Following in vivo APAP, LDK378 research buy a rapid decline in P-c-Src (not total Src) was observed in mitochondria but not cytoplasm. Knockdown of Sab with adeno-shSab prevented inactivation of mitochondrial c-Src after APAP in vivo. Next we found that DOK-4, Src binder (J Biol Chem. 280, 26383-96,2005), was expressed exclusively in mitochondria of hepatocytes. Liver mitochondria isolated after DOK-4 knockdown were resistant to direct P-JNK/ATP mediated inhibition of OCR. Knockdown of DOK-4 in vivo (adeno-shDOK-4) prevented de-phosphorylation
of active Src and markedly protected against APAP toxicity. Conclusions: The interaction of JNK with Sab leads to inactivation of intramitochondrial c-Src and impairment of respiration. The dephosphorylation of c-Src is dependent on DOK-4, a platform for binding of Src and Ptp. Knockdown of mitochondrial DOK-4 markedly protects against APAP hepatotoxicity. Therefore, dysregulation of intramitochon-drial c-Src kinase mediates the Sab dependent effects of JNK leading to impaired mitochondrial function in APAP toxicity. Disclosures: Neil Kaplowitz – Consulting: GlaxoSmithKline,
JNJ, Merck, Novartis, Hepregen, Takeda, Otsuka, Pfizer, Geron, Daiichi-Sanyo; Independent Contractor: Acetaminophen Litigation The following people have nothing to disclose: Sanda Win, Sorafenib mouse Tin A. Than Mechanisms of drug-induced liver injury (DILI) are incompletely understood. Hepatotoxicity from acetaminophen (APAP) poses widespread problems, including acute liver failure (ALF), and requires more therapeutic development. Although the Clostridium perfringens alpha toxin cell type-specific approach has typically been utilized for cytotoxic mechanisms, recent studies identified sharing of toxicity pathways and processes in a cell-agnostic manner. For instance, expression of N-methyl-D-aspartate receptors (NMDARs) is classically associated with excitoxic injury in neuronal tissues, e.g.,
ischemic or traumatic insults, Alzheimer’s, Parkinson’s, schizophrenia, etc., but rodent and human hepatocytes also expressed NMDAR activity after liver anoxia or injury. To determine whether NMDARs could contribute in APAP-induced hep-atotoxicity, we performed studies in cultured HuH-7 cells and primary mouse hepatocytes with or without NMDA and APAP, as well as NMDAR antgonists, MK801 or memantine. MTT assays were performed to assess cytotoxicity. Calcium fluxes were measured in hepatocytes with NMDA and NMDAR block-ers. Brain and liver tissues were examined for multiple NMDARs by RT-PCR, western, and immunostaining. C57BL/6 mice were used for studies with 500 mg/kg APAP with or without 30 mg/ kg memantine. Liver injury was evaluated by histology. We found HuH-7 cells and mouse hepatocytes expressed NMDARs and were sensitive to APAP-induced cytotoxicity, which was abolished by MK801 or memantine. In mouse hepatocytes, NMDA or quinolinic acid, another agonist of NMDAR, dose-de-pendently induced calcium fluxes, APAP alone did not directly stimulate calcium fluxes related to NMDARs.