Effects of adrenergic agents on the expression of zebrafish (Danio rerio) vitellogenin Ao1

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins, traditionally considered to be estrogen-responsive precursors of the major egg yolk proteins, expressed and synthesized mainly in hepatic tissue. The inducibility of VTGs has made them one of the most frequently used in vivo and in vitro biomarkers of exposure to estrogen-active substances. A significant level of zebrafish vtgAo1, a major estrogen responsive form, has been unexpectedly found in heart tissue in our present studies. Our studies on zebrafish cardiomyopathy, caused by adrenergic agonist treatment, suggest a similar protective function of the cardiac expressed vtgAo1. We hypothesize that its function is to unload surplus intracellular lipids in cardiomyocytes for "reverse triglyceride transportation" similar to that found in lipid transport proteins in mammals. Our results also demonstrated that zebrafish vtgAo1 mRNA expression in heart can be suppressed by both (x-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). Furthermore, the strong stimulation of zebrafish vtgAo1 expression in plasma induced by the beta-adrenergic antagonist, MOXIsylyl, was detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Such stimulation cannot be suppressed by taMOXIfen, an antagonist to estrogen receptors. Thus, Our present data indicate that the production of teleost VTG in vivo can be regulated not only by estrogenic agents, but by adrenergic signals as well. (c) 2009 Elsevier Inc. All rights reserved.

The first study on the effects of microcystin-RR on gene expression profiles of antioxidant enzymes and heat shock protein-70 in Synechocystis sp PCC6803

Microcystins are heptapeptide toxins produced by cyanobacteria. Microcystin-RR(MC-RR) is a common variant among the 80 variants identified so far. There have been many investigations documenting the toxic effects of microcystins on animals and higher plants, but little is known on the toxic effects of microcystins on algae, especially at molecular level. We studied the effects of MC-RR on gene expression profile of a few antioxidant enzymes and heat shock protein-70 (Hsp70) in Synechocystis sp. PCC6803. After two days post-exposure, a high dose toxin (5 mg/l, about 4.8 x 10(-3) mM) significantly increased expression levels of the genes gpx1, sodB, katG, acnB, gamma-TMTand dnaK2, while a relatively low dose toxin (1 mg/l, about 9.63 x 10(-4) mM) induced a moderate and slow increase of gene expression. Our results indicate that MC-RR could induce the oxidative stress in Synechocystis sp. PCC6803 and the increase in gene expression of antioxidant enzymes and Hsp70 might protect the organism from the oxidative damage. in addition, cell aggregation was observed during the early period of exposure, which might be a specific oxidative stress reaction to MC-RR. (C) 2008 Elsevier Ltd. All rights reserved.

Intraperitoneal injection of extracted microcystins results in hypovolemia and hypotension in crucian carp (Carassius auratus)

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.

Effects of tetrabrominated diphenyl ether and hexabromocyclododecanes in single and complex exposure to hepatoma HepG2 cells

This study was designed to determine cytotoxic effects of PBDE-47 and HBCDs individually or with a mixture of both compounds exposure to Hep G2 cells. The results showed PBDE-47 and HBCDs induced increase of nitric oxide synthase (NOS) activity, release of NO. dissipation of mitochondria membrane potential and cell apoptosis. Exposure to HBCDs induced ROS formation. Moreover, preincubation with PTIO (NO scavanger) and N-acetylcysteine (ROS scavanger) partially reversed cytotoxic effects of these compounds. The possible mechanism is that PBDE-47 and HBCDs could boost generation of NO and/or ROS, impact mitochondria, and result in start-ups of apoptosis program. Cells exposed to mixture of both compounds and each of them showed non-apoptotic rate significant difference, but the combination of them caused more adverse effects on cells. These results Suggest that PBDE-47 and HBCDs in single and complex exposure have the cytotoxic activity of anti-proliferation and induction of apoptosis in tumor cells in vitro. (C) 2008 Elsevier B.V. All rights reserved.

Plasma biochemical responses of the planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) to prolonged toxic cyanobacterial blooms in natural waters

The planktivorous filter-feeding silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are the attractive candidates for bio-control of plankton communities to eliminate odorous populations of cyanobacteria. However, few studies focused on the health of such fishes in natural water body with vigorous toxic blooms. Blood parameters are useful and sensitive for diagnosis of diseases and monitoring of the physiological status of fish exposed to toxicants. To evaluate the impact of toxic cyanobacterial blooms on the planktivorous fish, 12 serum chemistry variables were investigated in silver carp and bighead carp for 9 months, in a large net cage in Meiliang Bay, a hypereutrophic region of Lake Taihu. The results confirmed adverse effects of cyanobacterial blooms on two phytoplanktivorous fish, which mainly characterized with potential toxicogenomic effects and metabolism disorders in liver, and kidney dysfunction. In addition, cholestasis was intensively implied by distinct elevation of all four related biomarkers (ALP, GGT, DBIL, TBIL) in bighead carp. The combination of LDH, AST activities and DBIL, URIC contents for silver carp, and the combination of ALT. ALP activities and TBIL, DBIL. URIC concentrations for bighead carps were found to most strongly indicate toxic effects from cyanobacterial blooms in such fishes by a multivariate discriminant analysis. (C) 2009 Elsevier B.V. All rights reserved.

Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China

A previously unknown cyanophage, PaV-LD (Planktothrix agardhii Virus isolated from Lake Donghu), which causes lysis of the bloom-forming filamentous cyanobacterium P. agardhii, was isolated from Lake Donghu, Wuhan, China. PaV-LD only lysed P. agardhii strains isolated from Lake Donghu and not those isolated from other lakes. The PaV-LD particle has an icosahedral, non-tailed structure, ca. 70 to 85 nm (mean +/- SD = 76 +/- 6 nm) in diameter. PaV-LD was stable at freezing temperature, but lost its infectivity at temperatures >50 degrees C. Lysis of host cells was delayed about 3 d after the PaV-LD treatment with chloroform, and the virus was inactivated by exposure to low pH (<= 4). The latent period and burst size of the PaV-LD were estimated to be 48 to 72 h and about 340 infectious units per cell, respectively. The regrowth cultures of surviving host filaments were not lysed by the PaV-LD suspension. To our knowledge, this is the first isolation and cultivation of a virus infectious to the filamentous bloom-forming cyanobacterium Planktothrix from a freshwater lake.

Oxidative stress response after prolonged exposure of domestic rabbit to a lower dosage of extracted microcystins

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.

The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat

Deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, while some recent studies suggest that cardiogenic complication is also involved. So far, information on cardiotoxic effects of MC has been rare and the underlying mechanism is still puzzling. The present study examined toxic effects of microcystins on heart muscle of rats intravenously injected with extracted MC at two doses, 0.16LD(50) (14 mu g MC-LReq kg(-1) body weight) and 1LD(50) (87 mu g MC-LReq kg(-1) body weight). In the dead rats, both TTC staining and maximum elevations of troponin I levels confirmed myocardial infarction after MC exposure, besides a serious interstitial hemorrhage in liver. In the 1LD(50) dose group, the coincident falls in heart rate and blood pressure were related to mitochondria dysfunction in heart, while increases in creatine kinase and troponin I levels indicated cardiac cell injury. The corresponding pathological alterations were mainly characterized as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. MC administration at a dose of 1LD(50) not only enhanced activities and up-regulated mRNA transcription levels of antioxidant enzymes, but also increased GSH content. At both doses, level of lipid peroxides increased obviously, suggesting serious oxidative stress in mitochondria. Simultaneously. complex I and III were significantly inhibited, indicating blocks in electron flow along the mitochondrial respiratory chain in heart. In conclusion, the findings of this study implicate a role for MC-induced cardiotoxicity as a potential factor that should be considered when evaluating the mechanisms of death associated with microcystin intoxication in Brazil. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins

Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs oil proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mu g MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were Much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins. (c) 2008 Published by Elsevier Ltd.

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Distribution of microcystins in various organs (heart, liver, intestine, gonad, brain, kidney and lung) of Wistar rat via intravenous injection

The distribution of microcystins (MCs) in various tissues of Wistar rats was studied under laboratory conditions. Rats were injected intravenously (i.v.) with extracted MCs at a dose of 80 mu g MC-LRequivalent/kg body weight. MCs concentrations in various tissues were detected at 1, 2. 4, 6, 12 and 24 h post-injection using liquid chromatography-mass spectrometry (LC-MS). The highest concentration of MCs was found in kidney (0.034-0.295 mu g/g dry weight), followed by lung (0.007-0.067 mu g/g dry weight), stomach (0.010-0.058 mu g/g dry weight) and liver (0.003-0.052 mu g/g dry weight). The maximum MCs content in the whole body of rat, 2.9% of the injected dose, was observed at 2 h post-injection. MCs concentration was higher in kidney than in liver during the experiment, and two peaks of MCs concentration (at 2 and 24 h, respectively) were observed in kidney, indicating that MCs can be excreted directly via kidney of rat. Though heart, intestine, spleen, brain, gonad and stomach contained less than 0.2% of injected MCs during the whole experiment stage, the presence of MCs in these tissues represents potential damage to them. (c) 2008 Elsevier Ltd. All Fights reserved.

Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

In vivo studies on the toxic effects of microcystins on mitochondrial electron transport chain and ion regulation in liver and heart of rabbit

This study examined the toxic effects of microcystins on mitochondria of liver and heart of rabbit in vivo. Rabbits were injected i.p. with extracted microcystins (mainly MC-RR and -LR) at two doses, 12.5 and 50 MCLReq. mu g/kg bw, and the changes in mitochondria of liver and heart were studied at 1, 3,12, 24 and 48 h after injection. MCs induced damage of mitochondrial morphology and lipid peroxidation in both liver and heart. MCs influenced respiratory activity through inhibiting NADH dehydrogenase and enhancing succinate dehydrogenase (SDH). MCs altered Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities of mitochondria and consequently disrupted ionic homeostasis, which might be partly responsible for the loss of mitochondrial membrane potential (MMP). MCs were highly toxic to mitochondria with more serious damage in liver than in heart. Damage of mitochondria showed reduction at 48 h in the low dose group, suggesting that the low dose of MCs might have stimulated a compensatory response in the rabbits. (C) 2008 Elsevier Inc. All rights reserved.

Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

Perfluorooetanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship, were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of I mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential mechanisms of developmental toxicity. (C) 2008 Elsevier Inc. All rights reserved.

Gene expression profiles in liver of zebrafish treated with microcystin-LR

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptatoxin produced by cyanobacteria, which has tremendous negative impacts on fish, while its molecular mechanism behind remained unclear at present. Here, Affymetrix Zebrafish GeneChip was used to identify alterations in gene expression of zebrafish (Danio rerio) after MC-LR exposure. Among the 14,900 transcripts in the microarray, 273 genes were differentially expressed, in which 243 genes were elevated and 30 were decreased. According to GOstat analysis, MC-LR mainly influenced the cell cycle and mitogen-activated protein kinases (MAPK) signaling pathways. In addition, many immune-related genes were also influenced. These data suggest that MC-LR could promote tumorigenesis and cause immunotoxicity in fish. (C) 2008 Elsevier B.V. All rights reserved.