Using The Allium Test of 4-Methyl Umbelliferone: Physiological, Cytogenetic, Biochemical and Anatomical Evaluation

Yıl 2023, Cilt: 18 Sayı: 2, 110 - 128, 22.06.2023

Dilek Çavuşoğlu

https://doi.org/10.29233/sdufeffd.1217208

Öz

In this study, the dose dependent effects of exogenous 4-methyl umbelliferone (4-MU) on various physiological, cytogenetic, anatomical and biochemical parameters using Allium cepa L. as test material were researched. The physiological parameters examinated were germination percentage, root length, rootlet number and fresh weight; the cytogenetic parameters were micronucleus (MN) frequency, chromosome aberration (CA) and mitotic index (MI); the biochemical parameters were free proline content, malondialdehyde (MDA) level, catalase (CAT) and superoxide dismutase (SOD) activities. And the structural changes in root tip cells were investigated with anatomical sections. Structural changes in onion root tip cells were examined by taking anatomical sections. For these purposes, bulbs were divided into four groups as one control and three applications. The bulbs in the control group were treated with distilled water; the bulbs in the application groups were treated with 125 µM, 250 µM and 500 µM doses of 4-MU for 7 days. 4-MU application caused a decrease in the physiological parameters compared to the control group. This treatment created an increase in the frequency of MN and CA, and a reduce in the MI. In addition, 4-MU application induced an increase as dose-dependent in CAT and SOD activities and MDA and free proline contents compared to the control group. Moreover, after all 4-MU application, anatomical changes such as MN formation in epidermis cells, epidermis and cortex cell deformations, accumulation of some chemical compounds in cortex, unclear transmission tissue and necrose were identified and these root anatomical changes were found to reach maximum levels at 500 µM 4-MU. As a result, 4-MU had a negative effect on all the investigated parameters and it was determined that the Allium test material could be a useful bioindicator for monitoring these effects.

Anahtar Kelimeler

Allium cepa L., Antioxidant enzymes, Germination, Lipid peroxidation, 4-methyl umbelliferone, Cytogenetic

4-Metil Umbelliferon’un Allium Testi Kullanılarak; Fizyolojik, Sitogenetik, Biyokimyasal ve Anatomik Değerlendirilmesi

Öz

Bu çalışmada, test materyali olarak Allium cepa L. kullanılarak çeşitli fizyolojik, sitogenetik, anatomik ve biyokimyasal parametreler üzerine dışsal 4-metil umbelliferonun (4-MU) doza bağlı etkileri araştırıldı. İncelenen fizyolojik parametreler: çimlenme yüzdesi, kök uzunluğu, kökçük sayısı ve taze ağırlık; sitogenetik parametreler: mikronükleus (MN) sıklığı, kromozom anormalliği (KA) ve mitotik indeks (Mİ); biyokimyasal parametreler: serbest prolin içeriği, malondialdehit (MDA) düzeyi, katalaz (KAT) ve süperoksit dismutaz (SOD) aktiviteleridir. Soğan kök ucu hücrelerinde meydana gelen yapısal değişimler anatomik kesitler alınarak incelendi. Bu amaçlar için, soğanlar bir kontrol ve üç uygulama olmak üzere dört gruba ayrıldı. 7 gün süresince kontrol grubundaki soğanlar distile su ile muamele edilirken; uygulama gruplarındaki soğanlar 4-MU’nun 125 µM, 250 µM ve 500 µM dozları ile muamele edildi. 4-MU uygulaması kontrol grubuyla kıyaslandığında fizyolojik parametrelerde bir azalmaya neden oldu. Bu uygulama, KA ve MN sıklığında bir artış ile Mİ'de bir azalma meydana getirdi. Ayrıca, 4-MU muamelesi kontrol grubuyla karşılaştırıldığında KAT ve SOD aktiviteleri ile MDA ve serbest prolin içeriklerinde de doza bağlı olarak bir artışa neden oldu. Ayrıca, tüm 4-MU uygulamaları sonrasında, epidermis hücrelerinde MN oluşumu, epidermis ile korteks hücre deformasyonları, kortekste bazı kimyasal bileşiklerin birikmesi, belirgin olmayan iletim dokusu ve nekroz gibi kök anatomik değişimleri tespit edildi ve bu anatomik değişimlerin 500 µM 4-MU'da maksimum seviyelere ulaştığı bulundu. Sonuç olarak, 4-MU incelenen tüm parametreler üzerinde olumsuz bir etki gösterdi ve bu etkilerin izlenmesi için Allium test materyalinin yararlı bir biyoindikatör olabileceği tespit edildi.

Anahtar Kelimeler

Allium cepa L., Antioksidan enzimler, Çimlenme, Lipid peroksidasyonu, 4-metil umbelliferon, Sitogenetik

Kaynakça

• D. Srikrishna, C. Godugu, and P. K. Dubey, “A Review on Pharmacological Properties of Coumarins,” Mini-Rev. Chem., 18 (2), 113–141, 2018.
• M. Iranshahi, M. Askari, A. Sahebkar, and D. Adjipavlou-Litina, “Evaluation of antioxidant, anti-inflammatory and lipoxygenase inhibitory activities of the prenylated coumarin umbelliprenin,” DARU J. Pharm. Sci., 17 (2), 99–103, 2009.
• M. K. Bashir, Y. F. and M. K. Mustafa, “Oglah Antitumor, antioxidant, and antibacterial activities of glycosyl-conjugated compounds: A review,” Syst. Rev. Pharm., 11, 175–187, 2020.
• Y. F. Mustafa, E. T. Mohammed, and R. R. Khalil, “Antioxidant and antitumor activities of methanolic extracts obtained from Red Delicious and Granny Smith apples’ seeds,” Syst. Rev. Pharm., 11, 570–576, 2020.
• Y. Aoyama, T. Katayama, M. Yamamoto, H. Tanaka, and K. Kon, “ A new antitumor antibiotic product, demethylchartreusin. Isolation and biological activities,” J. Antibiot., 45 (6), 875–878, 1992.
• X. M. Peng, G. V. Damu, and C. Zhou, “Current developments of coumarin compounds in medicinal chemistry,” Cur. Pharm. Des., 19 (21), 3884–3930, 2013.
• G. H. Zhang, H. Zheng, M. Y. Guo, L. Du, G. J. Liu, and P. Wang, “Synthesis of polymeric fluorescent brightener based on coumarin and its performances on paper as light stabilizer, fluorescent brightener and surface sizing agent,” Appl. Surf. Sci., 367, 167–173, 2016.
• V. R. Mishra, and N. Sekar, “Photostability of coumarin laser dyes-A mechanistic study using global and local reactivity descriptors,” J. Fluoresc., 27, 1101–1108, 2017.
• M. J. Matos, L. Santana, E. Uriarte, O. Abreu, E. Molina Pe´rez, and E. Yordi, “Coumarins-An important class of phytochemicals,” in Phytochemicals-Isolation, Characterisation and Role in Human Health, A. Venket Rao and Leticia G. Rao, Eds., 2015, pp. 113–140.
• H. Wang, X. Lu, H. Yao, J. Feng, and R. Liu, “Research progress on application of coumarin and its derivatives,” Chem. Ind. Times., 23 (8), 40–43, 2009.
• F. Annunziata, C. Pinna, S. Dallavalle, L. Tamborini, and A. Pinto, “An overview of coumarin as a versatile and readily accessible scaffold with broad-ranging biological activities,” Int. J. Mol. Sci., 2 (13), 2020.
• M. Miranda, and A. Cuéllar, “Farmacognosia y productos naturales,” Fe´lix Varela, La Habana, Ed., Rev. Cuba. de Plantas Medicinales., 9, 34–49, 2001.
• K. N. Venugopala, V. Rashmi, and B. Odhav, “Review on natural coumarin lead compounds for their pharmacological activity,” Bio. Med. Res. Int., 963248, 2013.
• J. Choi, K. T. Liu, H. Ka, W. T. Jung, H. J. Jung, and H. J. Park, “Constituents of the essential oil of the Cinnamomum cassia stem bark and the biological properties,” Arch. Pharm. Res. (Seoul)., 24 (5), 418–423, 2001.
• F. Bourgaud, A. Hehn, R. Larbat, S. Doerper, E. Gontier, S. Kellner, and U. Matern, “Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes,” Phytochem. Rev., 5 (2), 293–308, 2006.
• S. Rosselli, A. M. Maggio, N. Faraone, V. Spadaro, S. L. Morris-Natschke, K. F. Bastow, K. H. Lee, and M. Bruno, “The cytotoxic properties of natural coumarins isolated from roots of Ferulago campestris (Apiaceae) and of synthetic ester derivatives of aegelinol,” Nat. Prod. Commun., 4 (12), 2009.
• G. Vialart, A. Hehn, A. Olry, K. Ito, C. Krieger, R. Larbat, C. Paris, B. Shimizu, Y. Sugimoto, M. Mizutani, and F. Bourgaud, “A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2’-hydroxylase activity (C2’H): a missing step in the synthesis of umbelliferone in plants,” Plant J., 70, 460–470, 2012.
• R. Ramalingam, and M. Vaiyapuri, “Effects of umbelliferone on lipid peroxidation and antioxidant status in diethylnitrosamine-induced hepatocellular carcinoma,” J. Acute Med., 3, 73–82, 2013.
• J. A. García-Vilas, A. R. Quesada, and M. Á. Medina, “4-Methylumbelliferone inhibits angiogenesis in vitro and in vivo,” J. Agric. Food Chem., 61, 4063–4071, 2013.
• S. Medimagh-Saidana, A. Romdhane, M. Daami-Remadi, H. Jabnoun-Khiareddine, D. Touboul, H. Ben Jannet, et al., “Synthesis and antimicrobial activity of novel coumarin derivatives from 4-methylumbelliferone,” Med. Chem. Res., 24 (8), 3247–3257, 2015.
• L. Pan, X. Li, H. Jin, X. Yang, and B. Qin, “Antifungal activity of umbelliferone derivatives: Synthesis and structure-activity relationships,” Microb. Pathog., 104, 110–115, 2017.
• L. Pan, X. Z. Li, Z. Q. Yan, H. R. Guo, and B. Qin, “Phytotoxicity of umbelliferone and its analogs: Structure-activity relationships and action mechanism,” Plant Physiol. Bioch., 97, 272–277, 2015.
• M. Deshmukh, P. Pawar, M. Joseph, M. Phalgune, U. Kashalkar, R. Deshpande, and R. Nirmala, “Efficacy of 4-methy-7-hydroxy coumarin derivatives against vector,” Indian J. Exp. Biol., 46 (11), 788–792, 2008.
• Y. Wei, S. Q. Li, and S. H. Hao, “New angular oxazole-fused coumarin derivatives: Synthesis and biological activities,” Nat. Prod. Res., 32 (15), 1824–1831, 2018.
• G. L. Liu, Y. Hu, X. H. Chen, G. X. Wang, and F. Ling, “Synthesis and anthelmintic activity of coumarin-imidazole hybrid derivatives against Dactylogyrus intermedius in goldfish,” Bioorg. Med. Chem. Lett., 26, 5039–5043, 2016.
• P. Arora, M. S. Ranawat, and N. Arora, “Synthesis and screening of some novel coumarin derivatives for antipsychotic activity,” Res. J. Pharm. Technol. , 5 (18), 968–972, 2012.
• K. Ostrowska, K. Młodzikowska, M. Głuch-Lutwin, A. Grybo´s, and A. Siwek, “Synthesis of a new series of aryl/heteroarylpiperazinyl derivatives of 8-acetyl-7-hydroxy-4-methylcoumarin with low nanomolar 5-HT 1A affinitie,” Eur. J. Med. Chem., 137, 108–116, 2017.
• S. T. Nam, Y. H. Park, H. W. Kim, H. S. Kim, D. Lee, M. B. Lee, Y. M. Kim, and W. S. Choi, “Suppression of IgE-mediated mast cell activation and mouse anaphylaxis via inhibition of Syk activation by 8-formyl-7-hydroxy-4-methylcoumarin, 4-8C,” Toxicol. Appl. Pharmacol., 332, 25–31, 2017.
• X. J. Hu, X. B. Wang, and L. Y. Kong, “Glucosidase inhibitors via green pathway: Biotransformation for bicoumarins catalyzed by Momordica charantia peroxidase,” J. Agric. Food Chem., 61, 1501–1508, 2013.
• E. Radwan, and I. A. Reem, “Anti-inflammatory screening and molecular modeling of some novel coumarin derivatives,” Molecules, 20, 5374–5391, 2015.
• Y. F. Mustafa, E. T. Mohammed, and R. R. Khalil, “Synthesis, characterization, and anticoagulant activity of new functionalized biscoumarins,” Egyptian J. Chem., 64, 4461–4468, 2021.
• L. Peruzzi, A. Carta, and F. Altinordu, “Chromosome diversity and evolution in Allium (Allioideae, Amaryllidaceae,” Plant Biosyst., 151, 212–220, 2017.
• T. K. Lim, “Modified stems, roots, bulbs,” in Springer, Netherlands, Edible Medicinal and Non-Medicinal Plants,” The Netherlands, 2015, pp. 124–203.
• M. Marrelli, V. Amodeo, G. Statti, and F. Conforti, “Biological properties and bioactive components of Allium cepa L.: focus on potential benefits in the treatment of obesity and related comorbidities,” Molecules, 24, 119–136, 2019.
• S. B. Tedesco, and I. V. H. D. Laughinghouse, “Boindicator of genotixicity. The Allium cepa test,” J. Environ. Contam., 138–156, 2012.
• G. Fiskesjo, “The Allium test as a standard in environ-1535 mental monitoring,” Hereditas, 102, 99–112, 1985.
• T. R. Chaparro, C. M. Botta, and E. C. Pires, “Biodegradability and toxicity assessment of bleach plant effluents treated anaerobically,” Water Sci. Technol., 62, 1312–1319, 2010.
• W. F. Grant, “Higher plant assays for the detection of chromosomal aberrations and gene mutations-a brief historical back ground on their use for screening and monitoring environmental chemicals,” Mutat. Res., 426, 107–112, 1999.
• K. Çavuşoğlu, and D. Çavuşoğlu, “Role of L-ornithine in mitigation salt stress in Allium cepa L.,” Bangl. J. Bot., 50(4), 1165–1171, 2019.
• P. C. Sharma, and P. K. Gupta, “Karyotypes in some pulse crops,” Nucleus, 25, 181–185, 1982.
• M. Fenech, W. P. Chang, M. Kirsch-Volders, N. Holland, S. Bonassi, and E. Zeiger, “HUMN (human micronucleus) project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures,” Mutation. Res., 534, 65–75, 2003.
• C. Beauchamp, and I. Fridovich, “Superoxide dismutase: improved assays and an assay applicable to acrylamide gels,” Anal. Biochem., 44, 276–287, 1971.
• J. Zou, J. Yue, W. Jiang, and D. Liu, “Effects of cadmium stress on root tip cells and some physiological indexes in Allium cepa var. agrogarum L.,” Acta Biol. Cracov. Ser. Bot., 54, 129–141, 2012.
• R. F. Beers, and I. W. Sizer, “Colorimetric method for estimation of catalase,” J. Biol. Chem., 195, 133–139, 1952.
• S. Ünyayar, A. Celik, F. O. Cekic, and A. Gozel, “Cadmium-induced genotoxicity, cytotoxicity and lipid peroxidation in Allium sativum and Vicia faba,” Mutagenesis, 21, 77–81, 2006.
• L. S. Bates, R. P. Waldren, and I. D. Teare, “Rapid determination of free proline for water stress studies,” Plant Soil., 39, 205-207, 1973.
• K. Çavuşoğlu, T. Kalefetoğlu Macar, O. Macar, D. Çavuşoğlu, and E. Yalçın, “Comparative investigation of toxicity induced by UV-A and UV-C radiation using Allium test,” Environ. Sci. Pollut. Res., 29, 33988–33998, 2022.
• D. Çavuşoğlu, O. Macar, T. Kalefetoğlu Macar, K. Çavuşoğlu, and E. Yalçın, “Mitigative effect of green tea extract against mercury (II) chloride toxicity in Allium cepa L. model,” Environ. Sci. Pollut. Res., 29, 27862–27874, 2022.
• M. R. Abenavoli, A. Nicolò, A. Lupini, S. Oliva, and A. Sorgonà, “Effects of different allelochemicals on root morphology of Arabidopsis thaliana,” Allelopathy J., 22, 245–252, 2008.
• X. Li, M. Y. Gruber, D. D., Hegedus, D. J, Lydiate, and M. J., Gao, “Effects of a Coumarin Derivate, 4-Methylumbelliferone, on Seed Germination and Seedling Establishment in Arabidopsis,” J. Chem. Ecol., 37, 880–890, 2011.
• R. A. Bernhard, “Some Studies of Coumarin and Coumarin Analogues as Germination Inhibitors of Radish Seeds,” Bot. Gaz., 17–21, 2016.
• H. Harashima, and A. Schnittger, “The integration of cell division, growth and differentiation,” Curr. Opin. Plant Biol., 13, 66–74, 2010.
• L. F. Andrade, L. C. Davide, and L. S. Gedraite, “The effect of cyanide compounds, fluorides, aluminum, and inorganic oxides present in spent pot liner on germination and root tip cells of Lactuca sativa,” Ecotoxicol. Environ. Saf., 73, 626–631, 2010.
• A. Acar, Z. Turkmen, K. Cavusoglu, and E. Yalcin, “Investigation of benzyl benzoate toxicity with anatomical, physiological, cytogenetic and biochemical parameters in vivo,” Caryologia, 73, 21–32, 2020.
• G. Öztürk, K. Çavusoglu, and E. Yalçın, “Dose-response analysis of potassium bromate-induced toxicity in Allium cepa L. meristematic cells,” Env. Sci. Pollut. Res., 27, 43312–43321, 2020.
• T. C. C. Fernandes, D. E. C. Mazzeo, and M. A. Marin Morales, “Mechanism of micronuclei formation in polyploidizated cells of A. cepa exposed to trifluralin herbicide,” Pest. Biochem. Physiol., 88, 252–259, 2007.
• N. A. Sutan, A. Popescu, C. Mihaescu, L. C. Soare, and M. V. Marinescu, “Evaluation of cytotoxic and genotoxic potential of the fungicide Ridomil in Allium cepa L.,” Analele Stiint Univ. Al I Cuza Iasi., 60, 5–12, 2014.
• M. Fenech, and J. W. Crott, “Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakage-fusion-bridge cycles in the cytokinesis block micronucleus assay,” Mutat. Res., 504, 131–136, 2002.
• C. Ruan, Y. Lian, and J. Lium, “Application of micronucleus test in Vicia faba in the rapid deletion of mutagenic environmental pollutants,” Chin J. Environ. Sci., 4, 56–58, 1992.
• M. Fenech, “The in vitro micronucleus technique,” Mutat. Res., 455, 81–95, 2000.
• H. Norppa, and G. C. Falck, “What do human micronuclei contain?,” Mutagenesis, 18, 221–233, 2003.
• D. Gisselsson, E. Palsson, C. Yu, F. Mertens, and N. Mandahl, “Mitotic instability associated with late genomic changes in bone and soft tissue tumours,” Cancer Lett., 206, 69–76, 2004.
• L. Luzhna, P. Kathiria, and O. Kovalchuk, “Micronuclei in genotoxicity assessment: from genetics to epigenetics and beyond,” Front. Genet., 4, 131, 2013.
• S. Türkoğlu, “Genotoxicity of five food preservatives tested on root tips of Allium cepa L.,” Mutat. Res., 626, 4–14, 2007.
• V. P. Kalcheva, A. P. Dragoeva, K. N. Kalchev, and D. D. Enchev, “Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells,” Genet. Mol. Biol., 32, 389–393, 2009.
• A. A. El-Ghamery, M. A. El-Kholy, and M. A. Abou El-Yousser, “Evaluation of cytological effects of Zn2+ in relation to germination and root growth of Nigella sativa L. and Triticum aestivum L.,” Mutat. Res., 537, 29–41, 2003.
• S. J. Neill, R. Desikan, A. Clarke, R. D. Hurst, and J. T. Hencock, “Hydrogen peroxide and nitric oxide as signaling molecules in plants,” J. Exp. Bot., 53, 1237–1247, 2002.
• E. Vranova, D. Inze, and F. Van Breusegen, “Signal transduction during oxidative stress,” J. Exp. Bot., 53, 1227–1236, 2002.
• A. K. Srivastava, and D. Singh, “Assessment of malathion toxicity on cytophysiological activity, DNA damage and antioxidant enzymes in root of Allium cepa model,” Sci. Rep., 10, 1–10, 2020.
• A. M. S. Soares, T. F. Souza, T. Jacinto, and O. L. T. Machado, “Effect of methyl jasmonate on antioxidative enzyme activities and on the contents of ROS and H2O2 in Ricinus communis leaves,” Braz. J. Plant Physiol., 22, 151–158, 2010.
• V. J. Odjegba, and R. A. Adeniran, “Bentazone herbicide induces genotoxic effect and physiological disorders in non-targeted Allium cepa L.,” Indian J. Plant Physiol., 20, 375–379, 2015.
• I. S. Fedina, and K. M. Benderliev, “Response of Scendesmus incrassatulus to salt stress as affected by methyl jasmonate,” Biol. Plant., 43, 625–627, 2000.
• D. R. Janero, “Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury,” Free Radic. Biol. Med., 9, 515–540, 1990.
• K. Shah, R. G. Kumar, S. Verma, and R. S. Dubey, “Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings,” Plant Sci., 161, 1135–1144, 2001.
• C. Dinakar, V. Abhaypratap, S. R. Yearla, A. S. Raghavendra, and K. Padmasree, “Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation,” Planta, 231, 461–474, 2010.
• E. Yarsan, “Lipid peroxidation event and its applications for prevention,” Van. Vet. J., 9, 89–95, 2014.
• M. Ashraf, and M. R. Foolad, “Roles of glycine betaine and proline in improving plant abiotic stress resistance,” Environ. Exp. Bot., 59, 206–216, 2007.
• S. T. Sadiqov, M. Akbulut, and V. Ehmedov, “Role of Ca2+ in drought stress signaling in wheat seedlings,” Biochem., 67, 491–497, 2002.
• R. Munns, and M. Tester, “Mechanisms of salinity tolerance,” Annu. Rev. Plant Biol., 59, 651-681, 2008.
• S. G. Kumar, A. M. Reddy, and C. Sudhakar, “NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance,” Plant Sci., 165, 1245–1251, 2003.
• C. A. Martinez, M. Maestri, and E. G. Lani, “In vitro salt tolerance and proline accumulation in Andean potato (Solanum spp.) differing in frost resistance,” Plant Sci., 116, 117–184, 2003.
• X. S. Wang, and J. G. Han, “Changes in proline content, activity, and active isoforms of antioxidative enzymes in two alfalfa cultivars under salt stress,” Agric. Sci. China., 8, 431–440, 2009.
• O. Çelik, and C. Atak, “The effect of salt stress on antioxidative enzymes and proline content of two Turkish tobacco varieties,” Turk. J. Biol., 36, 339–356, 2012.
• F. Boughalleb, R. Abdellaoui, M. Mahmoudi, and E. Bakhshandeh, “Changes in phenolic profile, soluble sugar, proline, and antioxidant enzyme activities of Polygonum equisetiforme in response to salinity,” Turk. J. Bot., 44, 25–35, 2020.
• J. Matysik, B. Alia-Bhalu, and P. Mohanty, “Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants,” Curr. Sci., 82, 525–532, 2002.