| Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits serine/threonine protein phosphatases types 1 and 2A in hepatocytes in vivo and in vitro, and disrupts actin microfilament (MF), microtubule (MT) and intermediate filament (IF) networks. The sequential ultrastructural and biochemical changes induced by MCLR were studied in the isolated perfused rat liver model. Livers were perfused with doses ranging from 0.1 to 5.0 {dollar}mu{dollar}g/ml for 5 to 40 min in order to characterize the earliest observable morphologic changes presumably induced by disorganization of the hepatocyte cytoskeleton, and to follow the progression of lesions over time. The earliest toxin-induced changes were observed at 0.1 {dollar}mu{dollar}g/ml after 15-20 min exposure, or at 0.3 {dollar}mu{dollar}g/ml after 5-10 min. Earliest lesions included loss of hepatocyte microvilli, widening of sinusoidal fenestrae with disruption of sinusoidal endothelium, dilation of bile canaliculi, widening of intercellular spaces, and the initial stages of hepatocyte separation. Lesions progressed in severity and extent with increasing dose and exposure time. The observed changes are likely to be attributable in large measure to altered hepatocyte cytoskeletal structure and function.; The effects of MCLR on the organization of MFs, IFs, and MTs in hepatocytes, renal epithelial cells and fibroblasts were compared using silver-enhanced gold labeling, and the sequence of toxin-induced changes was determined. Changes in fibroblasts and some hepatocytes were characterized initially by disorganization of IFs, followed rapidly by disorganization of MTs, with the progressive collapse of both networks around cell nuclei. The onset of changes in MF structure occurred later than with the other cytoskeletal elements in all cell types and at all doses. In many hepatocytes, however, and especially at later time points, changes were noted in MTs and MFs prior to effects on IFs. Changes in MTs had not been reported in cells exposed to MCLR prior to this study. The similarity of effects among different cell types suggests a common mechanism of action, once the toxin is able to gain entry into hepatocytes or non-hepatocytes. However, differences among hepatocytes in the sequence of cytoskeletal changes, and between hepatocytes and non-hepatocytes suggests that the toxin acts to destabilize the hepatocyte cytoskeleton at more than point. |
