Analytical and functional characterization of microcystins [Asp3]MC-RR and [Asp3,Dhb7]MC-RR: consequences for risk assessment?
The microcystin (MC) producing P. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, and raw water quality. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants [Asp3]MC-RR and [Asp3,Dhb7]MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability to quantify [Asp3]MC-RR, [Asp3,Dhb7]MC-RR, and MC-RR was compared to HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, and [Asp3]MC-RR were in the same range (1.9-3.8 and 0.45-0.75 nM). A 50-fold higher concentration of [Asp3,Dhb7]MC-RR (29.8 nM) was necessary to inhibit the PP2A by 50%. The PP1-IC50 of [Asp3,Dhb7]MC-RR was 22-fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capacity. Western blots demonstrated that [Asp3,Dhb7]MC-RR does not covalently bind to PP1. [Asp3,Dhb7]MC-RR has comparable in vivo LD50 values to MC-RR, despite a far lower PP-inhibiting capacity, suggesting that toxicodynamic and toxicokinetic characteristics of [Asp3,Dhb7]MC-RR are responsible for its high in vivo toxicity. The data demonstrate that cPPIA analysis of [Asp3,Dhb7]MC-RR-containing samples prevent reliable MC determination and lead to underestimation of potential toxicity.
Hoeger
S J
SJ
Environmental Toxicology, University of Konstanz, Germany.
Schmid
D
D
Blom
J F
JF
Ernst
B
B
Dietrich
D R
DR
D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D000458:Cyanobacteria / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005068:Eutrophication; D005618:Fresh Water / Q000382:microbiology*; D005858:Germany; D020128:Inhibitory Concentration 50; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D007928:Lethal Dose 50; D013058:Mass Spectrometry; D052998:Microcystins / Q000378:metabolism* / Q000633:toxicity*; D011506:Proteins / Q000037:antagonists & inhibitors; D018570:Risk Assessment; D018675:Toxicity Tests / Q000379:methods; D000097847:Protein Phosphatase Inhibitory Proteins
D047908:Intracellular Signaling Peptides and Proteins; D052998:Microcystins; D011506:Proteins; C021042:protein phosphatase inhibitor-1; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins