Dose- and Time-Dependent Inhibitory Effects of Mancozeb on Viability and Steroid Synthesis of Bovine Granulosa Cells In Vitro
DOI:
https://doi.org/10.71336/ijvar.770Keywords:
Bovine granulosa cells, Endocrine disruption, Mancozeb, Reproductive toxicology, Steroidogenesis, ViabilityAbstract
Mancozeb (MAN) is a widely used fungicide considered an endocrine disrupting compound with potential reproductive toxicity. This study aimed to evaluate the cytotoxic and endocrine-disrupting effects of MAN on bovine granulosa cells (bGCs) by assessing cell viability and steroid hormone secretion in vitro. Bovine granulosa cells were isolated from ovarian follicles of slaughtered healthy cows at a local abattoir. Cells were then cultured in DMEM/F12 medium and exposed to increasing concentrations of MAN (0 - 100 µM) for 12, 24, and 48 h. At the end of the experiment, cell viability was assessed using the MTT assay while estradiol (E2) and progesterone (P4) concentrations in culture media were measured using electrochemiluminescence immunoassay. Our results revealed that low concentrations of MAN (0.001–1 µM) did not significantly affect cell viability at any incubation period (p>0.05) while a transient increase in MTT activity was observed at 10 µM after 24 h exposure (p<0.05). In contrast, exposure to 100 µM MAN significantly reduced cell viability at all time points (p<0.001), accompanied by morphological deterioration and reduced cell confluency. Hormone analyses showed that 10 µM MAN did not significantly alter E2 or P4 secretion. However, 100 µM MAN significantly decreased progesterone production at both 24 and 48 h (p<0.001) and significantly reduced estradiol secretion after 48 h exposure (p<0.001). In conclusion, we showed for the first time that MAN exerts dose- and time-dependent inhibition of cellular metabolic activity and steroid secretion of bGC, indicating potential disruption of ovarian function.
References
Arikan S, Simsek O, Kalender H, Essiz D, Atmaca N, Kabakci R, Bilmen FS. 2017. Effects of acetamiprid, methomyl, and deltamethrin on steroidogenesis in cultured bovine luteal cells. Fresenius Environ. Bull., 26(5): 3280-3284.
Atmaca N, Arikan S, Essiz D, Kalender H, Simsek O, Bilmen FS, Kabakci R. 2018. Effects of mancozeb, metalaxyl and tebuconazole on steroid production by bovine luteal cells in vitro. Environ. Toxicol. Pharmacol., 59(2018): 114-118. https://doi.org/10.1016/j.etap.2018.03.009
Atuhaire A, Kaye E, Mutambuze IL, Matthews G, Friedrich T, Jørs E. 2017. Assessment of dithiocarbamate residues on tomatoes conventionally grown in Uganda and the effect of simple washing to reduce exposure risk to consumers. Environ. Health Insights, 111178630217712218. https://doi.org/10.1177/1178630217712218
Cao F, Souders II CL, Li P, Pang S, Liang X, Qiu L, Martyniuk CJ. 2019. Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae. Ecotoxicol. Environ. Saf., 170: 227-237. https://doi.org/10.1016/j.ecoenv.2018.11.110
Cecconi S, Paro R, Rossi G, Macchiarelli G. 2007. The effects of the endocrine disruptors dithiocarbamates on the mammalian ovary with particular regard to mancozeb. Curr. Pharm. Des., 13(29): 2989-3004. https://doi.org/10.2174/138161207782110516
Černohlávková J, Jarkovský J, Hofman J. 2009. Effects of fungicides mancozeb and dinocap on carbon and nitrogen mineralization in soils. Ecotoxicol. Environ. Saf., 72(1): 80-85. https://doi.org/10.1016/j.ecoenv.2008.07.001
Diamanti-Kandarakis E, Bourguignon J-P, Giudice LC, Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC. 2009. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr. Rev., 30(4): 293-342. https://doi.org/10.1210/er.2009-0002
Dinisri I, Kodikara S, Prasadani M, Pathirana I, Rathnayake C, Alexander B, Lee K-F, Kodithuwakku SP. 2021. Impairment of caprine oocyte maturation in vitro and alteration of granulosa cells functions by widely used fungicide mancozeb. Trop. Anim. Health Prod., 53(3): 406. https://doi:10.1007/s11250-021-02854-5
Girish B, Reddy PS. 2018. Forskolin ameliorates mancozeb‐induced testicular and epididymal toxicity in Wistar rats by reducing oxidative toxicity and by stimulating steroidogenesis. J. Biochem. Mol. Toxicol., 32(2): e22026. https://doi.org/10.1002/jbt.22026
Gore AC, Chappell VA, Fenton SE, Flaws JA, Nadal A, Prins GS, Toppari J, Zoeller RT. 2015. EDC-2: the endocrine society's second scientific statement on endocrine-disrupting chemicals. Endocr. Rev., 36(6): E1-E150. https://doi.org/10.1210/er.2015-1010
Hutt KJ, Albertini DF. 2007. An oocentric view of folliculogenesis and embryogenesis. Reprod. Biomed. Online, 14(6): 758-764. https://doi.org/10.1016/S1472-6483(10)60679-7
Iorio R, Castellucci A, Rossi G, Cinque B, Cifone MG, Macchiarelli G, Cecconi S. 2015. Mancozeb affects mitochondrial activity, redox status and ATP production in mouse granulosa cells. Toxicol. In Vitro, 30(1): 438-445. https://doi.org/10.1016/j.tiv.2015.09.018
Kabakci R, Clark KL, Plewes MR, Monaco CF, Davis JS. 2023. Perfluorooctanoic acid (PFOA) inhibits steroidogenesis and mitochondrial function in bovine granulosa cells in vitro. Environ. Pollut., 338122698. https://doi.org/10.1016/j.envpol.2023.122698
Kabakci R, Kaya A, Yigit AA, Varisli O. 2021. Assessment of tebuconazole exposure on bovine testicular cells and epididymal spermatozoa. Acta Vet. Hung., 69(2): 180-188. https://doi.org/10.1556/004.2021.00023
Kabakci R, Yigit AA. 2020. Effects of bisphenol A, diethylhexyl phthalate, and pentabrominated diphenyl ether 99 on steroid synthesis in cultured bovine luteal cells. Reprod. Domest. Anim., 55(6): 683-690. https://doi.org/10.1111/rda.13665
Liu Y, Wang Y-L, He S-w, Chen M-H, Zhang Z, Fu X-P, Fu B-B, Liao B-Q, Lin Y-H, Qi Z-Q. 2016. Protective effects of resveratrol against mancozeb induced apoptosis damage in mouse oocytes. Oncotarget, 8(4): 6233. https://doi.org/10.18632/oncotarget.14056
Mohammed TB, Donadeu FX. 2018. Bovine granulosa cell culture. in Mario Baratta (Ed.), Epithelial Cell Culture: Methods and Protocols. Humana Press, New York, USA, pp. 79-87.
Mohanty B, Pandey SP, Tsutsui K. 2017. Thyroid disrupting pesticides impair the hypothalamic-pituitary-testicular axis of a wildlife bird, Amandava amandava. Reprod. Toxicol., 7132-7141. https://doi.org/10.1016/j.reprotox.2017.04.006
Nordby K-C, Andersen A, Irgens LM, Kristensen P. 2005. Indicators of mancozeb exposure in relation to thyroid cancer and neural tube defects in farmers' families. Scand. J. Work. Environ. Health, 31(2): 89-96. https://doi.org/10.5271/sjweh.855
Palmerini MG, Belli M, Nottola SA, Miglietta S, Bianchi S, Bernardi S, Antonouli S, Cecconi S, Familiari G, Macchiarelli G. 2018. Mancozeb impairs the ultrastructure of mouse granulosa cells in a dose-dependent manner. J. Reprod. Dev., 64(1): 75-82. https://doi.org/10.1262/jrd.2017-143
Paro R, Tiboni GM, Buccione R, Rossi G, Cellini V, Canipari R, Cecconi S. 2012. The fungicide mancozeb induces toxic effects on mammalian granulosa cells. Toxicol. Appl. Pharmacol., 260(2): 155-161. https://doi.org/10.1016/j.taap.2012.02.005
Pocar P, Brevini T, Fischer B, Gandolfi F. 2003. The impact of endocrine disruptors on oocyte competence. Reproduction, 125(3): 313-325. https://doi.org/10.1530/rep.0.1250313
Rossi G, Palmerini MG, Macchiarelli G, Buccione R, Cecconi S. 2006. Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes. Reprod. Toxicol., 22(1): 51-55. https://doi.org/10.1016/j.reprotox.2005.11.005
Runkle J, Flocks J, Economos J, Dunlop AL. 2017. A systematic review of Mancozeb as a reproductive and developmental hazard. Environ. Int., 9929-9942. https://doi.org/10.1016/j.envint.2016.11.006
Sakr S, Okdah Y, El-Adly E. 2009. Effect of ginger (Zingiber officinale) on mancozeb fungicide induced testicular damage in albino rats. Aust. J. Basic Appl. Sci., 3(2): 1328-1333.
Tsagué Manfo FP, Chao W-F, Moundipa PF, Pugeat M, Wang PS. 2011. Effects of maneb on testosterone release in male rats. Drug Chem. Toxicol., 34(2): 120-128. https://doi.org/10.3109/01480545.2010.482589
Wang Z, Kottawatta KS, Kodithuwakku SP, Fernando TS, Lee Y-L, Ng EH, Yeung WS, Lee K-F. 2021. The fungicide Mancozeb reduces spheroid attachment onto endometrial epithelial cells through downregulation of estrogen receptor β and integrin β3 in Ishikawa cells. Ecotoxicol. Environ. Saf., 208111606. https://doi.org/10.1016/j.ecoenv.2020.111606
WHO. 1988. Dithiocarbamate pesticides, ethylenethiourea, and propylenethiourea: a general introduction. World Health Organization, Environ Health Criteria 78 Geneva.
Zago AM, Faria NM, Favero JL, Meucci RD, Woskie S, Fassa AG. 2022. Pesticide exposure and risk of cardiovascular disease: a systematic review. Glob. Public Health, 17(12): 3944-3966. https://doi.org/10.1080/17441692.2020.1808693
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