Fate of Planktothrix-derived toxins in aquatic food webs: A case study in Lake Mindelsee (Germany).

Blooms of the red, filamentous cyanobacterium Planktothrix rubescens occur frequently in pre-alpine lakes in Europe, often with concomitant toxic microcystin (MC) production. Trophic transfer of MCs has been observed in bivalves, fish, and zooplankton species, while uptake of MCs into Diptera species could facilitate distribution of MCs into terrestrial food webs and habitats. In this study, we characterized a Planktothrix bloom in summer 2019 in Lake Mindelsee and tracked possible trophic transfer and/or bioaccumulation of MCs via analysis of phytoplankton, zooplankton (Daphnia) and emergent aquatic insects (Chaoborus, Chironomidae and Trichoptera). Using 16 S rRNA gene amplicon sequencing, we found that five sequence variants of Planktothrix spp. were responsible for bloom formation in September and October of 2019, and these MC-producing variants, provisionally identified as P. isothrix and/or P. serta, occurred exclusively in Lake Mindelsee (Germany), while other variants were also detected in nearby Lake Constance. The remaining cyanobacterial community was dominated by Cyanobiaceae species with high species overlap with Lake Constance, suggesting a well-established exchange of cyanobacteria species between the adjacent lakes. With targeted LC-HRMS/MS we identified two MC-congeners, MC-LR and [Asp3]MC-RR with maximum concentrations of 45 ng [Asp3]MC-RR/L in lake water in September. Both MC congeners displayed different predominance patterns, suggesting that two different MC-producing species occurred in a time-dependent manner, whereby [Asp3]MC-RR was clearly associated with the Planktothrix spp. bloom. We demonstrate an exclusive transfer of MC-LR, but not [Asp3]MC-RR, from phytoplankton into zooplankton reaching a 10-fold bioconcentration, yet complete absence of these MC congeners or their conjugates in aquatic insects. The latter demonstrated a limited trophic transfer of MCs from zooplankton to zooplanktivorous insect larvae (e.g., Chaoborus), or direct transfer into other aquatic insects (e.g. Chironomidae and Trichoptera), whether due to avoidance or limited uptake and/or rapid excretion of MCs by higher trophic emergent aquatic insects.

PODO/TERT256 - A promising human immortalized podocyte cell line and its potential use for in vitro research at different oxygen levels.

Podocytes are of key interest for the prediction of nephrotoxicity as they are especially sensitive to toxic insults due to their central role in the glomerular filtration apparatus. However, currently, prediction of nephrotoxicity in humans remains insufficiently reliable, thus highlighting the need for advanced in vitro model systems using human cells with improved prediction capacity. Recent approaches for refining in vitro model systems focus on closely replicating physiological conditions as observed under the in vivo situation typical of the respective nephron section of interest. PODO/TERT256, a human immortalized podocyte cell line, were employed in a semi-static transwell system to evaluate its potential use as a human podocyte in vitro system for modelling potential human glomerular toxicity. Furthermore, the impact of routinely employed excessive oxygen tension (21 % - AtmOx), when compared to the physiological oxygen tensions (10 % - PhysOx) observed in vivo, was analyzed. Generally, cultured PODO/TERT256 formed a stable, contact-inhibited monolayer with typical podocyte morphology (large cell body, apical microvilli, finger-like cytoplasmic projections (reminiscent of foot processes), and interdigitating cell-cell junctions) and developed a size-selective filtration barrier. PhysOx, however, induced a more pronounced in vivo like phenotype, comprised of significantly larger cell bodies, significantly enhanced filtration barrier size-selectivity, and a remarkable re-localization of nephrin to the cell membrane, thus suggesting an improved in vitro replication of in vivo characteristics. Preliminary toxicity characterization with the known glomerulotoxin doxorubicin (DOX) suggested an increasing change in filtration permeability, already at the lowest DOX concentrations tested (0.01 µM) under PhysOx, whereas obvious changes under AtmOx were observed as of 0.16 µM and higher with a near all or nothing effect. The latter findings suggested that PODO/TERT256 could serve as an in vitro human podocyte model for studying glomerulotoxicity, whereby culturing at PhyOx tension appeared critical for an improved in vivo-like phenotype and functionality. Moreover, PODO/TERT256 could be incorporated into advanced human glomerulus systems in vitro, recapitulating microfluidic conditions and multiple cell types (endothelial and mesenchymal cells) that can even better predict human glomerular toxicity.

US regulations to curb alleged cancer causes are ineffectual and compromised by scientific, constitutional and ethical violations.

The 1958 Delaney amendment to the Federal Food Drug and Cosmetics Act prohibited food additives causing cancer in animals by appropriate tests. Regulators responded by adopting chronic lifetime cancer tests in rodents, soon challenged as inappropriate, for they led to very inconsistent results depending on the subjective choice of animals, test design and conduct, and interpretive assumptions. Presently, decades of discussions and trials have come to conclude it is impossible to translate chronic animal data into verifiable prospects of cancer hazards and risks in humans. Such conclusion poses an existential crisis for official agencies in the US and abroad, which for some 65 years have used animal tests to justify massive regulations of alleged human cancer hazards, with aggregated costs of $trillions and without provable evidence of public health advantages. This article addresses suitable remedies for the US and potentially worldwide, by critically exploring the practices of regulatory agencies vis-á-vis essential criteria for validating scientific evidence. According to this analysis, regulations of alleged cancer hazards and risks have been and continue to be structured around arbitrary default assumptions at odds with basic scientific and legal tests of reliable evidence. Such practices raise a manifold ethical predicament for being incompatible with basic premises of the US Constitution, and with the ensuing public expectations of testable truth and transparency from government agencies. Potential remedies in the US include amendments to the US Administrative Procedures Act, preferably requiring agencies to justify regulations compliant with the Daubert opinion of the Daubert ruling of the US Supreme Court, which codifies the criteria defining reliable scientific evidence. International reverberations are bound to follow what remedial actions may be taken in the US, the origin of current world regulatory procedures to control alleged cancer causing agents.

Physiological oxygen and co-culture with human fibroblasts facilitate in vivo-like properties in human renal proximal tubular epithelial cells.

Reliable prediction of compound mediated nephrotoxicity in humans is still unsatisfactory irrespective of the recent advancements in in silico, in vitro and in vivo models. Therefore, current in vitro approaches need refinement to better match the human in vivo situation, specifically with regard to the potential influence of other cell types (e.g. fibroblasts) and to the potential biases introduced by the excessive 21% O2 (AtmOx) as employed in routine cell culturing. We used a transwell co-culture model combining human renal proximal tubule epithelial cells (RPTEC/TERT1) and human fibroblasts (fHDF/TERT166) to compare the functional properties and expression of selected marker proteins at 21% O2 and at the physiologically normal 10% O2 tension (PhysOx) commensurate with in vivo conditions. Culturing at PhysOx and co-culturing with fibroblasts significantly improved epithelial barrier integrity, expression of transporters (e.g. aquaporin 2; OCT-MATE; MRP-OAT) and metabolism. Moreover, beyond culturing these human cells in co-culture for up to 41 days, we were able to demonstrate increased functionality of cation transport, as shown via ASP+ (OCT-MATE axis), and anion transport, as shown via LY (MRP-OAT axis). Thus, adjusting the in vitro system to near physiological conditions had a major impact on functionality and provides the basis for the future development of true flow-through microfluidic renal testing systems with better predictability of human renal proximal toxicity.

Investigation of microcystin conformation and binding towards PPP1 by molecular dynamics simulation.

Microcystins (MC) are a group of structurally similar cyanotoxins with currently 279 described structural variants. Human exposure is frequent by consumption of contaminated water, food or food supplements. MC can result in serious intoxications, commensurate with ensuing pathology in various organs or in rare cases even mortality. The current WHO risk assessment primarily considers MC-LR, while all other structural variants are treated as equivalent to MC-LR, despite that current data strongly suggest that MC-LR is not the most toxic MC, and toxicity can be very different for MC congeners. To investigate and analyse binding and conformation of different MC congeners, we applied for the first time Molecular Dynamics (MD) simulation to four MC congeners (MC-LR, MC-LF, [Enantio-Adda5]MC-LF, [ß-D-Asp3,Dhb7]MC-RR). We could show that ser/thr protein phosphatase 1 is stable in all MD simulations and that MC-LR backbone adopts to a second conformation in solvent MD simulation, which was previously unknown. We could also show that MC congeners can adopt to different backbone conformation when simulated in solvent or in complex with ser/thr protein phosphatase 1 and differ in their binding behaviour. Our findings suggest that MD Simulation of different MC congeners aid in understanding structural differences and binding of this group of structurally similar cyanotoxins.

Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC-MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake.

Variability in microcystin quotas during a Microcystis bloom in a eutrophic lake.

Microcystis is a bloom-forming genus of cyanobacteria with some genotypes that produce highly toxic microcystin hepatotoxins. In waterbodies where biological and physical factors are relatively homogenous, toxin quotas (the average amount of toxin per cell), at a single point in time, are expected to be relatively constant. In this study we challenged this assumption by investigating the spatial distribution of microcystin quotas at a single point in time on two separate occasions in a lake with a major Microcystis bloom. Microcystis cell concentrations varied widely across the lake on both sampling occasions (730- and 137-fold) together with microcystin quotas (148- and 362-fold). Cell concentrations and microcystin quotas were strongly positively correlated (R2 = 0.89, P < 0.001, n = 28; R2 = 0.67, P < 0.001, n = 25). Analysis of Microcystis strains using high-throughput sequencing of the 16S-23S rRNA intergenic spacer region showed no relationship between microcystin quota and the relative abundance of specific sequences. Collectively, the results of this study indicate an association between microcystin production and cell density that magnifies the potential for bloom toxicity at elevated cell concentrations.

Label-free identification and differentiation of different microplastics using phasor analysis of fluorescence lifetime imaging microscopy (FLIM)-generated data.

As plastic pollution is becoming an increasing worldwide problem, a variety of different techniques for the detection and in-depth characterization of plastics, including spectroscopy and chromatography methods, were introduced to the public. Recently we presented fluorescence lifetime imaging microscopy (FLIM) a new approach for the identification and characterization of microplastics using their fluorescence lifetime (τ) for differentiation. A very powerful extension of the recently established FLIM could be phasor analysis, which allows data representation in an interactive 2D graphical phasor plot thereby enabling a global view of the fluorescence decay in each pixel of the measured image. Microplastic particles generated from six different types of plastics were subjected to excitation wavelengths of 440 nm, upon which specific fluorescence lifetimes as well as the photon yield were determined using FLIM and phasor analysis. We could show that phasor analysis for FLIM with a laser pulse repetition frequency of 40 MHz was able to generate specific locations in the phasor plot for the plastics for fast differentiation, e.g. resulting in well-defined phasor plot positions for ABS at 3.019 ns, PPE at 6.239 ns, PET bottle from Germany at 2.703 ns and PET bottle from USA at 2.711 ns. Phasor analysis for FLIM proves to be a fast, label-free, and sensitive method for the identification and differentiation of plastics also with the aid of visualization variation enabling techniques such as heat treatment of plastics.

Examination of microcystin neurotoxicity using central and peripheral human neurons.

Microcystins (MC) are a group of cyanobacterial toxins that comprises MC-LF and other cyclic heptapeptides, best known as potent hepatotoxicants. Cell culture and epidemiological studies suggest that MC might also affect the nervous system when there is systemic exposure, e.g., via drinking water or food. We asked whether in vitro studies with human neurons could provide estimates on the neurotoxicity hazard of MC-LF. First, we used LUHMES neurons, a well-established test system for neurotoxicants and neuropathological processes. These central nervous system cells express OATP1A2, a presumed carrier of MC-LF, and we observed selective neurite toxicity in the µM range (EC20 = 3.3 µM ≈ 3.3 µg/mL). Transcriptome changes pointed towards attenuated cell maintenance and biosynthetic processes. Prolonged exposure for up to four days did not increase toxicity. As a second model, we used human dorsal root ganglia-like neurons. These peripheral nervous system cells represent parts of the nervous system not protected by the blood-brain barrier in humans. Toxicity was observed in a similar concentration range (EC20 = 7.4 µM). We conclude that MC-LF poses a potential neurotoxic hazard in humans. The adverse effect concentrations observed here were orders of magnitude higher than those presumed to be encountered after normal nutritional or environmental exposure. However, the low µM concentrations found to be toxic are close to levels that may be reached after very excessive algae supplement intake.

Is a Central Sediment Sample Sufficient? Exploring Spatial and Temporal Microbial Diversity in a Small Lake.

(1) Background: Paleolimnological studies use sediment cores to explore long-term changes in lake ecology, including occurrences of harmful cyanobacterial blooms. Most studies are based on single cores, assuming this is representative of the whole lake, but data on small-scale spatial variability of microbial communities in lake sediment are scarce. (2) Methods: Surface sediments (top 0.5 cm) from 12 sites (n = 36) and two sediment cores were collected in Lake Rotorua (New Zealand). Bacterial community (16S rRNA metabarcoding), Microcystis specific 16S rRNA, microcystin synthetase gene E (mcyE) and microcystins (MCs) were assessed. Radionuclide measurements (210Pb, 137Cs) were used to date sediments. (3) Results: Bacterial community, based on relative abundances, differed significantly between surface sediment sites (p < 0.001) but the majority of bacterial amplicon sequence variants (88.8%) were shared. Despite intense MC producing Microcystis blooms in the past, no Microcystis specific 16S rRNA, mcyE and MCs were found in surface sediments but occurred deeper in sediment cores (approximately 1950's). 210Pb measurements showed a disturbed profile, similar to patterns previously observed, as a result of earthquakes. (4) Conclusions: A single sediment core can capture dominant microbial communities. Toxin producing Microcystis blooms are a recent phenomenon in Lake Rotorua. We posit that the absence of Microcystis from the surface sediments is a consequence of the Kaikoura earthquake two years prior to our sampling.

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology.

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission's H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

Erratum to Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

In this manuscript, which appeared in ALTEX (2020), 37(1), 24-36, doi:10.14573/altex.1904031 , there were errors in Tables 1 and 3.

Erratum to Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

Machine learning prediction of cyanobacterial toxin (microcystin) toxicodynamics in humans.

Microcystins (MC) represent a family of cyclic peptides with approx. 250 congeners presumed harmful to human health due to their ability to inhibit ser/thr-proteinphosphatases (PPP), albeit all hazard and risk assessments (RA) are based on data of one MC-congener (MC-LR) only. MC congener structural diversity is a challenge for the risk assessment of these toxins, especially as several different PPPs have to be included in the RA. Consequently, the inhibition of PPP1, PPP2A and PPP5 was determined with 18 structurally different MC and demonstrated MC congener dependent inhibition activity and a lower susceptibility of PPP5 to inhibition than PPP1 and PPP2A. The latter data were employed to train a machine learning algorithm that should allow prediction of PPP inhibition (toxicity) based on MCs 2D chemical structure. IC50 values were classified in toxicity classes and three machine learning models were used to predict the toxicity class, resulting in 80-90% correct predictions.

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.

Simultaneous Detection of 14 Microcystin Congeners from Tissue Samples Using UPLC- ESI-MS/MS and Two Different Deuterated Synthetic Microcystins as Internal Standards.

Cyanobacterial microcystins (MCs), potent serine/threonine-phosphatase inhibitors, pose an increasing threat to humans. Current detection methods are optimised for water matrices with only a few MC congeners simultaneously detected. However, as MC congeners are known to differ in their toxicity, methods are needed that simultaneously quantify the congeners present, thus allowing for summary hazard and risk assessment. Moreover, detection of MCs should be expanded to complex matrices, e.g., blood and tissue samples, to verify in situ MC concentrations, thus providing for improved exposure assessment and hazard interpretation. To achieve this, we applied two synthetic deuterated MC standards and optimised the tissue extraction protocol for the simultaneous detection of 14 MC congeners in a single ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) run. This procedure was validated using plasma and liver homogenates of mice (male and female) spiked with deuterated MC standards. For proof of concept, tissue and plasma samples from mice i.p. injected with MC-LR and MC-LF were analysed. While MC-LF was detected in all tissue samples of both sexes, detection of MC-LR was restricted to liver samples of male mice, suggesting different toxicokinetics in males, e.g., transport, conjugation or protein binding. Thus, deconjugation/-proteinisation steps should be employed to improve detection of bound MC.

Functional transepithelial transport measurements to detect nephrotoxicity in vitro using the RPTEC/TERT1 cell line.

The kidney is a frequent target for organ-specific toxicity as a result of its primary function in controlling body fluids, for example, via resorption of amino acids, peptides, nutrients, ions, xenobiotics and water from the primary urine as well as excretion of metabolic waste products and hydrophilic and amphiphilic xenobiotics. Compounds exhibiting dose-limiting nephrotoxicity include drugs from highly diverse classes and chemical structures, e.g., antibiotics (gentamicin), chemotherapeutics (cisplatin), immunosuppressants (cyclosporine A and tacrolimus) or bisphosphonates (zoledronate). All of these compounds elicit nephrotoxicity primarily by injuring renal proximal tubule epithelial cells (RPTECs). However, prediction of a compound's nephrotoxic potential in humans to support early unmasking of risk-bearing drug candidates remains an unmet challenge, mainly due to the complex kidney anatomy as well as pronounced inter- and intraspecies differences and lack of relevant and validated human in vitro models. Accordingly, we used the recently established human RPTEC/TERT1 cell line to carry out toxicity studies with a focus on impairment of functional characteristics, i.e., transepithelial electrical resistance (TEER), vectorial transport of water, cations, and anions. Results were compared to real-time cytotoxicity assessments using cellular impedance (xCELLigence assay) and the routine cell viability readout (MTT). As expected, most toxins caused exposure time- and concentration-dependent cytotoxicity. However, for some compounds (cyclosporine A and tacrolimus), transport processes were strongly impaired in absence of a concomitant decrease in cell viability. In conclusion, these data demonstrate that functional parameters are important, highly sensitive and meaningful additional readouts for nephrotoxicity assessment in human renal proximal tubule epithelial cells.

Comparison of Aristolochic acid I derived DNA adduct levels in human renal toxicity models.

Aristolochic acid (AA) dependent human nephropathy results either from environmental exposure to Aristolochiaceae plant subspecies or their use in traditional phytotherapy. The toxic components are structurally related nitrophenanthrene carboxylic acids, i.e. Aristolochic acid I (AAI) and II (AAII). AAI is considered to be the major cause of Aristolochic acid nephropathy, characterized by severe renal fibrosis and upper urothelial cancer. Following enzymatic activation in kidney and/or liver, AAI metabolites react with genomic DNA to form persistent DNA adducts with purines. To determine whether AAI can be activated in human renal cells to form DNA adducts, we exposed telomerase immortalized renal proximal tubular epithelial cells (RPTEC/TERT1), the human embryonic kidney (HEK293) cell line, as well as primary human kidney cells (pHKC) to AAI in vitro. We modified an isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry (ID-UPLC-MS/MS) based method for the quantification of dA-AAI adducts in genomic DNA. In addition, time dependent accumulation of adducts in renal cortex and bladder tissue from AAI/II treated Eker rats were used to validate the detection method. AAI-induced toxicity in human renal cells was determined by dA-AAI adduct quantification, the impact on cell viability, and NQO1 expression and activity. Our findings demonstrated adduct formation in all cell lines, although only pHKC and RPTEC/TERT1 expressed NQO1. The highest adduct formation was detected in pHKC despite low NQO1 expression, while we observed much lower adduct levels in NQO1-negative HEK293 cells. Adduct formation and decreased cell viability correlated only weakly. Therefore, our data suggested that i.) enzymes other than NQO1 could be at least equally important for AA bioactivation in human renal proximal tubule cells, and ii.) the suggested correlation between adduct levels and viability appears to be questionable.

Toxic Cyanobacteria in Svalbard: Chemical Diversity of Microcystins Detected Using a Liquid Chromatography Mass Spectrometry Precursor Ion Screening Method.

Cyanobacteria synthesize a large variety of secondary metabolites including toxins. Microcystins (MCs) with hepato- and neurotoxic potential are well studied in bloom-forming planktonic species of temperate and tropical regions. Cyanobacterial biofilms thriving in the polar regions have recently emerged as a rich source for cyanobacterial secondary metabolites including previously undescribed congeners of microcystin. However, detection and detailed identification of these compounds is difficult due to unusual sample matrices and structural congeners produced. We here report a time-efficient liquid chromatography-mass spectrometry (LC-MS) precursor ion screening method that facilitates microcystin detection and identification. We applied this method to detect six different MC congeners in 8 out of 26 microbial mat samples of the Svalbard Archipelago in the Arctic. The congeners, of which [Asp³, ADMAdda5, Dhb7] MC-LR was most abundant, were similar to those reported in other polar habitats. Microcystins were also determined using an Adda-specific enzyme-linked immunosorbent assay (Adda-ELISA). Nostoc sp. was identified as a putative toxin producer using molecular methods that targeted 16S rRNA genes and genes involved in microcystin production. The mcy genes detected showed highest similarities to other Arctic or Antarctic sequences. The LC-MS precursor ion screening method could be useful for microcystin detection in unusual matrices such as benthic biofilms or lichen.

Canagliflozin mediated dual inhibition of mitochondrial glutamate dehydrogenase and complex I: an off-target adverse effect.

Recent FDA Drug Safety Communications report an increased risk for acute kidney injury in patients treated with the gliflozin class of sodium/glucose co-transport inhibitors indicated for treatment of type 2 diabetes mellitus. To identify a potential rationale for the latter, we used an in vitro human renal proximal tubule epithelial cell model system (RPTEC/TERT1), physiologically representing human renal proximal tubule function. A targeted metabolomics approach, contrasting gliflozins to inhibitors of central carbon metabolism and mitochondrial function, revealed a double mode of action for canagliflozin, but not for its analogs dapagliflozin and empagliflozin. Canagliflozin inhibited the glutamate dehydrogenase (GDH) and mitochondrial electron transport chain (ETC) complex I at clinically relevant concentrations. This dual inhibition specifically prevented replenishment of tricarboxylic acid cycle metabolites by glutamine (anaplerosis) and thus altered amino acid pools by increasing compensatory transamination reactions. Consequently, canagliflozin caused a characteristic intracellular accumulation of glutamine, glutamate and alanine in confluent, quiescent RPTEC/TERT1. Canagliflozin, but none of the classical ETC inhibitors, induced cytotoxicity at particularly low concentrations in proliferating RPTEC/TERT1, serving as model for proximal tubule regeneration in situ. This finding is testimony of the strong dependence of proliferating cells on glutamine anaplerosis via GDH. Our discovery of canagliflozin-mediated simultaneous inhibition of GDH and ETC complex I in renal cells at clinically relevant concentrations, and their particular susceptibility to necrotic cell death during proliferation, provides a mechanistic rationale for the adverse effects observed especially in patients with preexisting chronic kidney disease or previous kidney injury characterized by sustained regenerative tubular epithelial cell proliferation.