Antioxidant Defense of Betaine Against Oxidative Stress Induced by Ethanol in the Rat Testes

  • Masoud AlirezaeiEmail author
  • Gholamali Jelodar
  • Zeynab Ghayemi


Oxidative stress is one of the factors associated with decline in fertility and betaine has been shown to bear antioxidant and methyl donor properties in our recent studies. Thus, we designed the present study to examine antioxidant and methyl donor abilities of betaine in oxidative stress induced by ethanol in the rat testes. The adult male Sprague-Dawley rats were divided into four experimental groups and treated daily for 2 months as follows: control, ethanol (4 g/kg, orally), betaine (1.5 % of total diet, orally), and betaine plus ethanol (betaine, 1.5 % of total diet and after 120 min, ethanol 4 g/kg). Sperm motility and concentration significantly increased in betaine group when compared to the ethanol–treated rats. The main antioxidant enzyme (GPx) activity significantly increased (in order compensatory) in ethanol-treated rats when compared to betaine group while, antiperoxidative enzyme (CAT) activity significantly increased in betaine plus ethanol group as compared to ethanol-treated rats. Total homocysteine (tHcy) and TBARS concentration (as a lipid peroxidation marker) also significantly decreased in betaine and betaine plus ethanol groups as compared to ethanol-treated rats. Overall, methyl donor and antioxidant properties of betaine are promising and reduce the elevated tHcy and TBARS concentrations in betaine plus ethanol group. Therefore, betaine might be used as a potential therapy in hyperhomocysteinemia and oxidative stress induced by ethanol in alcoholism.


Betaine Testes Antioxidant status Ethanol Rat 



This research was financially supported by School of Veterinary Medicine-Shiraz University, Shiraz, Iran. We are most grateful to Saeedeh Ahmadi for the kind technical assistance; also like to thank M. Shoaei and R. Shirazi (the member and manager of Aryadalman Company, Tehran, Iran) for providing betaine (Betafine®).

Supplementary material

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  1. Alirezaei M, Saeb M, Javidnia K, Nazifi S, Khalighyan N, Saeb S (2010) Betaine reduction of hyperhomocysteinemia and enhancement of 5-hydroxyindoleacetic acid in ethanol-induced hyperhomocysteinemia in rabbits. Afr J Biochem Res 4(11):246–254Google Scholar
  2. Alirezaei M, Jelodar G, Niknam P, Ghayemi Z, Nazifi S (2011a) Betaine prevents ethanol-induced oxidative stress and reduces total homocysteine in the rat cerebellum. J Physiol Biochem 67:605–612PubMedCrossRefGoogle Scholar
  3. Alirezaei M, Saeb M, Javidnia K, Nazifi S, Saeb S (2011b) Hyperhomocysteinemia reduction in ethanol-fed rabbits by oral betaine. Comp Clin Pathol. doi: 10.1007/s00580-010-1110-6
  4. Alirezaei M, Kheradmand A, Heydari R, Tanideh N, Neamati S, Rashidipour M (2012) Oleuropein protects against ethanol-induced oxidative stress and modulates sperm quality in the rat testis. Mediterr J Nutr Metab :1–7. doi: 10.1007/s12349-011-0079-2
  5. Aurich JE, Schonherr U, Hoppe H, Aurich C (1997) Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology 48(2):185–192PubMedCrossRefGoogle Scholar
  6. Ball BA, Medina V, Gravance CG, Baumber J (2001) Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5 C. Theriogenology 56(4):577–589PubMedCrossRefGoogle Scholar
  7. Barak AJ, Beckenhauer HC (1988) The influence of ethanol on hepatic transmethylation. Alcohol Alcohol 23(1):73–77PubMedGoogle Scholar
  8. Barak AJ, Beckenhauer HC, Tuma DJ (1985) Ethanol feeding inhibits the activity of hepatic N5-methyltetrahydrofolate: homocysteine methyltransferase in the rat. IRCS Med Sci 13:760–761Google Scholar
  9. Barak AJ, Beckenhauer HC, Tuma DJ (2002) Methionine synthase a possible prime site of the ethanolic lesion in liver. Alcohol 26(2):65–67PubMedCrossRefGoogle Scholar
  10. Bauché F, Fouchard MH, Jégou B (1994) Antioxidant system in rat testicular cells. FEBS Lett 349(3):392–396PubMedCrossRefGoogle Scholar
  11. Bidulescu A, Chambless LE, Siega-Riz AM, Zeisel SH, Heiss G (2009) Repeatability and measurement error in the assessment of choline and betaine dietary intake: the atherosclerosis risk in communities (ARIC) study. Nutr J 8(1):14–20PubMedCrossRefGoogle Scholar
  12. Bleich S, Degner D, Sperling W, Bönsch D, Thürauf N, Kornhuber J (2004) Homocysteine as a neurotoxin in chronic alcoholism. Prog Neuropsychopharmacol Biol Psychiatry 28(3):453–464PubMedCrossRefGoogle Scholar
  13. Bottiglieri T (2005) Homocysteine and folate metabolism in depression. Prog Neuropsychopharmacol Biol Psychiatry 29(7):1103–1112PubMedCrossRefGoogle Scholar
  14. Cancel AM, Lobdell D, Mendola P, Perreault SD (2000) Objective evaluation of hyperactivated motility in rat spermatozoa using computer-assisted sperm analysis. Hum Reprod 15(6):1322PubMedCrossRefGoogle Scholar
  15. Chen NH, Reith ME, Quick MW (2004) Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch Eur J Physiol 447:519–531CrossRefGoogle Scholar
  16. Claiborne A (1985) Catalase activity. In: Greenwald RA (ed) CRC handbook of methods for oxygen radical research, Vol 1. CRC Press, Boca Raton, Florida, USA, pp 283–284Google Scholar
  17. Craig SA (2004) Betaine in human nutrition. Am J Clin Nutr 80(3):539–549PubMedGoogle Scholar
  18. Cravo ML, Gloria LM, Selhub J, Nadeau MR, Camilo ME, Resende MP, Cardoso JN, Leitao CN, Mira FC (1996) Hyperhomocysteinemia in chronic alcoholism: correlation with folate, vitamin B-12, and vitamin B-6 status. Am Soc Nutr 63:220–224Google Scholar
  19. Das SK, Vasudevan DM (2007) Alcohol-induced oxidative stress. Life Sci 81(3):177–187PubMedCrossRefGoogle Scholar
  20. Dinu D, Nechifor MT, Movileanu L (2006) Ethanol-induced alterations of the antioxidant defense system in rat kidney. J Biochem Mol Toxicol 19(6):386–395CrossRefGoogle Scholar
  21. Doreswamy K, Shrilatha B, Rajeshkumar T (2004) Nickel-induced oxidative stress in testis of mice: evidence of DNA damage and genotoxic effects. J Androl 25(6):103–996Google Scholar
  22. Edgar R, Domrachev M, Lash AE (2002) Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acid Res 30(1):207–210PubMedCrossRefGoogle Scholar
  23. Eichner ER, Hillman RS (1971) The evolution of anemia in alcoholic patients. Am J Med 50(2):218–232PubMedCrossRefGoogle Scholar
  24. Emanuele MA, Emanuele NV (1998) Alcohol’s effects on male reproduction. Alcohol Health Res World 22:195–201PubMedGoogle Scholar
  25. Emanuele MA, Emanuele N (2001) Alcohol and the male reproductive system. Alcohol Res Health 25(4):282–287PubMedGoogle Scholar
  26. Finkelstein JD (2007) Metabolic regulatory properties of S-adenosylmethionine and S-adenosylhomocysteine. Clin Chem Lab Med 45(12):1694–1699PubMedCrossRefGoogle Scholar
  27. Finkelstein JD, Kyle WE, Harris BJ (1971) Methionine metabolism in mammals. Regulation of homocysteine methyltransferases in rat tissue. Arch Biochem Biophys 146(1):84–92PubMedCrossRefGoogle Scholar
  28. Forges T, Monnier-Barbarino P, Alberto JM, Gueant-Rodriguez RM, Daval JL, Gueant JL (2007) Impact of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update 13(3):225–239PubMedCrossRefGoogle Scholar
  29. Ganesan B, Buddhan S, Anandan R, Sivakumar R, Anbinezhilan R (2009) Antioxidant defense of betaine against isoprenaline-induced myocardial infarction in rats. Mol Biol Rep 37(3):1319–1327PubMedCrossRefGoogle Scholar
  30. Golbahar J, Aminzadeh MA, Hamidi SA, Omrani GR (2005) Association of red blood cell 5-methyltetrahydrfoate folate with bone mineral density in postmenopausal Iranian women. Osteoporos Int 16(12):1894–1898PubMedCrossRefGoogle Scholar
  31. Halsted CH, Robles EA, Mezey E (1971) Decreased jejunal uptake of labeled folic acid (3H-PGA) in alcoholic patients: roles of alcohol and nutrition. N Eng J Med 285(13):701–706CrossRefGoogle Scholar
  32. Halsted CH, Villanueva JA, Devlin AM, Niemela O, Parkkila S, Garrow TA, Wallock LM, Shigenaga MK, Melnyk S, James SJ (2002) Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig. Proc Natl Acad Sci USA 99(15):10072–10077PubMedCrossRefGoogle Scholar
  33. Haubrich DR, Gerber NH (1981) Choline dehydrogenase. Assay, properties and inhibitors. Biochem Pharmacol 30(21):2993PubMedCrossRefGoogle Scholar
  34. Hemachand T, Shaha C (2003) Functional role of sperm surface glutathione S-transferases and extracellular glutathione in the haploid spermatozoa under oxidative stress. FEBS Lett 538(1–3):14–18PubMedCrossRefGoogle Scholar
  35. Herbert V, Zalusky R, Davidson CS (1963) Correlation of folate deficiency with alcoholism and associated macrocytosis, anemia, and liver disease. Ann Intern Med 58(6):977–988PubMedGoogle Scholar
  36. Ji C, Kaplowitz N (2003) Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology 124(5):1488–1499PubMedCrossRefGoogle Scholar
  37. Kanbak G, Arslan OC, Dokumacioglu A, Kartkaya K, Inal ME (2008) Effects of chronic ethanol consumption on brain synaptosomes and protective role of betaine. Neurochem Res 33(3):539–544PubMedCrossRefGoogle Scholar
  38. Karthikeyan G, Thachil A, Sharma S, Kalaivani M, Ramakrishnan L (2007) Elevated high sensitivity CRP levels in patients with mitral stenosis and left atrial thrombus. Int J Cardiol 122(3):252–254PubMedCrossRefGoogle Scholar
  39. Kasdallah-Grissa A, Mornagui B, Aouani E, Hammami M, Gharbi N, Kamoun A, El-Fazaa S (2006) Protective effect of resveratrol on ethanol-induced lipid peroxidation in rats. Alcohol Alcohol 41(3):236–239PubMedGoogle Scholar
  40. Kelly TLJ, Neaga OR, Schwahn BC, Rozen R, Trasler JM (2005) Infertility in 5,10-methylenetetrahydrofolate reductase (MTHFR)-deficient male mice is partially alleviated by lifetime dietary betaine supplementation. Biol Reprod 72(3):667–677PubMedCrossRefGoogle Scholar
  41. Kheradmand A, Alirezaei M, Asadian P, Alavi ER, Joorabi S (2009a) Antioxidant enzyme activity and MDA level in the rat testis following chronic administration of ghrelin. Andrologia 41(6):335–340PubMedCrossRefGoogle Scholar
  42. Kheradmand A, Taati M, Babaei H (2009b) The effects of chronic administration of ghrelin on rat sperm quality and membrane integrity. Anim Biol 59(2):159–168CrossRefGoogle Scholar
  43. Kheradmand A, Alirezaei M, Birjandi M (2010) Ghrelin promotes antioxidant enzyme activity and reduces lipid peroxidation in the rat ovary. Regul Pept 162(1–3):84–89PubMedCrossRefGoogle Scholar
  44. Kheradmand A, Dezfoulian O, Tarrahi MJ (2011) Ghrelin attenuates heat-induced degenerative effects in the rat testis. Regul Pept 167:97–104PubMedCrossRefGoogle Scholar
  45. Kim JM, Stewart R, Kim SW, Yang SJ, Shin IS, Yoon JS (2008) Predictive value of folate, vitamin B12 and homocysteine levels in late-life depression. Br J Psychiatry 192(4):268–274PubMedCrossRefGoogle Scholar
  46. Lee H, Kim JH, Chae YJ, Ogawa H, Lee MH, Gerton GL (1998) Creatine synthesis and transport systems in the male rat reproductive tract. Biol Reprod 58(6):1437–1444PubMedCrossRefGoogle Scholar
  47. Leevy CM, Baker H, Tenhove W, Frank O, Cherrick GR (1965) B-complex vitamins in liver disease of the alcoholic. Am J Clin Nutr 16:339–346PubMedGoogle Scholar
  48. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  49. Masella R, Vari R, D’Archivio M, Di Benedetto R, Matarrese P, Malorni W, Scazzocchio B, Giovannini C (2004) Extra virgin olive oil biophenols inhibit cell-mediated oxidation of LDL by increasing the mRNA transcription of glutathione-related enzymes. J Nutr 134(4):785–791PubMedGoogle Scholar
  50. McMartin KE, Collins TD, Eisenga BH, Fortney T, Bates WR, Bairnsfather L (1989) Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat. J Nutr 119(10):1490–1497PubMedGoogle Scholar
  51. Millian NS, Garrow TA (1998) Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch Biochem Biophys 356(1):93–98PubMedCrossRefGoogle Scholar
  52. Neamati S, Alirezaei M, Kheradmand A (2011) Ghrelin acts as an antioxidant agent in the rat kidney. Int J Pept Res Therap 17:239–245CrossRefGoogle Scholar
  53. Peltola V, Huhtaniemi I, Ahotupa M (1992) Antioxidant enzyme activity in the maturing rat testis. J Androl 13(5):450–455PubMedGoogle Scholar
  54. Peltola V, Mantyla E, Huhtaniemi I, Ahotupa M (1994) Lipid peroxidation and antioxidant enzyme activities in the rat testis after cigarette smoke inhalation or administration of polychlorinated biphenyls or polychlorinated naphthalenes. J Androl 15(4):353–361PubMedGoogle Scholar
  55. Peltola V, Huhtaniemi I, Metsa-Ketela T, Ahotupa M (1996) Induction of lipid peroxidation during steroidogenesis in the rat testis. Endocrinology 137(1):105–112PubMedCrossRefGoogle Scholar
  56. Ratnam S, Wijekoon EP, Hall B, Garrow TA, Brosnan ME, Brosnan JT (2006) Effects of diabetes and insulin on betaine-homocysteine S-methyltransferase expression in rat liver. Am J Physiol-Endocrinol Metab 290(5):E933–E939PubMedCrossRefGoogle Scholar
  57. Romero JJ, Tamura T, Halsted CH (1981) Intestinal absorption of [3H] folic acid in the chronic alcoholic monkey. Gastroenterology 80(1):99–102PubMedGoogle Scholar
  58. Sehirli O, Sener E, Sener G, Cetinel S, Erzik C, Yegen BC (2008) Ghrelin improves burn-induced multiple organ injury by depressing neutrophil infiltration and the release of pro-inflammatory cytokines. Peptides 29(7):1231–1240PubMedCrossRefGoogle Scholar
  59. Sher L, Oquendo MA, Grunebaum MF, Burke AK, Huang Y, Mann JJ (2007) CSF monoamine metabolites and lethality of suicide attempts in depressed patients with alcohol dependence. Eur Neuropsychopharmacol 17(1):12–15PubMedCrossRefGoogle Scholar
  60. Slow S, Lever M, Chambers ST, George PM (2009) Plasma dependent and independent accumulation of betaine in male and female rat tissues. Physiol Res 58:403–410PubMedGoogle Scholar
  61. Smith AM, Zeve DR, Grisel JJ, Chen WJA (2005) Neonatal alcohol exposure increases malondialdehyde (MDA) and glutathione (GSH) levels in the developing cerebellum. Dev Brain Res 160(2):231–238CrossRefGoogle Scholar
  62. Song Z, Zhou Z, Chen T, Hill D, Kang J, Barve S, McClain C (2003) S-adenosylmethionine (SAMe) protects against acute alcohol induced hepatotoxicity in mice. J Nutr Biochem 14(10):591–597PubMedCrossRefGoogle Scholar
  63. Sonmez M, Türk G, Yüce A (2005) The effect of ascorbic acid supplementation on sperm quality, lipid peroxidation and testosterone levels of male Wistar rats. Theriogenology 63(7):2063–2072PubMedCrossRefGoogle Scholar
  64. Srikanth V, Malini T, Arunakaran J, Govindarajulu P, Balasubramanian K (1999) Effects of ethanol treatment on epididymal secretory products and sperm maturation in albino rats. J Pharmacol Exp Therap 288(2):509–515Google Scholar
  65. Subbarao KV, Richardson JS, Ang LC (1990) Autopsy samples of Alzheimer’s cortex show increased peroxidation in vitro. J Neurochem 55(1):342–345PubMedCrossRefGoogle Scholar
  66. Sunden SLF, Renduchintala MS, Park EI, Miklasz SD, Garrow TA (1997) Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene. Arch Biochem Biophys 345(1):171–174PubMedCrossRefGoogle Scholar
  67. Tamura T, Halsted CH (1983) Folate turnover in chronically alcoholic monkeys. J Lab Clin Med 101(4):623–628PubMedGoogle Scholar
  68. Tamura T, Romero JJ, Watson JE, Gong EJ, Halsted CH (1981) Hepatic folate metabolism in the chronic alcoholic monkey. J Lab Clin Med 97(5):654–661PubMedGoogle Scholar
  69. Tremellen K (2008) Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 14(3):243–259PubMedCrossRefGoogle Scholar
  70. Turner TT, Lysiak JJ (2008) Oxidative stress: a common factor in testicular dysfunction. J Androl 29(5):488–498PubMedCrossRefGoogle Scholar
  71. Van Thiel DH (1983) Ethanol: its adverse effects upon the hypothalamic-pituitary-gonadal axis. J Lab Clin Med 101(1):21–33PubMedGoogle Scholar
  72. Villanueva JA, Halsted CH (2004) Hepatic transmethylation reactions in micropigs with alcoholic liver disease. Hepatology 39(5):1303–1310PubMedCrossRefGoogle Scholar
  73. Wallock-Montelius LM, Villanueva JA, Chapin RE, Conley AJ, Nguyen HP, Ames BN, Halsted CH (2007) Chronic ethanol perturbs testicular folate metabolism and dietary folate deficiency reduces sex hormone levels in the Yucatan micropig. Biol Reprod 76(3):455–465PubMedCrossRefGoogle Scholar
  74. Wu D, Cederbaum AI (2003) Alcohol, oxidative stress, and free radical damage. Alcohol Res Health 27:277–284PubMedGoogle Scholar
  75. Wu A, Chanarin I, Slavin G, Levi AJ (1975) Folate deficiency in the alcoholic—its relationship to clinical and haematological abnormalities, liver disease and folate stores. Br J Haematol 29(3):469–478PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Masoud Alirezaei
    • 1
    Email author
  • Gholamali Jelodar
    • 2
  • Zeynab Ghayemi
    • 2
  1. 1.Division of Biochemistry, School of Veterinary MedicineLorestan UniversityKhorram AbadIran
  2. 2.Department of Physiology, School of Veterinary MedicineShiraz UniversityShirazIran

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