title: 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 (H(2)O(2)) 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 H(2)O(2) and ActD/TNF-α incubation. These reactions were also suppressed by silibinin pretreatment. We conclude that the  anti-apoptotic activity of silibinin against OTA, H(2)O(2) 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, H(2)O(2) 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.
authors.affiliation: Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Schubert Street 81, Giessen D-35392, Germany. ebtisam_essid@yahoo.de
authors.forename: Ebtisam
authors.initials: E
authors.lastname: Essid
authors.forename: Yousef
authors.initials: Y
authors.lastname: Dernawi
authors.forename: Ernst
authors.initials: E
authors.lastname: Petzinger
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D003609:Dactinomycin / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D009793:Ochratoxins / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000633:toxicity*; D014466:Ultraviolet Rays
chemical_list: D000975:Antioxidants; D002843:Chromatin; D009793:Ochratoxins; D047888:Receptors, Tumor Necrosis Factor, Type I; D012838:Silymarin; D014409:Tumor Necrosis Factor-alpha; C025589:ochratoxin A; D003609:Dactinomycin; D000077385:Silybin; D008315:Malondialdehyde; D006861:Hydrogen Peroxide; D053148:Caspase 3