Evaluation of Drug-Induced Liver Injury in an In Vitro Oxidative Stress Inflammation System

Drug-induced liver injury (DILI) is a major concern in clinical studies as well as in post-marketing surveillance of drugs. Previous evidence suggests that drug exposure during periods of inflammation can increase an individual's susceptibility to toxicity. Inflammation caused by infections or endotoxins markedly activates NADPH (nicotinamide adenine dinucleotide phosphate) 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 (ROS). 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. Toxic pathways were also investigated using the "Accelerated Cytotoxicity Mechanism Screening" techniques. When a non-toxic H2O2 generating system (glucose/glucose oxidase) with peroxidase or Iron(II) [Fe(II)] (to simulate in vivo inflammation) was added to freshly isolated rat hepatocytes prior to the addition of the investigated drug, drug-induced cytotoxicity and ROS formation were increased. 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 (ESR) spectrometry spin trapping studies of 6-mercaptopurine metabolism by HRP (horseradish peroxidase)/H2O2 was also investigated. 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 oxidative stress inflammation system mimics the respiratory burst (release of H2O2) and release of myeloperoxidase by neutrophils or other immune cells (using horseradish peroxidase) or mimics the Fenton reaction, which could occur in vivo as immune cells also release Fe(II) during inflammation. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimetic, and several antioxidants, including DPPD (N,N'-diphenyl-p-phenylenediamine), mesna (2-mercaptoethanesulfonate), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and resveratrol (3,5,4'-trihydroxy-trans-stilbene) were also tested to determine their effect on drug-induced oxidative stress with or without the inflammation system. This system may provide a more robust in vitro pre-clinical tool for screening of drugs for potential hepatotoxicity associated with inflammation.Ph.D