RESUMEN
Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.
Asunto(s)
Microbiota , Microcistinas , Microcystis , Nitrógeno , Microcystis/metabolismo , Microcystis/crecimiento & desarrollo , Microcistinas/metabolismo , Nitrógeno/metabolismo , Aminoácidos/metabolismoRESUMEN
Accelerator mass spectrometry (AMS) is the method of choice for quantitation of low amounts of 14C-labeled biomolecules. Despite exquisite sensitivity, an important limitation of AMS is its inability to provide structural information about the analyte. This limitation is not critical when the labeled compounds are well-characterized prior to AMS analysis. However, analyte identity is important in other experiments where, for example, a compound is metabolized and the structures of its metabolites are not known. We previously described a moving wire interface that enables direct AMS measurement of liquid sample in the form of discrete drops or HPLC eluent without the need for individual fraction collection, termed liquid sample-AMS (LS-AMS). We now report the coupling of LS-AMS with a molecular mass spectrometer, providing parallel accelerator and molecular mass spectrometry (PAMMS) detection of analytes separated by liquid chromatography. The repeatability of the method was examined by performing repeated injections of 14C-labeled tryptophan, and relative standard deviations of the 14C peak areas were ≤10.57% after applying a normalization factor based on a standard. Five 14C-labeled amino acids were separated and detected to provide simultaneous quantitative AMS and structural MS data, and AMS results were compared with solid sample-AMS (SS-AMS) data using Bland-Altman plots. To demonstrate the utility of the workflow, yeast cells were grown in a medium with 14C-labeled tryptophan. The cell extracts were analyzed by PAMMS, and 14C was detected in tryptophan and its metabolite kynurenine.
Asunto(s)
Aminoácidos , Triptófano , Cromatografía Líquida de Alta Presión , Espectrometría de Masas/métodos , Cromatografía LiquidaRESUMEN
We have previously shown in a murine model of Non-alcoholic Fatty Liver Disease (NAFLD) that chronic, low-dose exposure to the Harmful Algal Bloom cyanotoxin microcystin-LR (MC-LR), resulted in significant hepatotoxicity including micro-vesicular lipid accumulation, impaired toxin metabolism as well as dysregulation of the key signaling pathways involved in inflammation, immune response and oxidative stress. On this background we hypothesized that augmentation of hepatic drug metabolism pathways with targeted antioxidant therapies would improve MC-LR metabolism and reduce hepatic injury in NAFLD mice exposed to MC-LR. We chose N-acetylcysteine (NAC, 40 mM), a known antioxidant that augments the glutathione detoxification pathway and a novel peptide (pNaKtide, 25 mg/kg) which is targeted to interrupting a specific Src-kinase mediated pro-oxidant amplification mechanism. Histological analysis showed significant increase in hepatic inflammation in NAFLD mice exposed to MC-LR which was attenuated on treatment with both NAC and pNaKtide (both p ≤ 0.05). Oxidative stress, as measured by 8-OHDG levels in urine and protein carbonylation in liver sections, was also significantly downregulated upon treatment with both antioxidants after MC-LR exposure. Genetic analysis of key drug transporters including Abcb1a, Phase I enzyme-Cyp3a11 and Phase II metabolic enzymes-Pkm (Pyruvate kinase, muscle), Pklr (Pyruvate kinase, liver, and red blood cell) and Gad1 (Glutamic acid decarboxylase) was significantly altered by MC-LR exposure as compared to the non-exposed control group (all p ≤ 0.05). These changes were significantly attenuated with both pNaKtide and NAC treatment. These results suggest that MC-LR metabolism and detoxification is significantly impaired in the setting of NAFLD, and that these pathways can potentially be reversed with targeted antioxidant treatment.
RESUMEN
Cyanotoxins called microcystins (MCs) are highly toxic and can be present in drinking water sources. Determining the structure of MCs is paramount because of its effect on toxicity. Though over 300 MC congeners have been discovered, many remain unidentified. Herein, a method is described for the putative identification of MCs using liquid chromatography (LC) coupled with high-resolution (HR) Orbitrap mass spectrometry (MS) and a new bottom-up sequencing strategy. Maumee River water samples were collected during a harmful algal bloom and analyzed by LC-MS with simultaneous HRMS and MS/MS. Unidentified ions with characteristic MC fragments (135 and 213 m/z) were recognized as possible novel MC congeners. An innovative workflow was developed for the putative identification of these ions. Python code was written to generate the potential structures of unidentified MCs and to assign ions after the fragmentation for structural confirmation. The workflow enabled the putative identification of eight previously reported MCs for which standards are not available and two newly discovered congeners, MC-HarR and MC-E(OMe)R.
Asunto(s)
Microcistinas , Espectrometría de Masas en Tándem , Cromatografía Liquida , Agua Dulce , Floraciones de Algas Nocivas , Microcistinas/análisisRESUMEN
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.
Asunto(s)
Hígado/química , Microcistinas/análisis , Animales , Cromatografía Liquida , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcistinas/farmacocinética , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Hígado/efectos de los fármacos , Microcistinas/toxicidad , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Estrés Oxidativo/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Hígado/patología , Masculino , Toxinas Marinas , Ratones , Ratones Endogámicos , Microcistinas/sangre , Microcistinas/orina , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/genética , Proteómica , Análisis de Supervivencia , Contaminantes Químicos del Agua/sangre , Contaminantes Químicos del Agua/orinaRESUMEN
Microcystins (MCs) appear during harmful algal blooms (HABs) in water sources worldwide, and represent a threat for humans and animals ingesting or inhaling MCs from the environment. Herein, treated rice husk (RH) was tested as a recyclable sorbent for removal of six MCs (MC-RR, MC-LR, MC-YR, MC-LA, MC-LF, and MC-LW) from water. RH was refluxed with hydrochloric acid and heated to 250⯰C to produce the sorbent material. Twenty milligrams of treated RH removed >95% of the MCs from a 30â¯mL solution containing 25⯵g/L of each MC. The adsorption of MCs onto RH follows the Freundlich isotherm model (R2â¯≥â¯0.9612) and pseudo-second-order kinetics (R2â¯≥â¯0.9996). More than 90% of MCs were removed within 5â¯min, and >95% were removed at equilibrium (in <40â¯min). Performance of the RH sorbent was evaluated by removing MCs from Lake Erie water collected during an algal bloom in 2017. The total concentration (extracellular plus intracellular) of six tested MCs in lake water ranged from 3.7 to 13,605.9⯵g/L, and removal of MCs by treated RH ranged from 100.0% to 71.8%, respectively. The removal capacity of RH for the six MCs from the lake water sample containing 13,605.9⯵g/L of MCs was 586⯵g per g of treated RH. After being used to extract MCs, the RH was heated to 560⯰C to produce silica nanoparticles. Therefore, treated RH enables rapid and efficient removal of MCs from water and it can be recycled for use as a raw material. Overall, treated RH can contribute to mitigation of environmental and health effects caused by MCs and reduce concerns for toxic waste disposal.
Asunto(s)
Agua Potable/análisis , Lagos/análisis , Microcistinas/análisis , Oryza/química , Contaminantes Químicos del Agua/análisis , Purificación del Agua/métodos , Adsorción , Agua Potable/microbiología , Floraciones de Algas Nocivas , Lagos/microbiología , Purificación del Agua/instrumentaciónRESUMEN
The protocols for solid-phase extraction (SPE) of six microcystins (MCs; MC-LR, MC-RR, MC-LA, MC-LF, MC-LW, and MC-YR) from mouse urine, mouse plasma, and human serum are reported. The quantification of those MCs in biofluids was achieved using HPLC-orbitrap-MS in selected-ion monitoring (SIM) mode, and MCs in urine samples were also quantified by ultra-HPLC-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimal conditions, the extraction recoveries of MCs from samples spiked at two different concentrations (1 µg/L and 10 µg/L) ranged from 90.4% to 104.3% with relative standard deviations (RSDs) ≤ 4.7% for mouse urine, 90.4-106.9% with RSDs ≤ 6.3% for mouse plasma, and 90.0-104.8% with RSDs ≤ 5.0% for human serum. Matrix-matched internal standard calibration curves were linear with R2 ≥ 0.9950 for MC-LR, MC-RR and MC-YR, and R2 ≥ 0.9883 for MC-LA, MC-LF, and MC-LW. The limits of quantification (LOQs) in spiked urine samples were â¼0.13 µg/L for MC-LR, MC-RR, and MC-YR, and â¼0.50 µg/L for MC-LA, MC-LF, and MC-LW, while the LOQs in spiked plasma and serum were â¼0.25 µg/L for MC-LR, MC-RR, and MC-YR, and â¼1.00 µg/L for MC-LA, MC-LF, and MC-LW. The developed methods were applied in a proof-of-concept study to quantify urinary and blood concentrations of MC-LR after oral administration to mice. The urine of mice administered 50 µg of MC-LR per kg bodyweight contained on average 1.30 µg/L of MC-LR (n = 8), while mice administered 100 µg of MC-LR per kg bodyweight had average MC-LR concentration of 2.82 µg/L (n = 8). MC-LR was also quantified in the plasma of the same mice. The results showed that increased MC-LR dosage led to larger urinary and plasma MC-LR concentrations and the developed methods were effective for the quantification of MCs in mouse biofluids.