Protective Effect of Glycine and Tri-Methyl Glycine (Betaine) Against Heavy Metals-Induced Oxidative Stress in Liver-Derived Post-Nuclear Supernatant (PNS)

Reza Heidari, Hamidreza Mohammadi, Asrin Ahmadi, Vahid Ghanbarinejad, Faraz Kasra, Amir Khosravi, Marziyeh Rashedinia


Heavy metals are environmental pollutants which pose toxicity toward biological systems. Most organs are susceptible to heavy metals-induced toxicity. Hence, finding protective agents against heavy metals-induced toxicity is valuable. The post-nuclear supernatant (PNS) has been accepted as an in vitro model for assessing xenobiotic-induced toxicity toward biological systems. Monitoring the toxic effects of a large number of xenobiotics in a short time is one of the superiorities of PNS system. The goal of the present study was to validate the PNS as an in vitro model for investigating the effect of heavy metals (Cd, Co, Cu, Fe, As, Hg, Cr, and Pb)-induced toxicity and evaluating the potential protective effects of glycine and betaine. Markers of oxidative stress including ROS formation, lipid peroxidation and glutathione content in addition of succinate dehydrogenase activity (MTT test) were monitored in the presence of heavy metals alone or in combination with glycine (1 mM) and betaine (100 µM). Our results suggest that PNS preparations can be used as an appropriate model for future investigation of xenobiotics-induced toxicity and estimation of the protective properties of different agents. Indeed, further evaluations in other experimental models could reveal the protective properties of betaine and glycine against heavy metals-induced organ injury.


Khan S, Cao Q, Zheng YM, Huang YZ, Zhu YG. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environ Pollut. 2008;152;686-692.

Johri N, Jacquillet G, Unwin R. Heavy metal poisoning: the effects of cadmium on the kidney. BioMetals. 2010;23;783-792.

Alloway BJ. Heavy metals in soils: Springer Science & Business Media; 1995 1995.

Förstner U, Wittmann GTW. Metal pollution in the aquatic environment: Springer Science & Business Media; 2012 2012.

Ingawale DK, Mandlik SK, Naik SR. Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): A critical discussion. Environ Toxicol Pharmacol. 2014;37;118-133.

Flora SJS. Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure. Oxid Med Cell Longev. 2009;2;191-206.

Ercal N, Gurer-Orhan H, Aykin-Burns N. Toxic Metals and Oxidative Stress Part I: Mechanisms Involved in Metal-induced Oxidative Damage. Curr Top Med Chem. 2001;1;529-539.

Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda KN. Toxicity, mechanism and health effects of some heavy metals. Interdiscip Toxicol. 2014;7;60-72.

Govil N, Chaudhary S, Waseem M, Parvez S. Postnuclear supernatant: an in vitro model for assessing cadmium-induced neurotoxicity. Biol Trace Elem Res. 2012;146;402-409.

Gupta R, Dubey DK, Kannan GM, Flora SJS. Concomitant administration of Moringa oleifera seed powder in the remediation of arsenic-induced oxidative stress in mouse. Cell Biol Int. 2007;31;44-56.

Heidari R, Jamshidzadeh A, Niknahad H, Safari F, Azizi H, Abdoli N, Ommati MM, Khodaei F, Saeedi A, Najibi A. The hepatoprotection provided by taurine and glycine against antineoplastic drugs induced liver injury in an ex vivo model of normothermic recirculating isolated perfused rat liver. Trend Pharm Sci. 2016;2;59-76.

Ommati MM, Tanideh N, Rezakhaniha B, Wang J, Sabouri S, Vahedi M, Dormanesh B, Koohi Hosseinabadi O, Rahmanifar F, Moosapour S, Akhlaghi A, Heidari R, Zamiri MJ. Is immunosuppression, induced by neonatal thymectomy, compatible with poor reproductive performance in adult male rats? Andrology. 2018;6;199-213.

Heidari R, Jafari F, Khodaei F, Shirazi Yeganeh B, Niknahad H. Mechanism of valproic acid‐induced Fanconi syndrome involves mitochondrial dysfunction and oxidative stress in rat kidney. Nephrology. 2018;23;351-361.

Heidari R, Babaei H, Ali Eghbal M. Mechanisms of methimazole cytotoxicity in isolated rat hepatocytes. Drug Chem Toxicol. 2012;1-9.

Abdoli N, Heidari R, Azarmi Y, Eghbal MA. Mechanisms of the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes. J Biochem Mol Toxicol. 2013;27;287-294.

Heidari R, Taheri V, Rahimi HR, Shirazi Yeganeh B, Niknahad H, Najibi A. Sulfasalazine-induced renal injury in rats and the protective role of thiol-reductants. Ren Fail. 2016;38;137-141.

Akram J, Hossein N, Reza H, Maryam A, Forouzan K, Mohammad Reza A, Omid F. Propylthiouracil-induced mitochondrial dysfunction in liver and its relevance to drug-induced hepatotoxicity. Pharm Sci. 2017;23;95-102.

Socci DJ, Bjugstad KB, Jones HC, Pattisapu JV, Arendash GW. Evidence that oxidative stress is associated with the pathophysiology of inherited hydrocephalus in the H-Tx rat model. Exp Neurol. 1999;155;109-117.

Ommati MM, Heidari R, Jamshidzadeh A, Zamiri MJ, Sun Z, Sabouri S, Wang J, Ahmadi F, Javanmard N, Seifi K, Mousapour S, Yeganeh BS. Dual effects of sulfasalazine on rat sperm characteristics, spermatogenesis, and steroidogenesis in two experimental models. Toxicol Lett. 2018;284;46-55.

Heidari R, Babaei H, Roshangar L, Eghbal MA. Effects of enzyme induction and/or glutathione depletion on methimazole-induced hepatotoxicity in mice and the protective role of N-acetylcysteine. Adv Pharm Bull. 2014;4;21-28.

Heidari R, Moezi L, Asadi B, Ommati MM, Azarpira N. Hepatoprotective effect of boldine in a bile duct ligated rat model of cholestasis/cirrhosis. PharmaNutrition. 2017;5;109-117.

Heidari R, Jamshidzadeh A, Keshavarz N, Azarpira N. Mitigation of Methimazole-Induced Hepatic Injury by Taurine in Mice. Sci Pharm. 2015;83;143-158.

Heidari R, Niknahad H, Sadeghi A, Mohammadi H, Ghanbarinejad V, Ommati MM, Hosseini A, Azarpira N, Khodaei F, Farshad O, Rashidi E, Siavashpour A, Najibi A, Ahmadi A, Jamshidzadeh A. Betaine treatment protects liver through regulating mitochondrial function and counteracting oxidative stress in acute and chronic animal models of hepatic injury. Biomed Pharmacother. 2018;103;75-86.

Jamshidzadeh A, Heidari R, Latifpour Z, Ommati MM, Abdoli N, Mousavi S, Azarpira N, Zarei A, Zarei M, Asadi B, Abasvali M, Yeganeh Y, Jafari F, Saeedi A, Najibi A, Mardani E. Carnosine ameliorates liver fibrosis and hyperammonemia in cirrhotic rats. Clin Res Hepatol Gastroenterol. 2017;41;424-434.

Ommati MM, Jamshidzadeh A, Heidari R, Sun Z, Zamiri MJ, Khodaei F, Mousapour S, Ahmadi F, Javanmard N, Yeganeh BS. Carnosine and Histidine Supplementation Blunt Lead-Induced Reproductive Toxicity through Antioxidative and Mitochondria-Dependent Mechanisms. Biol Trace Elem Res. 2018;In Press;1-12.

Katalinic V, Modun D, Music I, Boban M. Gender differences in antioxidant capacity of rat tissues determined by 2,2'-azinobis (3-ethylbenzothiazoline 6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays. Comp Biochem Physiol C Toxicol Pharmacol. 2005;140;47-52.

Hossein N, Akram J, Reza H, Narges A, Mohammad Mehdi O, Faezeh J, Mahdi Z, Behnam A. The postulated hepatotoxic metabolite of methimazole causes mitochondrial dysfunction and energy metabolism disturbances in liver. Pharm Sci. 2016;22;217-226.

Heidari R, Jafari F, Khodaei F, Shirazi Yeganeh B, Niknahad H. Mechanism of valproic acid-induced fanconi syndrome involves mitochondrial dysfunction and oxidative stress in rat kidney. Nephrology. 2017;23;351-361.

Alía M, Horcajo C, Bravo L, Goya L. Effect of grape antioxidant dietary fiber on the total antioxidant capacity and the activity of liver antioxidant enzymes in rats. Nut Res. 2003;23;1251-1267.

Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65;55-63.

Niknahad H, Jamshidzadeh A, Heidari R, Hosseini Z, Mobini K, Khodaei F, Ommati MM, Abdoli N, Keshavarz N, Bazyari M, Najibi A. Paradoxical effect of methimazole on liver mitochondria: In vitro and in vivo. Toxicol Lett. 2016;259;108-115.

Heidari R, Ghanbarinejad V, Mohammadi H, Ahmadi A, Ommati MM, Abdoli N, Aghaei F, Esfandiari A, Azarpira N, Niknahad H. Mitochondria protection as a mechanism underlying the hepatoprotective effects of glycine in cholestatic mice. Biomed Pharmacother. 2018;97;1086-1095.

Heidari R, Niknahad H, Jamshidzadeh A, Azarpira N, Bazyari M, Najibi A. Carbonyl traps as potential protective agents against methimazole-induced liver injury. J Biochem Mol Toxicol. 2015;29;173-181.

Niknahad H, Heidari R, Mohammadzadeh R, Ommati MM, Khodaei F, Azarpira N, Abdoli N, Zarei M, Asadi B, Rasti M, Yeganeh BS, Taheri V, Saeedi A, Najibi A. Sulfasalazine induces mitochondrial dysfunction and renal injury. Ren Fail. 2017;39;745-753.

Liu X, Song Q, Tang Y, Li W, Xu J, Wu J, Wang F, Brookes PC. Human health risk assessment of heavy metals in soil–vegetable system: A multi-medium analysis. Sci Total Environ. 2013;463-464;530-540.

Houston MC. The role of mercury and cadmium heavy metals in vascular disease, hypertension, coronary heart disease, and myocardial infarction. Altern Ther Health Med. 2007;13;S128.

Zheng N, Wang Q, Zhang X, Zheng D, Zhang Z, Zhang S. Population health risk due to dietary intake of heavy metals in the industrial area of Huludao city, China. Sci Total Environ. 2007;387;96-104.

Järup L. Hazards of heavy metal contamination. Br Med Bull. 2003;68;167-182.

Matés JM, Segura JA, Alonso FJ, Márquez J. Roles of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated mechanisms. Free Radic Biol Med. 2010;49;1328-1341.

Rybicki BA, Johnson CC, Uman J, Gorell JM. Parkinson's disease mortality and the industrial use of heavy metals in Michigan. Mov Disord. 1993;8;87-92.

Craig SAS. Betaine in human nutrition. Am J Clin Nut. 2004;80;539-549.

Day CR, Kempson SA. Betaine chemistry, roles, and potential use in liver disease. Biochim Biophys Acta. 2016;1860;1098-1106.

Detopoulou P, Panagiotakos DB, Antonopoulou S, Pitsavos C, Stefanadis C. Dietary choline and betaine intakes in relation to concentrations of inflammatory markers in healthy adults: the ATTICA study. Am J Clin Nut. 2008;87;424-430.

Dawczynski C, Schubert R, Jahreis G. Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chem. 2007;103;891-899.

Yin M, Zhong Z, Connor HD, Bunzendahl H, Finn WF, Rusyn I, Li X, Raleigh JA, Mason RP, Thurman RG. Protective effect of glycine on renal injury induced by ischemia-reperfusion in vivo. Am J Physiol. 2002;282;F417-F423.

Zhong Z, Wheeler MD, Li X, Froh M, Schemmer P, Yin M, Bunzendaul H, Bradford B, Lemasters JJ. L-Glycine: a novel antiinflammatory, immunomodulatory, and cytoprotective agent. Curr Opin Clin Nutr Metab Care. 2003;6;229-240.

Marsh DC, Vreugdenhil PK, Mack VE, Belzer FO, Southard JH. Glycine protects hepatocytes from injury caused by anoxia, cold ischemia and mitochondrial inhibitors, but not injury caused by calcium ionophores or oxidative stress. Hepatology. 1993;17;91-98.

Zhang K, Weinberg JM, Venkatachalam MA, Dong Z. Glycine Protection of PC-12 Cells Against Injury by ATP-Depletion. Neurochem Res. 2003;28;893-901.

Nissim I, Weinberg JM. Glycine attenuates Fanconi syndrome induced by maleate or ifosfamide in rats. Kidney Int. 1996;49;684-695.

Weinberg JM, Davis JA, Abarzua M, Rajan T. Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules. J Clin Invest. 1987;80;1446.

Bingül İ, Aydın AF, Başaran-Küçükgergin C, Doğan-Ekici I, Çoban J, Doğru-Abbasoğlu S, Uysal M. High-fat diet plus carbon tetrachloride-induced liver fibrosis is alleviated by betaine treatment in rats. Int Immunopharmacol. 2016;39;199-207.

Balkan J, Parldar FH, Dogru-Abbasoglu S, Aykaç-Toker G, Uysal M. The effect of taurine or betaine pretreatment on hepatotoxicity and prooxidant status induced by lipopolysaccharide treatment in the liver of rats. Eur J Gastroenterol Hepatol. 2005;17;917.

Wettstein M, Haussinger D. Cytoprotection by the osmolytes betaine and taurine in ischemia- reoxygenation injury in the perfused rat liver. Hepatology. 1997;26;1560-1566.

Hagar H, Medany AE, Salam R, Medany GE, Nayal OA. Betaine supplementation mitigates cisplatin-induced nephrotoxicity by abrogation of oxidative/nitrosative stress and suppression of inflammation and apoptosis in rats. Exp Toxicol Pathol. 2015;67;133-141.

Wang Z, Yao T, Pini M, Zhou Z, Fantuzzi G, Song Z. Betaine improved adipose tissue function in mice fed a high-fat diet: a mechanism for hepatoprotective effect of betaine in nonalcoholic fatty liver disease. Am J Physiol. 2010;298;G634-G642.

Oliva J, Bardag-Gorce F, Tillman B, French SW. Protective effect of quercetin, EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro. Exp Mol Pathol. 2011;90;295-299.

Banu MNA, Hoque MA, Watanabe-Sugimoto M, Matsuoka K, Nakamura Y, Shimoishi Y, Murata Y. Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress. J Plant Physiol. 2009;166;146-156.

Jung YS, Kim SJ, Kwon DY, Ahn CW, Kim YS, Choi DW, Kim YC. Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism. Food Chem Toxicol. 2013;62;292-298.



  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution 3.0 License.

Contact Information:
  • Trends in Pharmaceutical Sciences, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, IR, Iran.
    Tel/Fax: +98 71 32424128 Ext: 297
  • P.O. Box: 17345-1583 Shiraz, Iran
  • Web site:; 
  • pISSN: 2423-3722   eISSN: 2423-5652