Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

Abstract

Background & Aims Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. Methods Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and HPLC-ED analysis. Results We found that APAP-AD were detected at 1 hr, peaked at approximately 2 hrs, declined at 6 hrs and almost full removed at 24 hrs post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were co-localized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. Conclusions Our data indicate that APAP-AD are removed through selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

<b>3-methyadenine attenuates chloroform-induced hepatotoxicity via autophagy </b><b>activation </b>

“…Under some conditions autophagy exerts protective effects to promote cell survival, while under some other conditions autophagy is detrimental to the cell (24,28,33). Previous reports showed that in hepatotoxic models induced by acetaminophen and ethanol, induction of autophagy was protective and inhibition of autophagy was detrimental (5,20,26,27). Consistent with these reports, in our study we found that autophagy enhancement by 3-MA pro- subsequent production of pro-inflammatory cytokine, IL-1β.…”

Differential susceptibility to acetaminophen-induced liver injury in sub-strains of C57BL/6 mice: 6N versus 6J

“…Both damaged mitochondria (Ni et al, 2012a) and protein adducts (Ni et al, 2016) were shown to be removed by autophagy, an adaptive mechanism that protects against APAP hepatotoxicity. The consistently lower adducts in C57BL/6J mice could be caused by the reduced formation of adducts compared to C57BL/6N mice or could be the result of higher autophagic clearance.…”

Liver‐specific deficiency of unc‐51 like kinase 1 and 2 protects mice from acetaminophen‐induced liver injury

“…Autophagy has been thought to play a protective role against APAP hepatotoxicity through the removal of damaged organelles and alleviation of oxidative stress, and pharmaceutical inhibition of autophagy by leupeptin or chloroquine would render hepatocytes more sensitive to APAP‐induced necrotic cell death . Consistent with these findings, depletion of autophagy‐related gene Atg7 or SQSTM1/p62 in the liver strongly exaggerates AILI . However, probably attributed to the multiple functions of different autophagy‐related genes besides their roles in autophagy, liver‐specific knockout of Atg5 induced a paradoxically reduced liver injury during APAP treatment, which may be caused from a persistent activation of the Nrf2 antioxidant signaling pathway and increased hepatocyte proliferation during Atg5 loss …”