Ukr.Biochem.J. 2018; Volume 90, Issue 4, Jul-Aug, pp. 90-101


Nitrate and nitrite in drinking water affect antioxidant enzymes in erythrocytes of rats

E. Sierra-Campos1, M. A. Valdez-Solana1, M. I. Campos-Almazán1,
C. Avitia-Domínguez2, J. L. Hernández-Rivera1, J. A. De Lira-Sánchez1,
G. Garcia-Arenas3, A. Téllez-Valencia2

1Faculty of Chemical Sciences, Campus Gómez Palacio, University Juarez of Durango State, Gomez Palacio, Durango, Mexico;
2Faculty of Medicine and Nutrition, Campus Durango, University Juarez of Durango State, Durango, Mexico;
3Faculty of Health Sciences, Campus Gómez Palacio, University Juarez of Durango State, Gomez Palacio, Durango, Mexico

The present study evaluated the effect of short term intake of nitrite and nitrate drinking water on the antioxidant system and membrane damage of rat erythrocytes. Wistar rats were randomly divided into three groups as follows; the group I received only distilled water ad libitum; the group II was given water with nitrate (a dose of 124 mg/kg of nitrate-nitrogen) as drinking water and the group III was given nitrites dissolved in distilled water in a dose of 150 mg/kg for 7 days. At the end of the study, group III rats showed a significant decrease in activities of glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PDH) and catalase (CAT), while in group II rats, the activity of GPx and CAT were significantly reduced, but no significant changes in glutathione reductase activity and peroxynitrite levels were observed. On the other hand, malondialdehyde (MDA) was increased in both groups with respect to group I. Also, our major results indicate that all treatments changed methemoglobin levels and osmotic fragility in comparison to group I rats. The intensity of alterations was found more severe in rats of group III, followed by rats of group II. It can be concluded from these observations that nitrate or nitrite leads to alterations in the erythrocytes antioxidant defense status mainly throughout NADPH relate enzymes.

Keywords: , , , , ,


  1. Fawell J, Nieuwenhuijsen MJ. Contaminants in drinking water: Environmental pollution and health. Br Med Bull. 2003;68(1):199-208.  PubMed, CrossRef
  2. Fan AM, Steinberg VE. Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regul Toxicol Pharmacol. 1996 Feb;23(1 Pt 1):35-43. PubMed, CrossRef
  3. Medina-Morales M, Cano-Rios P. Contaminación por nitratos en agua, suelo y cultivos de la Comarca Lagunera. Revista Chapingo Serie Zonas Aridas. 2001;2(1):9-14.
  4. Singhal S, Gupta R, Goyle A. Comparison of antioxidant efficacy of vitamin E, vitamin C, vitamin A and fruits in coronary heart disease: a controlled trial. J Assoc Physicians India. 2001 Mar;49:327-31. PubMed
  5. Gladwin MT, Raat NJ, Shiva S, Dezfulian C, Hogg N, Kim-Shapiro DB, Patel RP. Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation. Am J Physiol Heart Circ Physiol. 2006 Nov;291(5):H2026-35. PubMed, CrossRef
  6. Sivilotti ML. Oxidant stress and haemolysis of the human erythrocyte. Toxicol Rev. 2004;23(3):169-88. PubMed, CrossRef
  7. Kurata M, Suzuki M, Agar NS. Antioxidant systems and erythrocyte life-span in mammals. Comp Biochem Physiol B. 1993 Nov;106(3):477-87. PubMed, CrossRef
  8. Vitak TY, Wasser SP, Nevo E, Sybirna NO. Enzymatic System of Antioxidant Protection of Erythrocytes in Diabetic Rats Treated with Medicinal Mushrooms Agaricus brasiliensis and Ganoderma lucidum (Agaricomycetes). Int J Med Mushrooms. 2017;19(8):697-708. PubMed, CrossRef
  9. Mladenov M, Gokik M, Hadzi-Petrushev N, Gjorgoski I, Jankulovski N. The relationship between antioxidant enzymes and lipid peroxidation in senescent rat erythrocytes. Physiol Res. 2015;64(6):891-6. PubMed
  10. Batina P, Fritsch P, de Saint Blanquat G, Mitjavila MT. In vitro kinetics of the oxidative reactivity of nitrate and nitrite in the rat erythrocyte. Food Addit Contam. 1990;7(Suppl 1):S145-9. PubMed, CrossRef
  11. Ansari FA, Ali SN, Mahmood R. Sodium nitrite-induced oxidative stress causes membrane damage, protein oxidation, lipid peroxidation and alters major metabolic pathways in human erythrocytes. Toxicol In Vitro. 2015 Oct;29(7):1878-86.  PubMed, CrossRef
  12. Shugaley IV, Lvov SN, Tselinsky IV, Baev VI. Effect of sodium nitrite poisoning on the activity of enzymes of anti-oxidant protection and peroxidation processes in mouse erythrocytes. Ukr Biokhim Zhurn. 1992 Mar-Apr;64(2):111-4. (In Russian). PubMed
  13. Bouaziz-Ketata H, Salah GB, Mahjoubi A, Aidi Z, Kallel C, Kammoun H, Fakhfakh F, Zeghal N. Hyparrhenia hirta: A potential protective agent against hematotoxicity and genotoxicity of sodium nitrate in adult rats. Environ Toxicol. 2015 Nov;30(11):1275-84. PubMed, CrossRef
  14. May JM, Qu ZC, Xia L, Cobb CE. Nitrite uptake and metabolism and oxidant stress in human erythrocytes. Am J Physiol Cell Physiol. 2000 Dec;279(6):C1946-54. PubMed, CrossRef
  15. Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB, Rassaf T, Maloney RE, Bharti A, Rodriguez J, Feelisch M. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol. 2005 Oct;1(5):290-7. PubMed, CrossRef
  16.  Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2007 Nov 27;104(48):19144-9.  PubMed, PubMedCentral, CrossRef
  17. Kleinbongard P, Dejam A, Lauer T, Jax T, Kerber S, Gharini P, Balzer J, Zotz RB, Scharf RE, Willers R, Schechter AN, Feelisch M, Kelm M. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med. 2006 Jan 15;40(2):295-302. PubMed, CrossRef
  18. Lu N, Chen C, He Y, Tian R, Xiao Q, Peng YY. The dual effects of nitrite on hemoglobin-dependent redox reactions. Nitric Oxide. 2014 Aug 31;40:1-9.  PubMed, CrossRef
  19. Giles AR. Guidelines for the use of animals in biomedical research. Thromb Haemost. 1987 Dec 18;58(4):1078-84. PubMed
  20. Miller LC, Tainter ML. Estimation of the ED50 and Its Error by Means of Logarithmic-Probit Graph Paper. Exp Biol Med. 1944;57(2):261-264. CrossRef
  21. Speijers GJA, Van Went GF, Van Apeldoorn ME, Montizaan GK, Janus JA, Canton JH, Van Gestel CAM, Van der Heijden CA, Heijna-Merkus E, Knaap AGAC, Luttik R, De Zwart D. Integrated criteria document nitrate: effects. Appendix to RIVM report nr. 758473007, National Institute of Public Health and Environmental Protection. Bilthoven, The Netherlands. 1987.
  22. Modificación a la Norma Oficial Mexicana NOM-127-SSA1-1994, Salud ambiental, agua para uso y consume humano. Límites permisibles de calidad y tratamiento a que debe someterse el agua para su potabilización. México. 2000.
  23. Nagababu E, Rifkind JM. Heme degradation during autoxidation of oxyhemoglobin. Biochem Biophys Res Commun. 2000 Jul 14;273(3):839-45. PubMed, CrossRef
  24. Hanson MS, Stephenson AH, Bowles EA, Sprague RS. Insulin inhibits human erythrocyte cAMP accumulation and ATP release: role of phosphodiesterase 3 and phosphoinositide 3-kinase. Exp Biol Med (Maywood). 2010 Feb;235(2):256-62. PubMed, PubMed, CrossRef
  25. Evelyn KA, Malloy HT. Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample blood. J Biol Chem. 1938;126:655-662.
  26. Sentürk UK, Gündüz F, Kuru O, Aktekin MR, Kipmen D, Yalçin O, Bor-Küçükatay M, Yeşilkaya A, Başkurt OK. Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats. J Appl Physiol. 2001 Nov;91(5):1999-2004. PubMed, CrossRef
  27. O’Dell BL, Browning JD, Reeves PG. Zinc deficiency increases the osmotic fragility of rat erythrocytes. J Nutr. 1987 Nov;117(11):1883-9. PubMed, CrossRef
  28. Huie RE, Padmaja S. The reaction of no with superoxide. Free Radic Res Commun. 1993;18(4):195-9. PubMed, CrossRef
  29. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121-6. PubMed, CrossRef
  30. Staal GE, Visser J, Veeger C. Purification and properties of glutathione reductase of human erythrocytes. Biochim Biophys Acta. 1969 Jul 8;185(1):39-48. PubMed, CrossRef
  31. Zinkham WH. The selective hemolytic action of drugs: clinical and mechanistic considerations. J Pediatr. 1967 Feb;70(2):200-9. PubMed, CrossRef
  32. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967 Jul;70(1):158-69. PubMed
  33. Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press. 1965.
  34. Dodge JT, Mitchel C, Hanahan DJ.  The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119-30. PubMed, CrossRef
  35. Lowry OH, Rosebrough NJ, Farr LA, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265-75. PubMed
  36. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-10. PubMed, CrossRef
  37. Joshi PK, Bose M, Harishi D. Haematological changes in the blood of Clarias batrachusn exposed to mercuric chloride. Ecotoxicol Enviro Monit. 2002;12(2):119-122.
  38. Duncan C, Dougall H, Johnston P, Green S, Brogan R, Leifert C, Smith L, Golden M, Benjamin N. Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nat Med. 1995 Jun;1(6):546-51. PubMed, CrossRef
  39. Kumar D, Branch BG, Pattillo CB, Hood J, Thoma S, Simpson S, Illum S, Arora N, Chidlow JH Jr, Langston W, Teng X, Lefer DJ, Patel RP, Kevil CG. Chronic sodium nitrite therapy augments ischemia-induced angiogenesis and arteriogenesis. Proc Natl Acad Sci USA. 2008 May 27;105(21):7540-5. PubMed, PubMedCentral, CrossRef
  40. Basic Information about Nitrite (Measured as Nitrogen) in Drinking Water. [Electronic resource]. – Regime of access:
  41. Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic fragility in cervical cancer patients. Clin Chim Acta. 2002 Dec;326(1-2):143-9.  PubMed, CrossRef
  42. Adenkola AY, Ayo JO. Effect of road transportation on erythrocyte osmotic fragility of pigs administered ascorbic acid during the harmattan season in Zaria, Nigeria. J Cell Anim Biol. 2009;3(1):4-8.
  43. Brzezińska-Slebodzińska E. Erythrocyte osmotic fragility test as the measure of defence against free radicals in rabbits of different age. Acta Vet Hung. 2001;49(4):413-9.  PubMed, CrossRef
  44. Misra HP, Fridovich I. The generation of superoxide radical during the autoxidation of hemoglobin. J Biol Chem. 1972 Nov 10;247(21):6960-2. PubMed
  45. Jain SK, Hochstein P. Polymerization of membrane components in aging red blood cells. Biochem Biophys Res Commun. 1980 Jan 15;92(1):247-54. PubMed, CrossRef
  46. Hochstein P, Jain SK. Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging. Fed Proc. 1981 Feb;40(2):183-8. PubMed
  47. Giulivi C, Hochstein P, Davies KJ. Hydrogen peroxide production by red blood cells. Free Radic Biol Med. 1994 Jan;16(1):123-9. PubMed, CrossRef
  48. Sadrzadeh SM, Graf E, Panter SS, Hallaway PE, Eaton JW. Hemoglobin. A biologic fenton reagent. J Biol Chem. 1984 Dec 10;259(23):14354-6. PubMed
  49. Borković SS, Saponjić JS, Pavlović SZ, Blagojević DP, Milosević SM, Kovacević TB, Radojicić RM, Spasić MB, Zikić RV, Saicić ZS. The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea. Comp Biochem Physiol C Toxicol Pharmacol. 2005 Aug;141(4):366-74. PubMed, CrossRef
  50. Shimizu N, Kobayashi K, Hayashi K. The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical. J Biol Chem. 1984 Apr 10;259(7):4414-8. PubMed
  51. Ansari FA, Ali SN, Mahmood R. Crocin protects human erythrocytes from nitrite-induced methemoglobin formation and oxidative damage. Cell Biol Int. 2016 Dec;40(12):1320-1331. PubMed, CrossRef
  52. Rober D, Mark L, Mitchell L. Investigation of the hereditary haemolytic anaemia: membrane and enzyme abnormalities. Churchill Livingstone, New York. 2001.
  53. Devasena T, Lalitha S, Padma K. Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis. Clin Chim Acta. 2001 Jun;308(1-2):155-61. PubMed, CrossRef
  54. Krishnamoorthy P, Sangeetha M. Hepatoprotective effect of vitamin C on sodium nitrite-induced lipid peroxidation in albino rats. Indian J Biochem Biophys. 2008;45:206-208.
  55. Marks PA. Enzymes of the pentose phosphate pathway. Methods Med Res. 1961;9:24-35. PubMed
  56. Sandhir R, Parwani S, Kiran R. Effect of Fenvalerate Exposure on Erythrocyte Antioxidant Status in Protein Malnourished Rats. Toxicol Environ Chem. 2002;83(1-4):99-106. CrossRef
  57. Carlström M, Persson AE, Larsson E, Hezel M, Scheffer PG, Teerlink T, Weitzberg E, Lundberg JO. Dietary nitrate attenuates oxidative stress, prevents cardiac and renal injuries, and reduces blood pressure in salt-induced hypertension. Cardiovasc Res. 2011 Feb 15;89(3):574-85. PubMed, CrossRef
  58. Zuckerbraun BS, Shiva S, Ifedigbo E, Mathier MA, Mollen KP, Rao J, Bauer PM, Choi JJ, Curtis E, Choi AM, Gladwin MT. Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth muscle proliferation via xanthine oxidoreductase-dependent nitric oxide generation. Circulation. 2010 Jan 5;121(1):98-109. PubMed, CrossRef
  59. Yang T, Peleli M, Zollbrecht C, Giulietti A, Terrando N, Lundberg JO, Weitzberg E, Carlström M. Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism. Free Radic Biol Med. 2015 Jun;83:159-66. PubMed, CrossRef
  60. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008 Feb;7(2):156-67. PubMed, CrossRef
  61. Chen B, Keshive M, Deen WM. Diffusion and reaction of nitric oxide in suspension cell cultures. Biophys J. 1998 Aug;75(2):745-54. PubMed, PubMedCentral, CrossRef
  62. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol. 1997;15(1):323-350. CrossRef
  63. Dedon PC, Tannenbaum SR. Reactive nitrogen species in the chemical biology of inflammation. Arch Biochem Biophys. 2004 Mar 1;423(1):12-22. PubMed, CrossRef
  64. Shafirovich V, Cadet J, Gasparutto D, Dourandin A, Geacintov NE. Nitrogen dioxide as an oxidizing agent of 8-oxo-7,8-dihydro-2′-deoxyguanosine but not of 2′-deoxyguanosine. Chem Res Toxicol. 2001 Feb;14(2):233-41. PubMed, CrossRef
  65. Brunelli L, Yermilov V, Beckman JS. Modulation of catalase peroxidatic and catalatic activity by nitric oxide. Free Radic Biol Med. 2001 Apr 1;30(7):709-14. PubMed, CrossRef
  66. Denicola A, Souza JM, Radi R. Diffusion of peroxynitrite across erythrocyte membranes. Proc Natl Acad Sci USA. 1998 Mar 31;95(7):3566-71. PubMed, PubMedCentral, CrossRef
  67. Marla SS, Lee J, Groves JT. Peroxynitrite rapidly permeates phospholipid membranes. Proc Natl Acad Sci USA. 1997 Dec 23;94(26):14243-8. PubMed, PubMedCentral, CrossRef
  68. Soszyński M, Bartosz G. Effect of peroxynitrite on erythrocytes. Biochim Biophys Acta. 1996 Oct 24;1291(2):107-14. PubMed
  69. Pandey KB, Rizvi SI. Biomarkers of oxidative stress in red blood cells. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011 Jun;155(2):131-6. PubMed, CrossRef

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