Field studies on the environmental factors in controlling microcystin production in the subtropical shallow lakes of the Yangtze River

Microcystin (MC) problem made more and more care about in China, intercellular MC (Int-MC) and cellular MC (Cel-MC) were important contents to reflect the producing-MC ability by cyanobacteria and by lakes. To study the correlations between Int-MC, Cel-MC concentration and biological and environmental factors, eight cyanobacterial blooming lakes were studied in the middle and lower reaches of the Yangtze River. Microcystin-RR (MC-RR) and Microcystin-LR (MC-LR) were the primary toxin variants in our data. From the linear correlations between MC and environmental factors, cellular-YR had significant correlation with most of chemical factors except total nitrogen (TN) and the ratio of total nitrogen and total phosphorus (TN/TP), most intracellular MC analogues had significant correlations with total dissolved nitrogen (TDN), ammonium (NH4+), nitrite (NO2-), TP, total dissolved phosphorus (TDP), Microcystis. From the canonal correspondence analysis, Int-MC concentrations were closely related with the chemical and biological factors, such as TP, total organic carbon (TOC), chlorophyll a (Chl a), Microcystis biomass, et al. While Cel-MC contents, especially Cel-RR and Cel-LR, were closely related with light environmental in the lakes such as water depth and transparence.

Deletion of yeast CWP genes enhances cell permeability to genotoxic agents

We have previously reported the development of a novel genotoxic testing system based on the transcriptional response of the yeast RNR3-lacZ reporter gene to DNA damage. This system appears to be more sensitive than other similar tests in microorganisms, and is comparable with the Ames test. In an effort to further enhance detection sensitivity, we examined the effects of altering major cell wall components on cell permeability and subsequent RNR3-lacZ sensitivity to genotoxic agents. Although inactivation of single CWP genes encoding cell wall mannoproteins had little effect, the simultaneous inactivation of both CWP1 and CWP2 had profound effects on the cell wall structure and permeability. Consequently, the RNR3-lacZ detection sensitivity is markedly enhanced, especially to high molecular weight compounds such as 4-nitroquinoline-N-oxide (> sevenfold) and phleomycin (> 13-fold). In contrast, deletion of genes encoding representative membrane components or membrane transporters had minor effects on cell permeability. We conclude that the yeast cell wall mannoproteins constitute the major barrier to environmental genotoxic agents and that their removal will significantly enhance the sensitivity of RNR-lacZ as well as other yeast-based genotoxic tests.

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.

Microcystin-RR induced apoptosis in tobacco BY-2 suspension cells is mediated by reactive oxygen species and mitochondrial permeability transition pore status

When tobacco BY-2 cells were treated with 60 mu g/mL MC-RR for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 mu g/mL MC-RR induced rapid apoptosis in tobacco BY-2 cells. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial membrane potential (Delta Psi(m)) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, blocked the decrease in Delta Psi(m) and subsequent cell apoptosis, indicating a critical role of ROS in serving as an important signaling molecule by causing a reduction of Delta Psi(m) and MC-RR-induced tobacco BY-2 cell apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of Delta Psi(m), as well as cell apoptosis when the cells were MC-RR stressed for 3 d, suggesting that PTP is involved in 60 mu g/mL MC-RR-induced tobacco cell apoptosis signalling process. Thus, we concluded that the mechanism of MC-RR-induced apoptosis signalling pathways in tobacco BY-2 cells involves not only the excess generation of ROS and oxidative stress, but also the opening of PTP inducing loss of mitochondrial membrane potential. (C) 2007 Published by Elsevier Ltd.

Dose-dependent effects of extracted microcystins on embryonic development, larval growth and histopathological changes of southern catfish (Silurus meridionalis)

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 mu g MC-LReq l(-1)). Higher MCs retarded egg development (2-10 h 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 mu g MC-LReql(-1) is close to a high concentration in natural cyanobacterial blooms, suggesting a possible existence of such toxic effects in eutrophic waters. (c) 2007 Elsevier Ltd. All rights reserved.

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.

The role of glutathione metabolism in tolerance of tobacco BY-2 suspension cells to microcystin-RR

This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 mu g/mL) for 0-6 days resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 days exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 days exposure. The GSH/GSSG ratio was much higher than control in 10 mu g/mL MC-RR treated group at day 4, but after 6 days exposure, the ratios in all treated groups were lower than that of the control group.

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.

Induction of hepatic enzymes and oxidative stress in Chinese rare minnow (Gobiocypris rarus) exposed to waterborne hexabromocyclododecane (HBCDD)

The objective of this study was to evaluate the sub-lethal toxicity of hexabromocyclododecane (HBCDD) in fish. Adult Chinese rare minnows as in vivo models were exposed to waterborne HBCDD from 1 to 500 mu g/l for 14, 28 and 42 days. Hepatic CYP1A1 (ethoxyresorufin-O-deethylase, EROD) and CYP2B1 (pentaoxyresorufin-O-depentylase, PROD) activities were measured. At the same time, molecular biomarkers of oxidative stress were also assayed in the brain, including reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances, TBARS), DNA damage and protein carbonyl, as well as superoxide dismutase (SOD) activity and glutathione (GSH) content. DNA damage was evaluated using the Comet assay on erythrocytes. Besides, the content of HBCDD in whole fish was determined after 42 days exposure. The results show that HBCDD could induce EROD and PROD at 500 mu g/l after 28 days exposure, and at 100 to 500 mu g/l after 42 days exposure (P < 0.05), respectively. ROS formation in fish brain was observed to be increased in both time- and dose-dependent manner due to HBCDD exposure. The significant increases in TBARS and protein carbonyl contents occurred in fish brain after 28 and 42 days exposure (P < 0.05). Significant DNA damage in erythrocytes by Comet assay was also found in the 100-500 mu g/l exposure groups (P < 0.05) after 42 days exposure. Moreover, significant depletion in brain GSH content occurred in all treated groups (P < 0.05) and apparent inhibition in SOD activity in brain was observed in the groups of 10-500 mu g/l concentrations during 42 days exposure. The results demonstrate that increasing duration of HBCDD exposure induced EROD and PROD activities, caused excess ROS formation, finally resulted in oxidative damage to lipids, proteins and DNA and decreased antioxidant capacities in fish. Chemical analysis of HBCDD in whole fish showed accumulation up to 654 mu g/g wet weight. (c) 2007 Elsevier B.V. All rights reserved.

Morphological variability between wild populations and inbred stocks of a Chinese minnow, Gobiocypris rarus

Gobiocypris rarus, a small, native cyprinid fish, is currently widely used in research on fish pathology, genetics, toxicology, embryology, and physiology in China. To develop this species as a model laboratory animal, inbred strains have been successfully created. In this study, to explore a method to discriminate inbred strains and evaluate inbreeding effects, morphological variation among three wild populations and three inbred stocks of G. rarus was investigated by the multivariate analysis of eight meristic and 30 morphometric characters. Tiny intraspecific variations in meristic characters were found, but these were not effective for population distinction. Stepwise discriminant analysis and cluster analysis of conventional measures and truss network data showed considerabe divergence among populations, especially between wild populations and inbred stocks. The average discriminant accuracy for all populations was 82.1% based on conventional measures and 86.4% based on truss data, whereas the discriminant accuracy for inbred strains was much higher. These results suggested that multivariate analyses of morphometric characters are an effective method for discriminating inbred strains of G. rarus. Morphological differences between wild populations and inbred strains appear to result from both genetic differences and environmental factors. Thirteen characters, extracted from stepwise discriminant analysis, played important roles in morphological differentiation. These characters were mainly measures related to body depth and head size.

The involvement of the antioxidant system in protection of desert cyanobacterium Nostoc sp against UV-B radiation and the effects of exogenous antioxidants

In this study, we found that UV-B radiation decreased photosynthetic activity and boosted lipid peroxidation of desert Nostoc sp., and exogenous chemicals (ascorbate acid (ASC), N-acetylcysteine (NAC), and sodium nitroprusside (SNP)) had obvious protective effects on photosynthesis and membranes under UV-B radiation. High-concentration SNP boosted the activities of antioxidant enzymes, but low-concentration SNP reduced the activities of antioxidant enzymes. Both NAC and ASC treatments of cells decreased activities of antioxidant enzymes. The results suggested that those chemicals possibly had different mechanisms of protection of algae cells against UV-B radiation. SNP might play double roles as a signal molecule in the formation of algae cell protection of Photosystem 11 under UV-B radiation and as a (reactive oxygen species) scavenger, while NAC and ASC might function as antioxidant reagents or precursors of other antioxidant molecules, which could protect cells directly against ROS initiated by UV-B radiation. (c) 2006 Elsevier Inc. All rights reserved.

Seasonal variations of estrogenic compounds and their estrogenicities in influent and effluent from a municipal sewage treatment plant in China

The seasonal variations of estrogenic compounds and the estrogenicities of influent and effluent were investigated by OF chemical analysis and in vitro assay in a municipal sewage treatment plant in Wuhan (China). The levels of eight estrogenic compounds, including 17 beta-estradiol (E-2) estrone (E-1), estriol (E-3) diethylstilbestrol (DES), 17 alpha-ethinylestradiol, nonylphenol (NP), 4-tert-octylphenol (OP), and bisphenol A (BPA), were measured by gas chromatography-mass spectrometry. Total estrogenic activity of sewage was quantitatively assessed using primary cultured hepatocytes of male Megalobrama amblycephala Yih using vitellogenin as a biomarker. The E-2 equivalents (EEQs) obtained from the chemical analysis were consistent with those measured by bioassay. The natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, as well as NP, were the main contributors of the total EEQs of influent and effluent in the present study. The levels of natural estrogens E-1 and E-3 in the influent and effluent were higher in winter than in summer, whereas the situation for NP and OP was the reverse. The levels of E-2, DES, and BPA varied little among different seasons. 17 alpha-Ethinylestradiol was not detected in the influent and effluent. The estrogenicities of the influent and of the primary and secondary effluents were all higher in summer than in winter. Estrogenic activities in winter mainly originated from natural (E-1, E-2, and E-3) and synthetic (DES) estrogens, whereas the increase of EEQs in summer was contributed by NP The results from chemical analysis and bioassay demonstrate that estrogenic compounds cannot be entirely removed by the existing sewage treatment process, which should be further improved to protect aquatic ecosystems and human health.

Oxidative damage in unfertilized eggs of Chinese rare minnow (Gobiocypris rarus) exposed to nonylphenol

In the present study, female Chinese rare minnows (Gobiocypris rarus) were used as in vivo models and exposed to nonylphenol (NP) at concentrations of 1 to 200 mu g/L for 21 d under semistatic conditions. Molecular biomarkers of oxidative stress were measured in unfertilized eggs and included reactive oxygen species (ROS), lipid peroxidation products (thiobarbituric acid-reactive substances [TBARS] and protein carbonyl), superoxide dismutase activity, and glutathione. Cathepsin D activity as an indicator of egg viability also was assayed. Nonylphenol induced ROS formation in unfertilized eggs in all exposed groups compared to the controls. The levels of protein carbonyl and TBARS in unfertilized eggs were significantly increased (p < 0.05) at 10 to 200 and 100 to 200 mu g/L, respectively. Good positive correlations were shown between ROS induction and levels of TBARS and protein carbonyl in eggs (R = 0.918, p < 0.05 and R = 0.784, p < 0.05, respectively). Superoxide dismutase activity in eggs was significantly inhibited (p < 0.05) in the 50 to 200 mu g/L exposure groups. Glutathione levels in eggs were significantly depleted (p < 0.05) at 100 to 200 mu g/L concentrations. In addition, ROS induction resulted in oxidative damage to lipid and protein in chorions. Significant reductions (p < 0.05) of the protein and lipid contents in chorions were both found in the 50 to 200 mu g/L exposure groups. A previous study found that NP exposure could lead to chorion thinning in zebra fish. Thus, the reductions in protein and lipid contents in chorion could be the reason for chorion thinning by NP exposure. Meanwhile, cathepsin D activity was significantly inhibited (p < 0.05) in all exposure groups. The results demonstrated that NP-induced oxidative stress could damage the chorion of unfertilized eggs and lead to a decline in gamete quality in female Chinese rare minnow.

Metals in surface sediments of large shallow eutrophic lake chaohu, china

Metal contents of surface sediments were analyzed temporally and spatially in Lake Chaohu, China. No obvious temporal variations were observed, which probably due to physio- and bio- mixing, e.g. wind and microbes, in this lake. Enrichment factor of some metals were generally greater than 1.0, suggesting significant anthropogenic impact on metal levels. Significantly positive correlations between concentrations of nutrient and metals indicated that the nutrients transported to this lake contributed, to some extent, to the enrichment of metals. The correlation between trace metals concentrations indicated the co-contamination of anthropogenically derived metal enrichment in surface sediment of Lake Chaohu.