Idiosyncratic, drug-induced liver injury (IDILI) occurs in a small fraction of susceptible patients and can be life threatening. Importantly, the current methods employed during preclinical safety evaluation of drug candidates fail to accurately identify those with IDILI liability before they reach the market. Accordingly, assays to identify drug candidates with the potential to cause IDILI early in the drug development process are greatly needed. Knowledge concerning mechanisms of IDILI is limited, but evidence in humans and animals implicates a role for immune mediators in the pathogenesis. Interestingly, several drugs associated with IDILI interact with cytokines, including tumor necrosis factor-alpha (TNF) and interferon gamma (IFN), in vitro to cause death of primary human hepatocytes and human-derived HepG2 cells. A major focus of this dissertation was to determine if cytotoxic synergy between drugs and cytokines can accurately classify drugs according to their IDILI liability. Indeed, cytotoxic synergy between drugs and TNF led to the generation of a statistical model that accurately classified a set of 24 drugs according to their IDILI potential. This result suggests a promising in vitro approach that is amenable to high throughput methodology and that could be used during preclinical safety evaluation to identify drug candidates with the potential to cause IDILI. Another major focus of this dissertation was to gain a deeper understanding of the signaling mechanisms underlying the cytotoxic interaction between IDILI-associated drugs and TNF and IFN. Along with antibiotics, NSAIDs are among the most frequent causes of IDILI. The cytotoxic interaction between NSAIDs with various IDILI liabilities and the two cytokines was investigated, and dichotomous roles for several mitogen activated protein kinases (MAPKs) were found. The findings suggest that NSAIDs associated with IDILI synergize with cytokines to cause HepG2 cell death that is driven by different kinase signaling mechanisms. The differences appear to be related to chemical structure and IDILI liability. The cytotoxic interaction between diclofenac (DCLF), an NSAID associated with IDILI, and TNF and IFN, was examined further. DCLF causes ER stress in HepG2 cells, which contributes to the cytotoxic interaction with TNF. Intracellular calcium (Ca++) dysregulation, ER stress and MAPK activation are closely linked cellular responses. DCLF is known to promote intracellular Ca++ dysregulation in hepatocytes. The contribution of free cytoplasmic Ca++ to the DCLF/cytokine interaction was examined. Chelation of intracellular free Ca++ with BAPTA/AM reduced DCLF-mediated activation of the ER stress sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK) and the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Importantly, BAPTA/AM and an inositol trisphosphate (IP3) receptor antagonist reduced the cytotoxic interaction between DCLF and cytokines, suggesting that Ca++ contributes to the cytotoxic interaction. Additionally, interdependence of the activation of JNK and ERK was found. These findings provide insight concerning the cytotoxic interaction between DCLF and cytokines. Additionally, these results raise the possibility that Ca++ contributes to the cytotoxic synergy between other drugs and the cytokines TNF and IFN, and might contribute to some cases of human IDILI.
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