Detection and Identification of Metabolites of Microcystins Formed in Vivo in Mouse and Rat Livers

Abstract

The hepatic metabolism of microcystins (MCs), potent cyclic peptide hepatotoxins produced by cyanobacteria, was studied by i.p. injection in mice and rats. An immunoaffinity purification method using an anti-MC-LR monoclonal antibody showed a remarkable effect on the removal of contaminants in the hepatic cytosol and enabled us to analyze MCs and their metabolites by HPLC and Frit-FAB LC/MS. At 3, 6, and 24 h post-injection of MC-RR, a small percent of the applied dose was detected in all of the mouse livers together with several metabolites. Among them, GSH and Cys conjugates of MC-RR were identified at 3 and 24 h, respectively, by comparison with the chemically prepared standards, indicating that the thiols of GSH and Cys nucleophilically bound to the Mdha moiety of MCs. Another metabolite was presumed to be formed by both epoxidation followed by hydrolysis and sulfate conjugation in the Adda moiety and GSH conjugation in the Mdha moiety. In rat livers, MC-LR showed almost the same behavior as that of MC-RR in mouse livers. These results suggest that the conjugation of GSH with MCs may play a role in the metabolic pathway leading to detoxification of MCs.

Quantitatively evaluating detoxification of the hepatotoxic microcystin-LR through the glutathione (GSH) pathway in SD rats

“…Glutathione (GSH), the major non-protein thiol abundant in the cells of most organisms, was demonstrated to play an important role in the detoxification of MCs in both mammals and aquatic organisms (Kondo et al 1996;Pflugmacher et al 1998Pflugmacher et al , 2001He et al 2012;Li et al 2014b). The glutathione and cysteine conjugates of MCs (MC-GSH and MC-Cys) were firstly synthesized under chemical conditions by Kondo et al (1992) with Frit-FAB liquid chromatographymass spectrometry (LC/MS), and then the resulting conjugates were identified as two metabolites of MCs in vivo in the livers of mouse and rat.…”

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography–tandem mass spectrometry

“…For MC-LR-GSH, the previous studies primarily focused on the qualitative analyses and no quantitative analyses have been developed [14,28,29]. Kondo et al [28] described the qualitative detection of the metabolites of microcystin-RR and -LR formed in vivo in mouse and rat liver by both HPLC and Frit-FAB LC/MS.…”

“…Kondo et al [28] described the qualitative detection of the metabolites of microcystin-RR and -LR formed in vivo in mouse and rat liver by both HPLC and Frit-FAB LC/MS. Pflugmacher et al [14] identified an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin MC-LR by HPLC and MALDI-TOF-MS. Ito et al [29] applied an immunostaining method to the study of absorption and distribution of MC-LR and its glutathione conjugate in different tissues in mice.…”

Quantitative liquid chromatography–tandem mass spectrometry method for determination of microcystin-RR and its glutathione and cysteine conjugates in fish plasma and bile

“…Previous studies have shown that GSH plays an important role in the detoxification of MCs in aquatic animals. The microcystin-glutathione conjugate has been demonstrated to form via glutathione S-transferase, which appeared to be the first step of detoxification, and then degraded to the cysteine conjugate [10][11][12][13]. However, these studies focused only on qualitative analyses.…”