Issues arising when interpreting the results of the protein phosphatase 2A inhibition assay for the monitoring of microcystins

Abstract

Microcystins, cyclic heptapeptidic hepatotoxins produced by a number of bloom forming freshwater cyanobacteria, are considered to represent a serious risk to public health through drinking and recreational water. A highly sensitive bioassay relying on the specific inhibition of the human protein phosphatase 2A was applied to the quantification of microcystins. A systematic approach based on the rational testing of seven purified mcyst variants as well as characterized environmental samples allowed to point out the limits and experimental bias associated with this assay. All the seven microcystin variants known as microcystins RR, YR, LR, LY, LA, LW and LF strongly inhibited the enzyme with IC 50 ranging between 0.29±0.02 and 0.84±0.07 nM for microcystins LW and YR, respectively. Using the model system of Microcystis aeruginosa PCC7820 axenic cultures and within the 1-year study of a Planktothrix agardhii bloom, the PP2A assay was shown to be strongly correlated to high-performance liquid chromatography (HPLC) coupled to ultra violet diode array detection. However the slope of the linear regression was significantly influenced by the sample composition, as confirmed by HPLC coupled to electrospray ionization mass spectrometry. A model based on pure additivity of mcyst effects was established to describe PP2A inhibition by standard mcyst mixtures, and fully agreed with experimental observations.