1. Introduction
Cellular senescence is a phenomenon that occurs after cell maturation. Senescent cells have no DNA replication and cannot proceed cell cycle while they are metabolically active [1]. According to various studies, the Reactive Oxygen Species (ROSs) have an important role in the induction of cellular senescence [2]. Acrylamide is an industrial chemical compound that can be found in carbohydrate-rich foods during their preparation at high temperatures [3]. Recently, acrylamide’s genotoxicity, immunotoxicity and carcinogenicity have attracted attentions. Various studies have indicated that oxidative stress is as an important mechanism in acrylamide toxicity [3, 4,  5].
Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is a flavonoid with high radical scavenging activity and antioxidant capacity [6]. These properties are potentially beneficial in protecting the stability of the genome. However, few information is available on the protective effect of Rutin on cellular senescence. Therefore, this study aims to investigate the capability of acrylamide in the induction of oxidative cellular senescence, and examine the anti-oxidative properties of Rutin attenuating cellular senescence in embryonic fibroblast cell line NIH3T3.
2. Methods
In this experimental study, NIH3T3 cells were incubated at 37 ℃ under humid atmosphere containing 5% CO2. After the third passage, the cells were seeded in culture plates and divided into following groups: control (Cells cultured in DMEM medium with 10% FBS, Pen/Strep and vehicle), positive control (cells exposed to 400 μM H2O2), acrylamide (cells treated with acrylamide 5 mM), Rutin + acrylamide (cells treated with 50 µM Rutin plus acrylamide), positive control + Rutin (cells treated with 50 µM Rutin plus H2O2). After cell treatment, MTT assay was used to evaluate the viability of the cells. Furthermore, senescence-associated β-galactosidase was evaluated by β-galactosidase staining kit and quantitative identification of β-galactosidase activity via ELISA kit (MyBioSource, CAT number: MBS703814). To assess the oxidative stress, lipid peroxidation and glutathione concentration were measured in cell suspensions’ homogenates. Malondialdehyde (MDA), as an end product of lipid peroxidation, was calculated by using thiobarbituric acid. Moreover, DTNB reagent was used for measuring glutathione concentration. Finally, the statistical significance was determined using one-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test. The P<0.05 was considered as the significance level [7].
3. Results
Acrylamide and H2O2 significantly decreased cell viability in comparison with the control group (P< 0.001). Furthermore, Rutin increased the acrylamide-treated cells’ viability at concentrations of 10, 25 and 50 µM (P< 0.05). At concentration above 50 µM, no any significant effect was reported. Hence, 50 µM concentration was selected as the most effective concentration of Rutin.
Staining of cells with β-galactosidase at pH=6 showed that acrylamide and H2O2 caused higher level of bluish-green color compared to the control group. On the other hand, the cells treated with Rutin showed a lower level of bluish-green color. Similarly, both acrylamide and H2O2 groups revealed a significant increase in the level of β-galactosidase activity compered to control group evaluated by ELISA kit. Treatment of cells by Rutin significantly decreased β-galactosidase activity in acrylamide-treated cells (P<0.05). Overall, the results showed that Rutin inhibited acrylamide-induced cellular senescence. 
After evaluation of oxidative stress parameters, an increased concentration of MDA was observed in cells treated by acrylamide and H2O2. Moreover, the amount of cellular glutathione in the acrylamide and H2O2 groups had a significant decrease compared to the control group (P<0.05). After treatment by Rutin, the Rutin + acrylamide group (compared to the acrylamide group) and the Rutin + H2O2 group (compared to the H2O2 group) showed a significantly higher concentration of cellular glutathione (P<0.05).
4. Conclusion
Acrylamide can induce cellular senescence via oxidative stress. Rutin, as a potent natural antioxidant, can efficiently decrease acrylamide-induced cellular senescence in NIH3T3 cell lines. Rutin may be effective in reducing the adverse effects of food impurities, such as acrylamide.

Ethical Considerations
Compliance with ethical guidelines
All procedures in this study were confirmed by the Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1397.413). Ethical principles are fully observed in this article. 

Funding
The present study received financial support from the Deputy for Research and Technology of Guilan University of Medical Sciences. 

Authors' contributions
All authors had contribution in preparing this article. Designing the experiments: Ehsan Zamani and Mehdi Evazalipour; Designing the methods: Ehsan Zamani, Reza Jafari-Shakib and Mehdi Evazalipour; Implementing the research: Forough Aghajani Torshkooh, Saye Gholampour and Mehdi Evazalipour; Preparing the initial draft: Ehsan Zamani and Forough Aghajani Torshkooh; Revising and reviewing the draft: and Mehdi Evazalipour and Ehsan Zamani.

Conflicts of interest
There are no conflicts of interest. 

Acknowledgements
This study was extracted from Ms. Sayeh Gholampour's PhD thesis and supported by the research council of Guilan University of Medical Sciences, Rasht, Iran.


References

1. Trougakos IP, Saridaki A, Panayotou G, Gonos ES. Identification of differentially expressed proteins in senescent human embryonic fibroblasts. Mechanisms of Ageing and Development. 2006; 127(1):88-92. [DOI:10.1016/j.mad.2005.08.009] [PMID]
2. Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science. 2005; 309(5733):481-4. [DOI:10.1126/science.1112125] [PMID]
3. Lu T, Finkel T. Free radicals and senescence. Experimental Cell Research. 2008; 314(9):1918-22. [DOI:10.1016/j.yexcr.2008.01.011] [PMID] [PMCID]
4. Friedman M. Chemistry, biochemistry, and safety of acrylamide. A review. Journal of Agricultural and Food Chemistry. 2003; 51(16):4504-26. [DOI:10.1021/jf030204+] [PMID]
5. Cao J, Liu Y, Jia L, Jiang LP, Geng CY, Yao XF, et al. Curcumin attenuates acrylamide-induced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. Journal of Agricultural and Food Chemistry. 2008; 56(24):12059-63. [DOI:10.1021/jf8026827] [PMID]
6. Jiang L, Cao J, An Y, Geng C, Qu S, Jiang L, et al. Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells. Toxicology in Vitro. 2007; 21(8):1486-92. [DOI:10.1016/j.tiv.2007.06.011] [PMID]
7. Jahani M, Shokrzadeh M, Vafaei-Pour Z, Zamani E, Shaki F. Potential role of cerium oxide nanoparticles for attenuation of diabetic nephropathy by inhibition of oxidative damage. Asian Journal of Animal and Veterinary Advances. 2016; 11(4):226-34. [DOI:10.3923/ajava.2016.226.234]                
8. Coccimiglio J, Alipour M, Jiang ZH, Gottardo C, Suntres Z. Antioxidant, antibacterial, and cytotoxic activities of the ethanolic Origanum vulgare extract and its major constituents. Oxidative Medicine and Cellular Longevity. 2016; 2016:1404505. [DOI:10.1155/2016/1404505] [PMID] [PMCID]
9. Shahidi F, Zhong Y. Measurement of antioxidant activity. Journal of Functional Foods. 2015; 18(Pt B):757-81. [DOI:10.1016/j.jff.2015.01.047]
10. Young IS, Woodside JV. Antioxidant in health and disease. Journal of Clinical Patholology. 2001; 54(3):176-86. [DOI:10.1136/jcp.54.3.176] [PMID] [PMCID]
11. Ozsoy N, Candoken E, Akev N. Implications for degenerative disorders: Antioxidative activity, total phenols, flavonoids, ascorbic acid, β-carotene and β-tocopherol in Aloe vera. Oxidative Medicine and Cellular Longevity. 2009; 2:315091. [DOI:10.4161/oxim.2.2.8493] [PMID] [PMCID]
12. Korkmaz A, Kolankaya D. Protective effect of rutin on the ischemia/reperfusion induced damage in rat kidney. Journal of Surgical Research. 2010; 164(2):309-15. [DOI:10.1016/j.jss.2009.03.022] [PMID]
13. Enogieru AB, Haylett W, Hiss DC, Bardien S, Ekpo OE. Rutin as a potent antioxidant: Implications for neurodegenerative disorders. Oxidative Medicine and Cellular Longevity. 2018; 2018:6241017. [DOI:10.1155/2018/6241017] [PMID] [PMCID]
14. Al-Rejaie SS, Aleisa AM, Sayed-Ahmed MM, Al-Shabanah OA, Abuohashish HM, Ahmed MM, et al. Protective effect of rutin on the antioxidant genes expression in hypercholestrolemic male Westar rat. BMC Complementary and Alternative Medicine . 2013; 13:136. [DOI:10.1186/1472-6882-13-136] [PMID] [PMCID]
15. Ma JQ, Liu CM, Yang W. Protective effect of rutin against carbon tetrachloride-induced oxidative stress, inflammation and apoptosis in mouse kidney associated with the ceramide, MAPKs, p53 and calpain activities. Chemico-Biological Interactions. 2018; 286:26-33. [DOI:10.1016/j.cbi.2018.03.003] [PMID]
16. Wei SM, Yan ZZ, Zhou J. Protective effect of rutin on testicular ischemia-reperfusion injury. Journal of Pediatric Surgery. 2011; 46(7):1419-24. [DOI:10.1016/j.jpedsurg.2010.09.044] [PMID]
17. Evazalipour M, Safarzadeh Kozani P, Safarzadeh Kozani P, Shabani S, Rezaei Soufi B, Zamani E. Acrylamide induced oxidative cellular senescence in embryonic fibroblast cell line (NIH3T3): A protection by carvacrol. Jundishapur Journal of Natural Pharmaceutical Products. 2021; 16(4):e109399. [DOI:10.5812/jjnpp.109399]
18. Sahinturk V, Kacar S, Vejselova D, Kutlu HM. Acrylamide exerts its cytotoxicity in NIH/3T3 fibroblast cells by apoptosis. Toxicol and Industrial  Health. 2018; 34(7):481-9. [DOI:10.1177/0748233718769806] [PMID]
19. Magalingam KB, Radhakrishnan A, Haleagrahara N. Protective effects of quercetin glycosides, rutin, and isoquercetrin against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in rat pheochromocytoma (PC-12) cells. International Journal of Immunopathology and Pharmacology. 2016; 29(1):30-9. [DOI:10.1177/0394632015613039] [PMID] [PMCID]
20. Patil SL, Swaroop K, Kakde N, Somashekarappa HM. In vitro protective effect of rutin and quercetin against radiation-induced genetic damage in human lymphocytes. Indian of Journal Nuclear Medicine. 2017; 32(4):289-95. [DOI:10.4103/ijnm.IJNM_30_17] [PMID] [PMCID]
21. Yoon S, Han S, Jeon KJ, Kwon S. Effects of collected road dusts on cell viability, inflammatory response, and oxidative stress in cultured human corneal epithelial cells. Toxicology Letters. 2018; 284:152-60. [DOI:10.1016/j.toxlet.2017.12.012] [PMID]
22. Baeeri M, Mohammadi-Nejad S, Rahimifard M, Navaei-Nigjeh M, Moeini-Nodeh S, Khorasani R, et al. Molecular and biochemical evidence on the protective role of ellagic acid and silybin against oxidative stress-induced cellular aging. Molecular and Cellular Biochemistry. 2018; 441(1):21-33. [DOI:10.1007/s11010-017-3172-0] [PMID]
23. Zamani E, Mohammadbagheri M, Fallah M, Shaki F. Atorvastatin attenuates ethanol-induced hepatotoxicity via antioxidant and anti-inflammatory mechanisms. Research in Pharmaceutical Sciences. 2017; 12(4):315-21. [DOI:10.4103/1735-5362.212049] [PMID] [PMCID]
24. Dorman HD, Surai P, Deans SG. In vitro antioxidant activity of a number of plant essential oils and phytoconstituents. Journal of Essential Oil Research. 2000; 12(2):241-8. [DOI:10.1080/10412905.2000.9699508]
25. Campisi J, Di Fagagna FDA. Cellular senescence: When bad things happen to good cells. Nature Reviews Molecular Cell Biology. 2007; 8(9):729-40. [DOI:10.1038/nrm2233] [PMID]
26. Romano AD, Serviddio G, de Matthaeis A, Bellanti F, Vendemiale G. Oxidative stress and aging. Journal of Nephrology. 2010; 23 Suppl 15:S29-36. [PMID]
27. Viña J, Borrás C, Miquel J. Theories of ageing. IUBMB Life. 2007; 59(4-5):249-54. [DOI:10.1080/15216540601178067] [PMID]
28. van Deursen JM. The role of senescent cells in ageing. Nature. 2014; 509(7501):439-46. [DOI:10.1038/nature13193] [PMID] [PMCID]
29. Debacq-Chainiaux F, Borlon C, Pascal T, Royer V, Eliaers F, Ninane N, et al. Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 signaling pathway. Journal of Cell Science. 2005; 118(4):743-58. [DOI:10.1242/jcs.01651] [PMID]
30. Llana-Ruiz-Cabello M, Gutiérrez-Praena D, Puerto M, Pichardo S, Jos Á, Cameán AM. In vitro pro-oxidant/antioxidant role of carvacrol, thymol and their mixture in the intestinal Caco-2 cell line. Toxicology in Vitro. 2015; 29(4):647-56. [DOI:10.1016/j.tiv.2015.02.006] [PMID]
31. Itahana K, Campisi J, Dimri GP. Mechanisms of cellular senescence in human and mouse cells. Biogerontology. 2004; 5(1):1-10. [DOI:10.1023/B:BGEN.0000017682.96395.10] [PMID]
32. Sellier C, Boulanger E, Maladry F, Tessier FJ, Lorenzi R, Nevière R, et al. Acrylamide induces accelerated endothelial aging in a human cell model. Food and Chemical Toxicology. 2015; 83:140-5. [DOI:10.1016/j.fct.2015.05.021] [PMID]
33. Kim DB, Shin GH, Kim JM, Kim YH, Lee JH, Lee JS, et al. Antioxidant and anti-ageing activities of citrus-based juice mixture. Food Chemistry. 2016; 194:920-7. [DOI:10.1016/j.foodchem.2015.08.094] [PMID]
34. Cui H, Kong Y, Zhang H. Oxidative stress, mitochondrial dysfunction, and aging. Journal of Signal Transduction. 2012; 2012:646354. [DOI:10.1155/2012/646354] [PMID] [PMCID]
35. Liguori I, Russo G, Curcio F, Bulli G, Aran L, Della-Morte D, et al. Oxidative stress, aging, and diseases. Clinical Interventions in Aging. 2018; 13:757-72. [DOI:10.2147/CIA.S158513] [PMID] [PMCID]
36. Leutner S, Eckert A, Müller W. ROS generation, lipid peroxidation and antioxidant enzyme activities in the aging brain. Journal of Neural Transmission. 2001; 108(8-9):955-67. [DOI:10.1007/s007020170015] [PMID]
37. Elblehi SS, El Euony OI, El-Sayed YS. Apoptosis and astrogliosis perturbations and expression of regulatory inflammatory factors and neurotransmitters in acrylamide-induced neurotoxicity under ω3 fatty acids protection in rats. NeuroToxicology. 2020; 76:44-57. [DOI:10.1016/j.neuro.2019.10.004] [PMID]
38. Yang Y, Zhang L, Jiang G, Lei A, Yu Q, Xie J, et al. Evaluation of the protective effects of Ganoderma atrum polysaccharide on acrylamide-induced injury in small intestine tissue of rats. Food & Function. 2019; 10(9):5863-72. [DOI:10.1039/C9FO01452G] [PMID]
39. Sastre J, Pallardó FV, Viña J. Glutathione, oxidative stress and aging. Age. 1996; 19(4):129-39. [DOI:10.1007/BF02434082]
40. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science. 1996; 273(5271):59-63. [DOI:10.1126/science.273.5271.59] [PMID]
41. Cudkowicz M, Sexton P, Ellis T, Hayden D, Gwilt P, Whalen J, et al. The pharmacokinetics and pharmacodynamics of procysteine in amyotrophic lateral sclerosis. Neurology. 1999; 52(7):1492-4. [DOI:10.1212/WNL.52.7.1492] [PMID]
42. Zamani E, Shokrzadeh M, Ziar A, Abedian-Kenari S, Shaki F. Acrylamide attenuated immune tissues’ function via induction of apoptosis and oxidative stress: Protection by L-carnitine. Human & Experimental Toxicology. 2018; 37(8):859-69. [DOI:10.1177/0960327117741753] [PMID]
43. Manouchehrabadi M, Farhadi M, Azizi Z, Torkaman-Boutorabi A. Carvacrol protects against 6-hydroxydopamine-induced neurotoxicity in in vivo and in vitro models of Parkinson’s disease. Neurotoxicity Research. 2020; 37(1):156-70. [DOI:10.1007/s12640-019-00088-w] [PMID]
44. Yang J, Guo J, Yuan J. In vitro antioxidant properties of rutin. LWT-Food Science and Technology. 2008; 41(6):1060-6. [DOI:10.1016/j.lwt.2007.06.010]