Evaluation of Genetic Damage and Antigenotoxic Effect of Ascorbic Acid in Erythrocytes of Orochromis niloticus and Ambystoma mexicanum Using Migration Groups as a Parameter
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemical and Physical Agents
2.2. Obtaining and Preparing the Sample
2.2.1. Ambystoma Mexicanum Blood Cells
2.2.2. Oreochromis niloticus Blood Cells
2.3. Blood Cell Treatment
2.3.1. EMS and UV-C-Induced Genetic Damage
2.3.2. Post-Treatment Antigenotoxic Effect of AA on Blood Cells Exposed to UV-C
2.4. Alkaline Comet Test
2.5. Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Uno, Y.; Kojima, H.; Omori, T.; Corvi, R.; Honma, M.; Schechtman, L.; Hayashi, M. JaCVAM-organized international validation study of the in vivo rodent alkaline comet assay for detection of genotoxic carcinogens: II. Summary of definitive validation study results. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 2015, 786–788, 45–76. [Google Scholar] [CrossRef] [PubMed]
- Koppen, G.; Azqueta, A.; Pourrut, B.; Brunborg, G.; Collins, A.R.; Langie, S.A.S. The next three decades of the comet assay: A report of the 11th International Comet Assay Workshop. Mutagenesis 2017, 32, 397–408. [Google Scholar] [CrossRef] [PubMed]
- Kirkland, D.; Uno, Y.; Luijten, M.; Beevers, C.; van Benthem, J.; Burlinson, B.K.; Lovell, D.P. In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT). Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 2019, 847, 403035. [Google Scholar] [CrossRef]
- Glei, M.; Schneider, T.; Schlörmann, W. Comet assay: An essential tool in toxicological research. Arch. Toxicol. 2016, 90, 2315–2336. [Google Scholar] [CrossRef] [PubMed]
- Martus, H.J.; Froetschl, R.; Gollapudi, B.; Honma, M.; Marchetti, F.; Pfuhler, S.; Schoeny, R.; Uno, Y.; Yauk, C.; Kirkland, D.J. Summary of major conclusions from the 7th International Workshop on Genotoxicity Testing (IWGT), Tokyo, Japan. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 2020, 852, 503134. [Google Scholar] [CrossRef] [PubMed]
- Rodríguez-Rey, A.; Noris-García, E.; Fundora-Torres, M.T. Principios y relevancia del ensayo cometa. Rev. Cuba. Investig. Bioméd. 2016, 35, 184–194. [Google Scholar]
- Singh, N.P. The comet assay: Reflections on its development, evolution and applications. Mutat. Res. Rev. Mutat. 2016, 767, 23–30. [Google Scholar] [CrossRef]
- Hughes, C.M.; Lewis, S.E.; Mckelvey-Martin, V.J.; Thompson, W. Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutat. Res. Fundam. Mol. Mech. Mutagenesis 1997, 374, 261–268. [Google Scholar] [CrossRef]
- Olive, P.L. The Comet Assay: An Overview of Techniques. In Situ Detection of DNA Damage. Methods Mol. Biol. 2002, 203, 179–194. [Google Scholar] [CrossRef]
- Olive, P.L.; Durand, R.E. Heterogeneity in DNA damage using the comet assay. Cytometry Part A 2005, 66, 1–8. [Google Scholar] [CrossRef]
- Nahon, P.; Allaire, M.; Nault, J.C.; Paradis, V. Characterizing the mechanism behind the progression of NAFLD to hepatocellular carcinoma. Hepatic Oncol. 2020, 7, HEP36. [Google Scholar] [CrossRef]
- Seidel, C.; Lautenschläger, C.; Dunst, J.; Müller, A.C. Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay. Radiat. Oncol. 2012, 7, 61. [Google Scholar] [CrossRef] [Green Version]
- Östling, O.; Johanson, K. Bleomycin, in Contrast to Gamma Irradiation, Induces Extreme Variation of DNA Strand Breakage from Cell to Cell. International Journal of Radiation Biology and Related Studies in Physics. Chem. Med. 1987, 52, 683–691. [Google Scholar] [CrossRef]
- Tronov, V.A.; Grin’ko, E.V.; Afanas’ev, G.G.; Filippovich, I.V. Study of DNA damage and heterogeneity of cells by a gel microelectrophoresis method. Biofizika 1994, 39, 810–819. [Google Scholar]
- Olive, P.L.; Banáth, J.P.; Durand, R.E. Detection of subpopulations resistant to DNA-damaging agents in spheroids and murine tumours. Mutat. Res. Fundam. Mol. Mech. Mutagenesis 1997, 375, 157–165. [Google Scholar] [CrossRef]
- Koppen, G.; Toncelli, L.; Triest, L.; Verschaeve, L. The comet assay: A tool to study alteration of DNA integrity in developing plant leaves. Mech. Ageing Dev. 1999, 110, 13–24. [Google Scholar] [CrossRef]
- Alvarez-Moya, C.; Reynoso-Silva, M.; Canales-Aguirre, A.A.; Chavez-Chavez, J.O.; Castañeda-Vázquez, H.; Feria-Velasco, A.I. Heterogeneity of genetic damage in cervical nuclei and lymphocytes in women with different levels of dysplasia and cancer-associated risk factors. Biomed. Res. Int. 2015, 2015, 293408. [Google Scholar] [CrossRef] [Green Version]
- Kobayashi, H. A comparison between manual microscopic analysis and computerized image analysis in the single cell gel electrophoresis. MMS Commun. 1995, 3, 103–115. Available online: https://scholar.google.com/scholar?hl=es&as_sdt=0%2C5&q=Kobayashi%2C+H.+%281995%29.+A+comparison+between+manual+microscopic+analysis+and+computerized+image+analysis+in+the+single+cell+gel+electrophoresis.+MMS+Commun.%2C+3%2C+103-115.&btnG= (accessed on 1 August 2021).
- Collins, A.R.; Ai-guo, M.; Duthie, S.J. The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. DNA Repair. 1995, 336, 69–77. [Google Scholar] [CrossRef]
- Collins, A.; Dušinská, M.; Franklin, M.; Somorovská, M.; Petrovská, H.; Duthie, S.; Fillion, L.; Panayiotidis, M.; Rašlová, K.; Vaughan, N. Comet assay in human biomonitoring studies: Reliability, validation, and applications. Environ. Mol. Mutagen. 1997, 30, 139–146. [Google Scholar] [CrossRef]
- Bolognesi, C.; Cirillo, S.; Chipman, J.K. Comet assay in ecogenotoxicology: Applications in Mytilus sp. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 2019, 842, 50–59. [Google Scholar] [CrossRef]
- Reynoso-Silva, M.; Álvarez-Moya, C.; Ramírez-Velasco, R.; Sámano-León, A.G.; Arvizu-Hernández, E.; Castañeda-Vásquez, H.; Ruíz-Lopez, M.A. Migration Groups: A Poorly Explored Point of View for Genetic Damage Assessment Using Comet Assay in Human Lymphocytes. Appl. Sci. 2021, 11, 4094. [Google Scholar] [CrossRef]
- Gonzáles, H.M.; Zamora, E.S. Manual Básico Para el Cuidado en Cautiverio del Axolote de Xochimilco (Ambystoma mexicanum); Editorial; Universidad Nacional Autónoma de México Instituto de Biología: Ciudad de Méxoco, México, 2014; pp. 18–25. Available online: http://www.ibiologia.unam.mx/barra/publicaciones/manual_axolotes.pdf (accessed on 1 January 2020).
- Barriga-Vallejo, C.; Aguilera, C.; Cruz, J.; Banda-Leal, J.; Lazcano, D.; Mendoza, R. Ecotoxicological Biomarkers in Multiple Tissues of the Neotenic Ambystoma spp. for a Non-lethal Monitoring of Contaminant Exposure in Wildlife and Captive Populations. Water Air Soil Pollut. 2017, 228, 1–11. [Google Scholar] [CrossRef]
- Møller, P. The comet assay: Ready for 30 more years. Mutagenesis 2018, 33, 1–7. [Google Scholar] [CrossRef] [PubMed]
- Close, B.; Banister, K.; Baumans, V.; Bernoth, E.M.; Bromage, N.; Bunyan, J.; Erhardt, W.; Flecknell, P.; Gregory, N.; Hackbarth, H.; et al. Recommendations for euthanasia of experimental animals: Part 2. Lab. Anim. UK 1997, 31, 1–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- NOM-062-ZOO-1999 Norma Oficial Mexicana. Especificaciones Técnicas para la Producción, Cuidado y Uso de los Animales de Laboratorio. México, D.F.: Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. 1999. Available online: https://www.fmvz.unam.mx/fmvz/principal/archivos/062ZOO.PDF (accessed on 11 February 2021).
- Speit, G.; Hartmann, A. The comet assay (single-cell gel test). In DNA Repair Protocols; Humana Press: Totowa, NJ, USA, 1999; pp. 203–212. [Google Scholar] [CrossRef]
- Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 1988, 175, 184–191. [Google Scholar] [CrossRef] [Green Version]
- Ma, T.H.; Cabrera, G.L.; Cebulska-Wasilewska, A.; Chen, R.; Loarca, F.; Vandenberg, A.L.; Salamone, M.F. Tradescantia stamen hair mutation bioassay. Mutat. Res. Fundam. Mol. Mech. Mutagenesis 1994, 310, 211–220. [Google Scholar] [CrossRef]
- Parcela, S. Sigmaplot (12.0) [Software Data Analysis]. Software. Systal. 2011. Available online: http://www.sigmaplot.co.uk/products/sigmaplot/produpdates/prod-updates18.php (accessed on 1 March 2021).
- Nau, R. Statgraphics (Versión 5) [Overview & tutorial guide]. Fuqua School of Business. Duke University. 2005. Available online: https://statgraphics.net/ (accessed on 1 April 2021).
- Kumaravel, T.; Jha, A.N. Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat. Res. Genet. Toxicol. Environ. Mutagenesis 2006, 605, 7–16. [Google Scholar] [CrossRef]
- Kodym, A.; Afza, R. Physical and Chemical Mutagenesis. In Plant Functional Genomics; Humana Press: Totowa, NJ, USA, 2003; pp. 189–202. [Google Scholar] [CrossRef]
- Wyatt, M.D.; Pittman, D.L. Methylating Agents and DNA Repair Responses: Methylated Bases and Sources of Strand Breaks. Chem. Res. Toxicol. 2006, 19, 1580–1594. [Google Scholar] [CrossRef] [Green Version]
- Gocke, E.; Bürgin, H.; Müller, L.; Pfister, T. Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate. Toxicol. Lett. 2009, 190, 254–265. [Google Scholar] [CrossRef]
- Buonanno, M.; Stanislauskas, M.; Ponnaiya, B.; Bigelow, A.W.; Randers-Pehrson, G.; Xu, Y.; Shuryak, I.; Smilenov, L.; Owens, D.M.; Brenner, D.J. 207-nm UV Light—A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. II: In-Vivo Safety Studies. PLoS ONE 2016, 11, e0138418. [Google Scholar] [CrossRef] [Green Version]
- Byrns, G.; Barham, B.; Yang, L.; Webster, K.; Rutherford, G.; Steiner, G.; Petras, D.; Scannell, M. The uses and limitations of a hand-held germicidal ultraviolet wand for surface disinfection. J. Occup. Environ. Hyg. 2017, 14, 749–757. [Google Scholar] [CrossRef]
- Narita, K.; Asano, K.; Morimoto, Y.; Igarashi, T.; Hamblin, M.R.; Dai, T.; Nakane, A. Disinfection and healing effects of 222-nm UVC light on methicillin-resistant Staphylococcus aureus infection in mouse wounds. J. Photochem. Photobiol. B Biol. 2018, 178, 10–18. [Google Scholar] [CrossRef]
- Alvarez-Moya, C.; Santerre-Lucas, A.; Zúñiga-González, G.; Torres-Bugarín, O.; Padilla-Camberos, E.; Feria-Velasco, A. Evaluation of genotoxic activity of maleic hydrazide, ethyl methane sulfonate, and N-nitroso diethylamine in Tradescantia. Salud Pública México 2001, 43, 6–12. Available online: http://www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S0036-36342001000600007 (accessed on 30 March 2022). [CrossRef]
- Olive, P.L.; Banáth, J.P. The comet assay: A method to measure DNA damage in individual cells. Nat. Protoc. 2006, 1, 23–29. [Google Scholar] [CrossRef]
- García-Lepe, U.O.; Cruz-Ramírez, A.; Bermúdez-Cruz, R.M. DNA repair during regeneration in Ambystoma mexicanum. Dev. Dynam. 2021, 250, 788–799. [Google Scholar] [CrossRef]
- Yen, G.C.; Duh, P.D.; Tsai, H.L. Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid. Food Chem. 2002, 79, 307–313. [Google Scholar] [CrossRef]
- Maeda, J.; Allum, A.J.; Mussallem, J.T.; Froning, C.E.; Haskins, A.H.; Buckner, M.A.; Miller, C.D.; Kato, T.A. Ascorbic Acid 2-Glucoside Pretreatment Protects Cells from Ionizing Radiation, UVC, and Short Wavelength of UVB. Genes 2020, 11, 238. [Google Scholar] [CrossRef] [Green Version]
- Miller, M.R.; Blair, J.B.; Hinton, D.E. DNA repair synthesis in isolated rainbow trout liver cells. Carcinogenesis 1989, 10, 995–1001. [Google Scholar] [CrossRef]
- Bailey, G.S.; Williams, D.E.; Hendricks, J.D. Fish Models for Environmental Carcinogenesis: The Rainbow Trout. Environ. Health Perspect. 1996, 104, 5–21. [Google Scholar] [CrossRef]
- Willett, K.L.; Lienesch, L.A.; Di Giulio, R.T. No detectable DNA excision repair in UV-exposed hepatocytes from two catfish species. Comp. Biochem. Physiol. Part C Toxicol. Pharm. 2001, 128, 349–358. [Google Scholar] [CrossRef]
- Kienzler, A.; Bony, S.; Devaux, A. DNA repair activity in fish and interest in ecotoxicology: A review. Aquat. Toxicol. 2013, 134–135, 47–56. [Google Scholar] [CrossRef]
- Nefić, H. The genotoxicity of vitamin C in vitro. Bosnian. J. Basic Med. Sci. 2008, 8, 141–146. [Google Scholar] [CrossRef] [Green Version]
Genotoxic Treatment | Ascorbic Acid Antigenotoxic Post-Treatment | |||||
---|---|---|---|---|---|---|
Number of Migration Groups | Migration Group Containing the Highest Comet Cells Number | Mean Tail Length in μm | Number of Migration Groups | Migration Group Containing the Highest Comet Cells Number | Mean Tail Length in μm | |
NC | 9.6 | 155 | 6.47 | 9.62 * | 155 | 6.47 |
EMS 2.5 mM | 14.87 | 108 * | 8.7 * | |||
EMS 5 mM | 16.87 | 118 * | 9.96 * | |||
EMS 10 mM | 14.12 | 106 | 9.2 * | |||
UV-C 1 min | 12.75 | 182 | 7.04 * | |||
UV-C 3 min | 14.87 | 130 | 8.19 * | |||
UV-C 5 min | 13.87 | 153 | 8.8 * | |||
PC (UV-C 5 min) | 13.87 | 10.87 | 153 | 7.04 | ||
UV-C, 5 min + AA 5 mM | 9.6 * | 178 * | 7.43 * | |||
UV-C, 5 min + AA 10 mM | 10.25 | 251 * | 7.33 | |||
UV-C, 5 min + AA 15 mM | 13.87 | 260 * | 8.28 * |
Genotoxic Treatment | Ascorbic Acid Antigenotoxic Post-Treatment | |||||
---|---|---|---|---|---|---|
Number of Migration Groups | Migration Group Containing the Highest Comet Cells Number | Mean Tail Length in μm | Number of Migration Groups | Migration Group Containing the Highest Comet Cells Number | Mean Tail Length in μm | |
NC | 13 | 123 | 10.61 | 16 | 123 | 10.59 * |
EMS 2.5 mM | 14 | 90 | 13.03 * | |||
EMS 5 mM | 11 | 105 | 12.74 * | |||
EMS 10 mM | 14 | 88 * | 13.71 * | |||
UV-C 1 min | 11 | 132 | 11.2 | |||
UV-C 3 min | 11 | 127 | 12.04 * | |||
UV-C 5 min | 11 | 95 | 12.94 * | |||
PC (UV-C 5 min) | 22 | 95 | 12.94 | |||
UV-C, 5 min + AA 5 mM | 19 * | 115 | 12.58 | |||
UV-C, 5 min + AA 10 mM | 19 * | 113 | 12.5 | |||
UV-C, 5 min + AA 15 mM | 19 * | 119 * | 11.79 * |
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Moya, C.A.; Silva, M.R.; Ramírez, L.B.; Radillo, J.d.J.V. Evaluation of Genetic Damage and Antigenotoxic Effect of Ascorbic Acid in Erythrocytes of Orochromis niloticus and Ambystoma mexicanum Using Migration Groups as a Parameter. Appl. Sci. 2022, 12, 7507. https://doi.org/10.3390/app12157507
Moya CA, Silva MR, Ramírez LB, Radillo JdJV. Evaluation of Genetic Damage and Antigenotoxic Effect of Ascorbic Acid in Erythrocytes of Orochromis niloticus and Ambystoma mexicanum Using Migration Groups as a Parameter. Applied Sciences. 2022; 12(15):7507. https://doi.org/10.3390/app12157507
Chicago/Turabian StyleMoya, Carlos Alvarez, Mónica Reynoso Silva, Lucia Barrientos Ramírez, and José de Jesús Vargas Radillo. 2022. "Evaluation of Genetic Damage and Antigenotoxic Effect of Ascorbic Acid in Erythrocytes of Orochromis niloticus and Ambystoma mexicanum Using Migration Groups as a Parameter" Applied Sciences 12, no. 15: 7507. https://doi.org/10.3390/app12157507