COMET ASSAY TO STUDY DNA DAMAGE CAUSED BY DIFFERENT STRESS INDUCERS IN THE RIBOFLAVIN OVERPRODUCER EREMOTHECIUM ASHBYII

COMET ASSAY TO STUDY DNA DAMAGE CAUSED BY DIFFERENT STRESS INDUCERS IN THE RIBOFLAVIN OVERPRODUCER EREMOTHECIUM ASHBYII SIMADRI DAS VISHAL YADAV RAJAGOPAL K VIJAYALAKSHMI S

Objectives: The present study focuses on investigating morphological changes and genotoxicity effects due to physical and chemical mutagens such as riboflavin, ethidium bromide (EtBr), pH, and ultraviolet (UV). Methods: Riboflavin doses were given at 0.2 mM. 0.4 mM, 0.8 mM, and 1.0 mM concentration, pH stress doses were given by adjusting the pH of the medium to pH 3.0, pH 5.0, and pH 7.0 whereas EtBr stress doses were 1.0 μL and 0.5 μL and UV radiation stress doses were given for 5 min, 10 min, 15 min. Our study reports at using a modified alkaline yeast comet assay methodology to detect DNA damages in Eremothecium ashbyii caused by stress inducers. Fluorescence microscopy was used to detect the comets and CaspLab software to quantify the intensity of DNA damages. Results and Conclusion: The metrics employed for the quantification of DNA damage under the specified stressful circumstances were, tail DNA, olive tail moment (OTM), and the tail moment (TM). The statistical analysis of the DNA damage doses with CaspLab parameters to detect DNA damage shows maximum DNA damages at pH 5.0 with the highest peak for OTM value followed by UV damage at 15 min. Our study reports the first case using the comet assay technique to detect the DNA damages in the filamentous E. ashbyii cells.
03 07 2025 69 74 http://creativecommons.org/licenses/by/4.0 10.22159/ajpcr.2025v18i3.53816 https://journals.innovareacademics.in/index.php/ajpcr/article/view/53816 https://journals.innovareacademics.in/index.php/ajpcr/article/download/53816/31640 https://journals.innovareacademics.in/index.php/ajpcr/article/download/53816/31640 10.1016/0006-291X(84)90411-X Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun. 1984 Jan;123(1):291-298. doi: 10.1016/0006-291x(84)90411-x 10.1385/MB:26:3:249 Collins AR. The comet assay for DNA damage and repair: Principles, applications, and limitations. Mol Biotechnol. 2004 Mar 26;26(3):249-61. doi: 10.1385/MB:26:3:249, PMID: 15004294 10.1002/yea.1820 Azevedo F, Maques F, Fokt H, Oliveria R, Johansson B. Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast. 2010 Sep 28;1:55-61. doi: 10.1002/yea.1820 Jugade RM, Masram CB. Polarographic studies of riboflavin and estimation in pharmaceutical formulations. Int J Chem Res. 2011 Sep 2;4:14-6. 10.5958/0974-360X.2018.00953.8 Jadhav SK, Vijayalakshmi S. Effect of stress inducer on the morphology of the riboflavin producer Eremothecium ashbyii. Res J Pharm Technol. 2018;11(12):5227-32. doi: 10.5958/0974- 360X.2018.00953.8 Shakya G, Goud C, Pajaniradje S, Rajagopalan R. Protective role of wheatgrass on oxidative stress in streptozotocin induced type 2 diabetic rats. Int J Pharm Pharm Sci. 2012 Apr 4;3:415-23. 10.1093/femsyr/foy114 Silva R, Aguiar TQ, Oliveira R, Domingues L. Light exposure during growth increases riboflavin production, reactive oxygen species accumulation and DNA damage in Ashbya gossypii riboflavin-overproducing strains. FEMS Yeast Res. 2018 Jan; 19(1):1-7. doi: 10.1093/femsyr/foy114 10.1002/(SICI)1097-0290(19991120)65:4<416::AID-BIT6>3.3.CO;2-Q Wongwicharn A, McNeil B, Harvey LM. Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D. Biotechnol Bioeng. 1999;65(4):416-24. doi: 10.1002/(sici)1097- 0290(19991120)65:4<416:aid-bit6>3.0.co;2-z, PMID: 10506417 10.1016/j.enzmictec.2003.07.008 Bai Z, Harvey LM, White S, McNeil B. Effects of oxidative stress on production of heterologous and native protein, and culture morphology in batch and chemostat cultures of Aspergillus niger (B1-D). Enzym Microb Technol. 2004 Jan;34(1):10-21. doi: 10.1016/j. enzmictec.2003.07.008 10.1111/j.1574-6968.2006.00200.x Ponts N, Pinson-Gadais L, Verdal-Bonnin MN, Barreau C, Richard- Forget F. Accumulation of deoxynivalenol and its 15-acetylated form is significantly modulated by oxidative stress in liquid cultures of Fusarium graminearum. FEMS Microbiol Lett. 2006 May;258(1):102-7. doi: 10.1111/j.1574-6968.2006.00200.x, PMID: 16630263 10.1016/j.mib.2008.10.008 Scott B, Eaton CJ. Role of reactive oxygen species in fungal cellular differentiations. Curr Opin Microbiol. 2008 Dec 11;11(6):488-93. doi: 10.1016/j.mib.2008.10.008, PMID: 18983937 10.1016/j.fgb.2012.06.006 Walther A, Wendland J. Yap1-dependent oxidative stress response provides a link to riboflavin production in Ashbya gosspyii. Fungal Genet Biol. 2012 Sep;49(9):697-707. doi: 10.1016/j.fgb.2012.06.006, PMID: 22750190 10.1146/annurev.phyto.43.040204.140214 Yu JH, Keller N. Regulation of secondary metabolism in filamentous fungi. Annu Rev Phytopathol. 2005;43:437-58. doi: 10.1146/annurev. phyto.43.040204.140214, PMID: 16078891 10.1007/s00253-007-1075-9 Schlösser T, Wiesenburg A, Gätgens C, Funke A, Viets U, Vijayalakshmi S, et al. Growth stress triggers riboflavin overproduction in Ashbya gossypii. Appl Microbiol Biotechnol. 2007 Jul;76(3):569-78. doi: 10.1007/s00253-007-1075-9, PMID: 17639374 10.1046/j.1462-2920.2001.00225.x Stahmann KP, Arst HN Jr., Althöfer H, Revuelta JL, Monschau N, Schlüpen C, et al. Riboflavin, overproduced during sporulation of Ashbya gossypii, protects its hyaline spores against ultraviolet light. Environ Microbiol. 2001 Sep 3;3(9):545-50. doi: 10.1046/j.1462- 2920.2001.00225.x, PMID: 11683864 10.1007/s10295-009-0647-3 Sugimoto T, Morimoto A, Nariyama M, Kato T, Park EY. Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production. J Ind Microbiol Biotechnol. 2010 Jan;37(1):57-64. doi: 10.1007/s10295-009-0647-3, PMID: 19826846 10.1007/978-1-61779-998-3_8 Oliveira R, Johansson B. Quantitative DNA damage and repair measurement with the yeast comet assay. Methods Mol Biol. 2012;920:101-9. doi: 10.1007/978-1-61779-998-3_8, PMID: 22941599 10.1007/s10565-007-9043-9 Kumaravel TS, Vihar B, Faux SP, Jha AN. Comet assay measurements: A perspective. Cell Biol Toxicol. 2009 Nov;25:53-64. doi: 10.1007/ s10565-007-9043-9 10.9735/0975-2862.2.1.18-22 Singh RK, Mishra SK, Kumar N, Singh AK. Assessment of DNA damage by comet assay in lymphocytes of workers occupationally exposed to petroleum fumes. Int J Genet. 2010;2(1):18-22. doi: 10.9735/0975- 2862.2.1.18-22 10.1371/journal.pone.0082363 Wang Q, Cui K, Espin-Garcia O, Cheng D, Qiu X, Chen Z, et al. Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis. PLoS One. 2013 Dec 8;8(12):e82363. doi: 10.1371/journal.pone.0082363, PMID: 24349265 10.1016/B978-0-12-409547-2.12381-9 Beedanagari S. Genetic toxicology. In: Chackalamannil S, Rotella D, Ward S, editors. Comprehensive Medicinal Chemistry. Vol. 3. Amsterdam: Elsevier; 2017. p. 195-203. doi: 10.1016/B978-0- 12-409547-2.12381-9