Reactive oxygen species-mediated cytotoxic and DNA-damaging mechanism of -hydroxycytidine, a metabolite of the COVID-19 therapeutic drug molnupiravir.

Molnupiravir is a prodrug of the antiviral ribonucleoside analogue -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 cytidine deaminase (CDA)-mediated ROS formation. In the present study, we investigated cell viability using the HL-60 human leukemia cell line and its HO-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 HO 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 HO generation during the redox reactions, suggesting that oxidative DNA damage induced by ROS plays an important role in molnupiravir-related cytotoxicity and mutagenicity.