Prolonged Sublethal Exposure to the Protein Phosphatase Inhibitor Microcystin-LR Results in Multiple Dose-Dependent Hepatotoxic Effects

Abstract

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.

Hepatic and intestine alterations in mice after prolonged exposure to low oral doses of Microcystin-LR

“…In agreement with previous reports (Solter et al, 1998;Ito et al, 2000;Sedan et al, 2013) we found higher levels of MC-LR in liver of mice treated with the highest dose.…”

“…Thus, our results indicate that prolonged and intermittent oral exposure at doses as low as 50 mg MC-LR/kg are capable of generating liver injury even when peripheral liver damage biomarkers remain unaltered. An important implication of these results from the clinical point of view, is that the measurement of plasma ALT, AST, and ALP is not the best choice of technique for detecting MC-LR exposure in a low-dose oral exposure scenario (Solter et al, 1998;Andrinolo et al, 2008). The experimental evidence supports the hypothesis that it is necessary to determine a range of parameters in addition to MC-LR plasma/urine quantification in order to more accurately evaluate microcystin exposure (Sedan et al, 2013).…”

“…The experimental data indicate that toxin accumulation in liver was 7-fold higher for mice treated with the highest dose (100 mg MC-LR/kg). A previous report (Solter et al, 1998) indicated that prolonged i.p. administration of doses of 16 and 32 mg MC-LR/kg resulted in 2.4% and 4.6% of total toxin administrated being detected in liver, respectively.…”

“…It was reported that protein phosphatase (PP1 and PP2A) inhibition and increased production of reactive oxygen species are the main deleterious effects caused by MC-LR (Yoshizawa et al, 1990;Ding et al, 1998). The hyperphosphorylation and pro-oxidative state induced by MC-LR leads to alterations in the cytoskeleton (Solter et al, 1998;Codd, 1996;Milutinovic et al, 2003), in mitogen-activated protein kinase (MAPK) activity (Pahan et al, 1998), glycogen storage (Guzman and Solter, 2002), and in mitochondrial structure and function (Ding and Ong, 2003), all damages that severely affect crucial cellular processes.…”

“…This type of exposure also resulted in biochemical alterations such as a decrease in antioxidant enzyme activities and increased lipid peroxidation in liver and kidney (Moreno et al, 2005). Chronic MC-LR exposure on the other hand is characterized by a completely different damage pattern involving hepatocyte alterations such as single-cell necrosis, cytosolic vacuolation, apoptosis, fibrosis, and tumor promotion (Ito et al, 1997;Solter et al, 1998;Guzman and Solter, 1999;Andrinolo et al, 2008;Lezcano et al, 2012). In addition, sub-chronic exposure to MC-LR induces liver and renal lipid peroxidation, alteration in enzymatic and non-enzymatic components of the antioxidant system, and modifications of the liver lipid profile (Andrinolo et al, 2008;Sedan et al, 2010).…”