Investigating the role of oxidative stress and oxidative signaling in drug-induced toxicity

The first aim of this dissertation was to investigate the effects of APAP-induced electrophilic stress on the two major redox nodes, thioredoxin and glutathione. Experiments were designed to monitor changes to either node following hepatotoxic doses of APAP in comparison to known indicators of oxidative stress and liver injury. Our results demonstrate that inhibition of thioredoxin reductase, the upstream regulator of the thioredoxin system, precedes histological changes associated with APAP toxicity. In addition, we identified N-acetyl-p-benzoquinone imine (NAPQI), the APAP reactive metabolite, as a potent inhibitor of thioredoxin reductase (TrxR), resulting in a pro-oxidant gain of function. Importantly, these findings were not observed with AMAP or its metabolism to reactive intermediates, suggesting a selective mechanistic link between electrophilic stress and downstream signaling triggered by some molecules such as APAP in drug-induced toxicity.;The second aim sought to characterize the effects of APAP-induced stress on redox signaling proteins that are dependent on maintenance of the thioredoxin system's redox equilibrium. We found evidence of oxidative stress occurring soon after exposure to APAP and affecting the oxidation status of peroxiredoxin, a redox system that ultimately depends on functional TrxR and thioredoxin for regeneration. Peroxiredoxin over-oxidation combined with an observed loss of thioredoxin disulfide reducing activity is suggestive of a significant role for the thioredoxin system in propagating APAP-induced oxidative stress and toxicity. Moreover, our findings suggest that mitochondrial thioredoxin oxidation and glutathionylation may contribute to the initiation of toxicity.;The third aim sought to identify oxidation-sensitive proteins that are affected by APAP-induced oxidative stress in vivo. Using a hepatotoxic dose of APAP, we were able to identify several proteins whose oxidative status changed in a time-dependent manner. Interestingly, many of these proteins have been identified previously as targets of APAP covalent binding, following biotransformation to its reactive intermediate NAPQI. These preliminary findings can serve to expand on our current understanding of redox sensitive proteins and their roles in drug-induced oxidative stress.;Together, these studies introduce a novel perspective from which we can elucidate mechanisms of drug-induced toxicity. The observations presented here demonstrate that redox signaling systems are not only involved in coordinating the cellular response to oxidative stress, but are themselves capable of initiating oxidative stress in response to electrophilic insult. These important findings suggest a crucial role for redox signaling in mechanisms of drug toxicity and further characterization will demonstrate their involvement with other toxicants. (Abstract shortened by UMI.)...