In this study, for the first time, changes in expressions of proteins and profiles of metabolites in liver of the small, freshwater fish Danio rerio (zebrafish) were investigated after long-term exposure to environmentally relevant concentrations of microcystin-LR (MC-LR). Male zebrafish were exposed via water to 1 or 10 μg MC-LR/L for 90 days, and iTRAQ-based proteomics and 1H NMR-based metabolomics were employed. Histopathological observations showed that MC-LR caused damage to liver, and the effects were more pronounced in fish exposed to 10 μg MC-LR/L. Metabolomic analysis also showed alterations of hepatic function, which included changes in a number of metabolic pathways, including small molecules involved in energy, glucose, lipids, and amino acids metabolism. Concentrations of lactate were significantly greater in individuals exposed to MC-LR than in unexposed controls. This indicated a shift toward anaerobic metabolism, which was confirmed by impaired respiration in mitochondria. Proteomics revealed that MC-LR significantly influenced multiple proteins, including those involved in folding of proteins and metabolism. Endoplasmic reticulum stress contributed to disturbance of metabolism of lipids in liver of zebrafish exposed to MC-LR. Identification of proteins and metabolites in liver of zebrafish responsive to MC-LR provides insights into mechanisms of chronic toxicity of MCs.

•MCLR could transfer from mother to offspring.•49 proteins with markedly altered expression were identified in the brain of rat pups.•MCLR may exert neurotoxicity by oxidative stress and endoplasmic reticulum stress.Recent studies showed that microcystins (MCs) can be transferred to offspring from their adults and exert notable neurotoxicity, but the exact mechanism is little known. In order to better understand cellular responses in brain tissues disrupted by prenatal transfer of MCs, this work mainly focuses on brain impairments of rat offspring. Pregnant SD rats were infused exposed to microcystin-LR (MCLR) at 10 μg/kg body weight (BW)/day or saline solution from gestational day 8 (GD8) to postnatal day 15 (PD15) of lactation. MCLR accumulation, the levels of malondialdehyde (MDA) and acetylcholine esterase (AChE) activity were detected. The results showed that MCLR enhanced toxin accumulation and MDA, but decreased GSH and the level of AChE activity in the brains of rat offspring. MCLR also caused changes to cerebrum ultrastructure, showing a sparse structure, distention of endoplasmic reticulum and swelling mitochondria. To explore the exact mechanisms, we used a proteomic analysis to identify global brain protein profiles. The proteomic results revealed that MCLR remarkably altered the abundance of 49 proteins that were involved in neurodevelopment, oxidative phosphorylation, cytoskeleton, metabolism, protein folding and degradation. Our results indicated that MCLR exerts neurotoxicity mainly by generating oxidative stress and endoplasmic reticulum stress.Biological significanceThe integration of proteomics and bioinformatics analyses revealed that perinatal exposure to MCLR can occur from mother to offspring and impair the brain of rat pups.MCLR has negative effects on the development of nervous system mainly by generating oxidative stress and endoplasmic reticulum stress.

•Development of a LC/MS/MS method for the analysis of MC-LR and its GSH metabolites in rat liver.•Pretreatment conditions were optimized to extract analytes efficiently.•Combined use of SPE columns increases analyte purity when isolated from a complex matrix.•This method has been validated with excellent results.•Time- and dosage-effect studies were conducted using rat liver.The roles of glutathione (GSH) and cysteine (Cys) in the detoxification of Microcystin-LR (MC-LR) have recently become a popular area of research. However, lacking analysis methods for MC-LR-GSH and MC-LR-Cys (two main GSH pathway metabolites) in mammals, elucidation of the detoxification mechanism and metabolic pathway of MC-LR in mammals is difficult. In this study, a novel method for the simultaneous quantitative analysis of MC-LR, MC-LR-GSH and MC-LR-Cys in rat liver was developed and validated. The analytes were simultaneously extracted from rat liver using 3 M sodium chloride solution containing 0.01 M EDTA-Na2-5% acetic acid, followed by solid-phase extraction (SPE) on Oasis HLB and silica cartridges and determination by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS/MS). Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR, MC-LR-GSH and MC-LR-Cys were obtained at three concentrations (0.2, 1.0 and 2.5 μg g−1 dry weight (DW)) with values ranging from 97.7 ± 4.2 to 98.7 ± 5.1%, 70.1 ± 4.8 to 71.1 ± 4.1% and 79.8 ± 3.5 to 81.4 ± 4.0%, respectively. The relative standard deviations (RSDs) of these compounds at 0.2, 1.0 and 2.5 μg g−1 DW were between 4.3% and 6.9%. The limits of detection (LODs) were 0.005, 0.007 and 0.006 μg g−1 DW and the limits of quantification (LOQs) were 0.017, 0.023 and 0.020 μg g−1 DW for MC-LR, MC-LR-GSH and MC-LR-Cys, respectively. Furthermore, this method was successfully applied to both time- and dosage-effect studies of MC-LR, MC-LR-GSH and MC-LR-Cys in vivo.

•A LC–MS/MS method for MC-RR and metabolites in plasma and bile was developed and validated.•Liquid–liquid and solid-phase extraction maximally enhance responses of analytes.•Limited biological liquid samples are important in the detoxification of MC-RR.•The developed assay was successfully utilized to examine toxicokinetics of MCs in plasma and bile.A rapid and sensitive liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for the simultaneous determination of microcystin-RR (MC-RR) and its glutathione and cysteine conjugates (MC-RR-GSH and MC-RR-Cys, respectively) in fish plasma and bile. The analytes were extracted using methanol, followed by an Oasis mixed-mode cation-exchange polymeric sorbent. The separation was performed on a reversed-phase Waters XBridge C18 column with the gradient mobile phase, consisting of water and acetonitrile (both acidified with 0.5‰ formic acid). Mean recoveries of MC-RR, MC-RR-GSH and MC-RR-Cys ranged from 80.7 to 93.7%, 81.1 to 93.1% and 80.3 to 93.2%, respectively, at three concentrations (0.2, 1.0 and 5.0 μg mL−1). Limits of detection (LODs) for MC-RR, MC-RR-GSH and MC-RR-Cys were 6, 12 and 9 ng mL−1, respectively. Limits of quantification (LOQs) were 15, 30 and 22.5 ng mL−1 for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. This method makes it feasible for the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in limited and complex biological fluid samples (such as plasma and bile, typically 50 μL), which were previously excluded or difficult to study due to the relatively large sample volumes.

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.

Off-flavors are among the most troublesome compounds in the environment worldwide. The lack of a viable theory for studying the sources, distribution, and effect of odors has necessitated the accurate measurement of odors from environmental compartments. A rapid and flexible microwave-assisted purge-and-trap extraction device for simultaneously determining five predominant odors, namely, dimethyltrisulfide, 2-methylisoborneol, geosmin, β-cyclocitral and β-ionone, from the primary sources and sinks is demonstrated. This instrument facilitates the extraction and concentration of odors from quite different matrices simultaneously. This device is a solvent-free automated system that does not require cleaning and is timesaving. The calibration curves of the five odor compounds showed good linearity in the range of 1–500 ng/L, with correlation coefficients above 0.999 (levels = 7) and with residuals ranging from approximately 77% to 104%. The limits of detection (S/N = 3) were below 0.15 ng/L in algae sample and 0.07 ng/g in sediment and fish tissue samples. The relative standard deviations were between 2.65% and 7.29% (n = 6). Thus the proposed design is ready for rapid translation into a standard analytical tool and is useful for multiple applications in the analysis of off-flavors.Highlights► In this study, a new online instrument was developed for the determination of odors. ► This instrument is an automated solvent free system and requires no clean-up step. ► Extraction and concentration of odors can be performed simultaneously. ► A method was developed by using the instrument for the analysis of odors from environment matrices. ► This method has been validated with excellent results.

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 μg 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.

In this study, bighead carp treated with two doses, i.e. 400 and 580 μg MC-LReq (Microcystin-LR equivalent)/kg bw. After dosing bighead carp with 400 and 580 ug MC-LReq/Kg bw, the mean concentrations of microcystins (MCs) was significantly higher in boiled muscle than unboiled controls. These results indicate that the potential threat of microcystins contaminated fish to humans has been underestimated. The increase in microcystins occurs by the release of phosphatase-bound microcystins by boiling.

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.Highlights► First simultaneously quantify MCs and their metabolites in bighead carp tissues. ► MCLR/RR-Cys are more abundant in kidney, suggesting their organotropism to kidney. ► MCs-GSH may act as a mid-metabolite and changed to MCs-Cys rapidly. ► Bighead carp can effectively metabolize MC-LR/RR into their cysteine conjugates. ► Cysteine conjugation is a vital mechanism for fish to resist toxic cyanobacteria.