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Acetaminophen Induces DNA Damage and ATM Mediated DNA Damage Repair in Mouse Hepatocytes
Author(s) -
Chu Christopher,
Williams Elaina,
Venter Juliet,
An Suyeon,
Petrescu Anca,
DeMorrow Sharon
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.03466
Subject(s) - dna damage , acetaminophen , oxidative stress , dna repair , comet assay , liver injury , viability assay , programmed cell death , pharmacology , toxicity , chemistry , biology , medicine , dna , cell , apoptosis , biochemistry
Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. While safe at therapeutic doses, it presents a risk for acute liver failure at higher dosages. Accounting for 50% of the cases of liver failure, drug‐induced liver toxicity is the most common cause of liver damage in western societies. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then contributes to the further progression of APAP‐induced liver injury. Methods Mouse hepatocytes (FL86B cells) were treated with APAP (1mM‐100mM) and viability was assessed. FL83B cells were then treated for 24 hours with 5mM APAP, a dose that gave 70% of viability after 24 hr. A DNA comet assay was performed on the treated cells to assay DNA damage. ATM, phospho‐ATM, phospho‐Chk2 and gH2AX were assessed via immunofluorescence. ATM, Chk2, and BP53 were assess via qPCR. To assess the effects that the removal of ATM has on APAP exposure, the FL86B cells were transfected with ATM siRNA for 48 hours prior to 24‐hour exposure of 5mM APAP. ATM and Chk2 were assessed via qPCR and ATM, phospho‐ATM, Chk2 and phospho‐Chk2 were assessed via immunofluorescence. Results APAP exposure to mouse hepatocytes showed a marked increase in the accumulation of DNA damage when compared to untreated hepatocytes. An increase in the markers for DNA double strand breaks (DSB), gH2AX and 53BP, as well the comet assay confirmed the accumulation of DNA damage after exposure to APAP. An increase of ATM and phosphor‐ATM and its downstream effector Chk2 and active form phospho‐Chk2 were increased after exposure to APAP. The removal of ATM attenuated the phosphorylation of ATM and Chk2 and enhanced cell viability with exposure to APAP. Conclusion Exposure to APAP induces DNA damage within hepatocytes. This elevated amount of DNA damage results in the activation of ATM and the DDR through Chk2. ATM‐mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM‐mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP‐induced liver damage.