Abstract
This study deals with the isolation and purification of an important variant of microcystins namely microcystin-RR (MCYST-RR) from Microcystis aeruginosa and reports its effects on mice liver protein profile and cellular functions. Protein profiling by 2-dimensional gel electrophoresis revealed changes in the number and accumulation of protein spots in liver of mice treated with different concentrations of MCYST-RR. Untreated (control) mice liver showed 368 protein spots while the number was 355, 348 and 332 in liver of mice treated with 200, 300 and 400 µg kg body wt−1 of MCYST-RR respectively. Altogether 102, 97, and 92 spots were differentially up-accumulated and 93, 91, and 87 spots were down- accumulated respectively with the treatment of 200, 300, 400 µg kg body wt−1. Eighteen differentially accumulated proteins present in all the four conditions were identified by MALDI-TOF MS. Of these eighteen proteins, 12 appeared to be involved in apoptosis/toxicological manifestations. Pathway analysis by Reactome and PANTHER database also mapped the identified proteins to programmed cell death/apoptosis clade. That MCYST-RR induces apoptosis in liver tissues was also confirmed by DNA fragmentation assay. Results of this study elucidate the proteomic basis for the hepatotoxicity of MCYST-RR which is otherwise poorly understood till date.
Highlights
Cyanobacteria inhabiting a wide spectrum of habitats including terrestrial, freshwater and marine are the most primitive oxygen-evolving Gram-negative photosynthetic prokaryotes[1]
Crude toxin was subjected to high performance liquid chromatography (HPLC) analysis wherein seven peaks with retention time (RT) of 1.8, 2.2, 7.4, 11.1, 14.2, 14.7 and 19.3 min appeared in the chromatogram (Fig. 1B)
HPLC analysis of both the fractions together with standard microcystin variants (RR and -LR) showed that the fraction having RT of 11.1 min resembled with the standard MCYST-RR and fraction with RT of 14.2 min to MCYST-LR
Summary
Cyanobacteria (blue-green algae) inhabiting a wide spectrum of habitats including terrestrial, freshwater and marine are the most primitive oxygen-evolving Gram-negative photosynthetic prokaryotes[1]. The hepatotoxic microcystins (MCYSTs) are the most widely distributed low molecular weight cyclic heptapeptide synthesized by different genera of cyanobacteria including Microcystis, Anabaena, Planktothrix, and Nostoc[2,5,6,7,8,9]. A few workers have studied proteomic changes following MCYST treatment and reported alterations in the number and expression of proteins[24,27,30]. Prompted by the above lacuna, we studied the changes in the proteome of the liver from the mice exposed to a toxic variant of microcystins, namely MCYST-RR employing 2-DE followed by MALDI-TOF MS analysis. We aimed to (a) isolate and purify MCYST-RR from Microcystis aeruginosa, (b) study the changes in the proteome of mouse liver following MCYST-RR treatment, and (c) assess the possible role of identified proteins in toxicity. The outcomes of this study may provide new insights for understanding how MCYST-RR can induce certain proteins and affect liver architecture leading to the death of mice
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