Inflammation-Enhanced Drug-Induced Liver Injury.

Drug-induced liver injury is a major concern in clinical studies as well as in post-marketing surveillance. Previous evidence suggests that drug exposure during periods of inflammation can increase an individual's susceptibility to toxicity. Inflammation caused by infections or endotoxin markedly activates NADPH oxidase that generates superoxide radicals by transferring electrons from NADPH. In the phagosome, superoxide radicals spontaneously form hydrogen peroxide (H2O2) and other reactive oxygen species. Neutrophils or Kupffer cells also release myeloperoxidase on activation. The aim of this study was to develop and validate an in vitro oxidative stress inflammation model to identify compounds that may increase hepatotoxicity during inflammation. When a non-toxic H2O2 generating system (glucose/glucose oxidase) with peroxidase or Fe(II) (to simulate in vivo inflammation) were added to the freshly isolated rat hepatocytes prior to the addition of the investigated drug which increased drug-induced cytotoxicity and ROS formation. This was reversed by 6-N-propyl-2-thiouracil (a peroxidase inhibitor) or desferoxamine (an Fe(II) chelator), respectively. Flutamide, nilutamide, nimesulide, methotrexate, and 6-mercaptopurine were found to form pro-oxidant radicals leading to oxidative stress and mitochondrial injury whereas azathioprine did not form any radicals with this inflammation system. Electron spin resonance spectrometry spin trapping studies of 6-mercaptopurine metabolism by HRP (horseradish peroxidase)/H2O2 was also investigated. A mixture of two radicals were trapped using DMPO (5,5-dimethyl-1-pyrroline-N-oxide) which were previously reported as purine-6-thiyl and superoxide radicals. This system may provide a more robust in vitro pre-clinical tool for screening of drugs for potential hepatotoxicity associated with inflammation.