Protective effects of nicotinamide mononucleotide on ethanol-induced DNA damage in L02 cells
DI Chunhong1, YIN Jie1, ZHONG Wenying1, ZHANG Yingying2, CAO Yuejia2, TAN Xiaohua2
1. Department of Laboratory Testing, Affiliated Hospital of Hangzhou Normal University, Hangzhou, Zhejiang 310015, China; 2. Hangzhou Normal University, Hangzhou, Zhejiang 311121, China
Abstract：Objective To investigate protective effects of nicotinamide mononucleotide (NMN) on ethanol-induced DNA damage in L02 cells, so as to provide the evidence for adjuvant therapy of NMN on alcoholic liver diseases. Methods L02 cells were pretreated with different concentrations of NMN (0, 1, 2, 4 and 8 mmol/L) for 6 h, and then were exposed to 0.4% ethanol for 12 h. The treated cells were divided into the control group, 0.4% ethanol group and different concentrations of NMN groups. Cell viability was analyzed using trypan blue staining for determining the concentration of NMN as a protective agent. The effects of NMN on ethanol-induced DNA damage in L02 cells were evaluated using immunofluorescence detection and reactive oxygen species (ROS) assay. L02 cells were exposed to 0.4% ethanol for 12 h, cultured in a medium containing a protective concentration of NMN, and divided into PBS group and NMN group. Cell viability was detected at 0, 2, 4, 8, 16 and 32 h, and the effects of NMN on repairing ethanol-induced DNA damage were evaluated by alkaline comet assay. Results The cell viability was lower in 0.4% ethanol group than than in the control group, and was higher in different concentrations of NMN groups than in 0.4% ethanol group (all P<0.05), with no significant difference in the cells viability between 4 mmol/L and higher concentrations of NMN groups and the control group (all P>0.05). Therefore, 4 mmol/L NMN was selected as a protective agent. The cell tail moments, relative immunofluorescence intensities of γH2AX and relative levels of ROS were higher in 0.4% ethanol group than in the control group, and lower in 4 mmol/L and higher concentrations of NMN groups than in 0.4% ethanol group (all P<0.05). The cell viability was increased and the cell tail moment was shortened with the increase of 4 mmol/L NMN intervention time; and the cell viability in 4 h and more of NMN groups were higher, and the cell tail moment were lower than that in PBS group (all P<0.05). Conclusions NMN attenuates DNA damage in a dose-dependent manner and promotes the repair of DNA damage in a time-dependent manner. NMN has a protective effect on ethanol-induced DNA damage in hepatocytes.
[1] HODSKINSON M R,BOLNER A,SATO K,et al.Alcohol-derived DNA crosslinks are repaired by two distinct mechanisms[J].Nature,2020,579(7800):603-608. [2] FORRESTER S J,KIKUCHI D S,HERNANDES M S,et al.Reactive oxygen species in metabolic and inflammatory signaling[J].Circ Res,2018,122(6):877-902. [3] 张真,洪颖,盖雅婷,等.双酚类化合物对BRL 3A肝细胞增殖、氧化应激和致突变作用研究[J].预防医学,2022,34(3):302-306. [4] KANG H,KIM M B,PARK Y K,et al.A mouse model of the regression of alcoholic hepatitis:monitoring the regression of hepatic steatosis,inflammation,oxidative stress,and NAD+ metabolism upon alcohol withdrawal[J/OL].J Nutr Biochem,2022,99[2024-04-12].https://doi.org/10.1016/j.jnutbio.2021.108852. [5] ALVES-FERNANDES D K,JASIULIONIS M G.The role of SIRT1 on DNA damage response and epigenetic alterations in cancer[J].Int J Mol Sci,2019,20(13):1-13. [6] LI M,YU X.The role of poly(ADP-ribosyl)ation in DNA damage response and cancer chemotherapy[J].Oncogene,2015,34(26):3349-3356. [7] YOSHINO J,BAUR J A,IMAI S I.NAD+ intermediates:the biology and therapeutic potential of NMN and NR[J].Cell Metab,2018,27(3):513-528. [8] ZHOU C X,LI Z X,DIAO H L,et al.DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors[J].Mutat Res,2006,604(1/2):8-18. [9] FATHI E,MESBAH-NAMIN S A,VIETOR I,et al.Mesenchymal stem cells cause induction of granulocyte differentiation of rat bone marrow C-kit+ hematopoietic stem cells through JAK3/STAT3,ERK,and PI3K signaling pathways[J].Iran J Basic Med Sci,2022,25(10):1222-1227. [10] 王泽泽,陈晋波,刘君瑶,等.活性氧在邻苯二甲酸单(2-乙基己基)酯所致心肌细胞DNA损伤中的作用[J].毒理学杂志,2022,36(1):10-14. [11] 吴帆,冯玲芳,陈俊斐,等.六价铬诱发rDNA拷贝数变异对不同细胞系DNA损伤反应的影响[J].预防医学,2023,35(5):374-379. [12] OKABE K,YAKU K,TOBE K,et al.Implications of altered NAD metabolism in metabolic disorders[J/OL].J Biomed Sci,2019,26[2024-04-12].https://doi.org/10.1186/s12929-019-0527-8. [13] QIU S T,SHAO S H,ZHANG Y H,et al.Comparison of protective effects of nicotinamide mononucleotide and nicotinamide riboside on DNA damage induced by cisplatin in HeLa cells[J/OL].Biochem Biophys Rep,2024,37[2024-04-12].https://doi.org/10.1016/j.bbrep.2024.101655. [14] JACKSON S P,BARTEK J.The DNA-damage response in human biology and disease[J].Nature,2009,461(7267):1071-1078. [15] HIROHASHI K,OHASHI S,AMANUMA Y,et al.Protective effects of Alda-1,an ALDH2 activator,on alcohol-derived DNA damage in the esophagus of human ALDH2*2(Glu504Lys)knock-in mice[J].Carcinogenesis,2020,41(2):194-202. [16] MIAO Y L,CUI Z K,GAO Q,et al.Nicotinamide mononucleotide supplementation reverses the declining quality of maternally aged oocytes[J/OL].Cell Rep,2020,32(5)[2024-04-12].https://doi.org/10.1016/j.celrep.2020.107987. [17] FANG E F,KASSAHUN H,CROTEAU D L,et al.NAD+ replenishment improves lifespan and healthspan in Ataxia telangiectasia models via mitophagy and DNA repair[J].Cell Metab,2016,24(4):566-581.
