REPLY:

Potential conflict of interest: Nothing to report. This work was supported by the National Natural Science Foundation of China (31971188). We thank Jaeschke and Ramachandran for their interest in our study. First, our findings of the phosphorylation of nuclear erythroid 2 p45‐related factor 2 (Nrf2) and diminished mRNAs of NAD(P)H:quinone oxidoreductase 1 (Nqo1), glutathione S‐transferase A3 (Gstα3), glutathione S‐transferase M1 (Gstm1), glutathione S‐transferase M5 (Gstm5), and aldo‐keto reductase 1C (AKR1C) by 6 hours after acetaminophen (APAP) treatment (300 mg/kg) (Supporting Fig. S2B),(1) indicate that the Nrf2/Antioxidant responsive element (ARE) cytoprotective system is impaired from a very early time point. No significant changes in the protein levels were detected at the 6‐hour time point. However, this is not unexpected given that these enzymes are known to have a long half‐life. Although our study only examined the expression of 5 ARE‐driven genes, >200 genes have so far been identified as Nrf2 target genes. As suggested in the editorial by Kaplowitz, Than, and Win,(2) further studies are required to define the roles of Nrf2‐regulated genes in APAP‐induced liver injury. In addition, we disagree with the suggestion that glutathione levels are a functional readout of ARE activation. Buthionine sulfoximine, an inhibitor of glutamate‐cysteine ligase, catalytic subunit, depletes hepatic glutathione to an extent similar to APAP in vivo but is unable to activate Nrf2.(3) Next, in our study, immunohistochemistry analysis showed slightly stronger staining of phosphorylated (P‐)Nrf2 in cells surrounding the necrotic areas in most liver sections from mice treated with APAP, although the contrast of the staining in some sections was not pronounced, possibly because of the low level of P‐Nrf2 protein expression. However, in the same liver sections, we observed substantial staining of Nqo1, Gstα3, Gstm1, Gstm5, and AKR1C in the hepatocytes surrounding necrotic areas, probably because of the strong hepatic expression level of these enzymes, suggesting that the malfunction of the Nrf2/ARE system is zone‐specific. These data will be submitted for publication in the near future. Last, we agree that regulation of mitochondrial oxidant stress by c‐Jun N‐terminal kinase is the critical downstream event in APAP‐induced liver injury.(4) Nrf2 has been recently recognized as a prominent player in mitochondria function. Interestingly, Barzegari et al. (2020) reported that the mitochondria‐targeted antioxidant mito‐TEMPO activates Nrf2.(5) However, the contribution of Nrf2 to the regulation of mitochondrial oxidant stress in APAP‐induced liver injury remains unclear and warrants further study.