Molnupiravir Metabolite--N⁴-Hydroxycytidine Causes DNA Damage in Hepatoblastoma Cells and Lung Large Cell Carcinoma Cellsin Vitro

Abstract ID 93586 Poster Board 210 N⁴-hydroxycytidine (NHC) is an active metabolite of molnupiravir, a novel antiviral ribonucleoside analog approved for the treatment of SARS-CoV-2 infection. Molnupiravir exerts antiviral activity by converting to NHC monophosphate and incorporating into viral RNA, causing a lethal mutation known as "error catastrophe" in virus. While the majority of studies suggest that NHC is unlikely to cause mutation in host cells, some conflicting data have been revealed. The study aimed to determine whether NHC causes DNA damage in human cells, including hepatoblastoma (HepG2) cells and lung large cell carcinoma (COR-L23) cells. Single-cell DNA break was detected by using the gel electrophoresis COMET assay. The fragmented DNA released out of the nuclei during electrophoresis was quantified as comet tail length and %DNA in tail. HepG2 cells treated with 20mM hydrogen peroxide showed increases in tail length and %DNA in tail compared to the control, indicating that these two parameters were suitable for quantification of DNA damage. The results showed that NHC exposure causes differential DNA damage to HepG2 and COR-L23 cells. NHC increased tail length and %DNA in tail to HepG2 cells in concentration-dependent (1–10 μM) and time-dependent (3–10 days) manners (two-way ANOVA, pTime < 0.0001; pConcentration < 0.0001 for tail length and %DNA in tail, respectively). On the contrary, NHC-induced DNA damage to COR-L23 cells is in concentration-dependent (1-10 μM), but not in time-dependent (3-10 days of exposure) manners (two-way ANOVA, pConcentration < 0.0001, and < 0.0001; pTime = 0.1557, and 0.3478; for tail length and %DNA in tail, respectively). In summary, NHC, an active metabolite of molnupiravir, causes DNA breaks in human hepatoblastoma cells and lung large cell carcinoma cells in vitro. The NHC-induced DNA damage effect of the two cancer cell lines including COR-L23 and HepG2 cells was compared to that effect in a normal cell line, human keratinocyte (HaCaT) cells, in the previously published work. The data suggested that the HaCaT cell line was relatively more sensitive to NHC-induced DNA damage compared to cancer cells. With the limitations of the in-vitro study, It has been suggested that molnupiravir treatment may produce differential therapeutic and toxicity responses in patients with and without cancer-underlying disease. The project is supported by Faculty of Medicine, Thammasat university. Grant agreement number ทป.2-36/2565 (to SS).