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Toxins 2012, 4(11), 1139-1156; doi:10.3390/toxins4111139
Article

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

1
, 2
 and 1,*
Received: 31 August 2012; in revised form: 24 October 2012 / Accepted: 25 October 2012 / Published: 2 November 2012
(This article belongs to the Special Issue Ochratoxins 2011-2012)
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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.
Keywords: ochratoxin A; tumor necrosis factor alpha; UV-C light; hydrogen peroxide; rat hepatocytes; DNA ladder; caspase-3; apoptosis; malondialdehyde; silibinin ochratoxin A; tumor necrosis factor alpha; UV-C light; hydrogen peroxide; rat hepatocytes; DNA ladder; caspase-3; apoptosis; malondialdehyde; silibinin
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Essid, E.; Dernawi, Y.; Petzinger, E. Apoptosis Induction by OTA and TNF-α in Cultured Primary Rat Hepatocytes and Prevention by Silibinin. Toxins 2012, 4, 1139-1156.

AMA Style

Essid E, Dernawi Y, Petzinger E. Apoptosis Induction by OTA and TNF-α in Cultured Primary Rat Hepatocytes and Prevention by Silibinin. Toxins. 2012; 4(11):1139-1156.

Chicago/Turabian Style

Essid, Ebtisam; Dernawi, Yousef; Petzinger, Ernst. 2012. "Apoptosis Induction by OTA and TNF-α in Cultured Primary Rat Hepatocytes and Prevention by Silibinin." Toxins 4, no. 11: 1139-1156.



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