Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue.
ABSTRACT: A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ? 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.
Project description:Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm3MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754?ng?mg-1 dw, followed by MC-LR (458?ng?mg-1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria).
Project description:Fast and reliable workflows are needed to quantitate microcystins (MCs), a ubiquitous class of hepatotoxic cyanotoxins, so that the impact of human and environmental exposure is assessed quickly and minimized. Our goal was to develop a high-throughput online concentration liquid chromatography tandem mass spectrometry (LC/MS/MS) workflow to quantitate the 12 commercially available MCs and nodularin in surface and drinking waters. The method run time was 8.5 min with detection limits in the low ng/L range and minimum reporting levels between 5 and 10 ng/L. This workflow was benchmarked by determining the prevalence of MCs and comparing the Adda-ELISA quantitation to our new workflow from 122 samples representing 31 waterbodies throughout Michigan. The frequency of MC occurrence was MC-LA > LR > RR > D-Asp³-LR > YR > HilR > WR > D-Asp³-RR > HtyR > LY = LW = LF, while MC-RR had the highest concentrations. MCs were detected in 33 samples and 13 of these samples had more than 20% of their total MC concentration from MCs not present in US Environmental Protection Agency (US EPA) Method 544. Furthermore, seasonal deviations between the LC/MS/MS and Adda-ELISA data suggest Adda-ELISA cross-reacts with MC degradation products. This workflow provides less than 24-h turnaround for quantification and also identified key differences between LC/MS/MS and ELISA quantitation that should be investigated further.
Project description:Microcystins (MCs) are included in drinking water and a family of cyclic heptapeptide hepatotoxins that have been implicated in the impairment of liver function in various animals. There is scarce information on the effect of MCs on cytokines and apoptotic gene expression and on whether MCs can induce inflammation and apoptosis in avian hepatic tissue. This study investigated the expression of genes related to proinflammatory interleukins, apoptosis, and antioxidant function in chicken liver tissues cultured in the presence of different doses of microcystin-leucine-arginine (MC-LR). Livers were collected from five hens and liver slices were placed in sterile tubes containing Dulbecco's medium supplemented with 0, 1, 10, or 100 ng/mL of MC-LR. After 6 h of cultivation, total RNA was extracted and quantitative PCR analysis was performed for interleukin genes (IL-1?, IL-6, and IL-8), TNF sf15, an apoptotic gene (caspase-3), and genes involved in antioxidant function ([catalase [CAT ], glutathione peroxidase [GSH-PX ], and superoxide dismutase [SOD]). Liver tissues in each group were fixed for histopathology. MC-LR downregulated the mRNA levels of IL-1?, IL-8, and TNF sf15 as compared to the control (0 ng/mL) in dose-dependent patterns; however, the differences were not significant. The expression of IL-6 in liver tissues exposed to 100 ng/mL of MC-LR was significantly (P<0.05) lower than that in tissues exposed to 1 ng/mL. In contrast, MC-LR upregulated the mRNA expression of caspase-3 and genes involved in antioxidant function in the liver tissues after 6 h, without the difference reaching statistical significance. Hepatocytes showed vacuolar degeneration and focal necrosis according to the dose of MC-LR. This study highlighted the risk of low doses of MC-LR in chicken liver. Moreover, MC-LR could modulate the transcriptional patterns of at least IL-6 in liver-tissue culture of chicken after 6 h of exposure.
Project description:Microcystins (MCs) are the most frequently found cyanobacterial toxins in freshwater systems. Many MC variants have been identified and variants differ in their toxicity. Recent studies showed that the variants MC-LW and MC-LF might be more toxic than MC-LR, the variant that is most abundant and mostly used for risk assessments. As little is known about the presence of these two variants in The Netherlands, we determined their occurrence by analyzing 88 water samples and 10 scum samples for eight MC variants ((dm-7-)MC-RR, MC-YR, (dm-7-)MC-LR, MC-LY, MC-LW and MC-LF) by liquid chromatography with tandem mass spectrometry detection. All analyzed MC variants were detected, and MC-LW and/or MC-LF were present in 32% of the MC containing water samples. When MC-LW and MC-LF were present, they contributed to nearly 10% of the total MC concentrations, but due to their suspected high toxicity, their average contribution to the total MC toxicity was estimated to be at least 45%. Given the frequent occurrence and possible high toxicity of MC-LW and MC-LF, it seems better to base health risk assessments on the toxicity contributions of different MC variants than on MC-LR concentrations alone.
Project description:Freshwater cyanobacteria produce highly toxic secondary metabolites, which can be transported downstream by rivers and waterways into the sea. Estuarine and coastal aquaculture sites exposed to toxic cyanobacteria raise concerns that shellfish may accumulate and transfer cyanotoxins in the food web. This study aims to describe the competitive pattern of uptake and depuration of a wide range of microcystins (MC-LR, MC-LF, MC-LW, MC-LY, [Asp3]-MC-LR/[Dha7]-MC-LR, MC-HilR) and nodularins (NOD cyclic and linear) within the common blue mussel Mytilus edulis exposed to a combined culture of Microcystis aeruginosa and Nodularia spumigena into the coastal environment. Different distribution profiles of MCs/NODs in the experimental system were observed. The majority of MCs/NODs were present intracellularly which is representative of healthy cyanobacterial cultures, with MC-LR and NOD the most abundant analogues. Higher removal rate was observed for NOD (?96%) compared to MCs (?50%) from the water phase. Accumulation of toxins in M. edulis was fast, reaching up to 3.4 ?g/g shellfish tissue four days after the end of the 3-days exposure period, with NOD (1.72 ?g/g) and MC-LR (0.74 ?g/g) as the dominant toxins, followed by MC-LF (0.35 ?g/g) and MC-LW (0.31 ?g/g). Following the end of the exposure period depuration was incomplete after 27 days (0.49 ?g/g of MCs/NODs). MCs/NODs were also present in faecal material and extrapallial fluid after 24 h of exposure with MCs the main contributors to the total cyanotoxin load in faecal material and NOD in the extrapallial fluid. Maximum concentration of MCs/NODs accumulated in a typical portion of mussels (20 mussels, ?4 g each) was beyond greater the acute, seasonal and lifetime tolerable daily intake. Even after 27 days of depuration, consuming mussels harvested during even short term harmful algae blooms in close proximity to shellfish beds might carry a high health risk, highlighting the need for testing.
Project description:Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15-17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.
Project description:Mast cells (MCs) are found in increased numbers at airway mucosal surfaces in asthmatic patients. Because human airway epithelial cells (HAECs) actively participate in airway inflammatory responses and are in direct contact with MCs in the mucosa, we hypothesized that HAEC-MC interactions may contribute to the differentiation and survival of MCs in the airway mucosa. Here, we show that HAECs express mRNA and protein for soluble and membrane-bound stem cell factor, releasing soluble stem cell factor into the cell culture supernatant at a concentration of 5.9 +/- 0.1 ng per 10(6) HAEC. HAECs were able to support MC survival in coculture in the absence of any exogenous cytokines for at least 4 d. Before the initiation of coculture, MCs were uniformly tryptase and chymase (MC(TC)) double positive, but by 2 d of coculture the majority of MCs expressed tryptase (MC(T)) alone. MCs supported in coculture generated low amounts of cysteinyl-leukotrienes (cys-LT) after FcepsilonRI-dependent activation (0.2 +/- 0.1 ng of cys-LT per 10(6) cells) and required priming with IL-4 and IL-3 during coculture to achieve a quantity of cys-LT generation within the range expected for human lung mucosal MC (26.5 +/- 16 ng of cys-LT per 10(6) cells). In these culture conditions, HAECs were able to direct mucosal MC protease phenotype, but T cell-derived Th2 cytokines were required for the expression of a functional airway MC eicosanoid phenotype. Thus, distinct cell types may direct unique aspects of reactive mucosal MC phenotype in the airways.
Project description:Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard for aquatic species. As MC congeners vary in their toxicity, the congener composition of a bloom primarily dictates the severity of adverse effects and appears primarily to be governed by toxicokinetics, i.e., whether transport of MCs occurs via organic anion-transporting polypeptides (Oatps). Differences in observed MC toxicity in various fish species suggest differential expression of Oatp subtypes leading to varying tissue distribution of the very same MC congener within different species. The objectives of this study were the functional characterization and analysis of the tissue distribution of Oatp subtypes in zebrafish (Danio rerio) as a surrogate model for cyprinid fish. Zebrafish Oatps (zfOatps) were cloned, and the organ distribution was determined at the mRNA level. zfOatps were transiently expressed in HEK293 cells for functional characterization using the Oatp substrates estrone-3-sulfate, taurocholate and methotrexate and specific MC congeners (MC-LR, MC-RR, MC-LF and MC-LW). Novel zfOatp isoforms were isolated. Among these isoforms, the organ-specific expression of zfOatp1d1 and of members of the zfOatp1f subfamily was identified. At the functional level, zfOatp1d1, zfOatp1f2, zfOatp1f3 and zfOatp1f4 transported at least one of the Oatp substrates, and zfOatp1d1, zfOatp1f2 and zfOatp1f4 were shown to transport MC congeners. MC-LF and MC-LW were generally transported faster than MC-LR and MC-RR. The subtype-specific expression of zfOatp1d1 and of members of the zfOatp1f subfamily as well as differences in the transport of MC congeners could explain the MC congener-dependent differences in toxicity in cyprinids.
Project description:Microcystins (MCs), the secondary metabolites of blue-green algae, are ubiquitous and major cyanotoxin contaminants. Besides the hepatopancreas/liver, the reproductive system is regarded as the most important target organ for MCs. Although reactive oxygen species (ROS) have been implicated in MCs-induced reproductive toxicity, the role of MCs in this pathway remains unclear. In the present study, Sertoli cells were employed to investigate apoptotic death involved in male reproductive toxicity of microcystin-LR (MC-LR). After exposure to various concentrations of MC-LR for 24 h, the growth of Sertoli cells was concentration-dependently decreased with an IC50 of ~32 ?g/mL. Mitochondria-mediated apoptotic changes were observed in Sertoli cells exposed to 8, 16, and 32 ?g/mL MC-LR including the increased expression of caspase pathway proteins, collapse of mitochondrial membrane potential (MMP), and generation of ROS. Pretreatment with a global caspase inhibitor was found to depress the activation of caspases, and eventually increased the survival rate of Sertoli cells, implying that the mitochondrial caspases pathway is involved in MC-LR-induced apoptosis. Furthermore, N-acetyl-l-cysteine attenuated the MC-LR-induced intracellular ROS generation, MMP collapse and cytochrome c release, resulting in the inhibition of apoptosis. Taken together, the observed results suggested that MC-LR induced apoptotic death of Sertoli cells by the activation of mitochondrial caspases cascade, while its effects on the ROS-mediated signaling pathway may contribute toward the initiation of mitochondrial dysfunction.
Project description:In the summer of 2018, six dogs exposed to a harmful algal bloom (HAB) of Microcystis in Martin County Florida (USA) developed clinicopathological signs of microcystin (MC) intoxication (i.e., acute vomiting, diarrhea, severe thrombocytopenia, elevated alanine aminotransferase, hemorrhage). Successful supportive veterinary care was provided and led to survival of all but one patient. Confirmation of MC intoxication was made through interpretation of clinicopathological abnormalities, pathological examination of tissues, microscopy (vomitus), and analytical MC testing of antemortem/postmortem samples (vomitus, blood, urine, bile, liver, kidney, hair). Gross and microscopic examination of the deceased patient confirmed massive hepatic necrosis, mild multifocal renal tubular necrosis, and hemorrhage within multiple organ systems. Microscopy of a vomitus sample confirmed the presence of Microcystis. Three analytical MC testing approaches were used, including the MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) technique, targeted congener analysis (e.g., liquid chromatography tandem-mass spectrometry of MC-LR), and enzyme-linked immunosorbent assay (ELISA). Total Adda MCs (as MMPB) were confirmed in the liver, bile, kidney, urine, and blood of the deceased dog. Urinalysis (MMPB) of one surviving dog showed a high level of MCs (32,000 ng mL-1) 1-day post exposure, with MCs detectable >2 months post exposure. Furthermore, hair from a surviving dog was positive for MMPB, illustrating another testable route of MC elimination in canines. The described cases represent the first use of urine as an antemortem, non-invasive specimen to diagnose microcystin toxicosis. Antemortem diagnostic testing to confirm MC intoxication cases, whether acute or chronic, is crucial for providing optimal supportive care and mitigating MC exposure.