Phosphoprotein analysis for investigation of in vivo relationship between protein phosphatase inhibitory activities and acute hepatotoxicity of microcystin-LR.
Microcystin-LR (MCLR) produced by freshwater cyanobacteria is a potent hepatotoxin and inhibits protein serine/threonine phosphatases 1 and 2A (PP1 and PP2A). Okadaic acid (OA) is a similar phosphatase inhibitor, which has less affinity to PP1 than PP2A. MCLR and OA behave similarly with primary culture hepatocytes with the induction of phosphorylation of the cytokeratins, morphological changes, and apoptosis. The purpose of this study was to investigate the in vivo relationship between the protein phosphatase inhibitory activities and the acute hepatotoxicity of MCLR compared to OA. MCLR and OA were intraperitoneally administrated to mice at approximately 220 microg/kg. After 30 min, the liver of only the MCLR-treated mouse was dark-colored and heavier than that of the control mouse. Subsequently, the phosphoproteins of the mouse liver were chemically modified with reversible biotinylation reagent and selectively analyzed by LC/MS/MS. Consequently, the phosphorylated Ser 354 of formyltetrahydrofolate dehydrogenase, which is an abundant enzyme in the liver cytoplasm, was observed in the MCLR- and the OA-treated mice 9.5 and 5.3 times more intensely than in the control mouse respectively, suggesting that MCLR and OA inhibited PP2A and induced the resulting phosphorylation. These results supported the hypothesis that the acute hepatotoxicity is possibly caused by the PP1 inhibition, and not by the PP2A inhibition.
Tachi
Masahiko
M
Graduate School of Environmental and Human Sciences, Meijo University, 150 Yagotoyama, Tempaku, Nagoya 468-8503, Japan. tachi.masahiko@tmrc.co.jp
Imanishi
Susumu Y
SY
Harada
Ken-Ichi
K
D000818:Animals; D000458:Cyanobacteria; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D052998:Microcystins / Q000633:toxicity*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000187:drug effects*
D052998:Microcystins; C078588:microcystin; D010749:Phosphoprotein Phosphatases