Apoptosis Induction by OTA and TNF-α in Cultured Primary Rat Hepatocytes and Prevention by Silibinin

Abstract

In cultures of primary rat hepatocytes, apoptosis occurred after application of 20 ng/mL tumor necrosis factor alpha (TNF-α). However, this was only in the presence of 200 ng/mL of the transcriptional inhibitor actinomycin D (ActD). This toxic effect was completely prevented in the presence of 25 µg/mL soluble TNF-α receptor I (sTNFR I) in the supernatant of hepatocyte cell cultures. Apoptosis also occurred after application of 12.5 µmol/L ochratoxin A (OTA). However, that was not prevented by up to 500 µg/mL sTNFR I, indicating that TNF-α/TNFR I is not involved in OTA mediated apoptosis in hepatocytes. The antioxidative flavanolignan silibinin in doses from 130 to 260 µmol/L prevented chromatin condensation, caspase-3 activation, and apoptotic DNA fragmentation that were induced by OTA, by 10 mmol/L hydrogen peroxide (H2O2) and by ultraviolet (UV-C) light (50 mJ/cm2), respectively. To achieve protection by silibinin, the drug was applied to the cell cultures for 2 h in advance. OTA stimulated lipid peroxidation on cultured immortalized rat liver HPCT cells, as was revealed by malondialdehyde (MDA) production. Lipid peroxidation occurred further by H2O2 and ActD/TNF-α incubation. These reactions were also suppressed by silibinin pretreatment. We conclude that the anti-apoptotic activity of silibinin against OTA, H2O2 and ActD/ TNF-α is caused in vitro by the antioxidative effects of the flavanolignan. Furthermore, cytotoxicity of the pro-apoptotic toxins was revealed by MTT-test. When applied separately, ActD and TNF-α showed no cytotoxic effects after 24 h, but were cytotoxic if applied in combination. The used concentrations of OTA, H2O2 and the dose of UV-C caused a substantial decrease in viability within 36 h that was prevented mostly by silibinin. We conclude that silibinin is a potent protective compound against apoptosis and cytotoxicity caused by OTA and the investigated compounds.

In Silico and In Vitro Studies of Mycotoxins and Their Cocktails; Their Toxicity and Its Mitigation by Silibinin Pre-Treatment

“…At these concentrations, silibinin at the concentration range from 0.9 to 109 µM was added to evaluate its preventive effect in extended range of recommended daily dose of the supplement. As previously published, the co-treatment with both silibinin and mycotoxins did not improve the cell growth, but pretreatment of the cells by silibinin for 2 h before mycotoxins addition protected the cells against mycotoxins-mediated apoptosis [41,42]. Thus, we chose this approach to evaluate the protective effect of silibinin against mycotoxins-induced cytotoxicity.…”

“…According to Naseer et al [38], silymarin showed better results compared to choline chloride (liver tonics) in lowering the AFB1-induced serum aminotransferase, creatinine, and blood urea nitrogen. Silibinin has received much attention, but the negation of mycotoxins toxicity by silibinin has only been achieved on primary rat hepatocytes, isolated rat Kupffer cells, calves, and mice [37,38,[40][41][42].…”

“…The strongest antagonism was found with the silibinin pretreatments at concentration of 13.6 µM for T-2 and 6.8 µM for HT-2 and DAS exposure,. The cytoprotective effect of silibinin, may be ascribed to its antioxidant and free-radical scavenger role [41]. Based on the results of Al-Anati et al [40], low silibinin dose (0.2 µM) reduced OTA-induced TNF-α level to 70% while the higher doses (1-26 µM) completely blocked OTA-induced TNF-α within 24 h. Furthermore, the protective effect of 130 µM silibinin against OTA cytotoxicity in hepatocyte cells was reported [41,42].…”

“…The cytoprotective effect of silibinin, may be ascribed to its antioxidant and free-radical scavenger role [41]. Based on the results of Al-Anati et al [40], low silibinin dose (0.2 µM) reduced OTA-induced TNF-α level to 70% while the higher doses (1-26 µM) completely blocked OTA-induced TNF-α within 24 h. Furthermore, the protective effect of 130 µM silibinin against OTA cytotoxicity in hepatocyte cells was reported [41,42]. The authors assumed that silibinin acts on the cell membranes to prevent the entry of toxic substances, stimulates protein synthesis, and accelerates regeneration processes.…”

Black Soybean Seed Coat Extract Prevents Hydrogen Peroxide-Mediated Cell Death via Extracellular Signal-Related Kinase Signalling in HepG2 Cells

“…Primary hepatocytes may be suitable for a liver model to speculate about antioxidant mechanisms in the liver (17), in which parts of liver functions reportedly decrease gradually (18), and, in view of animal welfare, sacrifice of animals to obtain primary hepatocytes should be replaced by other models if the models do not have crucial defects. As a model of mammalian hepatocytes, therefore, HepG2 human hepatoma cells were used in this study because HepG2 cells stably express MAPK and related proteins and show responses to oxidative stress similarly to those of in vivo studies despite the lack of some liver functions (6,10,19).…”