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Microcystins (MCs) are potent hepatotoxins produced by cyanobacteria in aquatic environments. MC-LR, a representative MC, strongly inhibits protein phosphatases 1 and 2A, leading to cell collapse in animal hepatocytes due to hyperphosphorylation of the cytoskeleton and apoptosis due to stimulation of the relevant systems. However, the molecular mechanisms and the metabolic pathways responsible for MC-LR toxicity are poorly understood. In the present study, we compared the cytotoxic effects of MC-LR in two cell lines: normal human hepatocytes (h-Nheps) and human hepatoma cell line HepG2. We also discussed the suitability of cellular assays for evaluating the toxicity of MCs. To obtain further insight into the molecular mechanism, the uptake, excretion, and intracellular distribution of MC-LR were analyzed using an antibody and assay kit targeting the catalytic subunit of protein phosphatase 2A (the PP2A assay kit). The responses toward MC-LR were distinctly different between the two cell lines. In HepG2 cells, MC-LR did not induce morphological change, did not reveal cytotoxicity, and accumulated to a lesser extent despite a slightly elevated expression of the MC transporter protein. MC-LR also did not alter the MC-binding potency of subcellular proteins. All these results indicate that HepG2 cells are inappropriate for the evaluation of MC-LR toxicity. The PP2A assay kits were useful not only for assessing PP2A-inhibitory potency, but also for determining the concentration of MCs in biological systems., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC(50) values for 21MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC(50) values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC(50) values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC(50) values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC(50) values were determined under the same experimental conditions, ensuring the quality of the data. The IC(50) values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.