DNA Damage Response Plays Critical Role In Liver Regeneration After Acetaminophen Overdose

Abstract

Acetaminophen (APAP) overdose is a leading cause of Acute Liver Failure (ALF). Previous studies have shown that liver regeneration followed by APAP overdose plays a critical role in survival. Here we show that significant DNA double strand break leading to inhibited DNA repair results in prolonged activation of cell cycle checkpoint delaying liver regeneration. We used a novel incremental dose model developed in our laboratory using a regenerating (300 mg/kg, APAP300) and a non‐regenerating (600 mg/kg, APAP600) dose of APAP to identify pathways involved in regeneration after APAP overdose. Marker of liver injury, regeneration and microarray‐based global gene expression changes were studied in male C57BL6 mice over time course of 0 to 72 hr following treatment with either APAP300 or APAP600. The ingenuity pathway analysis of microarray data revealed significant differences in DNA damage, replication and checkpoint related pathways between both doses of APAP. Western blot and immunofluorescence staining of pH2AX Ser139, a hallmark of DNA double strand break, showed prolonged phosphorylation of histone H2AX in mice treated with APAP600 than APAP300. DNA repair proteins involved in Non Homologous End Joining (NHEJ) pathway showed a rapid induction in APAP300 but were significantly suppressed in APAP600, indicating delay or lack of DNA repair. Further, extensive stabilization and activation of p53, the DNA damage Response (DDR) effector protein, was observed in APAP600 as compared to APAP300. p53 stabilization was accompanied by significantly higher and prolonged activation of several p53 target genes such as GADD45α,PAI1, BAX, p21 in APAP600 treated mice as compared to APAP300 treated mice. Furthermore, studies in p53KO mice showed significantly higher injury than WT mice after APAP300 treatment at initial time points. Interestingly, despite the higher injury, p53KO mice recovered similarly as the WT mice due to faster cell cycle resulting in much higher liver regeneration. These data illustrate that lack of prompt DSB repair response after APAP overdose leads to p53‐mediated prolonged growth arrest and replicative senescence. These results suggest that improving DNA repair process to rapidly decrease cell cycle checkpoint alert resulting in faster initiation of liver regeneration following APAP overdose may have therapeutic benefit.Support or Funding InformationThese studies were supported by NIH‐COBRE (P20 RR021940‐03), NIEHS Toxicology Training Grant (T32ES007079‐34) and NIDDK R0198414