To define reasons for the characteristic observation of active DNA synthesis but not cell division in residual hepatocytes in liver explants after ALF, we studied the effects of APAP on HuH-7 cells, mouse hepatocytes and intact mice. C57BL/6 mice were given LD50 dose of APAP i.p to induce ALF. Liver injury was characterized by encephalopathy,

liver test abnormalities, hepatic inflammation and perivenous necrosis, and mortality. Culture of HuH-7 cells or mouse hepatocytes with APAP in IC50 concentrations caused cytotoxicity as confirmed by MTT assays. Gene expression arrays from APAP-treated cells or mice showed disturbances in ATM signaling pathway and western blot of tissue and cell lysates confirmed ATM-related DNA damage responses (DDR), including pATM, pATR, pH2AX, pChek1 and pChek2 expression. ACP-196 order This DDR in the setting of ATM dysregulation was verified by Comet

assays with extensive double-strand DNA breaks. To evaluate greatest susceptibility of cell subpopulations to APAP, we analyzed HuH-7 cells by FACS, and found cells in S or G2/M were lost within 4 h, whereas cells in G0/G1 survived over long-term. This was confirmed when HuH-7 cells synchronized by hydroxyurea in late S were rapidly destroyed by APAP. By contrast, G0/G1 cells exposed to APAP stopped proliferating and failed to enter cell cycle, CYC202 despite removal of APAP from culture medium. These cells in G0/G1 displayed significant DNA damage, as indicated by gene expression arrays, pH2AX staining and Comet assays. Next, to determine whether APAP-induced arrest 上海皓元医药股份有限公司 of cell cycle could be reversed by G-CSF, which was previously found to improve outcomes in ALF, we performed further studies. Remarkably, after G-CSF treatment, HuH-7 cells exposed to APAP regained the ability to overcome

G0/G1 arrest and entered the cell cycle. Similarly, mice treated with G-CSF after induction of APAP toxicity showed improved survival and superior liver regeneration, with greater Ki67 expression compared with mice receiving APAP alone. This improvement correlated with less pH2AX staining and comet formation, indicating decreased DNA damage in G-CSFtreated animals. Conclusions: Actively cycling cells in S or G2/M were highly susceptible to APAP toxicity. By contrast, G0/G1 cells survived APAP-induced DNA damage but were prevented from cycling. The inability to reenter cell cycle will help explain failure of residual hepatocytes to regenerate liver in APAP-induced ALF. This molecular process should offer further new directions for therapeutic development in ALF. Disclosures: The following people have nothing to disclose: Preeti Viswanathan, Sriram Bandi, Sanjeev Gupta Background: Liver enlargement, due to accumulation of lipids and proteins in hepatocytes is common in heavy drinkers.