Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays

Abstract

In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARec provides a limit of detection (LOD) of 3.1 μg/L, PP1Rec and PP2AWild provide LODs of 0.6 and 0.5 μg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 μg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC50) values ranging from 1.4 to 359.3 μg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.

Effects of cyanobacterial toxins on the human gastrointestinal tract and the mucosal innate immune system

“…With more than 240 congeners, MC-LR is considered to be one of the most common, abundant and toxic structural variants, with a median lethal dose (LD50, acute oral toxicity) of 5-mg/kg bodyweight (bw) of orally exposed mice and a no-observed adverse effect level (NOAEL liver ) of 40 µg/kg/day for 13 weeks of repeated dose exposure (chronic toxicity) [2,47,[78][79][80][81][82]. Modifications of MC molecules, including modifications of Adda-methyldehydroalanine region as well as other parts, such as variable amino acid residues at positions 2 and 4, can lead to the reduction of PP1/2A inhibition potencies [62,63,[83][84][85][86][87]. However, the observed differences in acute in vivo toxicity of MCs [87] cannot be exclusively attributed to the differences in PP1/2A inhibition among different MC variants.…”

Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

“…We tested two MCs, MC-LR and MC-LF, as there is little known about the inhibition potency of MC variants with different physicochemical properties. MC-LR and MC-LF were chosen based on their estimated [ 34 ] and experimental [ 20 ] octanol-water partition coefficient, lethal concentration [ 20 ], and recombinant PP1 IC 50 [ 35 ]. In our study MC-LR and MC-LF inhibited PP1 with similar potency suggesting that the difference in hydrophobicity did not influence the toxicodynamics in the PPIA.…”

“…This is in agreement with several published studies demonstrating that MC-LR and MC-LF have similar IC 50 values for PP1 (1.2 and 1.8 µg/L [ 36 ], 1 and 3µg/L [ 37 ], 1.4 and 2.2 µg/L [ 38 ], for MC-LR and MC-LF, respectively). Recently a study using PP1 and p NPP to establish inhibition equivalency factors for different MC congeners in relation to MC-LR determined an IC 50 of 2.1 µg/L for MC-LR and an IC 50 of 359.3 µg/L for MC-LF [ 35 ]. The overall trend of lowest to highest IC 50 was as follows: MC-LR > MC-RR > MC-YR > MC-LY > MC-LW > MC-LF.…”

“…Usually, MC-LR is used as a standard to determine PP1 inhibitor concentration in terms of MC equivalents. While it is possible to use up to ten standard concentrations to estimate the amount of PP1 inhibitor present in a sample [ 27 , 35 ], quantitation is not necessary for these investigations and can be attempted with LC-MS/MS as a follow-up. While canine deaths due to cyanotoxin exposure are more likely to be reported, cattle may be at greater risk of cyanotoxin exposure due to their reliance on fresh water drinking ponds.…”

Application of cellular biosensors for detection of atypical toxic bioactivity in microcystin-containing cyanobacterial extracts

“…Additionally, immunobiotest ELIS, can be used as a screening method for estimation of microcystins concentrations (Metcalf and Codd, 2004;Lawton et al, 2010). Moreover, for the determination of biological activity (toxicity) of microcystins, the screening Protein Phosphatase Inhibition Assay was proposed (An and Carmichael, 1994;Sevilla et al, 2009;Garibo et al, 2014).…”