Rapid conversion and reversible conjugation of glutathione detoxification of microcystins in bighead carp (Aristichthys nobilis).

The glutathione and cysteine conjugates of microcystin (MC-GSH and MC-Cys, respectively) are two important metabolites in the detoxification of microcystins (MCs). Although studies have quantitated both conjugates, the reason why the amounts of MC-GSH are much lower than those of MC-Cys in various animal organs remains unknown. In this study, MC-RR-GSH and MC-RR-Cys were respectively i.p. injected into the cyanobacteria-eating bighead carp (Aristichthys nobilis), to explore the biotransformation and detoxification mechanisms of the two conjugates. The contents of MC-RR, MC-RR-GSH, MC-RR-Cys and MC-RR-N-acetyl-cysteine (MC-RR-Nac, the acetylation product of MC-RR-Cys) in the liver, kidney, intestine and blood of bighead carp in both groups were quantified via liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). In the MC-RR-GSH-treated group, the MC-RR-Cys content in the kidney increased 96.7-fold from 0.25 to 0.5h post-injection, demonstrating that MC-RR-GSH acts as a highly reactive intermediate and is rapidly converted to MC-RR-Cys. The presence of MC-RR in both MC-RR-GSH- and MC-RR-Cys-treated groups indicates, for the first time, that MC conjugation with the thiol of GSH/Cys is a reversible process in vivo. Total MC-RR concentrations dissociated from MC-RR-Cys were lower than those from MC-RR-GSH, suggesting that MC-RR-Cys is more capable of detoxifying MC-RR. MC-RR-Cys was the most effectively excreted form in both the kidney and intestine, as the ratios of MC-RR-Cys to MC-RR reached as high as 15.2, 2.9 in the MC-RR-GSH-treated group and 63.4, 19.1 in the MC-RR-Cys-treated group. Whereas MC-RR-Nac could not be found in all of the samples of the present study. Our results indicate that MC-RR-GSH was rapidly converted to MC-RR-Cys and then excreted, and that both glutathione and cysteine conjugates could release MC-RR. This study quantitatively proves the importance of the GSH detoxification pathway and furthers our understanding of the biochemical mechanism by which bighead carp are resistant to toxic cyanobacteria.