Molnupiravir Metabolite--N4 -hydroxycytidine Causes Cytotoxicity and DNA Damage in Mammalian Cells in vitro


  • Pholasit Chamod Biochemistry Division, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathum Thani, Thailand
  • Sutheera Sangsiri Pharmacology Division, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University
  • Krittin Tangjittham Faculty of Medicine, Thammasat University, Pathum Thani, Thailand
  • Pattranit Liu Faculty of Medicine, Thammasat University
  • Pakapon Mongkhonsakunrit Faculty of Medicine, Thammasat University
  • Jessada Pakotiprapha Faculty of Medicine, Thammasat University
  • Tanaboon Rimdusit Faculty of Medicine, Thammasat University
  • Onmanee Prajuabjinda Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University
  • Saovapak Poomirat Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University


N4 -hydroxycytidine, Molnupiravir, Cytotoxicity, DNA amage, Comet assay


N4-hydroxycytidine (NHC) is the active metabolite of molnupiravir—a new drug for COVID-19 treatment. NHC exerts antiviral activity by incorporating into SAR-CoV-2 RNA leading to false base-paring and lethal mutations to the virus. However, the risk of non-specific mutagenesis to host cells has been a concerned. The goal of this study is to detect cytotoxic activity and DNA damage induced by NHC in rapid-growing cells including human keratinocyte (HaCaT), and human adenocarcinomic alveolar basal epithelial (A549) cells in vitro by using sulforhodamine B (SRB) colorimetric and comet assays. NHC induced cytotoxicity in a concentration-dependent manner (0.1-30µM) in HaCaT and A549 cells. Half-maximal inhibitory concentration (IC50) values of NHC were lower in HaCaT compared to A549 cells after 3, 5, 10 days of exposure (4.40±0.09 vs 23.21±3.42, 5.82±0.91 vs 16.35±2.04, and 5.41±0.88 vs 13.83±2.05 µM, respectively), suggesting that the cytotoxic effect of NHC is more potent in HaCaT cells than in A549 cells. Significant increase in DNA damage parameters were observed in comet assay for HaCaT and A549 cells after exposure to NHC. NHC-induced DNA damage in HaCaT cells was concentration-dependent (1-10µM), and time-dependent (3-10 days). NHC-induced DNA damage in A549 cells was concentration-dependent (1-10µM), but not time-dependent (3-10days). Within the limitations of this in vitro study, we conclude that NHC could induce cytotoxic and DNA damage in mammalian cells at therapeutic and supratherapeutic concentrations. We propose caution in the use and supervision of molnupiravir, especially in patients with impaired xenobiotic clearance.


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Angélica JB, Gomes da Silva MM, Musungaie DB, et al. Molnupiravir for Oral Treatment of Covid-19 in Nonhospitalized Patients. New England Journal of Medicine. 2021;386(6):509-520.

Singh AK, Singh A, Singh R, Misra A. An updated practical guideline on use of molnupiravir and comparison with agents having emergency use authorization for treatment of COVID-19. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2022;16(2):102396.

Whitley R. Molnupiravir — A Step toward Orally Bioavailable Therapies for Covid-19. New England Journal of Medicine. 2021;386(6):592-593.

FitzGerald R, Dickinson L, Else L, et al. Pharmacokinetics of ß-d-N4-Hydroxycytidine, the Parent Nucleoside of Prodrug Molnupiravir, in Nonplasma Compartments of Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection. Clinical Infectious Diseases. 2022;75(1):e525-e528.

Kabinger F, Stiller C, Schmitzová J, et al. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nature Structural & Molecular Biology. 2021;28(9):740-746.

Gordon CJ, Tchesnokov EP, Schinazi RF, Götte M. Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template. J Biol Chem. 2021;297(1):100770.

Zhao Y, He G, Huang W. A novel model of molnupiravir against SARS-CoV-2 replication: accumulated RNA mutations to induce error catastrophe. Signal Transduction and Targeted Therapy. 2021;6(1):410.

Annex I: Conditions of use, condition for distribution and patients targeted and conditions for safety monitoring adressed to member states for unauthorised product Lagevrio (molnupiravir). In. Article 5(3). Science Medicines Health: European Medicines Agency; 2021.

Zhou S, Hill CS, Sarkar S, et al. β-d-N4-hydroxycytidine Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells. J Infect Dis. 2021;224(3):415-419.

Githaka JM. Molnupiravir Does Not Induce Mutagenesis in Host Lung Cells during SARS-CoV-2 Treatment. Bioinform Biol Insights. 2022;16:1-4.

Troth S, Butterton J, DeAnda CS, et al. Letter to the Editor in Response to Zhou et al. J Infect Dis. 2021;224(8):1442-1443.

Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A. In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J Ethnopharmacol. 2004;90(1):33-38.

Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82(13):1107-1112.

Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols. 2006;1(3):1112-1116.

Houghton P, Fang R, Techatanawat I, Steventon G, Hylands PJ, Lee CC. The sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived compounds for activities related to reputed anticancer activity. Methods. 2007;42(4):377-387.

Olive PL, Banáth JP. The comet assay: a method to measure DNA damage in individual cells. Nature Protocols. 2006;1(1):23-29.

Liao W, McNutt MA, Zhu W-G. The comet assay: A sensitive method for detecting DNA damage in individual cells. Methods. 2009;48(1):46-53.

Bajpayee M, Kumar A, Dhawan A. The Comet Assay: Assessment of In Vitro and In Vivo DNA Damage. Methods Mol Biol. 2019;2031:237-257.

Lu Y, Liu Y, Yang C. Evaluating In Vitro DNA Damage Using Comet Assay. J Vis Exp. 2017(128).

Petersen AB, Gniadecki R, Vicanova J, Thorn T, Wulf HC. Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes. Journal of Photochemistry and Photobiology B: Biology. 2000;59(1):123-131.

Linn S. DNA damage by iron and hydrogen peroxide in vitro and in vivo. Drug Metab Rev. 1998;30(2):313-326.

Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175(1):184-191.

Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol. 2004;26(3):249-261.

Assessment report: Use of molnupiravir for the treatment of COVID-19. In. Procedure under Article 5(3) of Regulation (EC) No 726/2004. European Medicines Agency: Science Medicines Health; 2022.

Australian product information-Lagevrio (molnupiravir) capsules. In. Australian government: Department of health and aged care; 2022:9.

Painter WP, Holman W, Bush JA, et al. Human Safety, Tolerability, and Pharmacokinetics of Molnupiravir, a Novel Broad-Spectrum Oral Antiviral Agent with Activity against SARS-CoV-2. Antimicrobial Agents and Chemotherapy. 2021;65(5):10.1128/aac.02428-02420.

National Center for Biotechnology Information. PubChem Compound Summary for CID 197020, N(4)-Hydroxycytidine. In. National Library of Medicine: National center for biotechnology infirmation.

Wallace KB, Bjork JA. Molnupiravir; molecular and functional descriptors of mitochondrial safety. Toxicol Appl Pharmacol. 2022;442:116003.

Pupo Correia M, Fernandes S, Filipe P. Cutaneous adverse reactions to the new oral antiviral drugs against SARS-CoV-2. Clin Exp Dermatol. 2022;47(9):1738-1740.

Santi Laurini G, Montanaro N, Motola D. Safety Profile of Molnupiravir in the Treatment of COVID-19: A Descriptive Study Based on FAERS Data. J Clin Med. 2022;12(1).

Fischer WA, 2nd, Eron JJ, Jr., Holman W, et al. A phase 2a clinical trial of molnupiravir in patients with COVID-19 shows accelerated SARS-CoV-2 RNA clearance and elimination of infectious virus. Sci Transl Med. 2022;14(628):eabl7430.

Law MF, Ho R, Law KWT, Cheung CKM. Gastrointestinal and hepatic side effects of potential treatment for COVID-19 and vaccination in patients with chronic liver diseases. World J Hepatol. 2021;13(12):1850-1874.

Speit G, Hartmann A. The comet assay: a sensitive genotoxicity test for the detection of DNA damage. Methods Mol Biol. 2005;291:85-95.

Keeratichamroen S, Lirdprapamongkol K, Svasti J. Mechanism of ECM-induced dormancy and chemoresistance in A549 human lung carcinoma cells. Oncol Rep. 2018;39(4):1765-1774.

Alfarouk KO, Stock CM, Taylor S, et al. Resistance to cancer chemotherapy: failure in drug response from ADME to P-gp. Cancer Cell Int. 2015;15:71.

Li Y-J, Lei Y-H, Yao N, et al. Autophagy and multidrug resistance in cancer. Chinese Journal of Cancer. 2017;36(1):52.

Miranda JA, McKinzie PB, Dobrovolsky VN, Revollo JR. Evaluation of the mutagenic effects of Molnupiravir and N4-hydroxycytidine in bacterial and mammalian cells by HiFi sequencing. Environmental and Molecular Mutagenesis. 2022;63(7):320-328.

Bian DJH, Sabri S, Abdulkarim BS. Interactions between COVID-19 and Lung Cancer: Lessons Learned during the Pandemic. Cancers (Basel). 2022;14(15).

Kobayashi H, Mori Y, Ahmed S, et al. Oxidative DNA Damage by N4-hydroxycytidine, a Metabolite of the SARS-CoV-2 Antiviral Molnupiravir. The Journal of Infectious Diseases. 2022;227(9):1068-1072.




How to Cite

Chamod, P. , Sangsiri, S., Tangjittham, K. , Liu, P. , Mongkhonsakunrit , P. , Pakotiprapha , J. , Rimdusit , T. , Prajuabjinda, O. and Poomirat, S. 2023. Molnupiravir Metabolite--N4 -hydroxycytidine Causes Cytotoxicity and DNA Damage in Mammalian Cells in vitro. Asian Medical Journal and Alternative Medicine. 23, 3 (Dec. 2023), 53–63.



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