Zinc attenuates ecstasy-induced apoptosis through downregulation of caspase-3 in cultured TM3 cells: An experimental study


Background: 3, 4-Methylenedioxymethamphetamine (MDMA) is commonly known as the most famous amphetamine derivative.

Objective: To evaluate the influence of zinc on MDMA-induced apoptosis and caspase- 3 gene expression in Leydig cell line (TM3).

Materials and Methods: Leydig cells were studied in differenet treatment groups regarding MDMA (0, 0.5, 1, 3, 5 mM) and zinc (0, 4, 8, 16, 32 μM). By the way, the effective concentration was determined to be 5 mM for MDMA and 8 μM for zinc. Then, TM3 cells were cultured in free medium as control (group I), medium containing MDMA (5 mM) (group II), zinc (8 μM) (group III), and zinc (8 μM) prior to MDMA (5 mM) (group IV) as well as in an untreated group (control). Cell viability was assessed at different times after cell culture by MTT assay. The mRNA expression level of caspase-3 was analyzed using real-time quantitative polymerase chain reaction.

Results: The cellular viability was significantly reduced in TM3 cells after 24 hr and 48 hr exposure time regarding different concentrations of MDMA as well as high concentration of zinc (16 and 32 μM). Cell viability was increased in the group that received zinc (8 μM) before addition of MDMA (5 mM) compared to the control and MDMA groups. The mean ± SE of fold was 22.40 ± 7.5, 0.06 ± 0.02, and 0.009 ± 0.003 in MDMA, zinc, and zinc + MDMA groups, respectively. The mean of caspase-3 mRNA level was significantly increased in the MDMA-treated group (5 mM), while the relative expression of caspase-3 gene was significantly decreased in the zinc (8 μM) + MDMA (5 mM) group compared with the MDMA (5 mM) group (p = 0.001).

Conclusion: Dietary intake of zinc has a protective effect against MDMA consumption in mouse.

Key words: Zinc, MDMA, Apoptosis, TM3 cells.

[1] Mohamed WMY, Ben Hamida S, Cassel JC, de Vasconcelos AP, Jones BC. MDMA: interactions with other psychoactive drugs. Pharmacol Biochem Behav 2011; 99: 759–774.

[2] Rochester JA, Kirchner JT. Ecstasy (3,4- methylenedioxymethamphetamine): history, neurochemistry, and toxicology. J Am Board Fam Pract 1999; 12: 137–142.

[3] Verrico CD, Miller GM, Madras BK. MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment. Psychopharmacology (Berl) 2007; 189: 489–503.

[4] Sprague JE, Banks ML, Cook VJ, Mills EM. Hypothalamicpituitary- thyroid axis and sympathetic nervous system involvement in hyperthermia induced by 3,4- methylenedioxymethamphetamine (Ecstasy). J Pharmacol Exp Ther 2003; 305: 159–166.

[5] Wetherell MA, Montgomery C. Basal functioning of the hypothalamic-pituitary-adrenal (HPA) axis and psychological distress in recreational ecstasy polydrug users. Psychopharmacology (Berl) 2014; 231: 1365–1375.

[6] Barenys M, Gomez-Catalan J, Camps L, Teixido E, de Lapuente J, Gonzalez-Linares J, et al. MDMA (ecstasy) delays pubertal development and alters sperm quality after developmental exposure in the rat. Toxicol Lett 2010; 197: 135–142.

[7] Barenys M, Macia N, Camps L, de Lapuente J, Gomez- Catalan J, Gonzalez-Linares J, et al. Chronic exposure to MDMA (ecstasy) increases DNA damage in sperm and alters testes histopathology in male rats. Toxicol Lett 2009; 191: 40–46.

[8] MacLeod J. The role of oxygen in the metabolism and motility of human spermatozoa. Am J Physiol 1943; 138: 512–518.

[9] Gharagozloo P, Aitken RJ. The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod 2011; 26: 1628–1640.

[10] Kao SH, Chao HT, Chen HW, Hwang TIS, Liao TL, Wei YH. Increase of oxidative stress in human sperm with lower motility. Fertil Steril 2008; 89: 1183–1190.

[11] Esteves SC, Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37: 5–15.

[12] Agarwal A, Allamaneni SSR. Free radicals and male reproduction. J Indian Med Assoc 2011; 109: 184–187.

[13] Khosrowbeygi A. [The role of oxidative stress in male infertility: A review]. Arak Medical University Journal 2013; 15: 94–103. (in Persian)

[14] Peak TC, Haney NM, Wang W, DeLay KJ, Hellstrom WJ. Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism. World J Stem Cells 2016; 8: 306–315.

[15] Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A. In vitro evaluation of cell/ biomaterial interaction by MTT assay. Biomaterials 1993; 14: 359–364.

[16] Pourmasumi S, Sabeti P, Rahiminia T, Mangoli E, Tabibnejad N, Talebi AR. The etiologies of DNA abnormalities in male infertility: An assessmentand review. Int J Reprod Biomed 2017; 15: 331–344.

[17] Montiel-Duarte C, Varela-Rey M, Osés-Prieto JA, López- Zabalza MJ, Beitia G, Cenarruzabeitia E, et al. 3,4- Methylenedioxymethamphetamine (“Ecstasy”) induces apoptosis of cultured rat liver cells. Biochim Biophys Acta 2002; 1588: 26–32.

[18] Tuerk MJ, Fazel N. Zinc deficiency. Curr Opin Gastroenterol 2009; 25: 136–143.

[19] Yan M, Song Y, Wong CP, Hardin K, Ho E. Zinc deficiency alters DNA damage response genes in normal human prostate epithelial cells. J Nutr 2008; 138: 667–673.

[20] Stefanidou M, Maravelias C, Dona A, Spiliopoulou C. Zinc: a multipurpose trace element. Arch Toxicol 2006; 80: 1–9.

[21] Prasad AS. Impact of the discovery of human zinc deficiency on health. J Am Coll Nutr 2009; 28: 257–265.

[22] Chasapis CT, Loutsidou AC, Spiliopoulou CA, Stefanidou ME. Zinc and human health: an update. Arch Toxicol 2012; 86: 521–534.

[23] Agarwal A, Virk G, Ong C, du Plessis SS. Effect of oxidative stress on male reproduction. World J Mens Health 2014; 32: 1–17.

[24] Pourhassanali N, Roshan-Milani S, Kheradmand F, Motazakker M, Bagheri M, Saboory E. Zinc attenuates ethanol-induced Sertoli cell toxicity and apoptosis through caspase-3 mediated pathways. Reprod Toxicol 2016; 61: 97–103.

[25] Carvalho M, Remião F, Milhazes N, Borges F, Fernandes E, Cen Monteiro Md, et al. Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro. Chem Res Toxicol 2004; 17: 623–632.

[26] Bora F, Yilmaz F, Bora T. Ecstasy (MDMA) and its effects on kidneys and their treatment: A review. Iran J Basic Med Sci 2016; 19: 1151–1158.

[27] Liechti ME. Effects of MDMA on body temperature in humans. Temperature 2014; 1: 192–200.

[28] Song BJ, Moon KH, Upreti VV, Eddington ND, Lee IJ. Mechanisms of MDMA (ecstasy)-induced oxidative stress, mitochondrial dysfunction, and organ damage. Curr Pharm Biotechnol 2010; 11: 434–443.

[29] Dickerson SM, Walker DM, Reveron ME, Duvauchelle CL, Gore AC. The recreational drug ecstasy disrupts the hypothalamic-pituitarygonadal reproductive axis in adult male rats. Neuroendocrinology 2008; 88: 95–102.

[30] Café C, Torri C, Bertorelli L, Tartara F, Tancioni F, Gaetani P, et al. Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex. Free Radic Biol Med 1995; 19: 853–857.

[31] Soleimani Asl S, Pourheydar B, Dabaghian F, Nezhadi A, Roointan A, Mehdizadeh M. Ecstasy-induced caspase expression alters following ginger treatment. Basic Clin Neurosci 2013; 4: 329–333.

[32] Montgomery T, Sitte H, McBean G. 4- Methylthioamphetamine (4-MTA) induces mitochondrialdependent apoptosis in SH-SY5Y cells independently of dopamine and noradrenaline transporters. BMC Pharmacology 2010; 10 (Suppl.): A22.

[33] Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochim Biophys Acta 2016; 1863: 2977–2992.

[34] Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 1995; 270: 96–99.

[35] Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh AJ. Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology 1995; 136: 5–12.

[36] Pentikäinen V, Erkkilä K, Suomalainen L, Otala M, Pentikäinen MO, Parvinen M, et al. TNFα down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis. J Clin Endocrinol Metab 2001; 86: 4480– 4488.

[37] Skulachev VP, Chistyakov VV, Jasaitis AA, Smirnova EG. Inhibition of the respiratory chain by zinc ions. Biochem Biophys Res Commun 1967; 26: 1–6.

[38] Maret W. The function of zinc metallothionein: a link between cellular zinc and redox state. J Nutr 2000; 130 (Suppl.): 1455S–1458S.