Idiosyncratic drug-induced liver injury (IDILI) is an adverse response to many pharmaceuticals representing a significant public health risk associated with significant morbidity and mortality in humans. IDILI is responsible for many of the FDA-imposed restrictions on drug therapies, representing additional regulatory and financial burdens. Despite the extensive efforts put forth to develop safe and effective drugs, the causes of IDILI are not well understood. Models of IDILI in animals have been developed recently in which coadministration of a nontoxic dose of an IDILI-associated drug and an otherwise nontoxic dose of bacterial lipopolysaccharide (LPS) precipitates hepatocellular injury. A key component to most of these drug-LPS hepatotoxicity models is a drug-mediated increase or prolongation of LPS-induced tumor necrosis factor-alpha (TNF) release in the plasma of animals that precedes the onset of toxicity. The focus of this dissertation was to model a drug-mediated increase in LPS-induced TNF release in vitro and study the underlying mechanisms responsible for the increased LPS-induced TNF release. Trovafloxacin (TVX), an antibiotic with IDILI liability, is hepatotoxic in mice when coadministered with LPS. TVX prolongs LPS-induced plasma TNF in mice, and this prolongation is required for hepatotoxicity. One hypothesis for the prolonged plasma is that TVX increased LPS-induced synthesis and release of TNF from inflammatory cells. Accordingly, a model of TVX/LPS coexposure was established in RAW 264.7 macrophage-like cells to recapitulate the increased LPS-induced TNF release. TVX increased LPS-induced TNF release in a concentration- and time-dependent manner. Analysis of the changes to upstream inducers of Tnf expression revealed that TVX activated mitogen activated protein kinases (MAPKs) ERK and JNK. The increased LPS-induced TNF release from RAW cells required ERK- or JNK-dependent signaling. The next group of studies tested the hypothesis that TVX increases LPS-induced TNF release due to eukaryotic topoisomerase poisoning, an off-target effect of TVX. An in silico analysis indicated that TVX could bind favorably to human topoisomerase, and TVX decreased human topoisomerase activity in a cell-free assay. In RAW cells, TVX induced a marker of DNA damage and activated ataxia telangiectasia-mutated Rad-3-related (ATR) kinase. ATR-dependent signaling was required for the TVX-mediated increase in LPS-induced TNF release. These results indicated that TVX poisoned topoisomerase in RAW cells and the resultant DNA damage led to ATR activation, which was required for the TVX-mediated increase in LPS-induced TNF release. In summary, the phenomenon of the TVX-mediated increase in LPS-induced TNF release in RAW cells in vitro recapitulated observations in vivo. The model in RAW cells was used to identify key signaling mechanisms which could increase LPS-induced plasma TNF in animals and identified a possible TVX-specific target in cells which could explain the IDILI liability associated with TVX in humans.
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