucial for fish growth. However, the underlying mechanisms remain unclear. Fish somatic growth relies on the white muscle growth. This study aimed to explore the effects and underlying mechanisms of Se on fish white muscle growth using a juvenile rainbow trout (Oncorhynchus mykiss) model. Fish were fed a basal diet unsupplemented or supplemented with selenium yeast at nutritional dietary Se levels (2 and 4 mg/kg Se, respectively) for 30 days. Results showed that dietary Se supplementation significantly enhanced trout somatic growth. Histological and molecular analysis of trout white muscle tissues at the vent level showed that dietary Se supplementation elevated the total cross-sectional area of white muscle, mean diameter of white muscle fibers, protein content, nuclei number, and DNA content of individual muscle fiber, and suppressed the activities of calpain system and ubiquitin-proteasome pathway. Overall, this study demonstrated that dietary Se within the nutritional range inhibits calpain- and ubiquitin-mediated protein degradation and promotes the fusion of myoblasts into the existed muscle fibers to promote the hypertrophic growth of white muscle, thereby accelerating the somatic growth of rainbow trout. Our results provide a mechanistic insight into the regulatory role of Se in fish growth.

In this study, the concentrations of 14 types of organochlorine pesticides (OCPs) and 7 types of polychlorinated biphenyls (PCBs) in four freshwater fish species (Ctenopharyngodon idella, Cyprinus carpio, Hypophthalmichthys molitrix and Hypophthalmichthys nobilis) collected from nine lakes in Anhui Province were determined. Among these contaminants, only hexachlorobenzene (HCB), heptachlor, hexachlorocyclohexane (β- and γ-HCH), dichlorodiphenyldichloroethylene (p,p'-DDE) and PCB 101 were detected, and HCB had the highest measured concentrations while the heptachlor showed the lowest concentrations. In the four fish species, C. carpio preferred to accumulate more OCPs and PCBs than C. idellus. Moreover, the health risk assessments demonstrated that consumption of these fish species may pose both non-carcinogenic and carcinogenic risks, especially for children at high exposure level (95th). As the concentrations of contaminants in ventral muscle were higher than that in dorsal muscle, the consumption of ventral muscle should be limited to avoid the potential risk of OCPs and PCBs.

This study aimed to investigate the formation of odor properties in surimi products by exploring changes in aroma compounds and their precursors at setting (40 °C) and gelling (90 °C) stages during surimi gel formation by solvent-assisted flavor evaporation, gas chromatography-mass spectrometry, and ultra performance liquid chromatography. Results revealed a gradual increase in the contents of most aldehydes, alcohols, and phenolics during surimi gel formation, while a decrease in the contents of (E)-2-pentenal, (E)-2-hexnenal, (Z)-4-heptenal, (E)-2-heptenal, and most ketones at gelling stage. During thermal process, 50 % decrease was observed in the contents of some unsaturated fatty acids (palmitoleic acid, oleic acid, etc.), and the contents of tyrosine, phenylalanine, methionine, isoleucine, and leucine decreased significantly (P 0.05) at gelling stage. Moreover, lipoxygenase activity reached 4.19-4.81 U/min·g at setting stage. Overall, amino acid degradation and lipid auto-oxidation mainly occurred at gelling stage to promote the formation of related compounds.

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.

Microcystin-LR (MC-LR) released by Microcystis blooms degradation usually co-exists with a chemical called nitrite, posing a serious harm to aquatic organisms. To assess the single and combined effects of MC-LR and nitrite on the reproductive endocrine system, a fully factorial experiment was designed and adult male zebrafish (Danio rerio) were exposed to 9 treatment combinations of MC-LR (0, 3, 30 μg/L) and nitrite (0, 2, 20 mg/L) for 30 d. The results showed that both MC-LR and nitrite caused concentration-dependent effects including the growth inhibition, decreased gonad index as well as testicular injuries with widen intercellular spaces and seminiferous epithelium deteriorations. And testicular pathological changes in the co-exposure groups of MC-LR and nitrite were similar but more serious than those in single-factor exposure groups. Concurrently, exposure to MC-LR or nitrite alone could significantly decrease T levels by downregulating gene expressions (gnrh2, lhβ, ar, lhr) in the hypothalamic-pituitary-gonadal-liver-axis (HPGL-axis), and there were significant interactions between MC-LR and nitrite on them. In contrast, E2 levels as well as transcriptional levels of cyp19a1b, cyp19a1a and vtg1 showed significant inductions with increasing MC-LR concentrations, indicating an estrogen-like effect of MC-LR. Our findings illustrated that co-exposure of MC-LR and nitrite synergistically cause reproductive dysfunction by interfering with the HPGL axis in male fish, which prompt us to focus more on the potential risks in fish reproduction and even population dynamics due to the wide occurrence of toxic cyanobacterial blooms.

Selenium (Se) is an essential trace element for fish growth and performs its physiological functions mainly through incorporating into selenoproteins. It is well known that dietary Se regulates fish growth by controlling the synthesis of deiodinase, a kind of selenoproteins. However, recently, as many as 41 selenoproteins have been characterized in teleost fish. We propose a hypothesis if other selenoproteins, besides deiodinase, could also involve in the regulation of fish growth. In the present study, rainbow trout (Oncorhynchus mykiss) were fed diets supplemented with or without graded levels of Se (2, 4 or 6 mg/kg, from selenium yeast, Se-yeast) for 10 weeks. At the end of the feeding trial, fish growth and the expressions of a total of 28 selenoprotein genes in tissues have been evaluated. Results showed that dietary Se-yeast supplementation significantly increased fish growth (P

In this study, crucian carp (Carassius auratus) was exposed to graded levels of waterborne Cr6+ (0, 0.1, 1 and 10 mg/L) for 28 days and subsequently transferred to clean water for 20 days. Fish tissues were collected every 4 days. The Cr6+ levels in tissues were determined and the potential health risks from consuming fish muscle and fish head were assessed. The results indicated that the Cr6+ contents in the head were always higher than those in muscle and that 10 mg/L Cr6+ treatment conferred risk in fish head consumption throughout the entire experimental period. A further kinetic study on the accumulation and depuration of Cr6+ in tissues indicated that compared to muscle, the higher Cr6+ content in fish head was attributed to the greater uptake rate (k1) and kinetic bioconcentration factor (BCFk), although the k1 and BCFk values in the muscle and head were lower than those in the liver, kidney, intestines and gills. The maximum Cr6+ concentrations in tissues at theoretical equilibrium (Cmax) ranged from 0.55 mg/kg in muscle to 112 mg/kg in intestines with exposure to 10 mg/L Cr6+. Moreover, the results of the distributions and transitions of Cr6+ in fish tissues demonstrated that fish head and muscle were short-term storage organs while the kidney was a long-term storage organ for Cr6+. Overall, compare to muscle, fish head has a higher capacity for Cr6+ accumulation, which poses a higher potential health risk to humans, especially to children, once the fish is contaminated by Cr6+.

In the present study, to comprehensively investigate the metal contamination in the fish of Anhui Province, four fish species, Ctenopharyngodon idella, Cyprinus carpio, Hypophthalmichthys molitrix, and Hypophthalmichthys nobilis, were collected from nine lakes, and the levels of Zn, Pb, Cr, Cu, Ni, As, Hg, and Cd in the fish muscle were determined. The results showed that the highest concentrations of Zn (7.791 mg/kg), Pb (0.522 mg/kg), Cr (0.030 mg/kg), and Cu (0.767 mg/kg) were found in Tiangang Lake, Xifei Lake, Tiangang Lake and Baidang Lake, respectively. However, metals Ni, As, Hg, and Cd were not detected in all fish samples. In the fish species, the metal bioaccumulation ability was decreased with the following order: C. idellus > H. molitrix > H. nobilis > C. carpio. Furthermore, the target hazard quotient (THQ) was used to assess the health risk via fish consumption. The results indicated for co-exposure; C. idellus would pose a health risk to children at high exposure level (95th) as THQ value was higher than 1. It should be pointed out that Pb contributes most to the total THQs (the ratio was 88%); thus, the contamination of Pb should be paid more attention. This field investigation combined with health risk assessment would provide useful information on the heavy metal pollution in Anhui Province.

The present study investigated the accumulation and depuration effects of hexavalent chromium (Cr6+) in ten tissues (gills, intestines, liver, kidney, blood, heart, bladder, spleen, skin and muscle) of the bighead carp (Aristichthys nobilis). Fish were exposed to graded levels of waterborne Cr6+ (0.01, 0.1, 1 and 5 mg/L) for 4, 7 and 14 days, and subsequently transferred to Cr6+-free water for 14 days. After 14-day exposure, a dose-dependent increase of Cr6+ has been observed in most tissues. While after 14-day depuration, Cr6+ contents were significantly decreased in various tissues except in kidney and spleen where Cr6+ contents significantly increased at the group of 5 mg/L. Considering that Cr6+ highly accumulated in gills, intestines, liver and kidney, the oxidative damage of Cr6+ on the four tissues were further investigated and found that the antioxidant response to Cr6+ were organ-specific. The results in this study indicated that a 14-day period is effective for accumulation and depuration of Cr6+ in bighead carp and there was no health risk of fish muscle consumption. Additionally, the delayed efflux of Cr6+ in the fish kidney and spleen indicates that high importance should be attached to them when evaluating the toxic effects and risk assessments of Cr6+.

Cyanobacterial blooms and their associated toxins pose a great threat to human beings. The situation is even worse for those whose drinking water source is a cyanotoxin-polluted water body. Therefore, efficient and safe treatments urgently need to be developed. The present study verified the application of vitamin C on the inhibition of toxic Microcystis aeruginosa. Our results showed that vitamin C drove the Fenton reaction and significantly sterilized cultures of M. aeruginosa. The algicidal activity of vitamin C was dependent on its involvement in iron (Fe) metabolism. Vitamin C enhanced iron absorption leading to high ferrous ion levels. The ferrous ion increased production of reactive oxygen species (ROS) by Fenton reaction, which play a crucial role in the killing process. Interestingly, vitamin C also dramatically decreased the release of microcystins. This study highlights the possible benefits of using a vitamin C-induced Fenton reaction to remove M. aeruginosa and microcystins from drinking water sources.

Background: Toxic cyanobacterial blooms result in the production of an organic biomass containing cyanotoxins (e.g. microcystins) and an elevated ammonia concentration in the water environment. The ingestion of toxic cyanobacteria and exposure to ammonia are grave hazards for fish. The present study assessed the effects of dietary toxic cyanobacteria and ammonia exposure on the flesh quality of blunt snout bream (Megalobrama amblycephala).; Results: Dietary toxic cyanobacteria and ammonia exposure had no impact on fish growth performance, fillet proximate composition and drip loss, whereas it significantly decreased fillet total amino acids, total essential amino acids, hardness and gumminess, and increased fillet ultimate pH as well as malondialdehyde content. However, there was no significant interaction between dietary toxic cyanobacteria and ammonia exposure on these parameters. Additionally, dietary toxic cyanobacteria significantly increased fillet initial pH, thaw loss and protein carbonyl content, whereas ammonia exposure did not.; Conclusion: The results of the present study indicate that dietary toxic cyanobacteria and ammonia exposure reduced the quality of blunt snout bream fillet. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

IgZ is considered to be the last immunoglobulin discovered in vertebrate species. In this study, the structure of secreted form of blunt snout bream (Megalabrama amblycephala) IgZ (sIgZ) heavy chain gene is VH-Cζ1-Cζ2-Cζ3-Cζ4, in which Cζ4 provides the specificity to the IgZ isotype. The deduced amino acid sequence of sIgZ shows highest similarity with that of Ctenopharyngodon idella (79%). The ontogeny of sIgZ gene expression showed a V-shape change pattern: decreased initially from unfertilized egg stage to 16-cell embryos and increased significantly from blastula stage, which exhibited maternal transmission effects. Compared with the juvenile fish, sIgZ mRNA level was higher in head kidney, spleen, trunk kidney, liver, intestine and gill of adult fish. In both juvenile and adult fish, sIgZ mRNA was detected in intestine and gill. Aeromonas hydrophila challenge experiment showed that sIgZ transcription significantly increased in skin, gill and intestine, indicating a prominent mucosal immune response. The results of Western blot also verified the protein alterations of sIgZ in mucosal organs in M. amblycephal. Our studies indicate a prominent role of IgZ in mucosa-associated lymphoid tissue immunity and further support the specialized role of IgZ in teleost mucosal immune responses.

Massive blooms of cyanobacteria frequently occur with microcystin (MC) production. Cyanobacteria are exposed to copper stresses such as copper algaecides which are often used to remove cyanobacterial blooms. However, copper increased the MC production of cyanobacteria, and the underlying mechanism remains unclear. The present study investigated the relationship between copper exposure (0.5 and 3 &micro, M) and MC synthesis in Microcystis aeruginosa PCC 7806. The study concluded that the content of intracellular MCs increased by nearly two times both in 0.5 and 3 &micro, M copper. High-throughput RNA sequencing (RNA-seq) provided evidence that copper mainly attacked Fe&ndash, S clusters, with evidence of changes in iron, sulfur, iron uptake regulators (fur), glutaredoxins and dehydratase genes. The transcription of numbers of genes implicated in iron uptake, MC synthesis and furA was also evaluated with quantitative real-time PCR (qRT-PCR). In these three Cu treatment groups, the amount of MCs increased as copper elevated. As the expression of mcyD gene was directly regulated by FurA and copper ions affected the expression of the FurA-related genes, we believed that MC synthesis genes were controlled by copper. This study has made a further understanding of the mechanism of the increase in MC synthesis of M. aeruginosa PCC 7806 treated with copper-based algaecides. We aimed to understand the mechanism of copper ion influencing the synthesis of MCs.

Cyanobacterial blooms and their secondary metabolites, microcystins (MCs), are not only toxic to aquatic organisms, but also to humans. MCs exert reproductive toxicity in female fish by affecting the oocyte development. However, the mechanism behind MC-LR interference in oocyte development remains largely unknown. In our study, adult female zebrafish were exposed to MC-LR (0, 1, 5, 20 μg/L) for 30 d. After exposure to MC-LR for 30 d, fertilized eggs from the treated females and healthy males were collected and cultured in water without MC-LR. Histomorphological observations showed pathological damage in the ovary after MC-LR exposure, which was mainly characterized by enlarged intercellular spaces, detachment of follicular cells from oocytes, and vacuolation of parenchymal tissues. The 20 μg/L MC-LR treatment caused a remarkable increase in the rate of the zebrafish oocytes germinal vesicle breakdown (GVBD) and a significant decrease in the levels of cyclic adenosine monophosphate (cAMP) and vitellogenin (VTG). In addition, the phosphorylation levels of the extracellular signal-regulated kinases (ERK) were elevated in ovaries from zebrafish exposed to 5 and 20 μg/L MC-LR, and cyclinB phosphorylation levels were also upregulated notably in the 20 μg/L MC-LR group. However, MC-LR exposure did not cause any change in the levels of cAMP-dependent protein kinase (PKA) protein and cdc2 phosphorylation in all the treatments. All the doses of MC-LR reduced the number of eggs, prematurely hatched the fertilized eggs and increased the abnormal rate of offspring generation. In summary, the present study demonstrates that MC-LR promotes oocyte maturation by activating the ERK1/2 and MPF signaling pathways, and cAMP is involved in this process.

The existence of microcystins (MCs), the secondary metabolite of cyanobacteria, has become a growing public health concern. Previous researches have proved that MCs can trigger endoplasmic reticulum stress (ERS), but the underlying mechanisms remain unclear. In the present study, adult female zebrafish were exposed to MC-LR (0, 1, 5 and 20 μg/L) for 30 d, and the offspring derived from the treated females and healthy males were cultured in water without MC-LR until 96 h post fertilization (hpf). Our data suggested that MC-LR causes a significant increase in the eif2s1a, atf4, and eif2ak3 transcription levels in the liver and ovary. The mRNA levels of atf4, atf6, bcl-2, hspa5, eif2s1a and eif2ak3 upregulated notably in the offspring. JNK phosphorylation level and cleaved-caspase3 protein expression elevated obviously in the liver and ovary, but had no remarkable change in the offspring. Furthermore, TUNEL results showed that MC-LR significantly induced apoptosis in the liver and ovary, while acridine orange (AO) staining indicated that MC-LR did not cause abnormal apoptosis in offspring. Concisely, the present study indicated that MC-LR leads to apoptosis through different ERS pathways in the liver, ovary and offspring, and also provides a new perspective for understanding the apoptosis caused by MC-LR.

Teleost skin serves as the first line of defense against invading pathogens, and contain a skin-associated lymphoid tissue (SALT) that elicit gut-like immune responses against antigen stimulation. Moreover, exposed to the water environment and the pathogens therein, teleost skin is also known to be colonized by diverse microbial communities. However, little is known about the interactions between microbiota and the teleost skin mucosal immune system, especially dynamic changes about the interactions under pathogen infection. We hypothesized that dramatic changes of microbial communities and strong mucosal immune response would be present in the skin of aquatic vertebrate under parasite infection. To confirm this hypothesis, we construct an infected model with rainbow trout (Oncorhynchus mykiss), which was experimentally challenged by Ichthyophthirius multifiliis (Ich). H & E staining of trout skin indicates the successful invasion of Ich and shows the morphological changes caused by Ich infection. Critically, increased mRNA expression levels of immune-related genes were detected in trout skin from experimental groups using qRT-PCR, which were further studied by RNA-Seq analysis. Here, through transcriptomics, we detected that complement factors, pro-inflammatory cytokines, and antimicrobial genes were strikingly induced in the skin of infected fish. Moreover, high alpha diversity values of microbiota in trout skin from the experimental groups were discovered. Interestingly, we found that Ich infection led to a decreased abundance of skin commensals and increased colonization of opportunistic bacteria through 16S rRNA pyrosequencing, which were mainly characterized by lose of Proteobacteria and increased intensity of Flavobacteriaceae. To our knowledge, our results suggest for the first time that parasitic infection could inhibit symbionts and offer opportunities for other pathogens' secondary infection in teleost skin.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. During the degradation of toxic cyanobacterial blooms, elevated ammonia and microcystins concentrations co-occur and exert toxicity on fish. Up to now, the combined effect of microcystins and ammonia on fish immunotoxicity has not been reported. The present study investigated immune responses of blunt snout bream (Megalabrama amblycephala) to dietary toxic cyanobacteria and ammonia exposure. Megalobrama amblycephala were exposed to solutions with different concentrations of NH3–N (0, 0.06, 0.12 mg/L) and fed with diets containing 15% and 30% of toxic cyanobacteria lyophilized powder for 30 d. The microcystins concentration in different organs of Megalobrama amblycephala was in the following sequence: head kidney > liver > intestine > gonad > spleen > gill > trunk kidney > brain > muscle > heart. In both head kidney and spleen, the MC-LR and MC-RR concentration increased significantly with increasing NH3–N concentration. It indicates that NH3–N maybe promote the accumulation of microcystins in immune organs of Megalobrama amblycephala. Meanwhile, broadened peripheral interspace of lymphocytes, nucleus shrivel and edematous mitochondria were observed in head kidney lymphocyte of toxic treatment fish. Moreover, there were significant interactions between dietary toxic cyanobacteria and ammonia exposure on head kidney macrophage phagocytosis activity, respiratory burst activities, total number of white blood cells and the transcriptional levels of sIgM, mIgD and sIgZ genes. Our data clearly demonstrated that dietary toxic cyanobacteria combined with ammonia exposure showed a synergistic effect on Megalobrama amblycephala immunotoxicity.

Animal studies provide strong evidence of positive associations between microcystins (MCs) exposure and reproductive toxicity, representing a threat to human reproductive health and the biodiversity of wild life. This paper reviews current knowledge of the reproductive toxicity of MCs, with regard to mammals, fishes, amphibians, and birds, mostly in males. Toxicity of MCs is primarily governed by the inhibition of protein phosphatases 1 and 2A (PP1 and PP2A) and disturbance of cellular phosphorylation balance. MCs exposure is related to excessive production of reactive oxygen species (ROS) and oxidative stress, leading to cytoskeleton disruption, mitochondria dysfunction, endoplasmic reticulum (ER) stress, and DNA damage. MCs induce cell apoptosis mediated by the mitochondrial and ROS and ER pathways. Through PP1/2A inhibition and oxidative stress, MCs lead to differential expression/activity of transcriptional factors and proteins involved in the pathways of cellular differentiation, proliferation, and tumor promotion. MC-induced DNA damage is also involved in carcinogenicity. Apart from a direct effect on testes and ovaries, MCs indirectly affect sex hormones by damaging the hypothalamic-pituitary-gonad (HPG) axis and liver. Parental exposure to MCs may result in hepatotoxicity and neurotoxicity of offspring. We also summarize the current research gaps which should be addressed by further studies.

Immunoglobulins (Igs), which bind antigens with high specificity, are essential molecules in adaptive immune system of jawed vertebrates. In this study, cDNA encoding the secreted form of the immunoglobulin heavy chain of IgM (sIgM) was cloned from the mesonephros of blunt snout bream (Megalabrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of sIgM heavy chain gene has 1961 nucleotides encoding a putative protein of 569 amino acids, constant region shares high amino acid identity with that of Ctenopharyngodon idella (80%), Carassius auratus langsdorfii (65%) and Danio rerio (59%). Multiple protein sequence alignment revealed that blunt snout bream sIgM was clustered with the homologues of cyprinid fish and constructed one clade. Using quantitative real-time PCR (qRT-PCR) analysis, the level of sIgM mRNA was determined, with a V-shape change pattern: decreased initially from unfertilized egg stage to 4 cells stage and increased from 16 cells stage to prelarva. This sharp drop indicates that sIgM mRNA is maternally transferred, and was continuously degraded until 16 cells stage. The drastic rising in sIgM level from blastula stage to prelarva might be attributed to embryonic stem cell differentiation procedure. Compared with juvenile fish, the expression of sIgM was significantly higher in pronephros, liver, spleen, gill and muscle of adult fish. After the injection of Aeromonas hydrophila, the expression pattern of sIgM was found first down-regulated at 4 h, then up-regulated and reached the peak at 7 d and 21 d in mesonephros, spleen, liver and gill, respectively.

Microcystins (MCs) released during cyanobacterial blooms exert varied toxicity on fish. Up to now, the reproductive toxicity of MCs on fish has rarely been reported. The present study investigated the reproductive toxicity of microcystin-LR (MC-LR) on male and female zebrafish (Danio rerio) by subchronic immersion in 1, 5, 20 μg/L for 30 d. After MC-LR exposure, the hatchability and the 17 beta-estradiol (E2) concentration in gonads significantly decreased in the 20 μg/L group. In the 5 and 20 μg/L groups, the whole body vitellogenin (VTG) levels significantly increased in females, while considerably decreased in males. The VTG1 transcriptional level significantly reduced in the liver of both female and male treated fish. Marked histological lesions were observed in the livers, ovaries and testes in MC-LR treated fish. Apoptotic rate in the ovaries significantly increased. Significant down-regulation of Bcl-2 transcriptional level was found in the gonads of all MC-LR treated fish, while marked up-regulation of Bax transcription level was determined in the 20 μg/L female treatment group, but a significant down-regulation in males. Although the transcriptional level of caspase-3 dropped in ovaries of the 5 and 20 μg/L treatment groups, the significant increase of caspase-3 activation levels in the ovaries and testes were detected. The present findings indicate that MC-LR exposure exerts diverse reproductive toxicity in zebrafish with females exhibiting more sensitivity than males. The present study also confirmed for the first time that MC-LR does not cause any estrogenic effects in adult zebrafish.

The present study evaluated the effects of dietary microbial phytase on the growth and gut health of hybrid tilapia (Oreochromis niloticus♀×Oreochromis aureus♂), focusing on the effect on intestinal histology, adhesive microbiota and expression of immune-related cytokine genes. Tilapia were fed either control diet or diet supplemented with microbial phytase (1000 U/kg). Each diet was randomly assigned to four groups of fish reared in cages (3×3×2 m). After 12 weeks of feeding, weight gain and feed conversion ratio of tilapia were not significantly improved by dietary microbial phytase supplementation. However, significantly higher level of P content in the scales, tighter and more regular intestinal mucosa folds were observed in the microbial phytase group and the microvilli density was significantly increased. The adhesive gut bacterial communities were strikingly altered by microbial phytase supplementation (0·41tnf-αandtgf-β) andhsp70. In addition, the gut microvilli height was significantly decreased in the phytase group. These results indicate that dietary microbial phytase may exert mixed effects on hybrid tilapia, and can guide our future selection of phytases as aquafeed additives – that is, eliminating those that can stimulate intestinal inflammation.

While microcystins (MCs) have been reported to exert reproductive toxicity on fish with a sex-dependent effect, the underlying mechanism has been rarely investigated. In the present study, zebrafish were exposed to 1, 5 and 20 μg/L MC-LR for 30 d. The gonad-somatic index declined in all treated males. 17β-estradiol (E2), testosterone (T), 11-keto testosterone (11-KT) and follicle-stimulating hormone (FSH) levels increased in serum from all treated females, while T, FSH and luteinizing hormone (LH) levels changed in all treated males. Histomorphological observation showed that MC-LR exposure evidently retarded oogenesis and spermatogenesis. Transcriptional changes of 22 genes of the hypothalamic-pituitary-gonad (HPG) axis exhibited sex-specific responses and the relationship between gene transcriptions and gametogenesis was evaluated by principle component analysis (PCA). Major contributors to PC1 (gnrh2, gnrhr3, ar, lhr, hmgra, hmgrb and cyp19a) were positively correlated with the number of post-vitellogenic oocytes, while PC1 (gnrh2, lhβ, erβ, fshr, cyp11a and 17βhsd) were positively correlated with the number of spermatozoa. The protein levels of 17βHSD and CYP19a were affected in both females and males. In conclusion, this study first investigated the sex-dependent effects of microcystins on fish reproduction and revealed some important molecular biomarkers related to gametogenesis in zebrafish suffered from MC-LR.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs), especially microcystin-LR (MC-LR), released during cyanobacterial blooms exert great toxicity on fish and even lead to massive death. The present study mainly investigated the pathological damage and immune response of spleen, gut and gill in zebrafish exposed to MC-LR. Fish were exposed to 0, 1, 5 and 20 μg/L of MC-LR for 30 d. In zebrafish exposed to 5 and 20 μg/L MC-LR, edematous mitochondria, deformation of the nucleus and compaction of chromatin were observed in lymphocyte of spleen; frayed gut villi, exfoliation of epithelial cells and widespread cell lyses were observed in intestines; hyperemia in gill lamellae, epithelial tissue edema and uplift and lamellar fusion were observed in gill. Varied changed gene expression was observed in spleen, intestine and gill of zebrafish. The transcriptional levels of IFN-1 and IL-8 in spleen significantly up-regulated in 20 μg/L group, and the transcription of IL-1β and TNFα in spleen increased in 1 μg/L MC-LR treated fish. In addition, the mRNA levels of IFN-1, IL-1β, IL-8, TGF-β and TNF-α dramatically increased in intestine and gill in all MC-LR treated groups. The present studies indicated that MC-LR exposure caused marked pathological damage, however, fish could adjust actively the expression of innate immune-related genes to resist the tissue damage. Our findings provided strong evidence of the recovery potential of fish exposed to microcystins.

Microcystin-LR (MCLR), released by toxic cyanobacterial blooms, has received worldwide concerns in the past decades for its hepatotoxicity. Recent studies show that microcystins (MCs) can be accumulated in immune organs and exert notable immunotoxicity. In order to better understand cellular responses in immune tissues disrupted by MCLR treatment, this work mainly focuses on the spleen impairments of rats. After a subchronic 50 d exposure (1 or 10 mu g/kg body weight per day), spleen index, MCLR accumulation, histological change and plasma lysozyme activity were detected in MCLR-treated rat. Results indicated that prolonged exposure of MCLR led to toxin accumulation and caused severe damage in spleen of rats, and eventually impaired the immune functions. To further our understanding of the toxic effects of MCLR on the spleen and the mechanisms behind it, a proteomic analysis was performed to determine the global effects of MCLR on splenetic protein levels. In total, 48 proteins were identified and showed a significant increase or decrease in abundance compared to the control after MCLR exposure. These proteins are mainly involved in immune response, oxidative stress, energetic metabolism and the cytoskeleton assembly, indicating that MCLR exerts complex toxic effects in rat spleen and jointly results in immunotoxicity. (C) 2012 Elsevier B.V. All rights reserved.

Microcystins (MCs) are highly liver-specific and evidenced as a liver tumor promoter. Oxidative stress is one of the most important toxicity mechanisms of MCs, which is tightly related to oxygen concentration. The effects of MCs on animals and cell lines in normoxia and the mechanisms have been well studied, but such effects in different oxygen conditions were still unclear. The aim of the present study was to explore the cellular response of the human hepatocellular carcinoma cell line (HepG2) to MC-LR exposure under hypoxic (1% O-2) and normoxic (21% O-2) conditions. We examined cell viability, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity and gene expression posttoxin exposure. Cell viability was increased by MC-LR in normoxia although decreased in hypoxia. MC-LR markedly induced MMP loss under hypoxic condition but only slightly MMP loss under normoxic condition. SOD activity was significantly induced by MC-LR in hypoxia, indicating prolonged oxidative stress. Inhibitory apoptosis protein (c-IAP2) was significantly up-regulated by MC-LR under normoxic condition, suggesting that c-IAP2 played an important role in the promotion of cell proliferation by MC-LR. These results indicate that MC-LR promotes cell proliferation under normoxic condition whereas induces cell apoptosis under hypoxic condition. Copyright (C) 2012 John Wiley & Sons, Ltd.

Microcystins (MCs) can cause evident hepatic apoptosis. In vitro studies indicated that uptake of MC by isolated hepatocytes was dramatically reduced as ambient temperature dropped, and some studies presented a hypothesis that differences in core body temperatures in animals result in diverse uptake of MC, as well as different toxic effects. Thus far, however, few in vivo studies have been conducted to investigate the effects of temperature on MC-induced hepatocyte apoptosis in fish, a typical poikilotherm. In the present study, zebrafish were treated with MC-LR, an MC metabolite, at three water temperatures (12, 22 and 32 degrees C), and evident differences in apoptotic profiles were observed. Damage to liver ultrastructures revealed temperature-dependent early-stage patterns of apoptosis. Flow-cytometric analysis indicated that hepatocyte apoptotic rates varied with a temperature-dependent effect. The transcription levels of some apoptosis-related genes were determined using quantitative real-time polymerase chain reaction, and significantly elevated gene expressions of P53, Bcl-2, Bax and caspase-3 were found in the 12 and 32 degrees C groups. Results of the present study indicate that different ambient temperatures can lead to various toxic effects of MCs on hepatic apoptosis in fish. Copyright (c) 2012 John Wiley & Sons, Ltd.

Hematological abnormalities or derangements have been demonstrated in patients suffering form microcystins (MCs) in hemodialysis unit in Caruaru, Brazil, 1996. While experimental study on hematological effect of microcystins has been rare and the underlying mechanisms are still puzzling. In the present study, microcystins were repeatedly intraperitoneally injected with a dose of 6 mu g/kg/day in rabbits (Oryctolagus cuniculus) for 14 days, and the prolonged effects of extracted microcystins on hematotoxicology were investigated. Significant decreases were observed in the hematological indices red blood cell counts, hematocrit, hemoglobin, and platelet count, while an obvious anemia occurred in rabbits after 14-day exposure. Moreover, red blood cell volume distribution width, mean corpuscular volume, and mean corpuscular hemoglobin did not vary significantly, indicating that rabbits suffered from normocytic anemia. In bone marrow, on the 14th day after toxin exposure, the frequency of micronucleus increased significantly, and the viability of bone marrow cells decreased markedly compared with the control. Serum erythropoietin levels declined on the 7th and 14th day, which suggested that the ability to regulate differentiation and maturation of erythrocytes was impaired. These results indicate that repeated exposure of microcystins can result in normocyte anemia, and the bone marrow injures and the sharp decreases of erythropoietin levels were responsible for the anemia. (C) 2011 Wiley Periodicals, Inc. Environ Toxicol 26: 472-479, 2011.

Microcystin-leucine-arginine (MCLR) is the most toxic and the most commonly encountered variant of microcystins (MCs) in aquatic environment, and it has the potential for developmental toxicity. A number of previous studies have described the developing toxicity of MCLR based on conventional toxicological indices. However, the molecular mechanisms by which it expresses its toxicity during the early development remain largely unknown. To further our understanding of mechanisms of action and identify the potential protein biomarkers for MCLR exposure, a proteomic analysis was performed on developing zebrafish embryos exposed to 0.5 mg/L MCLR until 96 hours post-fertilization. 2-DE combined with MS was employed to detect and identify the protein profiles. Results showed that 75 spots from the 0.5 mg/L MCLR condition showed a significant increase or decrease in abundance compared with the control. In total, 40 proteins were identified. These proteins were mainly included in process related to oxidative stress, energetic metabolism, and the cytoskeleton assembly. MCLR exposure also affects the expression of the subunits of protein phosphatases 2A. Furthermore, the proteomic and transcriptional analysis of nine proteins was determined by Western blot and quantitative real-time PCR due to their correlation with the known MCLR toxic mechanisms. The consistent and discrepant results between protein and mRNA levels indicated complicated regulatory mechanisms of gene expression in response to MCLR exposure.

It is well known that fish have stronger tolerance than mammals to microcystin (MC) exposure, and such a difference is attributed to their different core body temperatures. However, no in vivo study has been conducted to investigate the effects of temperature on MC-induced toxicity in fish, a typical poikilotherm. Tolerance and detoxification response of zebrafish treated with MC-LR were investigated under three temperatures. The LD(50) values evidently increased with a decline of the temperature (547, 260 and 176 mu g kg(-1) at 12,22 and 32 degrees C, respectively), indicating stronger tolerance of the fish at lower temperatures. Changes in the transcription of glutathione S-transferase (GST) isoforms in the fish were observed, and their sensitivity of response in the transcription of GST mRNA was on the order of 12 > 32 > 22 degrees C. We screened out several GST genes which were more delicate to solve the MC-LR exposure at different temperatures, i.e. GST rho1, al, p1 and theta1 in the 12 degrees C group, and GST zeta1 and p2 in the 22 and 32 degrees C groups. Our findings partly validate the hypothesis that high temperature enhances toxic effects of MCs on poikilotherms. Our studies also indicate that temperature-dependent toxic effects should be taken into account for field toxic assessment of microcystins in fish. Copyright (C) 2010 John Wiley & Sons, Ltd.

Microcystins (MCs) are the toxic molecules produced by common cyanobacterium in freshwater blooms. Their toxicities raise severe health issues in livestock and human beings. In current study, the immunotoxic effects of MC-LR were investigated in rabbit through evaluating the dynamics of white blood cell (WBC) numbers and cytokine production such as interleukin-3 (IL-3), IL-4, IL-6, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha). MCs at the high dose (50 mu g MC-LReq kg-1) significantly induced increase in the WBC number but decrease in the Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-3, IL-4, IL-6) production. In the low dose group(12.5 mu g MC-LReq kg-1), the number of WBC and the production of IFN-gamma, IFN-alpha, IL-4, IL-3, and IL-6 increased gradually in first 12 h, reach the peaks at 12 h, and dropped after 24 h. Significantly positive correlations were found between the cytokines production of IL-4 and IL-6, IFN-gamma and IFN-alpha, or IL-4 and IFN-gamma. In conclusion, MC-LR is able to disturb the rabbit immune system and there exists time-dose response relationship in the MC-LR-eliciting perturbation, which probably give a better insight into investigating the immunotoxicity mechanisms of MCs in vivo. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

A laboratory toxic experiment was conducted to examine dose-dependent effects of extracted microcystins (MCs) on embryonic development, larval growth and histopathological changes of southern catfish (Silurus meridionalis). Fertilized eggs were incubated in solutions with four concentrations of MCs (0, 1, 10, 100 microg MC-L Req l(-1)). Higher MCs retarded egg development (2-10h delays) and larval growth, reduced hatching rate (up to 45%), and caused high malformation rate (up to 15%) and hepatocytes damage (characterized by disorganization of cell structure and a loss of adherence between hepatocytes, cellular degeneration with vacuolar hepatocytes and marginal nuclei, even hepatocellular necrosis). A 10 microg MC-L Req l(-1) is close to a high concentration in natural cyanobacterial blooms, suggesting a possible existence of such toxic effects in eutrophic waters.

Purpose: The aim of this study was to investigate the role of de novo glutamine (Gln) synthesis in the proliferation of C6 glioma cells and its detection with 13N-ammonia. Methods: Chronic Gln-deprived C6 glioma (0.06C6) cells were established. The proliferation rates of C6 and 0.06C6 cells were measured under the conditions of Gln deprivation along with or without the addition of ammonia or glutamine synthetase (GS) inhibitor. 13N-ammonia uptake was assessed in C6 cells by gamma counting and in rats with C6 and 0.06C6 xenografts by micro–positron emission tomography (PET) scanning. The expression of GS in C6 cells and xenografts was assessed by Western blotting and immunohistochemistry, respectively. Results: The Gln-deprived C6 cells showed decreased proliferation ability but had a significant increase in GS expression. Furthermore, we found that low concentration of ammonia was sufficient to maintain the proliferation of Gln-deprived C6 cells, and 13N-ammonia uptake in C6 cells showed Gln-dependent decrease, whereas inhibition of GS markedly reduced the proliferation of C6 cells as well as the uptake of 13N-ammoina. Additionally, microPET/computed tomography exhibited that subcutaneous 0.06C6 xenografts had higher 13N-ammonia uptake and GS expression in contrast to C6 xenografts. Conclusion: De novo Gln synthesis through ammonia–glutamate reaction plays an important role in the proliferation of C6 cells. 13N-ammonia can be a potential metabolic PET tracer for Gln-dependent tumors.

Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study.

Toxic cyanobacterial blooms, which produce cyclic heptapeptide toxins known as microcystins, are worldwide environmental problems. On the other hand, the cyanobacteria protein (30–50%) has been recommended as substitute protein for aquaculture. The present laboratory study verified the feasibility of cyanobacteria protein substitution and risk assessment. Goldfish were fed diets supplemented lyophilised cyanobacteria powder for 16 weeks with the various doses: 0% (control), 10%, 20%, 30% and 40%. Low doses (10% and 20%) promoted growth whereas high doses (30% and 40%) inhibited growth. In cyanobacteria treated fish, the proximate composition of ash, crude fat content and crude protein content decreased in 16 weeks; the saturated fatty acid (SFA) content significantly increased; the n-3 polyunsaturated fatty acid content, collagen content and muscle pH significantly decreased; cooking loss percents increased significantly. Muscle fiber diameter and myofibril length were negatively correlation. Additionally, flavour compounds (e.g., amino acids, nucleotides, organic acids and carnosine) changed significantly in the treated fish and odour compounds geosmin and 2-methylisoborneol increased significantly. The estimated daily intake (EDI) of microcystins in muscle was close to or exceeded the World Health Organization (WHO) tolerable daily intake (TDI), representing a great health risk. Cyanobacterie is not feasible for protein sources use in aquaculture.

The outbreak of cyanobacterial blooms induces the production and release of microcystins (MCs) into water, representing a health hazard to aquatic organisms and even humans. Some recent studies have suggested that kidney is another important target organ of MCs except liver, however, the potential toxicity mechanisms are still unclear. In this study, we first investigated the collaborative effect of oxidative stress and cytoskeletal disruption in microcystin-induced apoptosis in CIK (Ctenopharyngodon idellus kidney) cells in vitro. CIK cells were treated with 0, 1, 10, and 100 mu g/L microcystin-LR (MC-LR) for 24 and 48 h. Cell viability was increased by MC-LR in 1 mu g/L group, while decreased in 100 mu g/L group at 48 h. Cell cycle assay showed that 1 and 10 mu g/L MC-LR induced cell cycle through G(1) into S and G(2)/M phases, while 100 mu g/L MC-LR reduced G2/M phase population. MC-LR markedly induced apoptosis in 10 and 100 mu g/L groups. Elevated reactive oxygen species (ROS) production, increased malondialdehyde (MDA) contents, decreased glutathione (GSH) levels, and modulated antioxidant enzymes including catalase (CAT) and superoxide dismutase (SOD) were observed in CIK cells exposed to MC-LR. These alterations were more pronounced at higher doses (10 and 100 mu g/L), indicating that oxidative stress was induced by MC-LR. Laser scanning confocal microscope observation showed aggregation and collapse of microfilaments (MFs) and microtubules (MTs) in CIK cells, and even loss of some cytoskeleton structure. Moreover, transcriptional changes of cytoskeletal genes (beta-actin, Ic3a, and keratin) were also determined, which have a high probability with cytoskeleton structure damage. Our data suggest that oxidative stress and cytoskeletal disruption may interact with each other and jointly lead to apoptosis and renal toxicity induced by MCs. (C) 2015 Elsevier B.V. All rights reserved.

Microcystins (MCs) are algal toxins produced intracellularly within the cyanobacteria cells. MCs exposure exerts great harm to the reproductive system of fish and deteriorates the quality of eggs and sperms, and has further adverse effects on early developmental stages of fish. Whether the MC toxicity can be parentally transmitted to offspring, even though the embryos and larvae are free of MC exposure? In the present study, adult zebrafish were continuously exposed to MC-LR (with dose of 1, 5 and 20 μg/L) for 30 days. After MC-LR exposure, fertilized eggs were collected and the following F1 generation was reared in water containing no MC-LR until 60 days post fertilization (dpf). In F1 offspring, both body weight and body length were evidently dropped. Some growth and immune related genes were detected using the real-time PCR. The transcriptional levels of these genes significantly decreased in F1 offspring of zebrafish whose parents were treated with 5 and 20 μg/L MC-LR. The activities of some antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) significantly dropped in 5 and 20 μg/L MC-LR groups, and the malondialdehyde (MDA) levels markedly increased in all the three treatment groups. Furthermore, distinct pathological changes in liver were observed in F1 zebrafish. Our findings show that the MC-LR exposure to parental zebrafish results in liver damage and evidently influences the growth and immune function in F1 offspring. We consider this damage as a parental transmission effect of microcystin toxicity. Further mechanism studies are necessary to elucidate this transmission effect.

Microcystins (MCs) are well-known cyanobacterial toxins produced in eutrophic waters and can act as potential carcinogens and have caused serious risk to human health. However, pleiotropic even paradoxical actions of cells exposure to MCs have been reported, and the mechanisms of MC-induced tumorigenesis and apoptosis are still unknown. In this study, we performed the first comprehensive in vitro investigation on carcinogenesis associated with nuclear factor kappa B (NF-kappa B) and its downstream genes in HeLa cells (Human cervix adenocarcinoma cell line from epithelial cells) exposure to MC-RR. HeLa cells were treated with 0, 20, 40, 60, and 80 mu g/mL MC-RR for 4, 8, 12, and 24 h. HeLa cells presented dualistic responses to different doses of MCs. CCK8 assay showed that MC-RR exposure evidently enhanced cell viability of HeLa cells at lower MCs doses. Cell cycle and apoptosis analysis revealed that lower MCs doses promoted G(1)/S transition and cell proliferation while higher doses of MCs induced apoptosis, with a dose-dependent manner. Electrophoretic mobility shift assay (EMSA) revealed that MC-RR could increase/decrease NF-kappa B activity at lower/higher MC-RR doses, respectively. Furthermore, the expression of NF-kappa B downstream target genes including c-FLIP, cyclinD1, c-myc, and c-IAP2 showed the same variation trend as NF-kappa B activity both at mRNA and protein levels, which were induced by lower doses of MC-RR and suppressed by higher doses. Our data verified for the first time that NF-kappa B pathway may mediate MC-induced cell proliferation and apoptosis and provided a better understanding of the molecular mechanism for potential carcinogenicity of MC-RR. (C) 2014 Elsevier Ltd. All rights reserved.

Microcystins (MCs) cause normocytic anemia in patients in a hemodialysis unit in Caruaru, Brazil in 1996, but the underlying mechanisms are still unclear. In the present study, Balb/c mice were intraperitoneally injected with microcystin-LR (MC-LR) at the doses of 0.5, 2 and 8 mu/kg body weight (bw) every 48 h for 30d. After the prolonged exposure of MC-LR, significant decreases of red blood cell count (RBC), hemoglobin (Hb) and hematocrit (Ht) were observed in 2 and 8 mu g/kg bw groups, but erythrocyte mean corpuscular volume (MCV) showed no significant changes. Significantly elevated micronucleus frequency was observed in bone marrow cells (BMCs) in all MC-LR treatments. The proliferation of BMCs significantly declined in both 2 and 8 mu g/kg bw groups. Serum levels of some hematopoietic growth factors significantly changed in 8 mu g/kg bw group, mainly including granulocyte-macrophage (GM-CSF), erythropoietin (EPO), interleukin-3 (IL-3) and TNF-alpha. The transcriptional levels of these 4 genes in BMCs were also significantly changed in 8 mu g/kg bw group. MC-LR exposure significantly increased the apoptosis rates in all MC-LR treatments. The present study indicates prolonged exposure of MC-LR induces normocytic anemia, and the disturbed hematopoietic growth factors and BMCs apoptosis are responsible for this normocytic anemia. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

An acute toxicological experiment was designed to characterize the sequence of renal ultrastructural changes with accumulated MCs in crucian carp injected intraperitoneally (i.p.) with extracted microcystins (mainly MC-RR and -LR) at two doses, 50 and 200 mu g MC-LReq. kg(-1) body weight. Quantitative and qualitative determinations of MCs in the kidney were conducted by HPLC and LC-MS, respectively. MC-RR content in kidney of crucian carp showed a time dose-dependent increase within 48 h post-injection, followed by a sharp decline afterward, while no MC-LR in kidney was detectable throughout the experiment. Ultrastructural changes in the kidney of crucian carp progressed with increasing accumulated MCs and exposure times within 48 h post-injection, whereas renal ultrastructural recovery of crucian carp in the 50 mu g MC-LReq. kg(-1) dose group was evident at 168 h post-injection. Our ultrastructural observation suggests that the membranous structure is the main action site of MCs in the kidney, among which mitochondria damage in the tubules is clearly an early, and presumably a critically important effect of MCs. The increases in blood urea nitrogen (BUN) and creatinine (CR) in both dose groups further revealed severe impairment occurred in the kidney of crucian carp. (C) 2013 Elsevier Ltd. All rights reserved.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs) released during cyanobacterial blooms exert toxicity on fish. Up to now, immunotoxicity of MCs on fish has been rarely reported. The present study investigated immune response of crucian carp (Carassius auratus) to cyanobacteria via chronic exposure in diet. Fish were fed with diets containing 20% (low dose group) and 40% (high dose group) of cyanobacteria lyophilized powder. After exposure of 30 d, a batch of assays was determined for assessing immunotoxicity of MCs. The head kidney and spleen indexes significantly increased in high dose group. Blood nitroblue tetrazolium activity in high dose group was nearly twice as much as that in control group with no cyanobacteria additive. Marked haemorrhage and hyperemia were observed in kidney and spleen in high dose group. The edematous mitochondria, deformation of the nucleus and compaction of chromatin occurred in lymphocytes of head kidney and spleen in both cyanobacteria groups. Lysozyme activity showed an obvious increase in low dose group but a sharp decrease in high dose group. Significant increase of macrophage bactericidal activity was detected in low dose group. The present findings indicate that via chronic diet exposure of different cyanobacteria levels, fish exhibit various immune responses. Fish immunity tends to proceed toward the direction of immunostimulative response at low MCs concentrations but toward the trend of immunosuppressive answer at high MCs concentrations.

Fish suffer from anemia and hypovolemic hypotensive shock after in vivo exposure with microcystins. However, except for in vivo causes for anemia and hypotension, an in vitro study of fish erythrocytes exposed to MC is necessary. For a better understanding of hematology toxicity of MC, the main aim of the present study was to investigate the toxic effects of microcystin on fish erythrocytes in vitro. Crucian carp erythrocytes were incubated in vitro with microcystin-LR (MC-LR) at doses of 0, 1, 10, 100 and 1,000 nM. The level of lipid peroxidate significantly increased in MC-LR treatment groups. Glutathione decreased after exposure to MC-LR. The activities of antioxidative enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase, were significantly increased after exposure with MC-LR. The hemolysis was significantly increased, while the activities of acetylcholinesterase, Na+–K+-ATPase and Ca2+–Mg2+-ATPase were significantly decreased. In addition, pathological alterations in agglomerated and jagged erythrocytes were observed in blood smears. The findings indicate that damages to erythrocytes should also be responsible for anemia and hypotensive shock or even death.

Circulatory responses of crucian carp injected intraperitoneally with extracted micro-cystins (MCs) were studied at sublethal and lethal doses (150 and 600 mu g MC kg(-1) body mass, respectively). Mean arterial blood pressure (MAP), heart rate, hematocrit (Hct), red blood cell (RBC) counts, and circulating blood volume (BV) were assayed at 0, 1, 3, 12, 24, and 48 h post-toxin administration. MAP decreased significantly in a dose-dependent manner over time. Within the 48-h test period, the lethal dose as well as the sublethal dose resulted in a steady decline of MAP without recovery. Heart rate significantly increased within 24 h post-injection as blood pressure significantly dropped, then showed a terminal decline to the control level. The dose-dependent decreases in BV and Hct were directly related to the drop in MAP. Intraperitoneal injection of a lethal dose of MCs led to hepatic and gill hemorrhage. Consequently, crucian carp given MCs suffered from hypovolemic hypotensive shock. (C) 2009 Elsevier Ltd. All rights reserved.

This study was conducted to investigate time-dependent changes in oxidative enzymes in liver of crucian carp after intraperitoneally injection with extracted microcystins 600 and 150 microg kg(-1) body weight. The results showed that activities of antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase generally exhibited a rapid increase in early phase (1-3 h post injection), but gradually decreased afterwards (12-48 h) compared with the control, with an evident time-dependent effect. These zigzag changes over time contributed a better understanding on oxidative stress caused by microcystins in fish.

Oxidative stress response after prolonged exposure to a low dose of microcystins (MCs) was studied in liver, kidney and brain of domestic rabbits. Rabbits were treated with extracted MCs (mainly MC-LR and MC-RR) at a dose of 2 MC-LReq. mu g/kg body weight or saline solution every 24 h for 7 or 14 days. During the exposure of MCs, increase of lipid peroxidation (LPO) levels were detected in all the organs studied, while antioxidant enzymes responded differently among different organs. The enzyme activities Of Superoxide dismutase (SOD). catalase (CAT) and glutathione reductase (GR) in liver decreased in the MCs treated animals. In brain, there were obvious changes in glutathione peroxidase (GPx) and GR, while only CAT was obviously influenced in kidney. Therefore, daily exposure at a lower dosage of MCs, which mimicked a natural route of MCs. could also induce obvious oxidative stress in diverse organs of domestic rabbits. The oxidative stress induced by MCs in brain was as serious as in liver and kidney, suggesting that brain may also be a target of MCs in mammals. And it seems that animals may have more time to metabolize the toxins or to form an adaptive response to reduce the adverse effects when exposed to the low dose of MCs. (C) 2008 Elsevier B.V. All rights reserved.