Abstract
Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.
Highlights
Some cyanobacterial species, such as those among the freshwater genera Microcystis, Planktothrix and Anabaena, produce secondary metabolites called microcystins [1]
We have previously shown that the amphipathicity of MC-LF and molecule with tryptophan (MC-LW) enabled the more hydrophobic toxin variants to associate with lipids in monolayer experiments to a larger extent compared to MC-LR [30]
These results suggest that MC-LF and MC-LW have clear cellular effects compared to MC-LR
Summary
Some cyanobacterial species, such as those among the freshwater genera Microcystis, Planktothrix and Anabaena, produce secondary metabolites called microcystins [1] These cyclic heptapeptides hepatotoxins are frequently reported worldwide and pose a threat to human health. When different microcystin variants have been evaluated and compared, differences regarding their toxicity have been shown [10,11,12,13]. Considering ingestion as a major exposure pathway, cell lines like Caco-2 cells that resemble the small intestine are appropriate for studying cellular effects of microcystins [27]. Due to the presence of both hydrophobic and hydrophilic amino acids in these biologically active peptides, we decided to take a closer look at their first contact with the cell membrane. We examined cell morphology, cell toxicity, cell proliferation and protein phosphatase inhibition of MC-LR, MC-LW and MC-LF on a Caco-2 cell line
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