Effects of adrenergic agonists on the extrahepatic expression of vitellogenin Ao1 in heart and brain of the Chinese rare minnow (Gobiocypris rarus)

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins and traditionally considered as the estrogen responsive precursors of the major egg yolk proteins. We identified five clones encoding VTGs, about 16% of the random EST clones from our constructed cDNA library from Chinese rare minnow liver tissue treated with 17 beta-estradiol (E2). Full-length vtgAo1 has been obtained based on the sequence information of four partial cDNA inserts by RACE. The inducibility of the vtgAo1 expression in liver by E2 was confirmed by RT-PCR. The presence of vtgAo1 transcripts have been observed primarily in liver. However. a significant level of the vtgAo1 was found in an unexpected location, heart, particularly in atrial cells by RT-PCR and whole mount in situ hybridization analyses. The vtgAo1 mRNA expression in heart and liver tissue could be suppressed by both alpha-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). The expression of VTG in the heart observed in the present studies suggested it may provide protection from surplus intracellular lipids in fish cardiomyocytes as triglyceride transport proteins do in mammals. The results also indicated that the production of teleost vtg in vivo can be regulated by riot only estrogenic agents, but adrenergic signals as well. (c) 2008 Elsevier B.V. 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.

Physiological Stress of High NH (4) (+) Concentration in Water Column on the Submersed Macrophyte Vallisneria Natans L.

The submersed macrophyte, Vallisneria natans L., was cultured in laboratory with NH (4) (+) -enriched tap water (1 mg L-1 NH4-N) for 2 months and the stressful effects of high ammonium (NH (4) (+) ) concentrations in the water column on this species was evaluated. The plant growth was severely inhibited by the NH (4) (+) supplement in the water column. The plant carbon and nitrogen metabolisms were disturbed by the NH (4) (+) supplement as indicated by the accumulation of free amino acids and the depletion of soluble carbohydrates in the plant tissues. The results suggested that high NH (4) (+) concentrations in the water column may hamper the restoration of submersed vegetation in eutrophic lakes.

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.

Spatial and temporal variations of microcystins in hepatopancreas of a freshwater snail from Lake Taihu

In this paper, spatial and temporal variations of three common microcystins (MC-RR, MC-YR, and MC-LR) in the hepatopancreas of a freshwater snail (Bellamya aeruginosa) were studied monthly in two bays of Lake Taihu. Microcystins (MCs) concentration in hepatopancreas was quantified by liquid chromatography-mass spectrometry (LC-MS). The MCs concentrations in hepatopancreas were higher at Site 1 than those at other sites, which was in agreement with the changes of intracellular MCs concentrations in the water column. There was a significant correlation between MCs concentrations in the hepatopancreas and that in the seston, suggesting that spatial variances of MCs; concentrations in hepatopancreas among the five sites were due to spatial changes of toxic Microcystis cells in the water column. PCCA indicates that in addition to Microcystis, other factors (e.g., water temperature) also substantially affected the accumulation of MCs in hepatopancreas of the snail. (C) 2008 Published by Elsevier Inc.

The effect of cyanobacterial crude extract on the transcription of GST mu, GST kappa and GST rho in different organs of goldfish (Carassius auratus)

The glutathione S-transferases play important roles in the detoxification of microcystin. Core-sequences of three classes of GST (mu, kappa and rho) were cloned from goldfish (Carassius auratus L) i.p. injected with cyanobacterial crude extract at two doses (50 and 200 mu g MC-LReq kg(-1) BW). The relative changes of the mRNA abundance in liver, kidney and intestine were analyzed by real-time PCR. The transcription of GST mu was inhibited in intestine at both doses and the transcription of GST kappa was inhibited from 12 to 48 h in kidney at both doses. The decreased transcription of GST rho was detected in all three organs at the high dose. It is suggested that transcription inhibition of GST rho might be significant in MCs toxicity at higher toxin concentration in omnivorous freshwater fish. Alteration in transcription of GSTs stimulated by MCs implicates an increased health risk to fish. (C) 2008 Published by Elsevier B.V.

Experimental studies on the effects of toxic Microcystis aeruginosa PCC7820 on the survival and reproduction of two freshwater rotifers Brachionus calyciflorus and Brachionus rubens

Blooms of Microcystis aeruginosa frequently occur in many eutrophic lakes in China, however, there is very little experimental study on the relationship between Microcystis and rotifers from Chinese waters. The effects of different concentrations of toxic M. aeruginosa PCC7820 on two common freshwater rotifers Brachionus calyciflorus and B. rubens were investigated in laboratory experiments. B. calyciflorus was able to utilize this strain of M. aeruginosa as a food source. However, M. aeruginosa suppressed the survival and reproduction of B. calyciflorus at the highest concentration (10(6) cells/ml) probably due to the inadequate nutrition. B. rubens was inhibited by toxic M. aeruginosa PCC7820 and the inhibition increased with the increasing Microcystis concentration. Our study indicates that the two rotifers have different sensitivities to toxic M. aeruginosa and that toxic cyanobacteria may affect zooplankton community structure by differentially inhibiting the different zooplankton taxa.

Cytotoxicity evaluation of three pairs of hexabromocyclododecane (HBCD) enantiomers on Hep G2 cell

Hexabromocyclododecanes (HBCDs) are additive brominated flame retardants mainly used in plastics and textiles. At the present time, these compounds are found in almost all environmental and human samples. In order to evaluate the environmental safety and health risk of HBCDs, the enantiomerically pure alpha-, beta-, and gamma-HBCD were prepared using high performance liquid chromatography (HPLC) on a PM-P-CD column and the cytotoxicities of their enantiomers were evaluated in Hep G2 cells. Results from the 3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), resazurin reduction and lactate dehydrogenase (LDH) release assays showed a good agreement that the order of cytotoxicity was gamma-HBCD >= beta-HBCD > alpha-HBCD, and that significantly lower cell viability and higher LDH release were observed in all (+)-enantiomers ((+) alpha-, (+) beta- and (+) gamma-HBCD) than the corresponding (-)-forms ((-) alpha-, (-) beta- and (-) gamma-HBCD). Additionally, the formation of reactive oxygen species (ROS) induced by these HBCD enantiomers were detected. The positive correlation between the LDH release and ROS formation demonstrated that the toxic mechanism might be mediated by oxidative damage. These results suggest that environmental and human health risks of HBCDs must be evaluated at the level of individual enantiomers. (C) 2008 Published by Elsevier Ltd.

Growth and antioxidant system of Escherichia coli in response to microcystin-RR

Microcystins are a kind of cyclic hepatoxins produced by many species of cyanobacteria. The toxic effects of microcystins on animals and plants have been well studied. However, the reports about the effects of microcystins on microbial cells are very limited. In present paper, Escherichia coli was undertaken to determine the effect of microcystin-RR. These results suggested that microcystin-RR could prolong the growth of E. coli when exposed to high concentrations of microcystin-RR and cause the accumulation of ROS and induce the oxidant stress for a short time. The antioxidant system protects E. coli from oxidative damage.

Kinetic study of the 2-methyl-3-methoxy-4-phenylbutanoic acid produced by oxidation of microcystin in aqueous solutions

Microcystins (MCs) are a family of related cyclic hepatotoxic heptapeptides, of which more than 70 types have been identified. The chemically unique nature of the C20 beta-amino acid, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca4,6-dienoic acid (Adda), portion of the MCs has been exploited to develop a strategy to analyze the entirety. Oxidation of MCs causes the cleavage of MC Adda to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB). In the present study, we investigated the kinetics of MMPB produced by oxidation of the most-often-studied MC variant, MC-LR (L = leucine, R = arginine), with permanganate-periodate. This investigation allowed insight regarding the influence of the reaction conditions (concentration of the reactants, temperature, and pH) on the conversion rate. The results indicated that the reaction was second order overall and first order with respect to both permanganate and MC-LR. The second-order rate constant ranged from 0.66 to 1.35 M/s at temperatures from 10 to 30 degrees C, and the activation energy was 24.44 kJ/mol. The rates of MMPB production can be accelerated through increasing reaction temperature and oxidant concentration, and sufficient periodate is necessary for the formation of MMPB. The initial reaction rate under alkaline and neutral conditions is higher than that under acidic conditions, but the former decreases faster than the latter except under weakly acidic conditions. These results provided new insight concerning selection of the permanganate-periodate concentration, pH, and temperature needed for the oxidation of MCs with a high and stable yield of MMPB.

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.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Detection of the hepatotoxic microcystins in 36 kinds of cyanobacteria Spirulina food products in China

Gel filtration chromatography, ultra-filtration, and solid-phase extraction silica gel clean-up were evaluated for their ability to remove microcystins selectively from extracts of cyanobacteria Spirulina samples after using the reversed-phase octadecylsilyl ODS cartridge for subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The reversed-phase ODS cartridge/silica gel combination were effective and the optimal wash and elution conditions were: H2O (wash), 20% methanol in water (wash), and 90% methanol in water (elution) for the reversed-phase ODS cartridge, followed by 80% methanol in water elution in the silica gel cartridge. The presence of microcystins in 36 kinds of cyanobacteria Spirulina health food samples obtained from various retail outlets in China were detected by LC-MS/MS, and 34 samples (94%) contained microcystins ranging from 2 to 163 ng g(-1) (mean=1427 ng g(-1)), which were significantly lower than microcystins present in blue green alga products previously reported. MC-RR-which contains two molecules of arginine (R)-(in 94.4% samples) was the predominant microcystin, followed by MC-LR-where L is leucine-(30.6%) and MC-YR-where Y is tyrose-(27.8%). The possible potential health risks from chronic exposure to microcystins from contaminated cyanobacteria Spirulina health food should not be ignored, even if the toxin concentrations were low. The method presented herein is proposed to detect microcystins present in commercial cyanobacteria Spirulina samples.