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.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Chronic effects of water-borne PFOS exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test

Perfluorooctane sulfonate (PFOS) is widely distributed and persistent in the environment and wildlife. The main aim of this study was to investigate the impact of long-term exposure to low concentrations of PFOS in zebrafish. Zebrafish fry (F-0, 14d post-fertilization, dpf) were exposed via the water for 70d to 0 (control), 10, 50 and 250 mu g L-1 PFOS, followed by a further 30d to assess recovery in clean water. The effects on survival and growth parameters and liver histopathology were assessed. Although growth suppression (weight and length) was observed in fish treated with high concentrations PFOS during the exposure period, no mortality was observed throughout the 70d experiment. Embryos and larvae (F-1) derived from maternal exposure suffered malformation and mortality. Exposure to 50 and 250 mu g L-1 PFOS could inhibit the growth of the gonads (GSI) in the female zebrafish. Histopathological alterations, primary with lipid droplets accumulation, were most prominently seen in the liver of males and the changes were not reversible, even after the fish were allowed to recover for 30d in clean water. The triiodothyronine (T-3)) levels were not significantly changed in any of the exposure groups. Hepatic vitellogenin (VTG) gene expression was significantly up-regulated in both male and female zebrafish, but the sex ratio was not altered. The overall results suggested that lower concentrations of PFOS in maternal exposure could result in offspring deformation and mortality. (c) 2008 Elsevier 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.

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.

Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2 -270 ng/g dry weight (DW), followed by heart (3 -100 ng/g DW) and kidney (13 -88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

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.

Distribution of toxins in various tissues of crucian carp intraperitoneally injected with hepatotoxic microcystins

An acute toxicity experiment was conducted to examine the distribution and depuration of microcystins (MCS) in crucian carp (Carassius aurutus) tissues. Fish were injected intraperitoneally with extracted MCs at a dose of 200 mu g MC-LR (where L = leucine and R = arginine) equivalent/kg body weight. Microcystin concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, and 48 h postinjection using liquid chromatography coupled with mass spectrometry. Microcystins were detected mainly in blood (3.99% of injected dose at 1 h), liver (1.60% at I h), gonad (1.49% at 3 h), and kidney (0.14% at 48 h). Other tissues, such as the heart, gill, gallbladder, intestine, spleen, brain, and muscle, contained less than 0.1% of the injected MCs. The highest concentration of MCs was found in blood (526-3,753 ng/g dry wt), followed by liver (103-1,656 ng/g dry wt) and kidney (279-1,592 ng/g dry wt). No MC-LR was detectable in intestine, spleen, kidney, brain, and muscle, whereas MC-RR was found in all examined fish tissues, which might result from organ specificity of different MCs. Clearance of MC-RR in brain tissue was slow. In kidney, the MC-RR content was negatively correlated with that in blood, suggesting that blood was important in the transportation of MC-RR to kidney for excretion.

Acute effects of microcystins on the transcription of antioxidant enzyme genes in crucian carp Carassius auratus

Recent evidences suggested that oxidative stress may play a significant role in the pathogenesis of MCs toxicity. In the present study, the acute effects of microcystins on the transcription of antioxidant enzyme genes were investigated in liver of crucian carp i.p.-injected with 50 mu g MC-LReq per kg body weight (BW). We reported the cDNA sequences for four kinds of antioxidant enzyme (GSH-PX, CAT, Cu/Zn SOD, and GR) genes, and evaluated the oxidant stress induced by MCs through analyzing the transcription abundance of antioxidant enzyme genes using real-time PCR method. The time-dependent change of relative transcription abundance and expression of the antioxiclant enzyme genes were determined at 1, 3, 12, 24, and 48 h. The transcription abundance varied among antioxiclant enzymes, with GSH-PX and GR down-regulation, and CAT and SOD significantly upregulation. Based on these data, we tentatively concluded that the oxidant stress was induced by MCs, and caused the different response of the antioxiclant enzyme genes. (c) 2008 Wiley Periodicals, Inc.

Preliminary hazard assessment of polychlorinated biphenyls, polybrominated diphenyl ethers, and polychlorinated dibenzo-p-dioxins and dibenzofurans to Yangtze finless porpoise in Dongting Lake, China

Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), a protected endangered species, is the sole freshwater subspecies of finless porpoise, living only in the middle and lower reaches of the Yangtze River, China, and its appended lakes. Its population has decreased sharply to 1,400 because of human activities, including environmental contamination. In the present study, polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) were determined in the blubber, liver, kidney, stomach, small intestine, and brains of five individual Yangtze finless porpoises collected from 1998 to 2004. The results showed PCB concentrations ranged from 0.06 to 1.89 mu g/g lipid weight in the organs and consisted mainly of penta-, hexa-. and decachlorinated biphenyls. The PBDE concentrations were between 5.32 and 72.76 ng/g lipid weight. Tetra-, penta-, and hexabrominated diphenyl ethers were the major homologues. The PCDD/F concentrations ranged from 65 to 1,563 pg/g lipid weight, and their predominant homologues were penta- and hexachlorinated dibenzofurans and hepta- and octachlorinated dibenzo-p-dioxins. The hazard quotients (HQs) based on toxic equivalency were determined to be greater than one in all individuals for PCBs, for PCDD/Fs, and for PCBs and PCDD/Fs In addition, HQs would be higher if PBDEs were included. The results suggest that reduction of environmental contamination may contribute greatly to protecting this highly endangered species.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01 M EDTA-Na-2-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5 mu g g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0 mu g g(-1) DW, with a limit of quantification (LOQ) of 0.02 mu g g(-1) DW. The limit of detection (LOD) of the method was 0.007 mu g g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR. (c) 2007 Elsevier B.V. All rights reserved.

Biochemical and ultrastructural changes of the liver and kidney of the phytoplanktivorous silver carp feeding naturally on toxic Microcystis blooms in Taihu Lake, China

Many experimental studies have documented the impact of microcystins (MC) on fish based on either intraperitoneal injection, or oral gavaging via the diet, but few experiments were conducted by MC exposure through natural food uptake in lakes. In this study, the phytoplanktivorous silver carp were stocked in a large pen set in Meiliang Bay of Taihu Lake where toxic Microcystis blooms occurred in the warm seasons. Fish samples were collected monthly and MC concentrations in liver and kidney of the fish were determined by LC-MS. The maximum MC concentrations in liver and kidney were present in July when damages in ultrastructures of the liver and kidney were revealed by electron microscope. In comparison with previous studies on common carp, silver carp showed less damage and presence of lysosome proliferation in liver and kidney. Silver carp might eliminate or lessen cell damage caused by MC through lysosome activation. Recovery in the ultrastructures of liver and kidney after Microcystis blooms was companied with a significant decrease or even disappearance of MC. Catalase and glutathione S-transferase in liver and kidney of silver carp during Microcystis blooms were significantly higher than before and after Microcystis blooms. The high glutathione pool in liver and kidney of silver carp suggests their high resistance to MC exposure. The efficient antioxidant defence may be an important mechanism of phytoplanktivorous fish like silver carp to counteract toxic Microcystis blooms. (C) 2007 Published by Elsevier Ltd.

An improved method for the extraction of low molecular weight organic acids in variable charge soils

Due to specific adsorption to variable charge soils, low molecular weight organic acids (LMWOAs) have not been sufficiently extracted, even if common extractants, such as water and 0.1 M sodium hydroxide (NaOH), were employed. In this work, the method for extracting LMWOAs in soils with 0.1 M NaOH was improved for variable charge soils; e.g. 1.0 M potassium fluoride (KF) with pH 4.0 was applied as an extractant jointed with 0.1 M NaOH based on its stronger ability to change the electrochemical properties of variable charge soils by specific adsorption. With the proposed method, the recoveries of oxalic, tartaric, malic, citric and fumaric acids were increased from 83 4, 93 1, 22 2, 63 +/- 5 and 84 +/- 3% to 98 +/- 2, 100 +/- 2, 85 +/- 2, 90 +/- 2 and 89 +/- 2%, respectively, compared with NaOH alone. Simultaneously, the LMWOAs in Agri-Udic Ferrosol with field moisture were measured with a satisfactory result.

Effect of dietary cyanobacteria on growth and accumulation of microcystins in Nile tilapia (Oreochromis niloticus)

A 12-week growth trial was conducted in a flow-through system to investigate the chronic toxic effect of dietary intake of cyanobacteria on growth, feed utilization and microcystins accumulation in Nile tilapia (Oreochromis niloticus L.) (initial body weight: 5.6 g). Six isonitrogenous and isocaloric diets were formulated to include different contents of cyanobacteria with the dietary microcystins increasing from 0 to 5460.06 ng/g diet. The results showed that dietary intake of cyanobacteria could increase the growth of tilapia while there are no impacts on feed conversion efficiency or mortality. Feeding rate was higher for the diets containing highest cyanobacteria. Microcystins were mostly accumulated in fish liver. The relationship between microcystins contents in muscle, liver, spleen and dietary intake could be described by quadratic equations. Microcystins content in the muscle of Nile tilapia in present study exceeded the upper limit of the tolerable daily intake (TDI) of microcystins suggested by the WHO (0.04 mu g/kg body weight/d). It is suggested that Nile tilapia fed on toxic cyanobacteria is not suitable for human food. (c) 2006 Elsevier B.V. All rights reserved.

In situ studies on the bioaccumulation of microcystins in the phytoplanktivorous silver carp (Hypophthalmichthys molitrix) stocked in Lake Taihu with dense toxic Microcystis blooms

The phytoplanktivorous silver carp is an important biomanipulation fish to control cyanobacterial blooms and is also a food fish with the greatest production in China. The accumulation of the hepatotoxic microcystins (MCs) determined by LC-MS in various organs of silver carp was studied monthly in Lake Taihu dominated by toxic Microcystis aeruginosa. Average recoveries of spiked fish samples were 78% for MC-RR and 81% for MC-LR. The highest content of MCs was found in the intestine (97.48 mu g g(-1) DW), followed by liver (6.84 mu g g(-1) DW), kidney (4.8 8 mu g g(-1) DW) and blood (1.54 mu g g(-1) DW), and the annual mean MC content was in the order of intestine > liver > kidney > blood > muscle > spleen > gallbladder > gill. Silver carp could effectively ingest toxic Microcystis cells (up to 84.4% of total phytoplankton in gut contents), but showed fast growth (from 141 g to 1759 g in I year in mean weight). Silver carp accumulated less microcystins in liver than other animals in the same site or other fish from different water bodies at similar level of toxin ingestion. There was possible inhibition of the transportation of the most toxic MC-LR across the gutwall. Muscle of silver carp in Lake Taihu should not be consumed during period of dense Microcystis blooms while viscera were risky for consumption in more months. (c) 2006 Elsevier B.V. All rights reserved.