Effects of thymoquinone on alpha-amanitin induced hepatotoxicity in human C3A hepatocytes

Abstract Thymoquinone (TQ) has shown hepatoprotective effects in various experimental studies. We aimed to investigate the possible beneficial effects of TQ regarding its prevention of alpha-amanitin induced hepatotoxicity in human C3A hepatocytes. After administering alpha-amanitin in a concentrations of 1 and 10µg/mL on the cells in a hepatocyte cell line, TQ was administered in various concentrations (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005 µg/mL). The MTT test was used to determine cell viability. For the groups given only TQ at various concentrations, the cell viability rates at 48 hours post-administration were found at 82.6, 98.3, 102.1, 102.5, 99.4, 99.4, 101.9 and 106.3%, respectively. For the group with 1μg/mL alpha-amanitin and various TQ concentrations, the cell viability rates were found at 74.6, 88.5, 87.4, 88.7, 85.7, 86.8, 88.4, and 92.9%, respectively. For the group with 10μg/mL alpha-amanitin and various TQ concentrations, the cell viability rates for each TQ subgroup were found at 65.2, 79.2, 81.4, 81.1, 81.8, 81.8, 82.2 and 91.9%, respectively. Our study is the first in vitro study that investigates TQ's effects on alpha-amanitin induced hepatotoxicity. Although TQ had beneficial effect in low doses did not significantly increase cell viability in liver damage due to alpha-amanitin toxicity.

The Effect of Glehnia Littoralis on Alpha-amanitin Induced Hepatotoxicity in a Murine Model

PURPOSE: Glehnia littoralis has been reported to have several pharmacological properties but no in vivo reports describing the protective effects of this plant on α-amanitin-induced hepatotoxicity have been published. α-Amanitin is a peptide found in several mushroom species that accounts for the majority of severe mushroom poisonings leading to severe hepatonecrosis. In our previous in vitro study, we found that α-amanitin induced oxidative stress, which may contribute to its severe hepatotoxicity. The aim of this study was to investigate whether Glehnia littoralis acetate extract (GLEA) has protective antioxidant effects on α-amanitin-induced hepatotoxicity in a murine model. METHODS: Swiss mice (n=40 in all groups) were divided into four groups (n=10/group). Three hours after giving α-amanitin (0.6 mg/kg, i.p.) to the mice, they were administered silibinin (50 mg/kg/d, i.p.) or Glehnia littoralis ethyl acetate extract (100 mg/kg/d, oral) therapies once a day for 3 days. After 72 hours of treatment, each subject was killed, cardiac blood was aspirated for hepatic aminotransferase measurement, and liver specimens were harvested to evaluate the extent of hepatonecrosis. The degree of hepatonecrosis was assessed by a pathologist blinded to the treatment group and divided into 4 categories according to the grade of hepatonecrosis. RESULTS: GLEA significantly improved the beneficial functional parameters in α-amanitin-induced hepatotoxicity. In the histopathological evaluation, the toxicity that was generated with α-amanitin was significantly reduced by GLEA, showing a possible hepatoprotective effect. CONCLUSION: In this murine model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Increases of aminotransferases and degrees of hepatonecrosis were attenuated by this antidotal therapy.

In vitro Protective Effects of Glehnia Littoralis on Alpha-amanitin Induced Hepatotoxicity

PURPOSE: Glehnia littoralis has been used to treat ischemic stroke, phlegm, cough, systemic paralysis, antipyretics and neuralgia. The pharmacological mechanisms of Glehnia littoralis include calcium channel block, coumarin derivatives, anticoagulation, anti-convulsive effect, as well as anti-oxidant and anti-inflammatory effects. Alpha-amanitin (α-amanitin) is a major toxin from extremely poisonous Amanita fungi. Oxidative stress, which may contribute to severe hepatotoxicity was induced by α-amanitin. The aim of this study was to investigate whether Glehnia littoralis ethyl acetate extract (GLEA) has the protective antioxidant effects on α-amanitin -induced hepatotoxicity. METHODS: Human hepatoma cell line HepG2 cells were pretreated in the presence or absence of GLEA (50, 100 and 200µg/ml) for 4 hours, then exposed to 60µmol/L of α-amanitin for an additional 4 hours. Cell viability was evaluated using the MTT method. AST, ALT, and LDH production in a culture medium and intracellular MDA, GSH, and SOD levels were determined. RESULTS: GLEA (50, 100 and 200µg/ml) significantly increased the relative cell viability by 7.11, 9.87, and 14.39%, respectively, and reduced the level of ALT by 10.39%, 34.27%, and 52.14%, AST by 9.89%, 15.16%, and 32.84%, as well as LDH by 15.86%, 22.98%, and 24.32% in culture medium, respectively. GLEA could also remarkably decrease the level of MDA and increase the content of GSH and SOD in the HepG2 cells. CONCLUSION: In the in vitro model, Glehnia littoralis was effective in limiting hepatic injury after α-amanitin poisoning. Its antioxidant effect is attenuated by antidotal therapy.