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Toxicological Profile of Microcystins : Determination of the Toxicokinetic and Toxicodynamic Mechanisms of Microcystins in Human Cells

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2024

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Although more than 250 microcystin congeners have been described, the toxicity of only a few are known and have been assessed using purified proteins, including only limited aspects of toxicity. Comparative studies including both toxicodynamic (serine/threonine phosphatase (PPP) inhibition and glutathione (GSH) conjugation) and toxicokinetic (cellular uptake via organic anion transporting polypeptides, OATPs) parameters are lacking so far. However, the mechanism of microcystin toxicity can only be fully understood by evaluating and combining both parameters. Therefore, the aim of this dissertation was to contribute to the understanding of each parameter, first in simplified but then in more complex but biologically more relevant cell systems. Using specifically synthesized microcystin congeners and purified PPPs, this study clearly demonstrated that the presence, length, and orientation of the unique and hydrophobic side chain Adda played a critical role in the microcystin-induced PPP inhibition mechanism. While the covalent bond formation, although inducing irreversibility, played a minor role in purified PPPs as well as in stably transfected HEK293 cell lines. Additionally, the individual transporting properties of OATP1B1 and OATP1B3 were studied. Therefore, stably transfected HEK293-OATP1B1, -OATP1B3, and control vector (CV) were exposed to 21 structurally different microcystin congeners covering the known spectrum of hydrophobicity, molecular weights and common as well as unusual modifications of the consensus structure. In this study, the positions (2) and (4) were demonstrated to determine the hydrophobicity of the microcystin molecule, thereby defining its OATP-mediated uptake. In this study, a clear correlation between the total hydrophobicity and OATP-mediated uptake was demonstrated and thereby allowing the classification of microcystins into three distinct groups: less toxic, moderately toxic, and very toxic. Moreover, position (2) was shown to be critical for OATP1B1-mediated, but not OATP1B3-mediated, microcystin uptake. Furthermore, it was shown that microcystin congener-specific toxicity in vivo is due to OATP-mediated uptake rather than to the different PPP inhibition capacities. This study provided preliminary insights into the potential role of GSH conjugation to microcystins by establishing three independent assays to simultaneously monitor cell viability (cytotoxicity), PPP activity and GSH response. The commonly accepted assumption that GSH conjugation to microcystin results in detoxification due to reduced toxicity was not supported as evidenced by total GSH depletion with subsequent microcystin exposure, while no significant changes in cytotoxicities and PPP activities were observed. These results were confirmed not only in simplified HEK293, but also in complex and biologically relevant human and mouse hepatocytes. Thereby, the microcystin-induced GSH response was shown to be significantly different between human and mouse hepatocytes, challenging the current risk assessment based solely on a mouse study. So far, microcystin toxicity studies have been limited to single exposures to high microcystin concentrations, mostly in either rodent or human but carcinogenic hepatocyte cell lines with questionable OATP expression and therefore critical biological relevance. This study demonstrated that the IHH cell line is a reliable and suitable cell line for (chronic) microcystin toxicity testing, as evidenced by the expression of hepatocyte-specific transporters, including the microcystin-relevant OATP1B1, OATP1B3, and MRP2.

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Fachgebiet (DDC)
570 Biowissenschaften, Biologie

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Microcystins, OATP1B1, OATP1B3, Toxicokinetics, Toxicodynamics, PPP2A, Phosphatases, PPPs

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ISO 690FOTLER, Regina, 2024. Toxicological Profile of Microcystins : Determination of the Toxicokinetic and Toxicodynamic Mechanisms of Microcystins in Human Cells [Dissertation]. Konstanz: University of Konstanz
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@phdthesis{Fotler2024Toxic-69964,
  year={2024},
  title={Toxicological Profile of Microcystins : Determination of the Toxicokinetic and Toxicodynamic Mechanisms of Microcystins in Human Cells},
  author={Fotler, Regina},
  address={Konstanz},
  school={Universität Konstanz}
}
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Prüfungsdatum der Dissertation

April 25, 2024
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Konstanz, Univ., Diss., 2024
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