Oxidative DNA Damage by N4-hydroxycytidine, a Metabolite of the SARS-CoV-2 Antiviral Molnupiravir

Abstract

Molnupiravir is an antiviral agent recently used for treating coronavirus disease 2019 (COVID-19). Here, we demonstrate that N4-hydroxycytidine (NHC), a molnupiravir metabolite, treated with cytidine deaminase (CDA) induced Cu(II)-mediated oxidative DNA damage in isolated DNA. A colorimetric assay revealed hydroxylamine generation from CDA-treated NHC. The site specificity of DNA damage also suggested involvement of hydroxylamine in the damage. Furthermore, Cu(I) and H2O2 play an important role in the DNA damage. We propose oxidative DNA damage via CDA-mediated metabolism as a possible mutagenic mechanism of NHC, highlighting the need for careful risk assessment of molnupiravir use in therapies for viral diseases, including COVID-19.

Keywords: N 4-hydroxycytidine; 8-dihydro-2′-deoxyguanosine; 8-oxo-7; DNA damage; cytidine deaminase; hydroxylamine; molnupiravir; reactive oxygen species.

Figure 1.

Damage to isolated DNA and the generation of hydroxylamine induced by NHC treated with CDA. A, Formation of 8-oxodG in calf thymus DNA by NHC treated with or without CDA in the presence of Cu(II). Reaction mixtures contained 100 µM/base calf thymus DNA, 100 µM CuCl₂, and the indicated concentrations of NHC with or without 16 hours CDA treatment at 37°C in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. Reaction mixtures were incubated at 37°C for 1 hour. After ethanol precipitation, the DNA was digested to nucleosides with nuclease P₁ and calf intestine phosphatase, then analyzed with an HPLC-ECD system. B, Generation of hydroxylamine from NHC treated with or without CDA. Reaction mixtures contained the indicated concentrations of NHC with or without 16 hours of CDA treatment at 37°C in 400 µL of 2.5% acetic acid. Hydroxylamine was oxidized to nitrite by iodine. The nitrite was measured by a spectrophotometric assay using N-(1-naphthyl)ethylenediamine as the color former. The absorbance was measured at 540 nm. C, Autoradiogram of ³²P-5′-end-labelled DNA fragments incubated with CDA-treated NHC in the presence of Cu(II). Reaction mixtures contained the ³²P-5′-end-labelled 309-bp fragment, 20 µM/base calf thymus DNA, 100 µM CuCl₂, and the indicated concentrations of NHC with 16 hours CDA treatment at 37°C in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37°C for 1 hour, the DNA fragments were treated with or without piperidine and then electrophoresed on a polyacrylamide gel. Presented data are the mean ± SD. *P < .05 versus 0 µM. Statistical significance was analyzed using a Student t test. Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; CDA, cytidine deaminase; DTPA, diethylenetriamine-N, N, N′, N′′, N′′-pentaacetic acid; HPLC-ECD, high performance liquid chromatograph equipped with an electrochemical detector; NHC, N⁴-hydroxycytidine.

Figure 2.

A possible mechanism for the oxidative DNA damage induced by NHC. A, Effects of ROS scavengers and a Cu(I) chelator on the DNA damage induced by CDA-treated NHC. Reaction mixtures contained the ³²P-5′-end-labelled 309-bp fragment, 20 µM/base calf thymus DNA, 100 µM CuCl₂, each scavenger or bathocuproine, and 50 µM NHC with 16 hours CDA treatment at 37°C in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37°C for 1 hour, the DNA fragments were treated with piperidine and then electrophoresed on a polyacrylamide gel. The concentrations of each scavenger and bathocuproine were 0.2 M ethanol, 0.1 M mannitol, 0.1 M sodium formate, 0.1 M methional, 50 U catalase, 250 µM bathocuproine, and 50 U SOD. B, Site specificity of the DNA damage induced by CDA-treated NHC or hydroxylamine. Reaction mixtures contained the ³²P-5′-end-labelled 309-bp fragment, 20 µM/base calf thymus DNA, 100 µM CuCl₂, and 150 µM NHC with 16 hours CDA treatment at 37°C (upper) or 20 µM hydroxylamine (lower) in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37°C for 1 hour, the DNA fragments were treated with piperidine and then electrophoresed on a polyacrylamide gel. C, Oxidative DNA damage resulted from CDA-mediated NHC metabolism and Cu(I)-hydroperoxo complex generation. Abbreviations: CDA, cytidine deaminase; DTPA, diethylenetriamine-N, N, N′, N′′, N′′-pentaacetic acid; NHC, N⁴-hydroxycytidine; ROS, reactive oxygen species; SOD, superoxide dismutase.