Reactive oxygen species-mediated cytotoxic and DNA-damaging mechanism of <i>N</i> ⁴ -hydroxycytidine, a metabolite of the COVID-19 therapeutic drug molnupiravir

Molnupiravir is a prodrug of the antiviral ribonucleoside analogue <i>N</i>⁴-hydroxycytidine (NHC), for use in the treatment of coronavirus disease 2019 (COVID-19). However, it is generally considered that NHC-triphosphate is incorporated into the host genome to induce mutations. In our previous preliminary report, we proposed oxidative DNA damage by NHC <i>via</i> cytidine deaminase (CDA)-mediated ROS formation. In the present study, we investigated cell viability using the HL-60 human leukemia cell line and its H₂O₂-resistant clone, HP100 cells. The survival rate was significantly reduced in HL-60 cells treated with NHC, but not in HP100 cells. LC-MS analysis revealed that uridine formation occurred from CDA-treated NHC, suggesting that CDA metabolizes NHC to uridine and hydroxylamine. We clarified mechanisms of CDA-mediated reactive oxygen species (ROS) generation and DNA damage by NHC using isolated DNA. CDA-treated NHC induced DNA damage in the presence of Cu(II). The DNA damage was enhanced by NADH addition and piperidine treatment. CDA-treated NHC and Cu(II) caused piperidine-labile sites at thymine, cytosine, and guanine, and the DNA cleavage pattern was similar to that of hydroxylamine. Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H₂O₂ and Cu(I). An indicator of oxidative DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanosine formation by CDA-treated NHC, was lower under hypoxic conditions than under normal conditions. Therefore, hydroxylamine, possibly produced from NHC treated with CDA, could induce metal-dependent H₂O₂ generation during the redox reactions, suggesting that oxidative DNA damage induced by ROS plays an important role in molnupiravir-related cytotoxicity and mutagenicity.