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Fulminant hepatocyte apoptosis in vivo following microcystin-LR administration to rats
1Department of Veterinary Pathobiology, Purdue University, West Lafayette, Indiana 47907, USA. shooser1@purdue.edu
Abstract:
Microcystin-LR (MCLR) is a cyanobacterial toxin responsible for human and livestock deaths worldwide. MCLR has also been implicated as a contributing factor in hepatocellular carcinoma. Following absorption, MCLR is taken up via a hepatocyte-specific bile acid carrier. Inside hepatocytes, MCLR selectively binds to protein phosphatases 1 and 2A, resulting in rapid, massive liver damage. However, the apoptotic nature of this toxicosis in rats has not been fully characterized as such at appropriate time points utilizing light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and electrophoresis of hepatic DNA. Rats were administered intraperitoneal saline or MCLR at 500 microg/kg (0.5 micromol/kg) and necropsied at 3 or 9 hours. Light microscopy at 3 hours revealed massive, widespread apoptotic necrosis of the majority of hepatocytes. Hepatocytes were rounded and disassociated, with cell shrinkage, increased eosinophilia, and margination of nuclear chromatin or pyknosis. The apoptotic index increased from 0.03% +/- 0.02% in controls to 205% +/- 12% in MCLR-treated animals (p < or = 0.0001). At 3 hours, transmission electron microscopy revealed hepatocellular changes typical of apoptotic necrosis: rounding and disassociation of hepatocytes, loss of microvilli, and margination and condensation of nuclear chromatin. Laddering of hepatic DNA by electrophoresis and widespread TUNEL staining of hepatocytes were consistent with apoptosis. These results demonstrate that in rats, hepatic damage caused by MCLR is due to extremely rapid induction and progression of apoptosis in virtually every hepatocyte in the liver. This model of fulminant hepatic necrosis should be useful for increased characterization and understanding of the relationship between protein phosphatase inhibition and apoptosis.
Insights
Microcystin-LR (MCLR), a cyanobacterial toxin, rapidly induces apoptosis in rat hepatocytes, causing massive liver damage. This study characterizes the apoptotic nature of MCLR toxicosis using microscopy and molecular techniques.
Area of Science:
- Hepatology
- Toxicology
- Cell Biology
Background:
- Microcystin-LR (MCLR) is a potent cyanobacterial toxin causing widespread mortality in humans and livestock.
- MCLR is linked to hepatocellular carcinoma and causes severe liver damage by inhibiting protein phosphatases 1 and 2A within hepatocytes.
Purpose of the Study:
- To comprehensively characterize the apoptotic mechanisms underlying Microcystin-LR-induced liver injury in a rat model.
- To investigate the temporal progression of hepatocellular apoptosis following MCLR exposure.
Main Methods:
- Rats were administered MCLR (500 µg/kg) or saline, with necropsies at 3 and 9 hours.
- Histopathological analysis using light and electron microscopy.
- Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis detection.
- Hepatic DNA electrophoresis to identify DNA fragmentation (laddering).
Main Results:
- Light microscopy revealed widespread apoptotic necrosis in hepatocytes within 3 hours, characterized by cell shrinkage and chromatin condensation.
- Electron microscopy confirmed apoptotic changes, including loss of microvilli and nuclear chromatin margination.
- TUNEL staining and DNA laddering provided molecular evidence of extensive apoptosis in MCLR-treated rats.
- The apoptotic index significantly increased in MCLR-exposed rats compared to controls (20.5% vs. 0.03%).
Conclusions:
- MCLR exposure triggers extremely rapid and widespread apoptosis in nearly all hepatocytes.
- This rapid apoptotic liver necrosis model is valuable for studying protein phosphatase inhibition and apoptosis.
- The findings highlight the critical role of apoptosis in MCLR-induced hepatotoxicity.