Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA(-/-)) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA(-/-)) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA(-/-) than in MsrA I hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA(-/-) than in MsrA(-/-) hepatocytes, while TXNRD1 depletion in both MsrA(-/-) and MsrA(-/-) cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA hepatocytes, while no significant change was observed in MsrA(-/-) cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRDI expression levels in APAP-treated MsrA(-/-) hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRDI expression. (C) 2017 Elsevier Inc. All rights reserved.