First Identification of the Hepatotoxic Microcystins in the Serum of a Chronically Exposed Human Population Together with Indication of Hepatocellular Damage

Hepatotoxic microcystins (MCs) are the most commonly reported cyanotoxins in eutrophic freshwaters. In 1996, human intoxications by MCs caused deaths of 76 patients at Caruaru dialysis centers in Brazil. So far, there have been no direct evidences of MC occurrence in human tissue in consequence of exposure to MC. In this study, we improved cleanup procedures for detecting MCs in serum sample using liquid chromatographymass spectrometry, and confirmed for the first time the presence of MCs in serum samples (average 0.39 ng/ml, which amounts to ca. 1/87 of the concentrations found in tissue samples of the Caruaru victims) of fishermen at Lake Chaohu. Daily intake by the fishermen was estimated to be in the range of 2.2-3.9 mu g MC-LReq, whereas the provisional World Health Organization tolerable daily intake (TDI) for daily lifetime exposure is 0.04 mu g/kg or 2-3 mu g per person. Moreover, statistical analysis showed closer positive relationships between MC serum concentrations and concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase than between the MC concentrations and other biochemical indicators. Thus, the data raise the question whether extended exposure in the range of the TDI or up to a factor of 10 above it may already lead to indication of liver damage. The results also demonstrate a risk of health effects from chronic exposure to MCs at least for populations with high levels of exposure, like these fishermen.

Effects of Arsenate on Microcystin Content and Leakage of Microcystis Strain PCC7806 Under Various Phosphate Regimes

Both arsenic pollution and eutrophication are prominent environmental issues when considering the problem of global water pollution. It is important to reveal the effects of arsenic species on cyanobacterial growth and toxin yields to assess ecological risk of arsenic pollution or at least understand naturally occurring blooms. The sensitivity of cyanobacteria to arsenate has often been linked to the structural similarities of arsenate and phosphate. Thus, we approached the effect of arsenate with concentrations from 10(-8) to 10(-4) M on Microcystis strain PCC7806 under various phosphate regimes. The present study showed that Microcystis strain PCC7806 was arsenate tolerant up to 10(-4) M. And such tolerance was without reference to both content of intra- and extra-cellular phosphate. It seems that arsenate involved the regulation of microcystin synthesis and cellular polyphosphate contributed to microcystin production of Microcystis responding to arsenate, since there was a positive linear correlation of the cellular microcystin quota with the exposure concentration of arsenate when the cells were not preconditioned to phosphate starvation. It is presumed that arsenate could help to actively export microcystins from living Microcystis cells when preconditioned to phosphate starvation and incubated with the medium containing 1 mu M phosphate. This study firstly provided evidence that microcystin content and/or release of Microcystis might be impacted by arsenate if it exists in harmful algal blooms. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24:97 94, 2009.

Changes in plasma thyroid hormones and cortisol levels in crucian carp (Carassius auratus) exposed to the extracted microcystins

The endocrine response of crucian carp injected intraperitoneally with extracted microcystins (MC) was investigated in this study. Fish were injected intraperitoneally either with 0.75% NaCl (control) and Microcystis extract corresponding to 150 and 600 mu g microcystins per kg body weight. The plasma levels of triiodothyronine (T-3), thyroxine (T-4), free triiodothyronine (FT3), free thyroxine (FT4), and cortisol were determined at 0, 1, 3, 12, 24. and 48 h post-administration of MC-containing extract. Treated fish displayed abnormal behaviors, Such as a startle response and disoriented swimming, as well as changes in ventilation rates. Plasma cortisol concentrations of fish in both dose groups significantly increased after administration of extracted MC and remained high throughout the experiment, which suggested that MC elicited a stress response in treated fish. The profiles of cortisol changes in treated fish appeared to be dose dependent, indicating that fish in the high dose group experienced greater MC-incluced disturbance. Mortality occurred after 12 h in the high dose group. Plasma levels of T-4, T-3, FT4, and FT3 did not vary significantly between the control fish. In contrast to this, fish exposed to MC-containing extract showed significant declines in T-3, FT4, and FT3 levels in a dose-depenclent manner throughout the experiment. Plasma T4 levels, however, did not vary significantly in the low dose group, whereas they decreased significantly it 48 h post injection in the high dose group. This study demonstrates that administration of microcystins-containing extract causes a stress response and reduces the plasma levels of thyroid hormones in crucian carp. These results illustrate that microcystins exerted potent effects on the endocrine system of crucian carp, through activating their hypothalamus-pituitary- interrenal axis and disturbing thyroid function. (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.

Effects of microcystin-RR on the antioxidant system of Bacillus subtilis

Microcystins are a kind of cyclic hepatotoxins produced by many cyanobacterial species. Many works have been done concerning, the toxic effects of microcystins on animals and plants. However, the reports about their effects on microbial cells are very limited. In the present paper, Bacillus subtilis (B. subtilis) was used to determine the dose- and time-effect of microcystin-RR, and the results showed that the activity of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) was significantly increased to that of control, when exposed to 5 or 10 mu g/ml microcystin-RR for 1 h. The contents of thiobarbituric acid-reactive sub-stances (TBARS) and glutathione (GSH) as well as glu-tathione reductase (GR) activity were obviously increased only when exposed to 10 mu g/ml microcystin-RR. For the time-effect of microcystin-RR on B. subtilis, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and TBARS, GSH contents in B. subtilis were at first significantly increased, and then subsequently de-creased. These results suggested that microcystin-RR could induce the oxidative stress of B. subtilis for a short period. The antioxidant system protects B. subtilis from oxidative damage.

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.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.

Effects of nonylphenol on the growth and microcystin production of Microcystis strains

Both organic pollution and eutrophication are prominent environmental issues concerning water pollution in the world. It is important to reveal the effects of organic pollutants on algal growth and toxin production for assessing ecological risk of organic pollution. Since nonylphenol (NP) is a kind of persistent organic pollutant with endocrine disruptive effect which exists ubiquitously in environments, NP was selected as test compound in our study to study the relationship between NP stress and Microcystis growth and microcystin production. Our study showed that responses of toxic and nontoxic Microcystis aeruginosa to NP stress were obviously different. The growth inhibition test with NP on M. aeruginosa yielded effect concentrations EbC50 values within this range of 0.67-2.96 mg/L. The nontoxic M. aeruginosa strains were more resistant to NP than toxic strains at concentration above 1 mg/L. Cell growth was enhanced by 0.02-0.2 mg/L NP for both toxic and nontoxic strains, suggesting a hormesis effect of NP on M. aeruginosa. Both toxic and nontoxic strains tended to be smaller with increasing NP. But with the increased duration of the experiment, both the cell size and the growth rate began to resume, suggesting a quick adaptation of M. aeruginosa to adverse stress. NP of 0.05-0.5 mg/L significantly promoted microcystin production of toxic strain PCC7820, suggesting that NP might affect microcystin production of some toxic M. aeruginosa in the field. Our study showed that microcystin excretion was species specific that up to 75% of microcystins in PCC7820 were released into solution, whereas > 99% of microcystins in 562 remained in algal cells after 12 days' incubation. NP also significantly influenced microcystin release into cultural media. The fact that NP enhanced growth and toxin production of M. aeruginosa at low concentrations of 0.02-0.5 mg/L that might be possibly found in natural freshwaters implies that low concentration of NP may favor survival of M. aeruginosa in the field and may play a subtle role in affecting cyanobacterial blooms and microcystin production in natural waters. (c) 2006 Elsevier Inc. All rights reserved.

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of blue-green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue-green algae meal. Diet 1 was used as control with no blue-green algae meal whereas the content in diets 2-6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow-through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3-6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish-fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish-fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).

The optimization of high performance liquid chromatographic method for analysis of trace microcystins in natural water bodies

The ratio of methanol., water and trifluoroacetic acid ( TFA) was regulated to change the polarity and the pH of the rinse solution and the eluent, so as to improve the high performance liquid chromatography HPLC) detection method for trace microcystines (MCs) in natural water bodies. The results showed that 40 % similar to 45 % methanol-water solution containing 0. 1 % TFA could get good effects on the rinse of impurity, and 70% methanol-water solution containing 0. 1% TFA could elute all the MCs in solid phase extraction ( SPE) cartridge ( C-18), In this way. it is suggested that, in analysis of environmental samples with high concentration of impurity, impurity should be washed with 40% similar to 45% methanol-water solution containing 0. 1% TFA, and MCs should be eluted with 70% similar to 100% methanol-water solution containing 0. 1% TFA.

Variation of microcystins in a lake for water supply

A site investigation was conducted to correlate the relationships between microcystins (MC) concentration and algal growth in Dianchi Lake in China. Laboratory experiments were undertaken to test the effects of sediment adsorption, photoirradiation and biodegradation on microcystins removal. Bioaccumulation of microcystins was also determined using silver carp fish. It was observed that MC concentrations varied in accordance with algae growth in Dianchi Lake. The results obtained in the laboratory demonstrated that the removal of MC with fresh sediments was less than 10%, photoirradiation removed more than 75% MC within two hours, and the biodegradation needed much longer time to produce substantial degradation of MC. The results suggest that bioaccumulation of microcystins in fish was not significant in Dianchi Lake.

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin-LR, -YR, and -RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the P-32 radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose-dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. (C) 2000 by John Wiley & Sons, Inc.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.