Laiyan Wu

Protein expression profiling in the zebrafish (Danio rerio) embryos exposed to the microcystin-LR.

Microcystin‐leucine‐arginine (MCLR) is the most toxic and the most commonly encountered variant of microcystins (MCs) in aquatic environment, and it has the potential for developmental toxicity. A number of previous studies have described the developing toxicity of MCLR based on conventional toxicological indices. However, the molecular mechanisms by which it expresses its toxicity during the early development remain largely unknown. To further our understanding of mechanisms of action and identify the potential protein biomarkers for MCLR exposure, a proteomic analysis was performed on developing zebrafish embryos exposed to 0.5 mg/L MCLR until 96 hours post‐fertilization. 2‐DE combined with MS was employed to detect and identify the protein profiles. Results showed that 75 spots from the 0.5 mg/L MCLR condition showed a significant increase or decrease in abundance compared with the control. In total, 40 proteins were identified. These proteins were mainly included in process related to oxidative stress, energetic metabolism, and the cytoskeleton assembly. MCLR exposure also affects the expression of the subunits of protein phosphatases 2A. Furthermore, the proteomic and transcriptional analysis of nine proteins was determined by Western blot and quantitative real‐time PCR due to their correlation with the known MCLR toxic mechanisms. The consistent and discrepant results between protein and mRNA levels indicated complicated regulatory mechanisms of gene expression in response to MCLR exposure.

Use of a Generalized Additive Model to Investigate Key Abiotic Factors Affecting Microcystin Cellular Quotas in Heavy Bloom Areas of Lake Taihu

Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation.

Metabolic Response to Oral Microcystin-LR Exposure in the Rat by NMR-Based Metabonomic Study

Microcystin-LR (MCLR), a potent hepatotoxin, is causing increased risks to public health. Although the liver is the main target organ of MCLR, the metabolic profiling of liver in response to MCLR in vivo remains unknown. Here, we comprehensively analyzed the metabolic change of liver and ileal flushes in rat orally gavaged with MCLR by 1H nuclear magnetic resonance (NMR). Quantification of hepatic MCLR and its glutathione and cysteine conjugates by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was conducted. Metabonomics results revealed significant associations of MCLR-induced disruption of hepatic metabolisms with inhibition of nutrient absorption, as evidenced by a severe decrease of 12 amino acids in the liver and their corresponding elevation in ileal flushes. The hepatic metabolism signature of MCLR was characterized by significant inhibition of tyrosine anabolism and catabolism, three disrupted pathways of choline metabolism, glutathione exhaustion, and disturbed nucleotide synthesis. Notably, substantial alterations of hepatic metabolism were observable even at the low MCLR-treated group (0.04 mg/kg MCLR), although no apparent histological changes in liver were observed in the low- and medium-dosed groups. These observations offered novel insights into the microcystin hepatotoxic mechanism at a functional level, thereby facilitating further assessment and clarification of human health risk from MCs exposure.

Quantitatively evaluating detoxification of the hepatotoxic microcystins through the glutathione and cysteine pathway in the cyanobacteria-eating bighead carp

Glutathione (GSH) and cysteine (Cys) conjugation have long been recognized to be important in the detoxification of microcystins (MCs) in animal organs, however, studies quantitatively estimating this process are rare, especially those simultaneously determining multiple toxins and their metabolites. This paper, for the first time, simultaneously quantified MC-LR (leucine arginine), MC-RR (arginine arginine), MCLR-GSH/Cys and MCRR-GSH/Cys in the liver, kidney, intestine and muscle of the cyanobacteria-eating bighead carp i.p. injected with two doses of MCs using liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). MCLR-Cys and MCRR-Cys content were much higher in kidney than in liver, intestine and muscle, suggesting the organotropism to kidney, while MCLR-GSH and MCRR-GSH were always below the detection limit. Bighead carp effectively metabolized MC-LR and MC-RR into the cysteine conjugates in kidney, as the ratios of MCLR-Cys to MC-LR and MCRR-Cys to MC-RR reached as high as 9.04 and 19.10, respectively. MC-LR and MC-RR were excreted mostly in the form of MCLR/RR-Cys rather than MCLR/RR-GSH, while MCs-GSH might act as mid-metabolites and changed to the more stable MCs-Cys rapidly. Cysteine conjugation of MCs appears to be an important biochemical mechanism for the cyanobacteria-eating fish to resist toxic cyanobacteria. A comparison of such detoxification mechanisms between fish and mammals would be interesting in the future studies.

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantitation of microcystin-RR and its metabolites in fish liver

A novel method for identification and quantification of microcystin-RR (MC-RR) and its metabolites (MC-RR-GSH and MC-RR-Cys) in the fish liver was developed and validated. These analytes were simultaneously extracted from fish liver using water containing EDTA with 5% acetic acid, followed by a mixed-mode cation-exchange SPE (Oasis MCX) and subsequently determined by liquid chromatography-electrospray ionization ion trap mass spectrometry (LC-ESI-ITMS). Extraction parameters including volume and pH of eluting solvents, were optimized. Best recoveries were obtained by using 10 mL of 15% ammonia solution in methanol. The mean recoveries at three concentrations (0.2, 1.0, and 5.0 microg g(-1) dry weight [DW]) for MC-RR, MC-RR-GSH and MC-RR-Cys were 93.6-99%, 68.1-73.6% and 90.0-95.2%, respectively. Method detection limit (MDL) were 4, 7 and 5 ng g(-1) DW for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. Limits of quantification (LOQs) for MC-RR, MC-RR-GSH and MC-RR-Cys were calculated to be 10, 18 and 13 ng g(-1) DW, respectively. Finally, this method was successfully applied to the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in the liver of bighead carp with acute exposure of MCs.

Rapid conversion and reversible conjugation of glutathione detoxification of microcystins in bighead carp (Aristichthys nobilis)

The glutathione and cysteine conjugates of microcystin (MC-GSH and MC-Cys, respectively) are two important metabolites in the detoxification of microcystins (MCs). Although studies have quantitated both conjugates, the reason why the amounts of MC-GSH are much lower than those of MC-Cys in various animal organs remains unknown. In this study, MC-RR-GSH and MC-RR-Cys were respectively i.p. injected into the cyanobacteria-eating bighead carp (Aristichthys nobilis), to explore the biotransformation and detoxification mechanisms of the two conjugates. The contents of MC-RR, MC-RR-GSH, MC-RR-Cys and MC-RR-N-acetyl-cysteine (MC-RR-Nac, the acetylation product of MC-RR-Cys) in the liver, kidney, intestine and blood of bighead carp in both groups were quantified via liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). In the MC-RR-GSH-treated group, the MC-RR-Cys content in the kidney increased 96.7-fold from 0.25 to 0.5h post-injection, demonstrating that MC-RR-GSH acts as a highly reactive intermediate and is rapidly converted to MC-RR-Cys. The presence of MC-RR in both MC-RR-GSH- and MC-RR-Cys-treated groups indicates, for the first time, that MC conjugation with the thiol of GSH/Cys is a reversible process in vivo. Total MC-RR concentrations dissociated from MC-RR-Cys were lower than those from MC-RR-GSH, suggesting that MC-RR-Cys is more capable of detoxifying MC-RR. MC-RR-Cys was the most effectively excreted form in both the kidney and intestine, as the ratios of MC-RR-Cys to MC-RR reached as high as 15.2, 2.9 in the MC-RR-GSH-treated group and 63.4, 19.1 in the MC-RR-Cys-treated group. Whereas MC-RR-Nac could not be found in all of the samples of the present study. Our results indicate that MC-RR-GSH was rapidly converted to MC-RR-Cys and then excreted, and that both glutathione and cysteine conjugates could release MC-RR. This study quantitatively proves the importance of the GSH detoxification pathway and furthers our understanding of the biochemical mechanism by which bighead carp are resistant to toxic cyanobacteria.

Quantitative analysis of glutathione and cysteine S-conjugates of microcystin-LR in the liver, kidney and muscle of common carp (Cyprinus carpio) in Lake Taihu

Tissue distribution of microcystin (MC)-LR-GSH, MC-LR-Cys and MC-LR of omnivorous fish in Lake Taihu was investigated. MC-LR and MC-LR-Cys were detected in liver, kidney and muscle. The concentration of MC-LR in liver and kidney was 0.052 μg g-1 DW and 0.067 μg g-1 DW, respectively. MC-LR-Cys appeared to be an important metabolite with average contents of 1.104 μg g-1 DW and 0.724 μg g-1 DW in liver and kidney, and the MC-LR-Cys/MC-LR ratio in liver and kidney reaching as high as 21.4 and 10.8. High MC-LR-Cys/MC-LR ratio and a significant correlation between MC-LR-Cys and MC-LR concentration in liver, suggest that liver is more active in detoxification of MC-LR by formation of MC-LR-Cys for omnivorous fish. Furthermore, there might be a balance between the accumulation and depuration/metabolism of MC-LR-Cys in kidney. The MC-LR-Cys can be formed in kidney directly, or transported from liver or other tissues, while the MC-LR-Cys in kidney might be dissociated to MC-LR or excreted. Although MC-LR and its metabolites were scarcely detected in muscle, it is necessary to investigate the distribution of toxic metabolites in edible muscle.