ROLE OF DNA DAMAGE RESPONSE IN LIVER REGENERATION AFTER APAP OVERDOSE INDUCED ALF

Abstract

Acetaminophen (APAP) overdose is a leading cause of acute liver failure (ALF) with limited treatment options. The mechanisms of APAP-induced liver injury include formation of a reactive metabolite N-acetyl-p-quinoneimine (NAPQI) and it’s covalent binding to protein, oxidative stress, mitochondrial damage and subsequent nuclear DNA damage leading to necrosis. The replacement of necrotic cells and restoration of liver function occurs through liver regeneration. Although several studies have shown that liver regeneration plays a crucial role in the final outcome of APAP-induced ALF patients, the mechanisms are not entirely known. DNA damage can activate the DNA damage response (DDR) – a signaling cascade that senses the damage and co-ordinates cell cycle progression with DNA repair and other cellular processes. Although APAP toxicity involves severe nuclear DNA damage, role of DDR in regulation of liver regeneration after APAP induced acute liver injury ALI has not been investigated. My hypothesis is that the DDR and p53- a DDR effector protein which induces cell cycle arrest, are activated after APAP overdose and connect liver injury response to initiation of liver regeneration. We studied DDR using incremental dose model with two different doses of APAP in mice [300 mg/kg (APAP300)-a regenerative dose and 600 mg/kg i.p. (APAP600)-a non-regenerative dose]. We began by analyzing microarray data obtained from the incremental dose model using the Ingenuity Pathway Analysis program. This analysis revealed significant differences in DNA damage, replication and checkpoint related pathways in regenerative and non-regenerative doses of APAP. We found that APAP overdose causes DNA Double Strand Break (DSB) in both groups of animals and it is sustained in mice treated with APAP600 relative to mice treated with APAP300. We also observed a subsequent increase in DNA repair proteins in APAP300 treated mice but not in APAP600 treated mice. The DNA repair pathway was significantly suppressed and p53 activation was significantly higher in mice treated with APAP600 as compared to APAP300. These data illustrate that delayed DSB repair response occurs after APAP overdose leading to prolonged growth arrest and may be a crucial mechanism involved in inhibition of liver regeneration. Next, we investigated roles of p53 in detail using WT and p53KO mice (C57BL/6J background) following APAP300 treatment. Remarkably, deletion of p53 resulted in a 3-fold higher liver injury when compared to WT mice implying a protective role for p53 in injury progression. Deletion of p53 did not affect APAP bioactivation however it delayed clearance of APAP protein adducts from liver. Intriguingly, despite higher injury p53KO mice recovered similarly as the WT mice due to faster liver regeneration. Global transcriptomic and molecular analysis revealed several mechanisms triggering higher progression of liver injury yet faster regeneration in p53KO animals after APAP300 treatment. Impaired metabolic homeostasis, GSH transsulfuration and reduced expression of mitochondrial complexes in p53KO mice resulted in higher liver injury. However, increased inflammatory signaling and proliferative signaling through AKT, ERK and mTOR pathways improved recovery in p53KO mice despite very high injury. These studies show that p53 plays a pleotropic role after APAP overdose where it prevents progression of liver injury by maintaining mitochondrial and metabolic homeostasis and also regulates initiation of liver regeneration through inflammatory and proliferative signaling. Overall, my studies comprehensively investigated roles of DDR and p53 in liver injury and regeneration after APAP overdose. These studies have uncovered novel mechanisms that connect cellular injury to initiation of liver regeneration after APAP-induced ALF, which will be useful for future therapeutic development.