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

Toxins

Ebtisam Essid¹  Yousef Dernawi² 

[1] Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Schubert Street 81, Giessen D-35392, Germany;Institute of Pathology, Faculty of veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Street 96, Giessen D-35392, Germany;

关键词: ochratoxin A;    tumor necrosis factor alpha;    UV-C light;    hydrogen peroxide;    rat hepatocytes;    DNA ladder;    caspase-3;    apoptosis;    malondialdehyde;    silibinin;   

DOI  :  10.3390/toxins4111139

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2012 by the authors; licensee MDPI, Basel, Switzerland.