The Role of TNF in Hepatotoxicity

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Tumor necrosis factor alpha (TNFalpha) has been demonstrated to be a primary mediator of endotoxin shock. One mechanism of TNF-induced injury is via the activation of NF-kB and the generation of reactive oxygen species. We have hypothesized that 2,3,7,8-tetrachlorodibenzodioxin (TCDD) -induced endotoxin hypersensitivity and subsequent induction of apoptosis may occur through modulation of TNFalpha signaling pathways. Studies using in vivo models of endotoxin hypersensitivity to examine the relationship between TNFalpha signaling and apoptosis are ongoing. We have characterized the kinetics of TCDD-induced hepatic damage in the liver by evaluating serum enzyme levels, quantifying apoptotic cells, and measuring alterations in gene expression at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. TCDD modulated endotoxin-mediated early expression of Fas, and upregulated expression of caspase 3 (day 10), NFkappaB (day 14), and TNFalpha (days 10 and 14). In addition, combined TCDD/endotoxin altered the kinetics of TCDD-induced hepatoxicity, with peak serum levels occurring 4 days earlier. TNFR1 and TNFR2 gene expression, IkappaBalpha and IkappaBbeta protein expression, and NFkappaB DNA-binding activity did not appear to be modulated by TCDD under the conditions of the study. Previous studies had shown that when rodents are treated with TCDD prior to endotoxin exposure a significant increase in toxicity occurs, and that inhibition of protein synthesis with cycloheximide blocks the TCDD-induced sensitivity to TNF in this model. Overexpression of caspases has been shown to induce apoptosis, and activation of the caspases appears to be necessary for development of the apoptotic phenotype. The protective effects of cycloheximide and alterations in TNFalpha, and NFkappaB gene expression suggest that TCDD treatment may result in alterations in TNF-induced activation of NFkappaB via the TNFalpha/TNFR2/TRAF pathway. The alterations in Fas and Caspase 3 gene expression indicate initiation of apoptosis and suggest involvement of the Fas Ligand/Fas/Caspase pathway.To investigate the mechanism by which TCDD alters NFkappaB expression we are currently examining whether TCDD alters the degradation of the IkappaB/NFkappaB complex at lower doses of endotoxin. In addition, we are attempting to clarify the role of Caspases 1 and 3 by separating and quantifying active and pro-active forms of the protease in treated and untreated animals.