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In vitro investigations on uptake and toxicity of cyanobacterial toxins

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FISCHER, Andreas, 2010. In vitro investigations on uptake and toxicity of cyanobacterial toxins [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Fischer2010vitro-12498, title={In vitro investigations on uptake and toxicity of cyanobacterial toxins}, year={2010}, author={Fischer, Andreas}, address={Konstanz}, school={Universität Konstanz} }

terms-of-use Fischer, Andreas eng 2012-12-02T23:25:03Z Fischer, Andreas Within their 3.3 to 3.5 billion years of existance cyanobacteria became the most diverse and widespread group of prokaryotes and adapted to nearly any given habitat throughout the world. During this period of time they developed an enormous variety of secondary metabolites with predominantly unknown physiological function. Some of these chemically and structurally very diverse compounds have been in the focus of international research ever since their toxic potential on other organisms became known.<br />Especially the MCs and more recently the CYNs have drawn major attention due to their frequent and widespread occurence and numerous in part fatal incidents of animal and human poisonings. MCs are cyclic heptapeptides comprising more than 80 congeners, whereas CYNs are classified as alkaloids. They consist of a sulphated and methylated tricyclic guanidino moiety linked to a hydroxymethyluracil.<br />The primary mechanism underlying the toxicity of MCs emerged to be the potent inhibition of serine/threonine-specific PPs, in particular PP1 and 2A. The unique amino acid Adda has thereby been identified as the toxic moiety of the MC molecule. However, isolated Adda was shown to be inhibitory ineffective on PP1 activity. Corresponding effects on PP2A were not determined.<br />Therefore, the first part of this study served to provide the lacking information: As expected, isolated Adda also failed to inhibit the activity of PP2A which corroborates the common assumption that PP inhibition by MCs and, hence, by the structural closely related NODs requires both the Adda moiety and the remaining structural units of the maternal toxins.<br />In contrast to MCs, the molecular mode of action of the geno- and cytotoxic CYNs (i.e. inhibition of protein synthesis and generation of (a) toxic metabolite(s) by CYP450 metabolization) is not completely understood and no information has so far been provided on its effects in human primary cells.<br />Furthermore, both toxins are known to primarily target the liver, but have been found to additionally affect other organs as well. Some members of the superfamily of organic anion transporting polypeptides (OATPs/SLCOs), that have been found to be expressed in nearly every organ/tissue, have been identified to be jointly responsible for the observed organotropism of the cell impermeant MCs (more precisely tritiated dihydro-MCLR and MCLR). Besides passive diffusion, these carriers or other members of the bile acid transport system have been suggested to also be involved in mediating the uptake of CYN.<br />Therefore, two further aims of the present study were:<br />to assess possible differences in the toxicokinetics (i.e. the uptake-mediated toxicity) of four different MCs (LR, RR, LW and LF) and as a prerequisite to compare the toxicodynamics (i.e. inhibition of serine/threonine-specific PPs) of these congeners<br />and to determine the cytotoxicity of CYN in primary human hepatocytes and liver OATP-expressing HEK293 cells to evaluate possible transport-dependent effects.<br />The experiments revealed that the four MC congeners investigated equipotently inhibit recombinant and cellular serine/threonine-specific PPs, but differed tremendously with regard to cytotoxicity in liver OATP-expressing HEK293 as well as primary human hepatocytes. Hence, these findings strongly suggest that MCs are taken up by OATP1B1 and OATP1B3 with varying affinities and lead to the assumption that the vast differences in the in vivo toxicity of MCs (e.g. MCLR and MCRR) can rather be attributed to congener-specific toxicokinetics than toxicodynamics.<br />In addition, primary hepatocytes appeared to be up to two orders of magnitude more susceptible to MCs induced cytotoxicity than the corresponding HEK293 OATP transfectants which highlights the importance to additionally employ primary cells in order to obtain physiologically more relevant results.<br />A further crucial finding was that MCLW and -LF elicited much higher toxicity in both primary human hepatocytes and OATP-expressing HEK293 cells than MCLR, the congener commonly considered as the most toxic. If this is found to be true for their in vivo toxicities as well, the current risk assessments and guideline values might have to be revised since those are based on MCLR toxicity only.<br />In contrast, OATP-associated toxicity was found to be far less relevant for CYN than for MCs. Cytotoxicity of CYN appeared to rather depend on the metabolic condition of the exposed cell types since the immortalized, hence, metabolically less active, OATP-expressing HEK293 cells were distinctly less susceptible than primary human hepatocytes. This suggests that the second mechanism in CYN totoxicity, i.e. the generation of (a) toxic metabolite(s) by CYP450 metabolization, has a higher impact at acute concentrations than the inhibition of protein synthesis which is supported by previous findings in primary mouse hepatocytes.<br />Moreover, the present data indicate that passive diffusion is indeed a mechanism in the uptake of CYN as the viability of control vector-transfected HEK293 cells was only marginally less reduced than that of the respective OATP transfectants.<br />The final scope of this study was to analyze cyanobacterial dietary supplements for contaminations with MCs and the excitotoxic amino acid BMAA in order to assess the risk emanating from this potential source of intoxication.<br />Since BMAA could neither be detected in Spirulina nor Aphanizomenon flos-aquae based supplements these products unlikely constitute a threat with regard to this toxin that has been suggested to be produced by the vast majority of cyanobacteria.<br />On the contrary, the analyses revealed MCs to be common contaminants of Aphanizomenon flos-aquae containing supplements. All of the samples were tested positively and approximately half of them exceeded the maximal acceptable concentration of 1 µg MCLR equivalents/g dw defined by the Oregon Health Division.<br />In conclusion, this study provides crucial findings on the toxicity of MCs that might have considerable implications on present risk assessments and guideline values and gives further insight on the uptake and toxicity of CYN. Moreover, it clearly demonstrates that MCs pose a serious health risk for consumers of cyanobacterial dietary supplements based on A. flos-aquae. 2011-04-12T07:48:17Z 2010 In vitro investigations on uptake and toxicity of cyanobacterial toxins

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