Investigation of cyto-genotoxic effects of a food sweetener Acesulfame potassium

Abstract

Acesulfame potassium (ACE-K) is an artificial sweetener widely used in many foods. This investigation assessed the cytotoxic effect of ACE-K using MTT assay in human hepatocellular carcinoma (HepG2) cell line and the genotoxic effect using chromosomal aberrations (CAs), micronucleus (MN), and comet assays in human lymphocytes. 7.5-240 μg/mL concentrations of ACE-K were applied to cells. ACE-K notably decreased the cell viability on HepG2 cells, especially at 120 and 240 μg/mL at 24 and 48 h. It also significantly reduced the mitotic index (MI) at 60, 120, and 240 μg/mL at both treatments (24 and 48 h) in human lymphocytes. The frequency of the CAs significantly increased at 60, 120, and 240 μg/mL for 48 h treatment compared to control. However, no difference was observed in the frequency of MN and nuclear division index (NDI) at all the treatments. ACE-K also induced comet tail length, tail intensity, and moment at 15 μg/mL in isolated human lymphocytes. Therefore, ACE-K showed a cytotoxic effect in HepG2 cells as well as human lymphocytes at higher concentrations. It also exhibits a mild genotoxic effect by increasing the frequency of CAs at long-term treatment and DNA damaging effect only at 15 μg/mL.

Keywords

• Acesulfame potassium
• Food sweetener
• MTT assay
• Chromosomal aberrations
• Micronucleus
• Comet assay

References

1. Ashok, I., Poornima, P.S., Wankhar, D., Ravindran, R., Sheeladevi, R. (2017). Oxidative stress evoked damages on rat sperm and attenuated antioxidant status on consumption of aspartame. International Journal of Impotence Research, 29(4), 164-170. https://doi.org/10.1038/ijir.2017.17
2. Bach, D-H., Zhang, W., Sood, A.K. (2019). Chromosomal Instability in Tumor Initiation and Development. Cancer Research, 79(16), 3995–4002. https://doi.org/10.1158/0008-5472.CAN-18-3235
3. Bandyopadhyay, A., Ghoshal, S., Mukherjee, A. (2008). Genotoxicity testing of low-calorie sweeteners: aspartame, acesulfame-K, and saccharin. Drug and Chemical Toxicology, 31(4), 447-457. https://doi.org/10.1080/01480540802390270
4. Belton, K., Schaefer, E., Guiney, P.D. (2020). A review of the environmental fate and effects of acesulfame‐potassium. Integrated Environmental Assessment and Management, 16(4), 421-437. https://doi.org/10.1002/ieam.4248
5. Cao, Y., Liu, H., Qin, N., Ren, X., Zhu, B., Xiaodong, X. (2020). Impact of food additives on the composition and function of gut microbiota: A review. Trends in Food Science & Technology, 99, 295-310. https://doi.org/10.1016/j.tifs.2020.03.006
6. Carocho, M., Morales, P., Ferreira, I.C. (2017). Sweeteners as food additives in the XXI century: A review of what is known, and what is to come. Food and Chemical Toxicology, 107, 302-317. https://doi.org/10.1016/j.fct.2017.06.046
7. Chappell, G.A., Wikoff, D.S., Doepker, C.L., Borghoff, S.J. (2020). Lack of potential carcinogenicity for acesulfame potassium–systematic evaluation and integration of mechanistic data into the totality of the evidence. Food and Chemical Toxicology, 141, 111375. https://doi.org/10.1016/j.fct.2020.111375
8. Chattopadhyay, S., Raychaudhuri, U., Chakraborty, R. (2014). Artificial sweeteners–a review. Journal of Food Science and Technology, 51(4), 611-621. https://doi.org/10.1007/s13197-011-0571-1

9. Choi, J.M., Oh, S.J., Lee, S.Y., Im, J.H., Oh, J.M., Ryu, C.S., et al. (2015). HepG2 cells as an in vitro model for evaluation of cytochrome P450 induction by xenobiotics. Archives of Pharmacal Research, 38(5), 691–704. https://doi.org/10.1007/s12272-014-0502-6 Cruz-Rojas, C., SanJuan Reyes, N., Fuentes Benites, M.P.A.G., Dublan García, O., Galar Martínez, M., Islas Flores, H, et al. (2019). Acesulfame potassium: Its ecotoxicity measured through oxidative stress biomarkers in common carp (Cyprinus carpio). Science of the Total Environment, 647, 772-784. https://doi.org/10.1016/j.scitotenv.2018.08.034
10. De Koning, L., Malik, V.S., Rimm, E.B., Willett, W.C., Hu, F.B., (2011). Sugar-sweetened and artificially sweetened beverage consumption and risk of type 2 diabetes in men. The American Journal of Clinical Nutrition, 93, 1321-1327. https://doi.org/10.3945/ajcn.110.007922
11. Dong, G., Li, X., Han, G., Du, L., Li, M. (2020). Zebrafish neuro-behavioral profiles altered by acesulfame (ACE) within the range of “no observed effect concentrations (NOECs)”. Chemosphere, 243, 125431. https://doi.org/10.1016/j.chemosphere.2019.125431
12. Dusinska, M., Mariussen, E., Rundén Pran, E., Hudecova, A.M., Elje, E., Kazimirova, A., et al. (2019). In vitro approaches for assessing the genotoxicity of nanomaterials. Qunwei Zhang (ed.), Nanotoxicity: Methods and Protocols, Methods in Molecular Biology. Nature, 1894, 83-122. https://doi.org/10.1007/978-1-4939-8916-4_6
13. Erikel, E., Yuzbasioglu, D., Unal, F. (2020). Genotoxic and antigenotoxic potential of amygdalin on isolated human lymphocytes by the comet assay. Journal of Food Biochemistry, 44(10), e13436. https://doi.org/10.1111/jfbc.13436
14. Eroglu, Y., Eroglu, H.E., Ilbas, A.I. (2007). Gamma ray reduces mitotik index in embriyonic roots of Hordeum vulgare L. Advanced Biomedical Research, 1(2), 26-28.
15. Evans, H.J. (1984). Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagentests in mutagen tests. In: B.J. Kilbey, M. Legator, W. Nichols, C. Ramel, (eds). Handbook of mutagenicity test procedures. Amsterdam: Elsevier Science Publishers, 405-427. https://doi.org/10.1016/B978-0-444-80519-5.50023-7
16. Fenech M. (2000). The in vitro micronucleus technique. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455(1-2), 81-95. https://doi.org/10.1016/S0027-5107(00)00065-8
17. Fenech, M., Knasmueller, S., Bolognesi, C., Holland, N., Bonassi, S., Kirsch-Volders, M. (2020). Micronuclei as biomarkers of DNA damage, aneuploidy, inducers of chromosomal hypermutation and as sources of pro-inflammatory DNA in humans. Mutation Research/Reviews in Mutation Research, 786, 108342. https://doi.org/10.1016/j.mrrev.2020.108342
18. Fındıklı, Z., Türkoglu, S., (2014). Determination of the effects of some artificial sweeteners on human peripheral lymphocytes using the comet assay. Journal of Toxicology and Environmental Health Sciences, 6(8), 147-153. https://doi.org/10.5897/JTEHS2014.0313
19. Fowler, S.P., Williams, K., Resendez, R.G., Hunt, K., Hazuda, H.P., Stern, M.P. (2008). Fueling the obesity epidemic? Artificially sweetened beverage use and longterm weight gain. Obesity, 16(8), 1894-1900. https://doi.org/10.1038/oby.2008.284
20. Fowler, S.P.G., (2016). Low-calorie sweetener use and energy balance: Results from experimental studies in animals, and large-scale prospective studies in humans. Physiology Behavior, 164, 517-523. https://doi.org/10.1016/j.physbeh.2016.04.047
21. Fung, T.T., Malik, V., Rexrode, K.M., Manson, J.E., Willett, W.C., Hu, F.B. (2009). Sweetened beverage consumption and risk of coronary heart disease in women. The American Journal of Clinical Nutrition, 89(4), 1037-1042. https://doi.org/10.3945/ajcn.2008.27140
22. Heredia-García, G., Gómez Oliván, L.M., Orozco Hernández, J.M., Luja Mondragón, M., Islas Flores, H., Juan Reyes, N. S., Galar-Martínez M., García-Medina, S., Dublán-García, O. (2019). Alterations to DNA, apoptosis and oxidative damage induced by sucralose in blood cells of Cyprinus carpio. Science of the Total Environment, 692, 411-421. https://doi.org/10.1016/j.scitotenv.2019.07.165
23. Hernández-Pérez, A.F., Jofre, F.M., Queiroz, S., de Arruda, P.V., Chandel, A.K., Graç, M.D., Almeida, F. (2020). Chapter 9: Biotechnological production of sweeteners. Verma, M., Chandel, A., (Eds). Biotechnological Production of Bioactive Compounds, Publisher: Elsevier, 261-292. https://doi.org/10.1016/B978-0-444-64323-0.00009-6
24. Ibi, D., Suzuki, F., Hiramatsu, M. (2018). Effect of AceK (acesulfame potassium) on brain function under dietary restriction in mice. Physiology & Behavior, 188, 291-297. https://doi.org/10.1016/j.physbeh.2018.02.024
25. Jain, A.K., Sorbhoy, R.K. (1988). Cytogenetical studies on the effects of some chlorinated pesticides. III. Concluding remarks. Cytologia, 53(3), 427-436. https://doi.org/10.1508/cytologia.53.427
26. Jeffrey, A.M., Williams, G.M. (2000). Lack of DNA-damaging activity of five non-nutritive sweeteners in the rat hepatocyte/DNA repair assay. Food and Chemical Toxicology, 38(4), 335-338. https://doi.org/10.1016/S0278-6915(99)00163-5
27. Joint FAO/WHO Expert Committee on Food Additives (JECFA), (1991). Thirty‐ seventh report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Geneva (CH). WHO Technical Report Series, No: 806, 20.
28. Kokotou, M.G., Asimakopoulos, A.G., Thomaidis, N.S. (2012). Artificial sweeteners as emerging pollutants in the environment: analytical methodologies and environmental impact. Analytical Methods, 4(10), 3057-3070. https://doi.org/10.1039/c2ay05950a
29. Li, D., O'Brien, J.W., Tscharke, B.J., Choi, P.M., Zheng, Q., Ahmed, F., et al. (2020). National wastewater reconnaissance of artificial sweetener consumption and emission in Australia. Environment International, 143, 105963. https://doi.org/10.1016/j.envint.2020.105963
30. Lindseth, G.N., Coolahan, S.E., Petros, T.V., Lindseth, P.D. (2014). Neurobehavioral effects of aspartame consumption. Research in Nursing & Health, 37(3), 185-193. https://doi.org/10.1002/nur.21595
31. Lutsey, P.L., Steffen, L.M., Stevens, J. (2008). Dietary intake and the development of the metabolic syndrome. Circulation, 117(6), 754-761. https://doi.org/10.1161/CIRCULATIONAHA.107.716159
32. Magnuson, B.A., Carakostas, M.C., Moore, N.H., Poulos, S.P., Renwick, A.G., (2016). Biological fate of low-calorie sweeteners. Nutrition Reviews, 74(11), 670-689. https://doi.org/10.1093/nutrit/nuw032
33. Mamur, S, Yuzbasioglu D, Yılmaz S, Erikel E, Unal F. (2018). Assessment of Cytotoxic and Genotoxic Effects of Enniatin-A In Vitro. Food Additives and Contaminants-Part A, 35(8), 1633-1644. https://doi.org/10.1080/19440049.2018.1486513
34. Mossman, T. (1983). Rapid colometric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal Immunol Methods, 65, 55-63. https://doi.org/10.1016/0022-1759(83)90303-4
35. Mukherjee, A., Chakrabarti, J. (1997). In vivo cytogenetic studies on mice exposed to acesulfame-K—a non-nutritive sweetener. Food and Chemical Toxicology, 35(12), 1177-1179. https://doi.org/10.1016/S0278-6915(97)85469-5
36. Mukhopadhyay, M., Mukherjee, A., Chakrabarti, J. (2000). In vivo cytogenetic studies on blends of aspartame and acesulfame-K. Food and Chemical Toxicology, 38(1), 75-77. https://doi.org/10.1016/S0278-6915(99)00115-5
37. Najam, K., Altaf, I., Ashraf, M., Rasheed, M.A., Saleem, F., Munir, N., et al. (2017). In vitro evaluation of mutagenicity and genotoxicity of sitagliptin alone and in combination with artificial sweeteners. Tropical Journal of Pharmaceutical Research, 16(8), 1841-1847. https://doi.org/10.4314/tjpr.v16i8.13
38. Oldfield, L.E., Roy, J.W., Robinson, C.E. (2020). Investigating the use of the artificial sweetener acesulfame to evaluate septic system inputs and their nutrient loads to streams at the watershed scale. Journal of Hydrology, 587, 124918. https://doi.org/10.1016/j.jhydrol.2020.124918
39. Palus, J., Rydzynski, K., Dziubaltowska, E., Wyszynska, K., Natarajan, A.T., Nilsson, R., et al. (2003). Genotoxic effects of occupational exposure to lead and cadmium. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 540(1), 19-28. https://doi.org/10.1016/S1383-5718(03)00167-0
40. Pintor, A.V.B., Queiroz, L.D., Barcelos, R., Primo, L.S.G., Maia, L.C., Alves, G.G. (2020). MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review. International Endodontic Journal, 53(10), 1348-1373. https://doi.org/10.1111/iej.13353
41. Schiano, C., Grimaldi, V., Scognamiglioi M., Costa, D., Soricelli, A., Nicoletti, G.F., et al. (2021). Soft drinks and sweeteners intake: Possible contribution to the development of metabolic syndrome and cardiovascular diseases. Beneficial or detrimental action of alternative sweeteners?. Food Research International, 142, 110220. https://doi.org/10.1016/j.foodres.2021.110220
42. Setayesh, T., Kundi, M., Nersesyan, A., Stopper, H., Fenech, M., Krupitza, G. et al. (2020). Use of micronucleus assays for the prediction and detection of cervical cancer: a meta-analysis. Carcinogenesis, 41(10), 1318-1328. https://doi.org/10.1093/carcin/bgaa087
43. Silva, M.R., Moya, C.Á., León, A.G.S., Velasco, R.R., Flores, A.M.C. (2018). Genotoxic activity of saccharin, acesulfame-K, stevia and aspartame-acesulfame-K in commercial form. Journal of Clinical Toxicology, 8(385), 2161-0495. https://doi.org/10.4172/2161-0495.1000385
44. Singh, N., Lubana, S.S., Arora, S., Sachmechi, I. (2020). A study of artificial sweeteners and thyroid cancer risk. Journal of Clinical Medicine Research, 12(8), 492. https://doi.org/10.14740/jocmr4258
45. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L. (1988). A simple technique forquantitation of low levels of DNA damage in individual cells. Experimental Cell Research, 175(1), 184-191. https://doi.org/10.1016/0014-4827(88)90265-0
46. Soffritti, M., Belpoggi, F., Esposti, D.D., Lambertini, L., Tibaldi, E., Rigano, A. (2006). First experimental demonstration of the multipotential carcinogenic effects of aspartame administered in the feed to Sprague-Dawley rats. Environmental Health Perspectives, 114(3), 379-385. https://doi.org/10.1289/ehp.8711
47. Souza, A.C.F., Yujra, V.Q., Pisani, L.P., Viana, de M.D.B., De Castro, G.M., Ribeiro, D.A. (2019). The use of single-cell comet assay on oral cells: a critical review. Anticancer Research, 39(8), 4011-4017. https://doi.org/10.21873/anticanres.13556
48. Stice, S.A., Beedanagari, S.R., Vulimiri, S.V., Bhatia, S.P., Mahadevan, B. (2019). Chapter 44: Genotoxicity Biomarkers: Molecular Basis of Genetic Variability and Susceptibility. In Biomarkers in Toxicology. Gupta, R., (Edt), Second Edition, Academic Press, 807-821. https://doi.org/10.1016/B978-0-12-814655-2.00044-X
49. Sturgeon, S.R., Hartge, P., Silverman, D.T., Kantor, A.F., Linehan, W.M., Lynch C., et al. (1994). Associations between bladder cancer risk factors and tumor stage and grade at diagnosis. Epidemiology, 5, 218-225. https://doi.org/10.1097/00001648-199403000-00012
50. Surrales, J., Xamena, N., Creus, A., Catalan, J., Norppa, H., Marcos, R. (1995). Induction of micronuclei by five pyrethroid insecticides in whole blood and isolated human lymphocytes cultures. Mutation Research/Genetic Toxicology, 341(3), 169-184. https://doi.org/10.1016/0165-1218(95)90007-1 van Eyk, A.D. (2015). The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells. Drug and Chemical Toxicology, 38(3), 318-327. https://doi.org/10.3109/01480545.2014.966381
51. van Tonder, A., Joubert, A.M., Cromarty, A.D. (2015). Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC Research Notes, 8(1), 1-10. https://doi.org/10.1186/s13104-015-1000-8
52. Vodicka, P., Musak, L., Vodickova, L., Vodenkova, S., Catalano, C., Kroupa, M., et al. (2018). Genetic variation of acquired structural chromosomal aberrations. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 836, 13-21. https://doi.org/10.1016/j.mrgentox.2018.05.014
53. Whitehouse, C.R., Boullata, J., McCauley, L.A. (2008). The potential toxicity of artificial sweeteners. Aaohn Journal, 56(6), 251–261. https://doi.org/10.1177/216507990805600604
54. World Health Organization (WHO), (1980). Toxicological evaluation of certain food additives. WHO Food Additives Series No 496. 16, 11.
55. Yang, Y., Liu, Z., Zheng, H., Zhu, S., Zhang, K., Li, X., et al. (2021). Sucralose, a persistent artificial sweetener in the urban water cycle: insights into occurrence, chlorinated byproducts formation, and human exposure. Journal of Environmental Chemical Engineering, 9(4), 105293. https://doi.org/10.1016/j.jece.2021.105293
56. Yuzbasioglu, D., Mahmoud, J. H., Mamur, S., Ünal, F. (2022). Cytogenetic effects of antidiabetic drug metformin. Drug and Chemical Toxicology, 45(2), 955-962. https://doi.org/10.1080/01480545.2020.1844226