The idiosyncratic nature, severity and poor diagnosis of drug-induced liver injury (DILI) make these reactions a major safety concern during drug development, as well as the most common cause for the withdrawal of drugs from the pharmaceutical market. Evidence suggests that aside from drug-induced direct damage to hepatocytes, an inflammatory innate immune response is triggered, which leads to the exacerbation and progression of liver injury. However, the role of damaged hepatocytes in the induction of inflammatory responses has not been investigated. We hypothesize that drug (more often reactive metabolite)-induced injury to hepatocytes causes the release of damage-associated molecular pattern (DAMP) molecules, which contribute to the progression of liver injury through pro-inflammatory activation of hepatic macrophages (Kupffer cells, KC). Acetaminophen (APAP)-induced liver injury in mice will be used as a model, and the specific aims of the proposed studies will be to determine i) whether APAP-stimulated hepatocytes release DAMP molecules that can activate hepatic KC, ii) - iv) whether HMGB-1, HSP-70, and uric acid represent key DAMP molecules released by APAP-stimulated hepatocytes. Mouse primiary hepatocytes will be isolated and treated with various concentrations of APAP in vitro. After various time points, the conditioned medium will be collected and used to stimulate KC, which will be isolated from mouse liver and purified by FACSorting. Activation of KC by the soluble factors released into the conditioned medium will be determined by measuring mRNA and protein levels of various pro- inflammatory cytokines and chemokines. The release of HMGB-1, HSP-70, and uric acid by APAP- challenged hepatocytes in vitro and in vivo will be determined by ELISA and immunohistochemistry. Pro- inflammatory activation of KC by HMGB-1, HSP-70 and uric acid will be investigated using recombinant proteins and cystal uric acid in vitro. Further, the roles of HMGB-1, HSP-70, and uric acid in causing the progression of APAP-induced liver damage through stimulation of KC will be determined using neutralizing anti-HMGB-1 and anti-HSP-70 antibodies, as well as allopurinol and uricase. The findings of the proposed research will contribute to our long-term objective of understanding the molecular and cellular mechanisms of DILI, and developing strategies to prevent these reactions and screen for drug candidates'potential of causing DILI.
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|Martin-Murphy, Brittany V; Holt, Michael P; Ju, Cynthia (2010) The role of damage associated molecular pattern molecules in acetaminophen-induced liver injury in mice. Toxicol Lett 192:387-94|