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.

The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation even in the absence of OTA. Treatment with 12.5 μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed DNA-laddering after 24–36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 μg/ml showed no apoptotic DNA-effects under these conditions. A low concentration of 26 μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas a five times higher concentration of silibinin (130 μmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions, caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12–24 h but was blocked by 130 μmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration of 12.5 μmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation times (≥36 h). In contrast, LPS up to 12.5 μg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis), also depending on the method of hepatocyte preparation. Silibinin at 130 μmol/l showed significant hepatoprotective and antiapoptotic effects against OTA-mediated cell damage on cultured rat hepatocytes.

The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 µmol/L OTA, or to 0.1 µg/mL LPS, for up to 24 h. OTA at 2.5 µmol/L and LPS at 0.1 µg/mL were not toxic to the tested cells as indicated by viability markers. TNF-a was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-a concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 µmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-a after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-a release via the pRaf/MEK 1/2–NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2-NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-a release upon OTA exposure.