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

International audienceCyanobacterial 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. Key Contribution: The use of deuterated microcystin standards and an improved extraction procedure using UPLC-MS/MS analytics, provides for the simultaneous detection of fourteen microcystin congeners. Thus, it allows more accurate quantitation of total microcystin load of a given sample in complex matrices like blood or tissue, and therefore better hazard interpretation

Die individuellen und kombinierten neurotoxischen Effekte cyanobakterieller Toxine

Wasserverunreinigungen durch toxische Cyanobakterien sind ein weltweit wachsendes Problem, das ein ernstes Risiko für Menschen darstellt. Am wahrscheinlichsten sind humane Vergiftungen möglich durch chronische Vergiftungen mit niedrigen Toxinkonzentrationen, zum Beispiel durch Kontaminationen von Trinkwasser, Nahrung (Fisch, Garnelen, Nahrungsergänzungsmittel auf Blau-Grün Algen Basis) oder durch Freizeitaktivitäten in Gewässern, die ein Massenvorkommen von toxischen Cyanobakterien aufweisen. Microcystine (MC) repräsentieren die am häufigsten vorkommende Gruppe von zyklischen Heptapeptiden mit mehr als 80 verschiedenen strukturellen Varianten (Kongenere). Es konnte gezeigt werden, dass MC-LR im Vergleich zu anderen Kongeneren sowohl in vitro als auch in vivo toxisch ist, wobei momentan vermutet wird, dass der Mechanismus der Toxizität hauptsächlich von der spezifischen und irreversiblen Inhibition von Serin/Threonin Protein Phosphatasen (PP) herrührt. Durch ihre chemische Struktur und Größe ist es MC nicht möglich, durch Zellmembranen hindurch zu diffundieren, sondern sie benötigen hierfür Transporter, sogenannte organic anion transporting polypeptides (Nager Oatp / Mensch OATP). Des Weiteren besitzen nicht alle Oatps/OATPs dieselbe Affinität und Kapazität für all diejenigen MC Kongenere, die während einer Cyanoblüte auftreten können. Deshalb ist die Verteilung, d.h. die Toxikokinetik von einzelnen bzw. mehreren Kongeneren, absolut abhängig von der spezifischen Expression von Oatps/OATPs im Gewebe und in dessen Zellen. Erste Anhaltspunkte nach humanen und tierischen (Wild- und Haustiere) MC Intoxikationen deuten auf eine Hepato-, Nephro- und Neurotoxizität hin. Tatsächlich findet man Oatps/OATPs in Hepatozyten und kortikalen Epithelzellen der Niere, die auch einzelne MC Kongenere transportieren können. Um eine Neurotoxizität induzieren zu können, sollten daher Oatps/OATPs nicht nur in der Blut-Hirn-Schranke, sondern auch in neuronalen Membranen exprimiert werden.Ziel dieser Arbeit war es zu untersuchen, ob MC potente Neurotoxine darstellen. Hauptaugenmerk wurde auf eine Neuronen spezifische sowie MC Kongener abhängige Toxizität gelegt. In ersten in vitro Versuchen mit primären murinen Whole Brain Cells (mWBCs) sollten Anhaltspunkte gesammelt werden, die auf eine generelle MC Kongener abhängige Neurotoxizität hindeuten. Für eine genauere Abschätzung sollte in weiteren Versuchen mit primären murinen Cerebellar Granule Neurons (mCGNs), d.h. primäre Neurone, die Präsenz von mOatps, ein MC Kongener abhängiger Transport, PP Inhibition sowie eine in Folge auftretende Neuritendegradation und Cytotoxizität (Nekrose/Apoptose) untersucht werden. Abschließend sollte ein erster in vivo Versuch mit Mäusen die beobachteten in vitro Effekte bestätigen.Zusammengefaßt zeigen die Daten eine mOatp abhängige Verteilung von MCs in mWBCs und mCGNs sowie einen MC Kongener anhängigen Cytotoxizitäts-Mechanismus, der MC-LF als den potentesten neurotoxischen MC Kongener darstellt. Da einzelne MC Kongenere sehr stark in ihrem neurotoxischen Potential variierten, sollte die momentane Risikobewertung, die nur auf MC-LR beruht, neu überarbeitet werden. Dies ist von höchster Wichtigkeit, da MC-LF häufig in Oberflächengewässern einer auftretenden Cyanoblüte zu finden ist. Folglich zeigen die Daten dieser Studie das Vorkommen eines neuen bis jetzt noch nicht in Betracht gezogenen Risikos durch MC für Mensch und Tier

Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells

Microcystins (MCs) are naturally occurring cyclic heptapeptides that exhibit hepato-, nephro- and possibly neurotoxic effects in mammals. Organic anion transporting polypeptides (rodent Oatp/human OATP) appear to be specifically required for active uptake of MCs into hepatocytes and kidney epithelial cells. Based on symptoms of neurotoxicity in MC-intoxicated patients and the presence of Oatp/OATP at the blood-brainbarrier (BBB) and blood-cerebrospinal-f1uid-barrier (BCFB) it is hypothesized that MCs can be transported across the BBB/BCFB in an Oatp/OATP-dependent manner and can induce toxicity in brain cells via inhibition of protein phosphatase (PP). To test these hypotheses. the presence of murine Oatp (rnOatp) in primary murine whole brain cells (mWBC) was invt'stigated at the mRNA and protein level. MC transport was tested by exposing mWBCs to three different MC-congeners (MC-LR, -LW, -LFJ with/without co-incubation with the OATP/Oatp-substrates taurocholate (TC) and bromosulfophthalein (BSP). Uptake of MCs and cytotoxicity was demonstrated via MC-Western blot analysis, immunocytochemistry, cell viability and PP inhibition assays. All MC congeners bound covalently and inhibited mWBC PP. MC-LF was the most cytotoxic congener followed by -LW and -LR. The lowest toxin concentration significantly reducing mWBC viability after 48 h exposure was 400 nM (MC-LF). Uptake of MCs into mWBCs was inhibited via co-incubation with excess TC (50 and 500 pM) and BSP (50 pM). MC-Western blot analys is demonstrated a concentration-dependent accumulation of MCs. In conclusion, the in vieTO data support the assumed MC-congener-dependent uptake in a mOatp-associated manner and cytotoxicity of MCs in primary murine whole brain cells

Microcystin congener– and concentration-dependent induction of Murine neuron apoptosis and Neurite degeneration

Cyanobacterial microcystins (MCs) represent a toxin group with > 100 variants, requiring active uptake into cells via organic anion–transporting polypeptides, in order to irreversibly inhibit serine/threonine-specific protein phosphatases. MCs are a human health hazard with repeated occurrences of severe poisonings. In the well-known human MC intoxication in Caruaru, Brazil (1996), patients developed signs of acute neurotoxicity, e.g., deafness, tinnitus, and intermittent blindness, as well as subsequent hepatotoxicity. The latter data, in conjunction with some animal studies, suggest that MCs are potent neurotoxins. However, there is little data to date demonstrating MC neuron-specific toxicity. MC exposure–induced cytotoxicity, caspase activity, chromatin condensation, and microtubule-associated Tau protein hyperphosphorylation (epitopes serine199/202 and serine396) were determined. Neurite degeneration was analyzed with confocal microscopy and neurite length determined using image analysis. MC-induced apoptosis was significantly increased by MC-LF and MC-LW, however, only at high concentrations (≥ 3MyM), whereas significant neurite degeneration was already observed at 0.5MyM MC-LF. Moreover, sustained hyperphosphorylation of Tau was observed with all MC congeners. The concentration- and congener-dependent mechanisms observed suggest that low concentrations of MC-LF and MC-LW can induce subtle neurodegenerative effects, reminiscent of Alzheimer’s disease type human tauopathies, and thus should be taken more seriously with regard to potential human health effects than the apical cytotoxicity (apoptosis or necrosis) demonstrated at high MC concentrations

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro : a comparison of primary human hepatocytes and OATP-transfected HEK293 cells

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate