Assessing the Reducing Effect of Coenzyme Q10 on Carbendazim-Induced Testicular Tissue Dysfunction Through Modulation of miR-202-5p/Apoptosis Signaling in Rats: A Histological and Immunohistochemical Study

Articles in Press

Document Type : Research Article

Authors

• Sobhan Karimi ¹
• Ali Louei Monfared ¹
• Hassaneen Sharoot ²

¹ Department of Histology, Faculty of Veterinary Sciences, Ilam University, Ilam, Iran.

² Department of Anatomy and Histology, College of Veterinary Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq.

10.22067/ijvst.2025.89476.1411

Abstract

Widespread application of carbendazim (Carb) in agriculture and veterinary is a major environmental concern because of its residues that disrupt spermatogenesis. Recently, coenzyme Q10 (CoQ10) supplementation has demonstrated various health benefits due to its anti-apoptosis and anti-inflammatory nature. Thus, the present study aimed to investigate the possible mechanistic pathway of CoQ10 in Carb-induced reproductive dysfunction in male rats. Adult male Wistar rats were orally exposed to Carb (150 mg/kg) singly or in combination with CoQ10 (200 mg/kg). The rats received their treatments daily for 9 weeks. At the end of the work, the testis specimens were excised for histological (H & E staining), immunohistochemical, hormonal, and molecular (real-time quantitative PCR) assessments. The Carb group showed adverse testicular alterations confirmed by immunostaining and demonstrated a significant upregulation of Bax and Caspase-3 expression, while exhibiting a notable reduction in the immunopositivity of Bcl-2 protein within the testes of rats. Real-time PCR analysis revealed that Carb treatment decreased the expression of miR-202-5p with a concomitant decline in concentrations of testosterone and LH hormones.  Conversely, in Carb-treated rats, co-treatment with CoQ10 restored the tissue architecture, hormonal disturbance, and declined apoptotic index to near control level. In addition, high expression of miR-202-5p was observed in the Carb + CoQ10 group, and testicular tissues returned to nearly normal histological architecture. We concluded that Carb causes adverse testicular alterations via miR-202-5p/apoptosis pathway, and CoQ10 may prove useful in combating Carb-induced adverse effects via its anti-apoptotic and gene regulatory effects.

Keywords

• Histology
• Testis
• Carbendazim
• Rat
• Coenzyme Q10

Main Subjects

• Histology

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References

1.    Zhou T, Guo T, Wang Y, Wang A, Zhang M. Carbendazim: Ecological risks, toxicities, degradation pathways and potential risks to human health. Chemosphere. 2023;314:137723. Doi: 10.1016/j.chemosphere.2022.137723.
2.    Verma S, Srivastava A. Cyto-genotoxic consequences of carbendazim treatment monitored by cytogenetical analysis using Allium root tip bioassay. Environ Monit Assess. 2018;190(4):238. Doi: 10.1007/s10661-018-6616-4. 
3.    Hashim M, Al-Attar AM, Alomar MY, Shaikh Omar AM, Alkenani NA, Abu Zeid IM. Alleviation of carbendazim toxicity effect by Moringa oleifera oil and Linum usitatissimum L. oil on testes of male rats: Physiological, histological and in silico study. Saudi J Biol Sci. 2024;31(2):103921. Doi: 10.1016/j.sjbs.2023.103921.
4.    Owumi SE, Nowozo SO, Najophe ES. Quercetin abates induction of hepatic and renal oxidative damage, inflammation, and apoptosis in carbendazim-treated rats. Toxicol Res Appl. 2019;3:1-8.
5.    Ebedy YA, Elshazly MO, Hassan NH, Ibrahim MA, Hassanen EI. Novel insights into the potential mechanisms underlying carbendazim-induced hepatorenal toxicity in rats. J Biochem Mol Toxicol. 2022;36(8):e23079. doi: 10.1002/jbt.23079. 
6.    Rajeswary S, Kumaran B, Ilangovan R, Yuvaraj S, Sridhar M, Venkataraman P, Srinivasan N, Aruldhas MM. Modulation of antioxidant defense system by the environmental fungicide carbendazim in Leydig cells of rats. Reprod Toxicol. 2007;24(3-4):371-80. Doi: 10.1016/j.reprotox.2007.03.010.
7.    Liu J, Zhang P, Zhao Y, Zhang H. Low dose carbendazim disrupts mouse spermatogenesis might be through estrogen receptor related histone and DNA methylation. Ecotoxicol Environ Saf. 2019;176:242-9. Doi: 10.1016/j.ecoenv.2019.03.103.
8.    Sharma M., Maheshwari N., Khan F.H., Mahmood R. Carbendazim toxicity in different cell lines and mammalian tissues. J Biochem Mol Toxicol. 2022;36(12):e23194.
9.    Zari AT, Al-Attar MA. Therapeutic effects of olive leaves extract on rats treated with a sublethal concentration of carbendazim. Eur Rev Med Pharmacol Sci. 2011;15:413-26.
10.    Abolaji AO, Awogbindin IO, Adedara IA, Farombi EO. Insecticide chlorpyrifos and fungicide carbendazim, common food contaminants mixture, induce hepatic, renal, and splenic oxidative damage in female rats. Hum Exp Toxicol. 2017;36(5):483-93.
11.    Patil NV, Lonare MK, Sharma M, Deshmukh S, Gupta K, Sharma SK. Ameliorative effect of quercetin on neurotoxicogical alterations induced by carbendazim: oxidative stress, biochemicals, and histopathology. Proc Natl Acad Sci India Sect B: Biol Sci. 2022;2022:1-14.
12.    Patil SV, Mohite BV, Marathe KR, Salunkhe NS, Marathe V, Patil VS. Moringa tree, gift of nature: a review on nutritional and industrial potential. Curr Pharmacol Rep. 2022;8:262-280.
13.    Moffit JS, Bryant BH, Hall SJ, Boekelheide K. Dose-dependent effects of sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis. Toxicol Pathol. 2007;35(5):719-27. Doi: 10.1080/01926230701481931.
14.    Adedara IA, Vaithinathan S, Jubendradass R, Mathur PP, Farombi EO. Kolaviron prevents carbendazim-induced steroidogenic dysfunction and apoptosis in testes of rats. Environ Toxicol Pharmacol. 2013;35(3):444-53. Doi: 10.1016/j.etap.2013.01.010.
15.    Sakr SA, Shalaby SY. Carbendazim-induced testicular damage and oxidative stress in albino rats: ameliorative effect of licorice aqueous extract. Toxicol Ind Health. 2014;30(3):259-67. Doi: 10.1177/0748233712456059.  
16.    Raizner AE. Coenzyme Q10. Methodist Debakey Cardiovasc J. 2019;15(3):185-191. Doi: 10.14797/mdcj-15-3-185.
17.    Karimi F, Khodabandeh Z, Nazari F, Dara M, Masjedi F, Momeni-Moghaddam M. Post-Weaning Exposure to Sunset Yellow FCF Induces Changes in Testicular Tight and Gap Junctions in Rats: Protective Effects of Coenzyme Q10. Reprod Sci. 2023;30(10):2962-72. Doi: 10.1007/s43032-023-01240-w.
18.    Erol B, Bozlu M, Hanci V, Tokgoz H, Bektas S, Mungan G. Coenzyme Q10 treatment reduces lipid peroxidation, inducible and endothelial nitric oxide synthases, and germ cell-specific apoptosis in a rat model of testicular ischemia/reperfusion injury. Fertil Steril. 2010;93:280-2.
19.    Mirmalek SA, Gholamrezaei Boushehrinejad A, Yavari H, Kardeh B, Parsa Y, Salimi-Tabatabaee SA, Yadollah-Damavandi S, Parsa T, Shahverdi E, Jangholi E. Antioxidant and anti-inflammatory effects of coenzyme Q10 on L-arginine-induced acute pancreatitis in rat. Oxidative Med Cell Longev. 2016;2016:5818479.
20.    Gules Ö, Kum Ş, Yıldız M, Boyacıoğlu M, Ahmad E, Naseer Z, Eren Ü. Protective effect of coenzyme Q10 against bisphenol-A-induced toxicity in the rat testes. Toxicol Ind Health. 2019;35(7):466-481. Doi: 10.1177/0748233719862475.
21.    El-Khadragy M, Al-Megrin WA, AlSadhan NA, Metwally DM, El-Hennamy RE, Salem FEH, Kassab RB, Abdel Moneim AE. Impact of Coenzyme Q10 Administration on Lead Acetate-Induced Testicular Damage in Rats. Oxid Med Cell Longev. 2020;2020:4981386. Doi: 10.1155/2020/4981386.
22.    Zheng B, Zhou Q, Guo Y, Shao B, Zhou T, Wang L, Zhou Z, Sha J, Guo X, Huang X. Establishment of a proteomic profile associated with gonocyte and spermatogonial stem cell maturation and differentiation in neonatal mice. Proteomics. 2014;14(2-3):274-85. Doi: 10.1002/pmic.201300395.
23.    Barbu MG, Thompson DC, Suciu N, Voinea SC, Cretoiu D, Predescu DV. The Roles of MicroRNAs in Male Infertility. Int J Mol Sci. 2021;22(6):2910. Doi: 10.3390/ijms22062910. 
24.    Asadpour R, Mofidi Chelan E. Using microRNAs as molecular biomarkers for the evaluation of male infertility. Andrologia. 2022;54(2):e14298. Doi: 10.1111/and.14298. 
25.    Chen J, Gao C, Lin X, Ning Y, He W, Zheng C, Zhang D, Yan L, Jiang B, Zhao Y, Alim Hossen M, Han C. The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis. Development. 2021;148(24):dev199799. Doi: 10.1242/dev.199799.
26.    Fernández-Pérez D, Brieño-Enríquez MA, Isoler-Alcaraz J, Larriba E, & Del Mazo J. MicroRNA dynamics at the onset of primordial germ and somatic cell sex differentiation during mouse embryonic gonad development. RNA. 2018;24(3):287-303.
27.    Papaioannou MD, Nef S. microRNAs in the testis: Building up male fertility. J Androl. 2010;31(1):26-33.  Doi: 10.2164/jandrol.109.008128.
28.    Le Moal J, Rolland M, Goria S, Wagner V, De Crouy-Chanel P, Rigou A, De Mouzon J, Royère D. Semen quality trends in French regions are consistent with a global change in environmental exposure. Reproduction. 2014;147(4):567-74. Doi: 10.1530/REP-13-0499. 
29.    Mehrpour O, Karrari P, Zamani N, Tsatsakis AM, Abdollahi M. Occupational exposure to pesticides and consequences on male semen and fertility: a review. Toxicol Lett. 2014;230(2):146-56. Doi: 10.1016/j.toxlet.2014.01.029.
30.    Yu G, Guo Q, Xie L, Liu Y, Wang X. Effects of subchronic exposure to carbendazim on spermatogenesis and fertility in male rats. Toxicol Ind Health. 2009;25(1):41-7. Doi: 10.1177/0748233709103033.
31.    Li H, Zhang P, Zhao Y, Zhang H. Low doses of carbendazim and chlorothalonil synergized to impair mouse spermatogenesis through epigenetic pathways. Ecotoxicol Environ Saf. 2020;188:109908. Doi: 10.1016/j.ecoenv.2019.109908.
32.    Eid RA, Abadi AM, El-Kott AF, Zaki MSA, Abd-Ella EM. The antioxidant effects of coenzyme Q10 on albino rat testicular toxicity and apoptosis triggered by bisphenol A. Environ Sci Pollut Res Int. 2023;30(14):42339-42350. Doi: 10.1007/s11356-022-24920-7. 
33.    Gou C, Zhou Z, Chen Z, Wang K, Chen C, Chen B, Pan N, He X. Studies on improving semen quality and increasing pregnancy chances through the in vitro addition of L-carnitine and coenzyme Q10 to semen in patients with asthenozoospermia. Basic Clin Androl. 2022;32(1):17.
34.    Alahmar AT, Sengupta P. Impact of Coenzyme Q10 and Selenium on Seminal Fluid Parameters and Antioxidant Status in Men with Idiopathic Infertility. Biol Trace Elem Res. 2021;199(4):1246-1252. Doi: 10.1007/s12011-020-02251-3.
35.    Mancini A, Balercia G. Coenzyme Q(10) in male infertility: physiopathology and therapy. Biofactors. 2011;37(5):374-80. Doi: 10.1002/biof.164.
36.    El-Khadragy M, Al-Megrin WA, AlSadhan NA, Metwally DM, El-Hennamy RE, Salem FEH, Kassab RB, Abdel Moneim AE. Impact of Coenzyme Q10 Administration on Lead Acetate-Induced Testicular Damage in Rats. Oxid Med Cell Longev. 2020;2020:4981386. Doi: 10.1155/2020/4981386.
37.    Arafa EA, Hassanein EHM, Ibrahim NA, Buabeid MA, Mohamed WR. Involvement of Nrf2-PPAR-γ signaling in Coenzyme Q10 protecting effect against methotrexate-induced testicular oxidative damage. Int Immunopharmacol. 2024;129:111566. Doi: 10.1016/j.intimp.2024.111566. 
38.    Chen J, Cai T, Zheng C, Lin X, Wang G, Liao S, Wang X, Gan H, Zhang D, Hu X, Wang S, Li Z, Feng Y, Yang F, Han C. MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins. Nucleic Acids Res. 2017;45(7):4142-4157. Doi: 10.1093/nar/gkw1287.
39.    Dabaja AA, Mielnik A, Robinson BD, Wosnitzer MS, Schlegel PN, Paduch DA. Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis. Basic Clin Androl. 2015;25:2. Doi: 10.1186/s12610-015-0018-z.
40.    Sakurai K, Mikamoto K, Shirai M, Iguchi T, Ito K, Takasaki W, Mori K. MicroRNA profiles in a monkey testicular injury model induced by testicular hyperthermia. J Appl Toxicol. 2016;36(12):1614-1621. Doi: 10.1002/jat.3326.
41.    Mohammed BT, Donadeu FX. Localization and in silico-based functional analysis of miR-202 in bull testis. Reprod Domest Anim. 2022;57(9):1082-1087. Doi: 10.1111/rda.14159.
42.    Yang C, Yao C, Tian R, Zhu Z, Zhao L, Li P, Chen H, Huang Y, Zhi E, Gong Y, Xue Y, Wang H, Yuan Q, He Z, Li Z. miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling. Mol Ther Nucleic Acids. 2019;14:1-19. Doi: 10.1016/j.omtn.2018.10.012.
43.    Wainwright EN, Jorgensen JS, Kim Y, Truong V, Bagheri-Fam S, Davidson T, Svingen T, Fernandez-Valverde SL, McClelland KS, Taft RJ, Harley VR, Koopman P, Wilhelm D. SOX9 regulates microRNA miR-202-5p/3p expression during mouse testis differentiation. Biol Reprod. 2013;89(2):34. Doi: 10.1095/biolreprod.113.110155. 
44.    Garcia MS, Cavalcante DNC, Araújo Santiago MDS, de Medeiros PDC, do Nascimento CC, Fonseca GFC, Le Sueur-Maluf L, Perobelli JE. Reproductive toxicity in male juvenile rats: Antagonistic effects between isolated agrochemicals and in binary or ternary combinations. Ecotoxicol Environ Saf. 2021;209:111766. Doi: 10.1016/j.ecoenv.2020.111766.
45.    Alghamdi SA. Effect of Nigella sativa and Foeniculum vulgare seeds extracts on male mice exposed to carbendazim. Saudi J Biol Sci. 2020;27(10):2521-2530. doi: 10.1016/j.sjbs.2020.04.016. 
46.    Kennedy C, Okanya P, Nyariki JN, Amwayi P, Jillani N, Isaac AO. Coenzyme Q10 nullified khat-induced hepatotoxicity, nephrotoxicity and inflammation in a mouse model. Heliyon. 2020;6(9):e4917. Doi: 10.1016/j.heliyon.2020.e04917.
47.    Carrasco J, Anglada FJ, Campos JP, Muntane J, Requena MJ, Padillo J. The protective role of coenzyme Q10 in renal injury associated with extracorporeal shockwave lithotripsy: a randomised, placebo-controlled clinical trial. BJU Int. 2014;113(6):942-950. Doi: 10.1111/bju.12485.
48.    Bakhtiary Z, Shahrooz R, Ahmadi A, Soltanalinejad F. Protective effect of ethyl pyruvate on testicular histology and fertilization potential in cyclophosphamide treated mice. Vet Res Forum. 2020;11(1):7-13. Doi: 10.30466/vrf.2018.91253.2047.
49.    Abd-Elkareem M, Abd El-Rahman MAM, Khalil NSA et al. Antioxidant and cytoprotective effects of Nigella sativa L. seeds on the testis of monosodium glutamate challenged rats. Sci Rep. 2021;11:13519.  Doi: 10.1038/s41598-021-92977-4.