Hepatocytes microcystins

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

Hepatotoxins include microcystins, which are cyclic heptapeptides (Fig. 5.1a) and cylindrospermopsin, a sulfated guanidinium alkaloid (Fig. 5. lb). Microcystins bind to certain protein phosphatases responsible for regulating the distribution of cytoskeletal proteins (Zurawell et al. 2005 Leflaive and Ten-Hage 2007). Hepatocytes exposed to microcystins eventually undergo cellular deformation, resulting in intra-hepatic bleeding and, ultimately, death (Carmichael 2001 Batista et al. 2003). In contrast, cylindrospermopsin appears to have a different mode of activity, possibly involving inhibition of protein or nucleotide synthesis (Codd et al. 1999 Froscio et al. 2003 Reisner et al. 2004). Nevertheless, microcystins are the most common cyanotoxins isolated from cyanobacterial blooms (Sivonen and Jones 1999). 

These natural toxins are heptapeptides produced by cyanobacteria, which are associated with algal blooms. These substances are a hazard to wild and farm animals and sometimes humans who come in contact with contaminated water. There are a number of these toxins, some of which such as microcystin LR are hepatotoxic, causing damage to both hepatocytes and endothelial cells. The toxins have some unusual structural features, incorporating three D-amino acids and two very unusual ones, namely, methyldehydro alanine (Mdha) and amino-methoxy-trimethyl-phenyl-decadi-enoic acid (Adda) (Fig. 7.26). 

Matsushima-Nishiwaki, R., Shidoji, Y., Nishiwaki, S., Yamada, T., Moriwaki, H., and Muto, Y. 1995. Suppression by carotenoids of microcystin-induced morphological changes in mouse hepatocytes. Lipids 30, 1029-1034. 

Other bioassays reported in the literature using rat (Aune and Berg 1986 Heinze 1996 Fladmark 1998) or salmon (Fladmark 1998) hepatocytes have been proposed as a monitoring tool for the peptide hepatotoxins. However, operational difficulties in water testing laboratories (e.g., preparation of cell suspensions) and limitations of sensitivity in the case of rat hepatocytes preclude then-use on a routine basis for water samples. Responses in these systems may well correlate with mammalian toxicity, thereby producing a measirre of toxicity in microcystin-LR toxicity equivalents if microcystin-LR were used for calibration. They may, therefore, be an attractive option in the futrrre with further development. 

Changes in intracellular calcium homeostasis produced by active metabolites of xenobiotics may cause disruption of the dynamic cytoskeleton. There are a few toxins that cause disruption of the cytoskeleton through mechanisms independent of biotransformation. Microcystin is one of these toxins. Microcystin is produced by the cyanobacterium Microcystis aeruginosa. Similar toxins are produced by other species of cyanobacteria. The hepatocyte is the specific target of microcystin, which enters the cell through a bile-acid transporter. Microcystin covalently binds to serine/threonine protein phosphatase, leading to the hyperphosphorylation of cytoskeletal proteins and deformation of the cytoskeleton (Treinen-Moslen, 2001). 

A rather close relationship between the apoptosis induced by microcystin-LR and caspase-3 activity was observed. Hepatocytes are extremely sensitive to microcystins and nodularin, as can be deduced from the quick apparition of cellular apoptotic changes such as superficial budding and shrinkage of cytosol and nucleus. Analogous findings were obtained in other kinds of cells, like Swiss 3T3 fibroblasts or promyelocytic IPC.81 cells, provided that the toxins were microinjected. However, in caspase-3 deficient MCF-7 cells, apoptosis developed slowly and was independent of the ZVAD, a compound that ordinarily inhibited apoptosis without affecting the hyperphosphorylation caused by PP inhibition [147]. 

When applied to intact cells, different factors should be considered. The microcystins, cantharidin, and endothal are not permeable across the plasmalem-ma but may be taken up by hepatocytes via the bile acid transport system (Eriksson et al., 1990). A derivative of endothal, endothal thioanhydride, is permeable across the cell membrane (M. Hirano and F. Er-dddi, unpublished observations). Thus permeability across lipid bilayers is one consideration. In addition, the potency of inhibitors used with intact cells always appears less than the in vitro assays. The potency with intact cells is often 10- to 100-fold less sensitive. For example, with 3T3 fibroblasts, external concentrations above 10 nM were required to elicit shape changes (Chartier et al., 1991). This difference in dose dependence could be due to inefficient uptake by the cells or preferential localization of the inhibitor with lipids. Cohen et al. (1989) suggested that the higher concentrations required with intact cells reflect the intracellular concentration of the targeted phosphatase. 

Protein phosphatase inhibition has been correlated with the onset of acute microcystin-induced hepatotoxicosis in mice (70) and with microfilament reorganization and cell deformation in isolated hepatocytes (65). Inhibition of these enzymes causes hyperphosphorylation of numerous cytosolic and cytoskeletal proteins in isolated hepatocytes exposed to microcystin (63,65,71). It has recently been shown that at higher concentrations similar morphological effects are produced also in non-hepatocytes (72). 

The toxicity exerted by PTX in several cell types indicates that the hepatocytes are not the only cellular model sensitive to this group of toxins. Therefore, unlike the fresh water toxin microcystin, hepatotoxicity of which is due to unique transport mechanisms present in the hepa-tocyte plasma membrane [40,41], PTXs hepatotoxicity in vivo seems to be determined in part by... 

Fastner, J. et al., Cyanotoxin occurrence in Germany Microcystins and hepatocyte toxicity, in Cyanotoxins—Ocurrence, Causes, Consequences, Chorus, I., Ed., Springer, Berlin, 2001, 22. 

After ingestion, microcystins are released from cyano-bacterial cells and are absorbed into the portal circulation from the small intestine via bile acid transporters in the intestinal wall. Microcystins are then accumulated in hepatocytes via similar bile acid transporters on hepato-cyte membranes (Hooser et al., 1991). Microcystins irreversibly inhibit serine/threonine protein phosphatases 1 and 2A (Yoshizawa et al., 1990). Microcystin-LR may also bind to AP synthase, leading to hepatocyte apoptosis (Mikhailov et al., 2003). 

Protein phosphatases are ubiquitous. They are foxmd in all tissues and across species as diverse as mammals, plants, and bacteria, and they play a critical role in the regulation of multiple cellular metabolic pathways. Protein phosphatases reverse tiie active state of kinases through the hydrolytic removal of tiie phosphoryl group from kinases. The protein phosphatases inhibited by microcystins have broad substrate specificity and play roles in the regulation of a wide range of cellular fxmc-tions. Protein phosphatase 2A is highly conserved and is a major downregulator of active protein kinases in eukaryotic cells. Toxic effects in hepatocytes and other... 

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