Tetraconazole-induced Programmed Cell Death in Schizosaccharomyces pombe

Year 2021, , 833 - 843, 31.12.2021

Hızlan Hıncal Ağuş Ahsen Çetin İrem Naz Yalçın

https://doi.org/10.35193/bseufbd.963547

Abstract

Tetraconazole, a systemic triazole fungicide, shows potential toxic effects in agriculture and human health. Therefore, its cytotoxic effects and accompanying mechanisms should be unraveled. S. pombe (ED666) was used in this study, as a unicellular biology and toxicology model. Cells were grown on standard media and all treatments were done at 30 C and shaking at 180 rpm 1-10 mg/L tetraconazole induced a dose-dependent cell death. Apoptosis was monitored by DAPI ve AO/EB staining. Excessive ROS production and mitochondrial impairment were shown by DCFDA/NBT assays and Rhodamine 123 staining, which were supported by increased expressions of superoxide dismutases and glutathione peroxidase. Involvement of one of the potential apoptotic genes, Cnx1, in apoptosis was shown by increased transcription whereas two other potential genes, Pca1 and Aif1, were not affected by tetraconazole treatment. In conclusion, tetraconazole-induced cytotoxicity and underlying mechanisms which were mediated via ROS damage and mitochondrial dysregulation (Cnx1-driven) were clarified in S. pombe.

Keywords

Schizosaccharomyces Pombe, Tetraconazole, Apoptosis, Cnx1, Oxidative Damage

Supporting Institution

İstanbul Yeni Yüzyıl Üniversitesi Mütevelli Heyeti

Thanks

This work was supported by Istanbul Yeni Yuzyil University. We specially thanks to Aysegul Topal-Sarikaya, Bedia Palabiyik for providing S. pombe cells, Sinem Tunçer Gurbanov and Emre Yoruk for consumables and chemicals.

Schizosaccharomycespombe’deTetrakonazol Kaynaklı Programlı Hücre Ölümü

Abstract

Sistemik triazol bir fungisit olan tetrakonazol tarımda ve insan sağlığında potansiyel toksik etkiler göstermektedir. Bu yüzden, sitotoksik etkileri ve eşlik eden mekanizmaları açığa çıkarılmalıdır. Bu çalışmada, tek hücreli biyoloji ve toksikoloji modeli olarak S. pombe (ED666) kullanılmıştır. Hücreler standart medyumda büyütülmüş, muameleler 30 C’de ve 180 rpm hızda çalkalamalı olarak yapılmıştır. 1-10 mg/L tetrakonazol doz-bağımlı hücre ölümüne sebep olmuştur. Apoptoz DAPI ve AO/EB boyamasıyla görüntülenmiştir. Aşırı ROS üretimi ve mitokondriyel bozulma DCFDA/NBT deneyleri ve Rhodamin 123 boyamasıyla gösterilmiş, bu sonuçlar da süperoksitdismutazlar ve glutatyonperoksidaz ifadelerindeki artışlarla desteklenmiştir. Potansiyel apoptotik genlerden biri olan Cnx1’in apoptozla ilişkisi transkripsiyonundaki artışla gösterilirken, diğer iki potansiyel gen, Pca1 ve Aif1 tetrakonazolden etkilenmemiştir. Sonuç olarak, tetrakonazol kaynaklı apoptoz ile, ROS hasarı ve mitokondriyel düzensizliğin (Cnx1-yoluyla) aracılık etmiş olduğu mekanizmalar S. pombe’de açıklığa kavuşturulmuştur.

Keywords

Schizosaccharomyces Pombe, Tetrakonazol, Apoptoz, Cnx1, Oksidatif Hasar

References

• Gavarkar, P. S., Adnaik, R. S., & Mohite, S. K. (2013). An Overview of Azole Antifungals. International Journal of Pharmaceutical Sciences and Research, 4, 4083–4089.
• Templeton, I. E., Thummel, K. E., Kharasch, E. D., Kunze, K. L., Hoffer, C., Nelson, W. L., & Isoherranen, N. (2008). Contribution of Itraconazole Metabolites to Inhibition of CYP3A4 In Vivo. Clinical Pharmacology & Therapeutics, 83, 77–85.
• Vermeer, L. M. M., Isringhausen, C. D., Ogilvie, B. W., & Buckley, D. B. (2016). Evaluation of Ketoconazole & Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters. Drug Metabolism and Disposition, 44, 453–459.
• Shirasaka, Y., Sager, J. E., Lutz, J. D., Davis, C., & Isoherranen, N. (2013). Inhibition of CYP2C19 and CYP3A4 by Omeprazole Metabolites and Their Contribution to Drug-Drug Interactions. Drug Metabolism and Disposition, 41, 1414–1424.
• Mishra, A., Malakar, A., Biswal, H. T., Barman, M. K., & Krishnamoorthy, G. (2015). Interactions of a few azole derivatives with a transport protein: role of heteroatoms. Journal of Molecular Recognition, 28, 299–305.
• Banerjee, K., Oulkar, D. P., Patil, S. H., Dasgupta, S., & Adsule, P. G. (2008). Degradation kinetics and safety evaluation of tetraconazole and difenoconazole residues in grape. Pest Management Science, 64, 283–289.
• Tong, Z., Dong, X., Yang, S., Sun, M., Gao, T., Duan, J., & Cao, H. (2019). Enantioselective effects of the chiral fungicide tetraconazole in wheat: Fungicidal activity and degradation behavior. Environmental Pollution, 247, 1–8.
• Carelli, A., Farina, G., Gozzo, F., Merlini, L., & Kelly, S. L. (1992). Interaction of tetraconazole and its enantiomers with cytochrome P450 from Ustilago maydis. Pesticide Science, 35, 167–170.
• Emami, S., Tavangar, P., & Keighobadi, M. (2017). An overview of azoles targeting sterol 14α-demethylase for antileishmanial therapy. European Journal of Medicinal Chemistry, 135, 241–259.
• Warrilow, A. G. S., Price, C. L., Parker, J. E., Rolley, N. J., Smyrniotis, C. J., Hughes, D. D., Thoss, V., Nes, W. D., Kelly, D. E., Holman, T. R., & Kelly, S. L. (2016). Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa. Scientific Reports, 6, 27690.
• Lv, Q., Yan, L., & Jiang, Y. (2016). The synthesis, regulation, and functions of sterols in Candida albicans: Well-known but still lots to learn. Virulence, 7, 649–659.
• Office of Pesticide Programs, U. (2006). Pesticide fact sheet for tetraconazole. U.S. EPA [online], https://www.epa.gov/pesticides (Accessed April 4, 2019).
• Office of Pesticide Programs, U. (2007). Tetraconazole: Human-Health Risk Assessment for Proposed Uses on Soybean, Sugar Beet, Peanut, Pecan, and Turf. U.S. EPA [online], https://www.epa.gov/pesticides (Accessed April 4, 2019).
• Authority, A. P. and V. M. (2005). Evaluation of the new active Tetraconazole in the product Domark 40ME Fungicide, [online] http://fluoridealert.org/wp-content/pesticides/tetraconazole.2005.report.australia.pdf (Accessed April 5, 2019).
• Daniel, S. L., Hartman, G. L., Wagner, E. D., & Plewa, M. J. (2007). Mammalian Cell Cytotoxicity Analysis of Soybean Rust Fungicides. Bulletin of Environmental Contamination and Toxicology, 78, 474–478.
• El-Sherief, H. A. M., Youssif, B. G. M., Bukhari, S. N. A., Abdel-Aziz, M., & Abdel-Rahman, H. M. (2018). Novel 1,2,4-triazole derivatives as potential anticancer agents: Design, synthesis, molecular docking and mechanistic studies. Bioorganic Chemistry, 76, 314–325.
• Ahmad, K., Khan, M. K. A., Baig, M. H., Imran, M., & Gupta, G. K. (2018). Role of Azoles in Cancer Prevention and Treatment: Present and Future Perspectives. Anti-Cancer Agents in Medicinal Chemistry, 18, 46–56.
• Filho, R. I., Gonzaga, D. T. G., Demaria, T. M., Leandro, J. G. B., Costa, D. C. S., Ferreira, V. F., Sola-Penna, M., de C. da Silva, F., & Zancan, P. (2018). A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties. Current Topics in Medicinal Chemistry, 18, 1483–1493.
• Sidrim, J. J. C., de Maria, G. L. & Paiva, M. D. et al. (2021). Azole-Resilient Biofilms and Non-wild Type C. Albicans Among Candida Species Isolated from Agricultural Soils Cultivated with Azole Fungicides: an Environmental Issue?. Microbial Ecology. https://doi.org/10.1007/s00248-021-01694-y
• Demuyser L & Van Dijck P. (2019) Can Saccharomyces cerevisiae keep up as a model system in fungal azole susceptibility research? Drug Resistance Updates. 42, 22-34.
• Martins, D., Nguyen, D. & English, A.M. (2019) Ctt1 catalase activity potentiates anti fungalazoles in the emerging opportunistic pathogen Saccharomyces cerevisiae. Scientific Reports, 9, 9185.
• Hagan, I. M., Grallert, A., & Simanis, V. (2016). Analysis of the Schizosaccharomyces pombe Cell Cycle. Cold Spring Harbor Protocols, 2016, pdb.top082800.
• Koyama, M., Nagakura, W., Tanaka, H., Kujirai, T., Chikashige, Y., Haraguchi, T., Hiraoka, Y., & Kurumizaka, H. (2017). In vitro reconstitution and biochemical analyses of the Schizosaccharomyces pombe nucleosome. Biochemical and Biophysical Research Communications, 482, 896–901.
• Lin, S. J., & Austriaco, N. (2014). Aging and cell death in the other yeasts, Schizosaccharomyces pombe and Candida albicans. FEMS Yeast Research, 14, 119–135.
• Agus, H. H., Sengoz, C. O., & Yilmaz, S. (2019). Oxidative stress-mediated apoptotic cell death induced by camphor in sod1-deficient Schizosaccharomyces pombe. Toxicology Research, 8, 216–226.
• Madeo, F., Herker, E., Wissing, S., Jungwirth, H., Eisenberg, T., & Fröhlich, K.-U. (2004). Apoptosis in yeast. Current Opinion in Microbiology, 7, 655–660.
• Sajiki, K., Hatanaka, M., Nakamura, T., Takeda, K., Shimanuki, M., Yoshida, T., Hanyu, Y., Hayashi, T., Nakaseko, Y., & Yanagida, M. (2009). Genetic control of cellular quiescence in S. pombe. Journal of Cell Science, 122, 1418–29.
• Lock, A., Rutherford, K., Harris, M. A., & Wood, V. (2018). PomBase: The Scientific Resource for Fission Yeast. Methods in Molecular Biology, 1757, 49–68.
• Liu, M., Huang, Y., Wen, H., & Qiu, G. (2015). Comparing Cell Toxicity of Schizosaccharomyces pombe Exposure to Airborne PM2.5 from Beijing and Inert Particle SiO2. Huan Jing Ke Xue= Huanjing Kexue, 36, 3943–51.
• Olayanju, B., Hampsey, J. J., & Hampsey, M. (2015). Genetic analysis of the Warburg effect in yeast. Advances in Biological Regulation, 57, 185–192.
• Carmona-Gutierrez, D., Reisenbichler, A., Heimbucher, P., Bauer, M. A., Braun, R. J., Ruckenstuhl, C., Büttner, S., Eisenberg, T., Rockenfeller, P., Fröhlich, K.-U., Kroemer, G., & Madeo, F. (2011). Ceramide triggers metacaspase-independent mitochondrial cell death in yeast. Cell Cycle, 10, 3973–3978.
• Natter, K., & Kohlwein, S. D. (2013). Yeast and cancer cells - common principles in lipid metabolism. Biochimica et Biophysica Acta, 1831, 314–326.
• Villahermosa, D., Knapp, K., & Fleck, O. (2017). A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents. Current Genetics, 63, 1081–1091.
• Castro, C., Flores, D.-L., Cervantes-Vásquez, D., Vargas-Viveros, E., Gutiérrez-López, E., & Muñoz-Muñoz, F. (2019). An agent-based model of the fission yeast cell cycle. Current Genetics, 65, 193–200.
• Vishwanatha, A., & D’Souza, C. J. M. (2017). Multifaceted effects of antimetabolite and anticancer drug, 2-deoxyglucose on eukaryotic cancer models budding and fission yeast. IUBMB Life, 69, 137–147.
• Carmona-Gutierrez, D., Bauer, M. A., Zimmermann, A., Aguilera, A., Austriaco, N. et al. (2018). Guidelines and recommendations on yeast cell death nomenclature. Microbial Cell, 5, 4-31.
• Emami, P & Ueno, M. (2021). 3,3’-Diindolylmethane induces apoptosis and autophagy in fission yeast. BioRxiv. (preprint). DOI: https://doi.org/10.1101/2021.08.05.455326
• Falcone, C. & Mazzoni, C. (2016). External and internal triggers of cell death in yeast. Cellular and Molecular Life Sciences, 73, 2237–2250.
• Du, L., Yu, Y., Chen, J., Liu, Y., Xia, Y., Chen, Q., & Liu, X. (2007). Arsenic induces caspase-and mitochondria-mediated apoptosis in Saccharomyces cerevisiae. FEMS Yeast Research, 7, 860–865.
• Chazotte, B. (2011). Labeling nuclear DNA using DAPI. Cold Spring Harbor Protocols, 2011, pdb.prot5556.
• Pajaniradje, S., Mohankumar, K., Pamidimukkala, R., Subramanian, S., & Rajagopalan, R. (2014). Antiproliferative and apoptotic effects of sesbania grandiflora leaves in human cancer cells. BioMed Research International, 2014, 474953.
• Agus, H. H., Kok, G., Derinoz, E., Oncel, D., & Yilmaz, S. (2020). Involvement of Pca1 in ROS-mediated apoptotic cell death induced by alpha-thujone in the fission yeast (Schizosaccharomyces pombe). FEMS Yeast Research, 20, foaa022.
• Azad, G. K., Singh, V., Mandal, P., Singh, P., Golla, U., Baranwal, S., Chauhan, S., & Tomar, R. S. (2014). Ebselen induces reactive oxygen species (ROS)-mediated cytotoxicity in Saccharomyces cerevisiae with inhibition of glutamate dehydrogenase being a target. FEBS Open Bio.,4, 77–89.
• Kwolek-Mirek, M., & Zadrag-Tecza, R. (2014). Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Research, 14, 1068–1079.
• Yörük, E. (2018). Tetraconazole Leads To Alterations In Fusarium Graminearum At Different Molecular Levels. Applied Ecology and Environmental Research, 16, 6155–6167.
• Salucci, S., Burattini, S., Falcieri, E., & Gobbi, P. (2015). Three-dimensional apoptotic nuclear behavior analyzed by means of Field Emission in Lens Scanning Electron Microscope. European Journal of Histochemistry: EJH, 59, 2539.
• Mutoh, N., Kitajima, S., & Ichihara, S. (2011). Apoptotic Cell Death in the Fission Yeast Schizosaccharomyces pombe Induced by Valproic Acid and Its Extreme Susceptibility to pH Change. Bioscience, Biotechnology, and Biochemistry, 75, 1113–1118.
• Muñoz, M., Cedeño, R., Rodríguez, J., Van Der Knaap, W. P. W., Mialhe, E., & Bachère, E. (2000). Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei. Aquaculture, 191, 89–107.
• Zhu, S., Luo, F., Zhu, B., & Wang, G.-X. (2017). Toxicological effects of graphene oxide on Saccharomyces cerevisiae. Toxicology Research, 6, 535–543.
• Schnabel, D., Salas-Vidal, E., Narváez, V., del Rayo Sánchez-Carbente, M., Hernández-García, D., Cuervo, R., & Covarrubias, L. (2006). Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death. Developmental Biology, 291, 291–299.
• Barroso, G., Taylor, S., Morshedi, M., Manzur, F., Gaviño, F., & Oehninger, S. (2006). Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations. Fertility and Sterility, 85, 149–154.
• Baracca, A., Sgarbi, G., Solaini, G., & Lenaz, G. (2003). Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. Biochimica et Biophysica Acta, 1606, 137–146.
• Guérin, R., Arseneault, G., Dumont, S., & Rokeach, L. A. (2008). Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast. Molecular Biology of the Cell, 19, 4404–20.
• Low, C. P., & Yang, H. (2008). Programmed cell death in fission yeast Schizosaccharomyces pombe. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1783, 1335–1349.
• Lin, S. J., & Austriaco, N. (2014). Aging and cell death in the other yeasts, Schizosaccharomyces pombe and Candida albicans. FEMS Yeast Research, 14, 119–135.
• Sevrioukova, I. F. (2011). Apoptosis-inducing factor: structure, function, and redox regulation. Antioxidants & Redox Signaling, 14, 2545–2579.
• Azzopardi, M., Farrugia, G., & Balzan, R. (2017). Cell-cycle involvement in autophagy and apoptosis in yeast. Mechanisms of Ageing and Development, 161, 211–224.
• Yardımcı, B. K. (2020). Imidazole Antifungals: A Review of Their Action Mechanisms on Cancerous Cells. International Journal of Secondary Metabolite, 2020, 139–159.
• Zhang, C., Lai, S. H., Yang, H. H., Xing, D. G., Zeng, C. C., Tang, B., Wan, D., & Liu, Y. J. (2017). Photoinduced ROS regulation of apoptosis and mechanism studies of iridium(iii) complex against SGC-7901 cells. RSC Advances, 7, 17752–17762.
• Macar, O. (2021). Multiple toxic effects of tetraconazole in Allium cepa L. meristematic cells. Environmental Science and Pollution Research, 28, 10092–10099.
• Wang, J., Luo, B., Li, X., Lu, W., Yang, J., Hu, Y., Huang, P., & Wen, S. (2017). Inhibition of cancer growth in vitro and in vivo by a novel ROS-modulating agent with ability to eliminate stem-like cancer cells. Cell Death & Disease, 8, e2887.
• Bhat, A. H., Dar, K. B., Anees, S., Zargar, M. A., Masood, A., Sofi, M. A., & Ganie, S. A. (2015). Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight. Biomedicine & Pharmacotherapy, 74, 101–110.
• Li, J., Liu, X., Zhang, Y., Tian, F., Zhao, G., Yu, Q., Jiang, F., & Liu, Y. (2012). Toxicity of nano zinc oxide to mitochondria. Toxicology Research, 1, 137.
• Lim, H. W., Kim, S. J., Park, E. H., & Lim, C. J. (2007). Overexpression of a metacaspase gene stimulates cell growth and stress response in Schizosaccharomyces pombe. Canadian Journal of Microbiology, 53, 1016–1023.
• Chaves, S. R., Rego, A., Martins, V. M., Santos-Pereira, C., Sousa, M. J., & Côrte-Real, M. (2021). Regulation of Cell Death Induced by Acetic Acid in Yeasts. Frontiers in Cell and Developmental Biology, 9, 642375.
• Amigoni, L., Frigerio, G., Martegani, E., & Colombo, S. (2016). Involvement of Aif1 in apoptosis triggered by lack of Hxk2 in the yeast Saccharomyces cerevisiae. FEMS Yeast Research, 16, fow016.
• Muzaffar, S., & Chattoo, B. B. (2017). Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae. Apoptosis, 22, 463–474.
• Ağuş, H. H., Yilmaz, S., & Şengöz, C. O. (2019). Crosstalk between autophagy and apoptosis induced by camphor in Schizosaccharomyces pombe. Turkish Journal of Biology, 10.3906/biy-1908-11.
• Núñez, A., Dulude, D., Jbel, M., & Rokeach, L. A. (2015). Calnexin is essential for survival under nitrogen starvation and stationary phase in Schizosaccharomyces pombe. PloS One, 10, e0121059.