Deoxyribonuclease 1 aggravates acetaminophen-induced liver necrosis in male CD-1 mice

Abstract

An overdose of acetaminophen (APAP) (N-acetyl-p-aminophenol) leads to hepatocellular necrosis induced by its metabolite N-acetyl-p-benzoquinone-imine, which is generated during the metabolic phase of liver intoxication. It has been reported that DNA damage occurs during the toxic phase; however, the nucleases responsible for this effect are unknown. In this study, we analyzed the participation of the hepatic endonuclease deoxyribonuclease 1 (DNASE1) during APAP-induced hepatotoxicity by employing a Dnase1 knockout (KO) mouse model. Male CD-1 Dnase1 wild-type (WT) (Dnase1 ؉/؉ ) and KO (Dnase1 ؊/؊ ) mice were treated with 2 different doses of APAP. Hepatic histopathology was performed, and biochemical parameters for APAP metabolism and necrosis were investigated, including depletion of glutathione/glutathione-disulfide (GSH؉GSSG), ␤-nicotinamide adenine dinucleotide (NADH؉NAD ؉ ), and adenosine triphosphate (ATP); release of aminotransferases and Dnase1; and occurrence of DNA fragmentation. As expected, an APAP overdose in WT mice led to massive hepatocellular necrosis characterized by the release of aminotransferases and depletion of hepatocellular GSH؉GSSG, NADH؉NAD ؉ , and ATP. These metabolic events were accompanied by extensive DNA degradation. In contrast, Dnase1 KO mice were considerably less affected. In conclusion, whereas the innermost pericentral hepatocytes of both mouse strains underwent necrosis to the same extent independent of DNA damage, the progression of necrosis to more outwardly located cells was dependent on DNA damage and only occurred in WT mice. Dnase1 aggravates APAP-induced liver necrosis. A cetaminophen, a widely used analgesic, is one of the most commonly overdosed pharmaceuticals. 1 Overdose leads to liver necrosis caused by the electrophilic metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which is generated via oxidation of acetaminophen (APAP) by a microsomal cytochrome P450 containing mixed-function oxidase system. 2 Subsequent detoxication of NAPQI by cellular glutathione leads to depletion of glutathione/glutathione-disulfide (GSHϩGSSG) followed by NAPQI accumulation and induction of necrosis due to protein arylation and severe oxidative stress. 2,3 Nuclear accumulation of Ca 2ϩ and DNA damage have been described to occur during APAPinduced cytotoxicity of murine hepatocytes in vitro and in vivo. 4-6 DNA damage seems to be an important event, because APAP toxicity was prevented by known inhibitors of Ca 2ϩ -dependent endonucleases, including Ca 2ϩ -chelators such as aurintricarboxylic acid, glycol ether diamine tetra-acetic acid, and the Ca 2ϩ -calmodulin antagonist chlorpromazine. 5,7,8 However, the nucleases involved in APAP-induced DNA damage have not yet been identified.Deoxyribonuclease 1 (DNASE1), a Ca 2ϩ /Mg 2ϩ -dependent secreted endonuclease with a pH optimum of approximately 7.5, hydrolyzes double-stranded DNA generating 5Ј-phospho-tri-and/or tetra-oligonucleotides. 9,10 DNASE1 gene expression has been demonstrated in a number of different organs of the urogenital and ...

Caspase activation is associated with spontaneous recovery from acute liver failure

“…31 In models of AAP-induced liver damage, which is a major cause of human ALF, some studies showed apoptosis after drug exposure of mice and mouse hepatocytes, whereas other reports indicated that necrosis is the principal mode of liver damage. 32,33 These observations might be related to our findings. Using two independent methods, we demonstrate for the first time that Predictive discrimination of (A) caspase-generated CK-18 fragments and (B) caspase-3/7 activity as determined by receiver operating characteristics (ROC) plot analysis.…”

“…36 In models of AAP-induced cytotoxicity it was also demonstrated that nonapoptotic calcium-dependent endonucleases, such as DNase-1, mediate TUNEL reactivity and DNA fragmentation. 33 These data clearly suggest that TUNEL reactivity, which is often used to identify apoptosis but also occurs in necrosis, certainly overestimates the role of apoptosis in models of ALF. 26,27 Moreover, ATP depletion-induced necrosis resulting from severe mitochondrial dysfunction is consistent with the high lactate levels that develop in critically ill ALF patients and are associated with poor outcome.…”

“…35 In mouse models, it has been shown that an AAP overdose leads to massive hepatocellular necrosis characterized by the depletion of glutathione, reduced nicotinamide adenine dinucleotide, and ATP. 32,33 When ATP depletion was prevented, however, necrosis was blocked, whereas caspase-dependent apoptosis increased. 36 In models of AAP-induced cytotoxicity it was also demonstrated that nonapoptotic calcium-dependent endonucleases, such as DNase-1, mediate TUNEL reactivity and DNA fragmentation.…”

Regulation of Apoptotic Endonucleases by EndoG

“…However, in response to stress, endonucleases become activated and are released from cellular compartments to reach nuclear DNA, thus completely destroying it within a few hours after cell injury or cell death. Therefore, inactivation of endonucleases before cell/tissue injury is usually cytoprotective (Basnakian et al, 2005;Napirei et al, 2006;Apostolov et al, 2007Apostolov et al, , 2009Apostolov et al, , 2011.…”

Cytotoxic Endonucleases: New Targets for Prostate Cancer Chemotherapy

“…On the other hand, their participation in cell death in general after tissue injury seems crucial and evidence of this is overwhelming. Recent studies demonstrated that inactivation of the endonucleases causes protection of normal and cancer cells against a variety of injuries in vitro and in vivo Basnakian et al, 2005;Napirei et al, 2006;Yin et al, 2007), suggesting that the endonucleases are essential for and mechanistically linked to injuryrelated cell death. In addition to causing cell death itself, the endonuclease are certainly essential for clean up after cell death, removal of DNA from blood plasma, and destroying "foreign" DNA from bacteria and viruses consumed by cells (Buzder et al, 2009).…”