Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,ß-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

An assessment of a biosensor system for the quantification of microcystins in freshwater cyanobacterial blooms.

Microcystin-producing cyanobacterial blooms are a global issue threatening drinking water supplies and recreation on lakes and beaches. Direct measurement of microcystins is the only way to ensure waters have concentrations below guideline concentrations; however, analyzing water for microcystins takes several hours to days to obtain data. We tested LightDeck Diagnostics' bead beater cell lysis and two versions of the quantification system designed to give microcystin concentrations within 20 min and compared it to the standard freeze-thaw cycle lysis method and ELISA quantification. The bead beater lyser was only 30 % effective at extracting microcystins compared to freeze-thaw. When considering freeze-thaw samples analyzed in 2021, there was good agreement between ELISA and LightDeck version 2 (n = 152; R2 = 0.868), but the LightDeck slightly underestimated microcystins (slope of 0.862). However, we found poor relationships between LightDeck version 2 and ELISA in 2022 (n = 49, slopes 0.60 to 1.6; R2 < 0.6) and LightDeck version 1 (slope = 1.77 but also a high number of less than quantifiable concentrations). After the quantification issues are resolved, combining the LightDeck system with an already-proven rapid lysis method (such as microwaving) will allow beach managers and water treatment operators to make quicker, well-informed decisions.

Aerosolized Cyanobacterial Harmful Algal Bloom Toxins: Microcystin Congeners Quantified in the Atmosphere.

Cyanobacterial harmful algal blooms (cHABs) have the potential to adversely affect public health through the production of toxins such as microcystins, which consist of numerous molecularly distinct congeners. Microcystins have been observed in the atmosphere after emission from freshwater lakes, but little is known about the health effects of inhaling microcystins and the factors contributing to microcystin aerosolization. This study quantified total microcystin concentrations in water and aerosol samples collected around Grand Lake St. Marys (GLSM), Ohio. Microcystin concentrations in water samples collected on the same day ranged from 13 to 23 µg/L, dominated by the d-Asp3-MC-RR congener. In particulate matter <2.5 µm (PM2.5), microcystin concentrations up to 156 pg/m3 were detected; the microcystins were composed primarily of d-Asp3-MC-RR, with additional congeners (d-Asp3-MC-HtyR and d-Asp3-MC-LR) observed in a sample collected prior to a storm event. The PM size fraction containing the highest aerosolized MC concentration ranged from 0.44 to 2.5 µm. Analysis of total bacteria by qPCR targeting 16S rDNA revealed concentrations up to 9.4 × 104 gc/m3 in aerosol samples (≤3 µm), while a marker specific to cyanobacteria was not detected in any aerosol samples. Concentrations of aerosolized microcystins varied even when concentrations in water were relatively constant, demonstrating the importance of meteorological conditions (wind speed and direction) and aerosol generation mechanism(s) (wave breaking, spillway, and aeration systems) when evaluating inhalation exposure to microcystins and subsequent impacts on human health.

Elucidating microbial mechanisms of microcystin-LR degradation in Lake Erie beach sand through metabolomics and metatranscriptomics.

As one of five Laurentian Great Lakes, Lake Erie ranks among the top freshwater drinking sources and ecosystems globally. Historical and current agriculture mismanagement and climate change sustains the environmental landscape for late summer cyanobacterial harmful algal blooms, and consequently, cyanotoxins such as microcystin (MC). Microcystin microbial degradation is a promising mitigation strategy, however the mechanisms controlling the breakdown of MCs in Lake Erie are not well understood. Pelee Island, Ontario, Canada is located in the western basin of Lake Erie and the bacterial community in the sand has demonstrated the capacity of metabolizing the toxin. Through a multi-omic approach, the metabolic, functional and taxonomical signatures of the Pelee Island microbial community during MC-LR degradation was investigated over a 48-hour period to comprehensively study the degradation mechanism. Cleavage of bonds surrounding nitrogen atoms and the upregulation of nitrogen deamination (dadA, alanine dehydrogenase, leucine dehydrogenase) and assimilation genes (glnA, gltB) suggests a targeted isolation of nitrogen by the microbial community for energy production. Methylotrophic pathways RuMP and H4MPT control assimilation and dissimilation of carbon, respectively and differential abundance of Methylophilales indicates an interconnected role through electron exchange of denitrification and methylotrophic pathways. The detected metabolites did not resolve a clear breakdown pathway, but rather the diversity of products in combination with taxonomic and functional results supports that a variety of strategies are applied, such as epoxidation, hydroxylation, and aromatic degradation. Annual repeated exposure to the toxin may have allowed the community to adaptatively establish a novel pathway through functional plasticity and horizontal gene transfer. The culmination of these results reveals the complexity of the Pelee Island sand community and supports a dynamic and cooperative metabolism between microbial species to achieve MC degradation.

Microcystin congeners in Lake Erie follow the seasonal pattern of nitrogen availability.

Cyanobacteria harmful algal blooms produce many toxic secondary metabolites called cyanotoxins. The most studied group of cyanotoxins are microcystins (MC), with over 300 congeners reported. MC-LR is the most studied congener because of its abundance and toxicity. Recent toxicology studies suggest that more hydrophobic MC congeners such as MC-LA, MC-LF, and MC-LW may be less abundant but up to seven times more toxic than MC-LR, whereas, MC-RR's toxicity is only one-fifth that of MC-LR. Hence, understanding the environmental stressors that change the MC congener profile is critical to assessing the negative impact on environmental and human health. A two-year field and experimental study investigated seasonal and spatial changes of MC congener profiles in the western basin of Lake Erie. Both studies showed that nitrogen enrichment favored the production of nitrogen-rich MC-RR (C49H75N13O12). The field study showed that nitrogen depletion favored the low-nitrogen MC-LA (C46H67N7O12). MC-LR (a medium N level, C49H75N10O12) accounted for ∼30% to 50% of the total MC concentration and was stable across nitrogen concentrations. Using the relative toxicity and concentrations of each MC congener, both LC-MS/MS and ELISA overestimated the toxicity early bloom (July) and underestimated it late bloom (September). On 24 July 2019, highly toxic MC-LW and MC-LF were detected at nearshore stations with relative toxicity exceeding drinking water standards. This study demonstrated that the less toxic, high nitrogen MC-RR dominated under nitrogen-replete conditions in the early season, whereas the more toxic, less nitrogen MC-LA dominated under nitrogen-limited conditions later in the season.

Intraspecific genetic variation is critical to robust toxicological predictions of aquatic contaminants.

Environmental risk assessment is a critical tool for protecting aquatic life and its effectiveness is predicated on predicting how natural populations respond to contaminants. Yet, routine toxicity testing typically examines only one genotype, which may render risk assessments inaccurate as populations are most often composed of genetically distinct individuals. To determine the importance of intraspecific variation in the translation of toxicity testing to populations, we quantified the magnitude of genetic variation within 20 Daphnia magna clones derived from one lake using whole genome sequencing and phenotypic assays. We repeated these assays across two exposure levels of microcystins, a cosmopolitan and lethal aquatic contaminant produced by harmful algal blooms. We found considerable intraspecific genetic variation in survival, growth, and reproduction, which was amplified by microcystins exposure. Finally, using simulations we demonstrate that the common practice of employing a single genotype to calculate toxicity tolerance failed to produce an estimate within the 95% confidence interval over half of the time. These results illuminate the importance of incorporating intraspecific genetic variation into toxicity testing to reliably predict how natural populations will respond to aquatic contaminants.

Microcystin concentrations and congener composition in relation to environmental variables across 440 north-temperate and boreal lakes.

Understanding the environmental conditions and taxa that promote the occurrence of cyanobacterial toxins is imperative for effective management of lake ecosystems. Herein, we modeled total microcystin presence and concentrations with a broad suite of environmental predictors and cyanobacteria community data collected across 440 Canadian lakes using standardized methods. We also conducted a focused analysis targeting 14 microcystin congeners across 190 lakes, to examine how abiotic and biotic factors influence their relative proportions. Microcystins were detected in 30 % of lakes, with the highest total concentrations occurring in the most eutrophic lakes located in ecozones of central Canada. The two most commonly detected congeners were MC-LR (61 % of lakes) and MC-LA (37 % of lakes), while 11 others were detected more sporadically across waterbodies. Congener diversity peaked in central Canada where cyanobacteria biomass was highest. Using a zero-altered hurdle model, the probability of detecting microcystin was best explained by increasing Microcystis biomass, Daphnia and cyclopoid biomass, soluble reactive phosphorus, pH and wind. Microcystin concentrations increased with the biomass of Microcystis and other less dominant cyanobacteria taxa, as well as total phosphorus, cyclopoid copepod biomass, dissolved inorganic carbon and water temperature. Collectively, these models accounted for 34 % and 70 % of the variability, respectively. Based on a multiple factor analysis of microcystin congeners, cyanobacteria community data, environmental and zooplankton data, we found that the relative abundance of most congeners varied according to trophic state and were related to a combination of cyanobacteria genera biomasses and environmental variables.

Quantification of microcystin production and biodegradation rates in the western basin of Lake Erie.

Cyanobacterial biomass forecasts currently cannot predict the concentrations of microcystin, one of the most ubiquitous cyanotoxins that threaten human and wildlife health globally. Mechanistic insights into how microcystin production and biodegradation by heterotrophic bacteria change spatially and throughout the bloom season can aid in toxin concentration forecasts. We quantified microcystin production and biodegradation during two growth seasons in two western Lake Erie sites with different physicochemical properties commonly plagued by summer Microcystis blooms. Microcystin production rates were greater with elevated nutrients than under ambient conditions and were highest nearshore during the initial phases of the bloom, and production rates were lower in later bloom phases. We examined biodegradation rates of the most common and toxic microcystin by adding extracellular stable isotope-labeled microcystin-LR (1 µg L-1), which remained stable in the abiotic treatment (without bacteria) with minimal adsorption onto sediment, but strongly decreased in all unaltered biotic treatments, suggesting biodegradation. Greatest biodegradation rates (highest of -8.76 d-1, equivalent to the removal of 99.98% in 18 h) were observed during peak bloom conditions, while lower rates were observed with lower cyanobacteria biomass. Cell-specific nitrogen incorporation from microcystin-LR by nanoscale imaging mass spectrometry showed that a small percentage of the heterotrophic bacterial community actively degraded microcystin-LR. Microcystin production and biodegradation rates, combined with the microcystin incorporation by single cells, suggest that microcystin predictive models could be improved by incorporating toxin production and biodegradation rates, which are influenced by cyanobacterial bloom stage (early vs. late bloom), nutrient availability, and bacterial community composition.

As We Drink and Breathe: Adverse Health Effects of Microcystins and Other Harmful Algal Bloom Toxins in the Liver, Gut, Lungs and Beyond.

Freshwater harmful algal blooms (HABs) are increasing in number and severity worldwide. These HABs are chiefly composed of one or more species of cyanobacteria, also known as blue-green algae, such as Microcystis and Anabaena. Numerous HAB cyanobacterial species produce toxins (e.g., microcystin and anatoxin-collectively referred to as HAB toxins) that disrupt ecosystems, impact water and air quality, and deter recreation because they are harmful to both human and animal health. Exposure to these toxins can occur through ingestion, inhalation, or skin contact. Acute health effects of HAB toxins have been well documented and include symptoms such as nausea, vomiting, abdominal pain and diarrhea, headache, fever, and skin rashes. While these adverse effects typically increase with amount, duration, and frequency of exposure, susceptibility to HAB toxins may also be increased by the presence of comorbidities. The emerging science on potential long-term or chronic effects of HAB toxins with a particular emphasis on microcystins, especially in vulnerable populations such as those with pre-existing liver or gastrointestinal disease, is summarized herein. This review suggests additional research is needed to define at-risk populations who may be helped by preventative measures. Furthermore, studies are required to develop a mechanistic understanding of chronic, low-dose exposure to HAB toxins so that appropriate preventative, diagnostic, and therapeutic strategies can be created in a targeted fashion.

Identification of Novel Microcystins Using High-Resolution MS and MSn with Python Code.

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.

Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging.

Cyanotoxins can be found in water and air during cyanobacterial harmful algal blooms (cHABs) in lakes and rivers. Therefore, it is very important to monitor their potential uptake by animals and humans as well as their health effects and distribution in affected organs. Herein, the distribution of hepatotoxic peptide microcystin-LR (MC-LR) is investigated in liver tissues of mice gavaged with this most common MC congener. Preliminary matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging experiments performed using a non-automated MALDI matrix deposition device and a MALDI-time-of-flight (TOF) mass spectrometer yielded ambiguous results in terms of MC-LR distribution in liver samples obtained from MC-LR-gavaged mice. The tissue preparation for MALDI-MS imaging was improved by using an automated sprayer for matrix deposition, and liver sections were imaged using an Nd:YAG MALDI laser coupled to a 15 Tesla Fourier-transform ion cyclotron resonance (FT-ICR)-mass spectrometer. MALDI-FT-ICR-MS imaging provided unambiguous detection of protonated MC-LR (calculated m/z 995.5560, z = +1) and the sodium adduct of MC-LR (m/z 1017.5380, z = +1) in liver sections from gavaged mice with great mass accuracy and ultra-high mass resolution. Since both covalently bound and free MC-LR can be found in liver of mice exposed to this toxin, the present results indicate that the distribution of free microcystins in tissue sections from affected organs, such as liver, can be monitored with high-resolution MALDI-MS imaging.

The Lake Erie HABs Grab: A binational collaboration to characterize the western basin cyanobacterial harmful algal blooms at an unprecedented high-resolution spatial scale.

Monitoring of cyanobacterial bloom biomass in large lakes at high resolution is made possible by remote sensing. However, monitoring cyanobacterial toxins is only feasible with grab samples, which, with only sporadic sampling, results in uncertainties in the spatial distribution of toxins. To address this issue, we conducted two intensive "HABs Grabs" of microcystin (MC)-producing Microcystis blooms in the western basin of Lake Erie. These were one-day sampling events during August of 2018 and 2019 in which 100 and 172 grab samples were collected, respectively, within a six-hour window covering up to 2,270 km2 and analyzed using consistent methods to estimate the total mass of MC. The samples were analyzed for 57 parameters, including toxins, nutrients, chlorophyll, and genomics. There were an estimated 11,513 kg and 30,691 kg of MCs in the western basin during the 2018 and 2019 HABs Grabs, respectively. The bloom boundary poses substantial issues for spatial assessments because MC concentration varied by nearly two orders of magnitude over very short distances. The MC to chlorophyll ratio (MC:chl) varied by a factor up to 5.3 throughout the basin, which creates challenges for using MC:chl to predict MC concentrations. Many of the biomass metrics strongly correlated (r > 0.70) with each other except chlorophyll fluorescence and phycocyanin concentration. While MC and chlorophyll correlated well with total phosphorus and nitrogen concentrations, MC:chl correlated with dissolved inorganic nitrogen. More frequent MC data collection can overcome these issues, and models need to account for the MC:chl spatial heterogeneity when forecasting MCs.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.

Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue.

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.

Harmful Algal Bloom Toxins in Aerosol Generated from Inland Lake Water.

Harmful algal blooms (HABs) caused by cyanobacteria in freshwater environments produce toxins (e.g., microcystin) that are harmful to human and animal health. HAB frequency and intensity are increasing with greater nutrient runoff and a warming climate. Lake spray aerosol (LSA) released from freshwater lakes has been identified on lakeshores and after transport inland, including from lakes with HABs, but little is known about the potential for HAB toxins to be incorporated into LSA. In this study, freshwater samples were collected from two lakes in Michigan: Mona Lake during a severe HAB with microcystin concentrations (>200 µg/L) well above the Environmental Protection Agency (EPA) recommended "do not drink" level (1.6 µg/L) and Muskegon Lake without a HAB (<1 µg/L microcystin). Microcystin toxins were identified in freshwater, as well as aerosol particles generated in the laboratory from Mona Lake water by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at atmospheric concentrations up to 50 ± 20 ng/m3. Enrichment of hydrophobic microcystin congeners (e.g., microcystin-LR) was observed in aerosol particles relative to bulk freshwater, while enrichment of hydrophilic microcystin (e.g., microcystin-RR) was lower. As HABs increase in a warming climate, understanding and quantifying the emissions of toxins into the atmosphere is crucial for evaluating the health consequences of HABs.

Microplastic ingestion by quagga mussels, Dreissena bugensis, and its effects on physiological processes.

The impacts of microplastic particulates in benthic freshwater organisms have been largely unexplored despite abundant plastic accumulation in the sediments of these systems. We investigated the uptake of plastic particles by benthic filter feeding quagga mussels (Dreissena bugensis) and associated toxicity exhibited through impacts on mortality, filtration rate, reproduction and oxygen consumption. Matrix Assisted Laser Desorption/Ionization Imaging Mass Spectrometry (MALDI-IMS) technology was used to assess the microplastic inclusion. For this purpose, quagga mussels were exposed to four treatments ranging from 0.0 to 0.8 g/L of a high density fluorescent red polyethylene powder in the size range of 10-45 µm for 24-h, and the targeted endpoints were quantified. Identification of several micrograms of microplastics in the digestive tract suggests rapid clearance from the water column by filtering. At the higher concentrations, about 95% of the microplastics ingested remained in the mussels after 24-h. Microplastics were found in the gills which correlated with decreasing filtration rate at higher microplastic concentrations. Despite large-scale ingestion, plastic exposure did not affect survivorship, reproduction rates, or oxygen consumption in the period examined. MALDI-IMS identified unique mass spectra that correlated with microplastic inclusion. This research suggests that microplastics can impair feeding through decreased filtration rates of filter feeding organisms, potentially resulting in a reduction of overall fitness over time and that MALDI-IMS may have the potential to identify microplastics and changes in tissue at the borders of plastic inclusion.

Dhb Microcystins Discovered in USA Using an Online Concentration LC-MS/MS Platform.

Based on current structural and statistical calculations, thousands of microcystins (MCs) can exist; yet, to date, only 246 MCs were identified and only 12 commercial MC standards are available. Standard mass spectrometry workflows for known and unknown MCs need to be developed and validated for basic and applied harmful algal bloom research to advance. Our investigation focuses on samples taken in the spring of 2018 from an impoundment fed by Oser and Bischoff Reservoirs, Indiana, United States of America (USA). The dominant cyanobacterium found during sampling was Planktothrix agardhii. The goal of our study was to identify and quantify the MCs in the impoundment sample using chemical derivatization and mass spectrometry. Modifying these techniques to use online concentration liquid chromatography tandem mass spectrometry (LC-MS/MS), two untargeted MCs have been identified, [d-Asp3, Dhb7]-MC-LR and [Dhb7]-MC-YR. [Dhb7]-MC-YR is not yet reported in the literature to date, and this was the first reported incidence of Dhb MCs in the United States. Furthermore, it was discovered that the commercially available [d-Asp3]-MC-RR standard was [d-Asp3, Dhb7]-MC-RR. This study highlights a workflow utilizing online concentration LC-MS/MS, high-resolution MS (HRMS), and chemical derivatization to identify isobaric MCs.

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease.

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.

Permanganate Oxidation of Microcystin-LA: Kinetics, Quantification, and Implications for Effective Drinking Water Treatment.

Permanganate pretreatment of drinking water is effective in transforming dissolved, noxious contaminants and in reducing halogenated by-products. Permanganate targets specific compounds such as taste and odor compounds, disinfection precursors, manganese, and natural organic contaminants that are not removed readily by conventional treatment alone. Cyanobacterial blooms (cHABs) can increase disinfection by-product precursors as well as the cyanotoxin, microcystin (MC), a potent liver toxin. MC toxicity is conferred by a unique, conserved amino acid, Adda, that inhibits protein phosphatase 1 and 2A. Although over 150 MC congeners have been reported, thousands of MCs are statistically possible. Over the last ten years, one congener, MC-LA, has been reported with increasing frequency, making it one of the most common cyanotoxins identified in North American freshwater systems; yet its oxidation has not been widely studied. Frequently, Adda specific enzyme-linked immunosorbent assay (ELISA) and protein phosphatase inhibition assay (PPIA) are used to quantitate total MCs to evaluate treatment efficiency and exposure. Anecdotal reports suggest that MC degradation products can cause interference with the Adda-ELISA. MC-LA was used as the model MC compound in this study. PPIA quantitation of MC-LA in water agreed with liquid chromatography high resolution mass spectrometry (LC/HRMS), whereas the ELISA quantitation did not agree with LC/HRMS quantitation. We determined the second order rate constant for MC-LA as 118 ± 9 M-1 s-1, the activation energy to be 21.2 kJ mol-1, and the rate to be independent of pH between pH 6 and 9. Ten oxidation products (OPs) were observed by LC/HRMS and three primary reaction pathways are proposed. The reaction pathways were used to explain differences in the quantification by Adda-ELISA, HRMS, and PPIA. The oxohydroxylation of MC-LA produced two major OPs, C46H67N7O14 [M+H] + = 942.4819 and C46H69N7O15 [M+H]+ =960.4925. Major OPs may contain an unmodified Adda and are the likely cause of interference with the Adda-ELISA. Several governmental agencies recommend the use of the Adda-ELISA to determine the MC quantitation for treatment efficiency and customer exposure; yet our results suggest that these or other OPs interfere with the Adda-ELISA causing artificially high values for total MCs.

Perspectives on Harmful Algal Blooms (HABs) and the Cyberbiosecurity of Freshwater Systems.

Harmful Algal Blooms (HABs) have been observed in all 50 states in the U.S., ranging from large freshwater lakes, such as the Great Lakes, to smaller inland lakes, rivers, and reservoirs, as well as marine coastal areas and estuaries. In 2014, a HAB on Lake Erie containing microcystin (a liver toxin) contaminated the municipal water supply in Toledo, Ohio, providing non-potable water to 400,000 people. Studying HABs is complicated as different cyanobacteria produce a range of toxins that impact human health, such as microcystins, saxitoxin, anatoxin-a, and cylindrospermopsin. HABs may be increasing in prevalence with rising temperatures and higher nutrient runoff. Consequently, new tools and technology are needed to rapidly detect, characterize, and respond to HABs that threaten our water security. A framework is needed to understand cyber threats to new and existing technologies that monitor and forecast our water quality. To properly detect, assess, and mitigate security threats on water infrastructure, it is necessary to envision water security from the perspective of a cyber-physical system (CPS). In doing so, we can evaluate risks and research needs for cyber-attacks on HAB-monitoring networks including data injection attacks, automated system hijacking attacks, node forgery attacks, and attacks on learning algorithms. Herein, we provide perspectives on the research needed to understand both the threats posed by HABs and the coupled cyber threats to water security in the context of HABs.