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.

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.

Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from Nostoc sphaeroides

Nostoc sphaeroides Kuetzing has been used as a traditional medicine in China to treat a variety of ailments. This research identified the antioxidant activities of polysaccharide extract from Nostoc sphaeroides. The extract, which contains 46.2% carbohydrates, exhibited an effective scavenging capability on superoxide radical, hydroxyl radicals in non site-specific as well as site-specific assays, and also performed lipid peroxidation inhibition in a dose-dependent manner. Polysaccharide extract had no 1,1-diphenyl-2-picrylhydrazyl radical scavenging potential at all test concentrations. Activities of superoxide dismutase, catalase, and glutathione peroxidase in human embryo kidney 293 cells were increased effectively when Nostoc sphaeroides extract was applied. These results suggested that the use of N. sphaeroides in treating ailments may be based on the antioxidant capacities of polysaccharide composition.

Antioxidant enzyme activities of Microcystis aeruginosa in response to nonylphenols and degradation of nonylphenols by M-aeruginosa

The aim of this study was to examine the effects of chemical nonylphenols (NPs) on the antioxidant system of Microcystis aeruginosa strains. The degradation and sorption of NPs by M. aeruginosa were also evaluated. High concentrations of NPs (1 and 2 mg/l) were found to cause increases in superoxidase dismutase (SOD) and glutathione-S-transferase (GST) activities and in glutathione (GSH) levels. These results suggest that toxic stress manifested by elevated SOD and GST levels and GSH contents may be responsible for the toxicity of NPs to M. aeruginosa and that the algal cells could improve their antioxidant and detoxification ability through the enhancement of enzymatic and nonenzymatic prevention substances. The observed elevations in GSH levels and GST activities were relatively higher than those in SOD activities, indicating that GSH and GST contributed more in eliminating toxic effects than SOD. Low concentrations of NPs (0.05-0.2 mg/l) enhanced cell growth and decreased GST activity in algal cells of M. aeruginosa, suggesting that NPs may have acted as a protecting factor, such as an antioxidant. The larger portion of the NPs (> 60%) disappeared after 12 days of incubation, indicating the strong ability of M. aeruginosa to degrade the moderate persistent NP compounds. The sorption ratio of M. aeruginosa after a 12-day exposure to low nominal concentrations of NPs (0.02-0.5 mg/l) was relatively high (> 30%). The fact that M. aeruginosa effectively resisted the toxic effects of NPs and strongly degraded these pollutants indicate that M. aeruginosa cells have a strong ability to adapt to variations in environmental conditions and that low and moderate concentrations of organic compounds may favor its survival. Further studies are needed to provide detailed information on the fate of persistent organic pollutants and the survival of algae and to determine the possible role of organic pollutants in the occurrence of water blooms in eutrophic lakes.

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 mu g MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 mu g g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 mu g g(-1) dry weight at 48 h, indicating a deputation of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis. (C) 2007 Elsevier Inc. 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.

Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell line

Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo-p-dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genescytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3 beta-hydroxysteroid dehydrogenase (3PHSD2), 17 beta-hydroxysteroid dehydrogenase (17 beta HSD1 and 17 beta HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3PHSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo-p-dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption.

Difterentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system. (c) 2006 Elsevier B.V. All rights reserved.

First report of aphantoxins in China - waterblooms of toxigenic Aphanizomenon flos-aquae in Lake Dianchi

The oligohaline cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs (A. flos-aquae) has been reported in several countries to produce paralytic shellfish poisons (PSPs) or protracted toxic effects. In the past years, A. flos-aquae blooms have occurred annually in the eutrophic Lake Dianchi (300 km(2) in area, located in southwestern China). Material from natural blooms dominated by A. flosaquae was collected and lyophilized. Acute toxicity testing was performed by mouse bioassay using extracts from the lyophilized material. Clear symptoms of PSPs, intoxications were observed. To confirm the production of PSPs, a strain of A. flos-aquae (DC-1) was isolated and maintained in culture. Histopathological effects were studied by examining the organ damages using transmission electron microscopy (TEM). Slight hepatocytic damage with swollen mitochondria was found. The ultrastructural pulmonary lesions were characterized by distortied nuclei and indenting of karyotheca, together with degeneration and tumefaction of mitochondria and endoplasmic reticulum. Control animals injected with acetic acid did not exhibit histopathological damage in any organ. Toxic effects of cultured algal cells on enzymatic systems in the mouse were studied using sublethal doses of extracts. Significant glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) increases, together with decrease of the glutathione (GSH) level, were measured. These results indicated a potential role of PSPs intoxicating and metabolizing in the test animals. HPLC-FLD and LC/MS analysis of extracts from cultured material demonstrated the PSP toxins produced by A. flos-aquae bloom. To the best of our knowledge, this is the first study reporting chemically and toxicologically confirmed PSP toxins related to A. flosaquae in China. (c) 2005 Elsevier Inc. All rights reserved.

Identification of differentially expressed genes in Tetrahmena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization

The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.

Responses of antioxidant system in Arabidopsis thaliana suspension cells to the toxicity of microcystin-RR

Microcystins are cyclic heptapeptide hepatoxins produced by many species of cyanobacteria. The toxic effects and mechanism of microcystins on animals have been well studied both in vivo and in vitro. It was also reported that microcystins had adverse effects on plants. However, to our knowledge, there is no information about the toxic effects and mechanism of microcystins on plant suspension cells. In this study, Arabidopsis thaliana suspension cells were exposed to a range dose of microcystin-RR. Lipid peroxidation, a main manifestation of oxidative damage, was studied and a time- and dose-dependent increase in malondiadehyde was observed. In contrast, glutathione (GSH) levels in the cells decreased after 48 h treatment with 1 and 5 mg/L of microcystin-RR. The activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly after 48 h exposure to I and 5 mg/L of microcystin-RR, but glutathione S-transferase (GST) activity showed no difference compared with the control. These results clearly indicate that microcystin-RR is able to cause oxidative damage in A. thaliana suspension cells. Decrease of GSH content and increases of SOD and CAT activities reveal that the antioxidant system may play an important role in eliminating or alleviating the toxicity of microcystin-RR. The possible toxicity mechanism of microcystin-RR on the A. thaliana suspension cells is also discussed in this paper. (C) 2005 Elsevier Ltd. All rights reserved.

Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption

So far, little is known on the distribution of hepatotoxic microcystin (MC) in various organs of bivalves, and there is no study on MC accumulation in bivalves from Chinese waters. Distribution pattern and seasonal dynamics of MC-LR, -YR and -RR in various organs (hepatopancreas, intestine, visceral mass, gill, foot, and rest) of four edible freshwater mussels (Anodonta woodiana, Hyriopsis cumingii, Cristaria plicata, and Lamprotula leai) were studied monthly during Oct. 2003-Sep. 2004 in Lake Taihu with toxic cyanobacterial blooms in the summer. Qualitative and quantitative determinations of MCs in the organs were done by LC-MS and HPLC. The major toxins were present in the hepatopancreas (45.5-55.4%), followed by visceral mass with substantial amount of gonad (27.6-35.5%), whereas gill and foot were the least (1.8-5.1%). The maximum MC contents in the hepatopancreas, intestine, visceral mass, gill, foot, and rest were 38.48, 20.65, 1.70, 0.64, 0.58, and 0.61 mu g/g DW, respectively. There were rather good positive correlation in MC contents between intestines and hepatopancreas of the four bivalves (r = 0.75-0.97, p < 0.05). There appeared to be positive correlations between the maximum MC content in the hepatopancreas and the delta(13)C (r = 0.919) or delta(15)N (r = 0.878) of the foot, indicating that the different MC content in the hepatopancreas might be due to different food ingestion. A glutathione (GSH) conjugate of MC-LR was also detected in the foot sample of C. plicata. Among the foot samples analyzed, 54% were above the provisional WHO tolerable daily intake (TDI) level, and the mean daily intakes from the four bivalves were 8-23.5 times the TDI value when the bivalves are eaten as a whole, suggesting the high risk of consuming bivalves in Lake Taihu. (C) 2005 Wiley Periodicals, Inc.

Microcystin-RR-induced accumulation of reactive oxygen species and alteration of antioxidant systems in tobacco BY-2 cells

Microcystins are cyclic heptapeptide hepatoxins produced by cyanobacteria. It has been shown that microcystins have adverse effects on animals and on plants as well. Previous researches also indicated that microcystins were capable of inducing oxidative damage in animals both in vivo and in vitro. In this study, tobacco BY-2 suspension cell line was applied to examine the effects of microcystin-RR on plant cells. Cell viability and five biochemical parameters including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPX) and peroxide dismutase (POD) were investigated when cells were exposed to 50 mg/L microcystin-RR. Results showed that microcystin-RR evoked decline of the cell viability to approximately 80% after treating for 144 h. ROS levels, POD and GPX activities of the treated cells were gradually increased with a time dependent manner. Changes of SOD and CAT activities were also detected in BY-2 cells. After 168 h recovery, ROS contents, POD, GPX and CAT activities returned to normal levels. These results suggest that the microcystin-RR can cause the increase of ROS contents in plant cells and these changes led to oxidant stress, at the same time, the plant cells would improve their antioxidant abilities to combat mirocystin-RR induced oxidative injury. (c) 2005 Elsevier Ltd. All rights reserved.

Growth and antioxidant system of the cyanobacterium Synechococcus elongatus in response to microcystin-RR

Microcystins, one type of the cyanobacterial toxins, show a broad range of hazardous effects on other organisms. Most of the researches on the toxic effects of microcystins have involved in animals and higher plants. Little work, however, has been done on evaluating the mechanisms of microcystin toxicity on algae. In this study, the toxicological effects of microcystin-RR (MC-RR) on the cyanobacterium Synechococcus elongatus were investigated. For this purpose, six physio-biochemical parameters (cell optical density, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST)) were tested in algal cells when exposed to 100 mug(-1) microcystin-RR. The results showed that the growth of Synechococcus elongatus ( expressed as optical density) was significantly inhibited compared with the control. At the same time, the treated algae exhibited a pronounced increase in production of ROS and MDA after 6 days exposure to microcystin-RR. Signi. cant changes in GSH levels and GSH-Px, GSH activities were also detected in algal cells, with higher values being observed in the toxin treated algae after 6 days exposure. GST activities in the treated algae exhibited a decline after exposure and rapid augmentation on day 3, thereafter, they kept at a high level when compared to the control group. GSH contents and GSH-Px activities were also significantly raised in the toxin-treated algae cells from day 3, but they showed a sharp decrease on day 4, which was the onward of cell proliferation. These results suggested that oxidative stress manifested by elevated ROS levels and MDA contents might be responsible for the toxicity of microcystin to Synechococcus elongatus and the algal cells could improve their antioxidant ability through the enhancement of enzymatic and non-enzymatic preventive substances.

Physiological and biochemical microcystin-RR toxicity to the Synechococcus elongatus

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 mug (.) L-1 significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F-v/F-m) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article. (C) 2004 Wiley Periodicals, Inc.