Microcystin-LR Induced Reactive Oxygen Species Mediate Cytoskeletal Disruption and Apoptosis of Hepatocytes in Cyprinus carpio L.

Abstract

Microcystins (MCs) are a group of cyclic hepatotoxic peptides produced by cyanobacteria. Microcystin-LR (MC-LR) contains Leucine (L) and Arginine (R) in the variable positions, and is one of the most common and potently toxic peptides. MC-LR can inhibit protein phosphatase type 1 and type 2A (PP1 and PP2A) activities and induce excessive production of reactive oxygen species (ROS). The underlying mechanism of the inhibition of PP1 and PP2A has been extensively studied. The over-production of ROS is considered to be another main mechanism behind MC-LR toxicity; however, the detailed toxicological mechanism involved in over-production of ROS in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the hydroxyl radical (•OH) was significantly induced in the liver of carp after a relatively short-term exposure to MC-LR. The elevated reactive oxygen species (ROS) production may play an important role in the disruption of microtubule structure. Pre-injection of the antioxidant N-acetyl-cysteine (NAC) provided significant protection to the cytoskeleton, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction. In addition, the elevated ROS formation induced the expression of apoptosis-related genes, including p38, JNKa, and bcl-2. A significant increase in apoptotic cells was observed at 12 - 48 hours. Our study further supports evidence that ROS are involved in MC-LR induced damage to liver cells in carp, and indicates the need for further study of the molecular mechanisms behind MC-LR toxicity.

Mechanisms of Microcystin-induced Cytotoxicity and Apoptosis

“…Our previous study also showed that pre-treatment of BSO enhanced the level of oxidative stress and promoted MC-induced apoptosis [55]. MCinduced oxidative stress and apoptosis were also observed in studies on aquatic animals both in vivo [73][74][75] and in vitro [56,[76][77][78].…”

“…These elements not only provide cellular architecture to maintain cell shape, cell division, migration, adhesion and contraction/relaxation, but also play a central role in signal transduction. Both in vivo and in vitro studies have shown that exposure of human, rat and fish hepatocytes to MC-LR induce alteration, aggregation, breakdown, reorganization and collapse of the cytoskeleton, loss of intercellular contacts and consequently disruption of cellular architecture [75,[100][101][102]. Cytoskeletal alterations induced by MC-LR were also reflected in plants, Vicia faba [103] and Ceratophyllum demersum [104].…”

“…However, all of these effects were significantly attenuated by the antioxidants, NAC and vitamin C, suggesting that ROS generation triggered by MC-LR is a key intracellular event that contributes to an induction of p38-MAPK activation and tau phosphorylation. Pre-injection of the antioxidant NAC also provided significant protection to the cytoskeleton system in the liver of common carp (Cyprinus carpio L.) exposed to MC-LR, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction [75].…”

Zebrafish Abcb4 is a potential efflux transporter of microcystin-LR

“…These toxic MCs are primarily accumulated in the liver and mediated by organic anion transporters, such as members of the OATP superfamily which are highly expressed in hepatocytes (Fischer et al, 2005). Upon entry into cells, MCs specially bind to and inhibit protein phosphatases 1 and 2A (PP-1 and PP2A) (MacKintosh et al, 1990) and can increase the formation of reactive oxygen species (ROS) (Nong et al, 2007;Jiang et al, 2013), thereby influencing the regulation of cellular protein phosphorylation and finally cause loss of liver architecture, dysfunction and necrosis (Cazenave et al, 2006). P-glycoproteins are versatile efflux transporters for a wide range of substrates with broad specificity, including endo-and exogenous hydrophobic amphipathic compounds such as physiological metabolites, therapeutic drugs and toxic xenobiotics (Sharom, 2011).…”

A review of reproductive toxicity of microcystins

“…These elements exert a wide range of impacts on reproductive events including sperm maturation and motility, oocyte maturation, fertilization and early embryo development [116]. It has been documented that both protein phosphatase inhibition and oxidative stress induced by MCs may lead to cytoskeletal damage [117][118][119][120][121][122]. Gácsi et al [77] showed that apoptotic shrinkage in Chinese hamster ovary (CHO-K1) cells was associated with the shortening and loss of MFs and with a concentration-dependent depolymerization of MTs.…”