Choice of DMEM, formulated with or without pyruvate, plays an important role in assessing the in vitro cytotoxicity of oxidants and prooxidant nutraceuticals

  • H. Babich
  • E. J. Liebling
  • R. F. Burger
  • H. L. Zuckerbraun
  • A. G. Schuck
Article

Abstract

There is much interest in the positive health effects of nutraceuticals, in particular, polyphenols, which have both antioxidant and prooxidant characteristics. Pyruvate, a scavenger of hydrogen peroxide, is a component in some, but not in all, commercial formulations of cell culture media, Dulbecco’s modified Eagle’s medium in particular. This study showed that the cytotoxicities to human fibroblasts of hydrogen peroxide, tert-butyl hydroperoxide, and various prooxidant nutraceuticals were lessened in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to the same medium but formulated without pyruvate. Intracellular glutathione was unaffected in cells treated with hydrogen peroxide in Dulbecco’s modified Eagle’s medium formulated with pyruvate, as compared to medium formulated without pyruvate. In these studies, intracellular glutathione was analyzed in acid-soluble cell extracts by determining the oxidation of reduced glutathione by 5,5′-dithiobis(2-nitrobenzoic acid) to glutathione disulfide, with the formation of the yellow chromagen, 5-thio-2-nitrobenzoic acid, measured spectrophotometrically at 412 nm and by the visualization of reduced glutathione in cells stained with the fluorescent dye, Cell Tracker™ Green 5-chloromethylfluorescein diacetate. A survey of various cell culture media, formulated with and without pyruvate, confirmed that the level of added hydrogen peroxide was greatly lessened in those media formulated with pyruvate. This study suggested that the pyruvate status of Dulbecco’s modified Eagle’s medium be specified in the experimental design, especially in studies involving oxidative stress.

Keywords

Pyruvate Oxidants Prooxidants Nutraceuticals In vitro toxicity 

References

  1. Ahmad N.; Gupta S.; Mukhtar H. Green tea polyphenol epigallocatechin-3-gallate differentially mediates nuclear factor κB in cancer cells versus normal cells. Arch. Biochem. Biophys. 376: 338–346; 2000. doi:10.1006/abbi.2000.1742.PubMedCrossRefGoogle Scholar
  2. Azam S.; Hadi N.; Khan N. U.; Hadi S. M. Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: implications for anticancer properties. Toxicol. In Vitro 18: 555–561; 2004. doi:10.1016/j.tiv.2003.12.012.PubMedCrossRefGoogle Scholar
  3. Babich H.; Gottesman R. T.; Liebling E. J.; Schuck A. G. Theaflavin-3-gallate and theaflavin-3′-gallate, polyphenols in black tea with prooxidant properties. Basic Clin. Pharmacol. Toxicol. 103: 66–74; 2008. doi:10.1111/j.1742-7843.2008.00232.x.PubMedCrossRefGoogle Scholar
  4. Babich H.; Pinsky S. M.; Muskin E. T.; Zuckerbraun H. L. In vitro cytotoxicity of a theaflavin mixture from black tea to malignant, immortalized, and normal cells from the human oral cavity. Toxicol. In Vitro 20: 677–688; 2006. doi:10.1016/j.tiv.2005.09.017.PubMedCrossRefGoogle Scholar
  5. Borenfreund E.; Babich H.; Martin-Alguacil N. Rapid chemosensitivity assay with human normal and tumor cells in vitro. In Vitro Cell. Dev. Biol. 26: 1030–1034; 1990. doi:10.1007/BF02624436.PubMedCrossRefGoogle Scholar
  6. Bunger R.; Mallet R. T.; Hartman D. A. Pyruvate-enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart. Near-complete prevention of reperfusion contractile failure. Eur. J. Biochem. 180: 221–233; 1989. doi:10.1111/j.1432-1033.1989.tb14637.x.PubMedCrossRefGoogle Scholar
  7. Chai P. C.; Long L. H.; Halliwell B. Contribution of hydrogen peroxide to the cytotoxicity of green tea and red wine. Biochem. Biophys. Res. Commun. 304: 650–654; 2003. doi:10.1016/S0006-291X(03)00655-7.PubMedCrossRefGoogle Scholar
  8. Chan M. M.; Soprano K. J.; Weinstein K.; Fong D. Epigallocatechin-3-gallate delivers hydrogen peroxide to induce death of ovarian cancer cells and enhances their cisplatin susceptibility. J. Cell. Physiol. 207: 389–396; 2006. doi:10.1002/jcp.20569.PubMedCrossRefGoogle Scholar
  9. Decker D. E. Phenolics: prooxidants or antioxidants. Nutr. Rev. 55: 396–398; 1997.PubMedCrossRefGoogle Scholar
  10. Dringen R.; Pawlowski P. G.; Hirrlinger J. Peroxide detoxification by brain cells. J. Neurosci. Res. 79: 157–165; 2005. doi:10.1002/jnr.20280.PubMedCrossRefGoogle Scholar
  11. Fernandez-Gomez F. J.; Pastor M. D.; Garcia-Martinez E. M.; Melero-Fernandez de Mera R.; Gou-Fabregas M.; Gomez-Lazaro M.; Calvo S.; Soler R. M.; Galindo M. F.; Jordan J. Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating Akt signaling pathway and increasing glutathione peroxidase expression. Neurobiol. Dis. 24: 296–307; 2006. doi:10.1016/j.nbd.2006.07.005.PubMedCrossRefGoogle Scholar
  12. Fukumoto L. R.; Mazza G. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48: 3597–3604; 2000. doi:10.1021/jf000220w.PubMedCrossRefGoogle Scholar
  13. Garcia C. K.; Goldstein J. L.; Pathak R. K.; Anderson R. G.; Brown M. S. Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates. Implications for the Cori cycle. Cell 76: 865–873; 1994. doi:10.1016/0092-8674(94)90361-1.PubMedCrossRefGoogle Scholar
  14. Griffin F. M.; Ashland G.; Capizzi R. L. Kinetics of phototoxicity of Fischer’s medium for L5178Y leukemic cells. Cancer Res. 41: 2241–2248; 1981.PubMedGoogle Scholar
  15. Hagar H.; Ueda N.; Shah S. V. Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Am. J. Physiol. 271: F209–F215; 1996.PubMedGoogle Scholar
  16. Halliwell B. Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett 540: 3–6; 2003. doi:10.1016/S0014-5793(03)00235-7.PubMedCrossRefGoogle Scholar
  17. Hegde K. R.; Varma S. D. Prevention of oxidative stress to the retina by pyruvate. Ophthalmologica 222: 194–198; 2008. doi:10.1159/000126083.PubMedCrossRefGoogle Scholar
  18. Inoue S.; Ito K.; Yamamoto K.; Kawanishi S. Caffeic acid causes metal-dependent damage to cellular and isolated DNA through H2O2 formation. Carcinogenesis 13: 1497–1502; 1992. doi:10.1093/carcin/13.9.1497.PubMedCrossRefGoogle Scholar
  19. Jagtap J. C.; Chandele A.; Chopde B. A.; Shastry P. Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line SK-N-MC. J Chem Neuroanat 26: 109–113; 2003. doi:10.1016/S0891-0618(03)00037-1.PubMedCrossRefGoogle Scholar
  20. Lambert J. D.; Kwon S.-J.; Hong J.; Yang C. S. Salivary hydrogen peroxide generation produced by holding or chewing tea polyphenols in the oral cavity. Free Rad. Res. 41: 850–853; 2007. doi:10.1080/10715760601091659.CrossRefGoogle Scholar
  21. Lapidot T.; Walker M. D.; Kanner J. Can apple antioxidants inhibit tumor cell proliferation? Generation of H2O2 during interaction of phenolic compounds with cell culture media. J. Agric. Food Chem. 50: 3156–3160; 2002a. doi:10.1021/jf011522g.PubMedCrossRefGoogle Scholar
  22. Lapidot T.; Walker M. D.; Kanner J. Antioxidant and prooxidant effects of phenolics on pancreatic β-cells in vitro. J. Agric. Food Chem. 50: 7220–7225; 2002b. doi:10.1021/jf020615a.PubMedCrossRefGoogle Scholar
  23. Lee K.; Hur H. J.; Lee H. J.; Lee C. Y. Antiproliferative effects of dietary phenolic substances and hydrogen peroxide. J. Agric. Food Chem. 53: 1990–1995; 2005. doi:10.1021/jf0486040.PubMedCrossRefGoogle Scholar
  24. Long L. H.; Clement M. V.; Halliwell B. Artifacts in cell culture: Rapid generation of hydrogen peroxide on addition of (−)-epigallocatechin, (−)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem. Biophys. Res. Commun. 273: 50–53; 2000. doi:10.1006/bbrc.2000.2895.PubMedCrossRefGoogle Scholar
  25. Lu J.; Ho C.-T.; Ghai G.; Chan K. Y. Differential effects of theaflavin monogallates on cell growth, apoptosis, and cox-2 gene expression in cancerous versus normal cells. Cancer Res. 60: 6465–6471; 2000.PubMedGoogle Scholar
  26. MacMichael G. The adverse effects of UV and short-wavelength visible radiation on tissue culture. Am. Biotechnol. Lab. 4: 30–31; 1986.Google Scholar
  27. Mallet R. T. Pyruvate: metabolic protector of cardiac performance. Proc. Soc. Exp. Biol. Med. 223: 136–148; 2000. doi:10.1046/j.1525-1373.2000.22319.x.PubMedCrossRefGoogle Scholar
  28. Nakagawa H.; Hasumi K.; Woo J.-T.; Nagai K.; Wachi M. Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (−)-epigallocatechin gallate. Carcinogenesis 25: 1567–1574; 2004. doi:10.1093/carcin/bgh168.PubMedCrossRefGoogle Scholar
  29. Nath K. A.; Enright H.; Nutter L.; Fischereder M.; Zou J. N.; Hebbel R. P. Effect of pyruvate on oxidant injury to isolated and cellular DNA. Kidney Intern. 45: 166–176; 1994. doi:10.1038/ki.1994.20.CrossRefGoogle Scholar
  30. Park S. M.; Jung H. C.; Koak I. S.; Na H. Y.; Woo S. J.; Jung J. S.; Kim Y. K. Oxidant-induced cell death in renal epithelial cells: differential effects of inorganic and organic hydroperoxides. Pharmacol. Toxicol. 92: 43–50; 2003. doi:10.1034/j.1600-0773.2003.920108.x.PubMedCrossRefGoogle Scholar
  31. Roques S. C.; Landrault N.; Teissedre P.-L.; Laurent C.; Besancon P.; Rouanet J.-M.; Cappriccio B. Hydrogen peroxide generation in Caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid. Free Rad. Res. 36: 593–599; 2002. doi:10.1080/10715760290025979.CrossRefGoogle Scholar
  32. Sakagami H.; Arakawa H.; Maeda M.; Satoh K.; Kadofuku T.; Fukuchi K.; Gomi K. Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants. Anticancer Res. 21: 2633–2642; 2001.PubMedGoogle Scholar
  33. Schuck A. G.; Ausubel M. A.; Zuckerbraun H. L.; Babich H. Theaflavin-3,3′-digallate, a component of black tea: An inducer of oxidative stress and apoptosis. Toxicol. In Vitro 22: 598–609; 2008. doi:10.1016/j.tiv.2007.11.021.PubMedCrossRefGoogle Scholar
  34. Shostak A.; Gotloib Z.; Wajsbrot R. V. Protective effect of pyruvate upon cultured mesothelial cells exposed to 2 mM hydrogen peroxide. Nephron. 84: 362–366; 2000. doi:10.1159/000045612.PubMedCrossRefGoogle Scholar
  35. Spierenburg G. T.; Oerlemans F. T. J. J.; van Laarhoven J. P. R. M.; de Bruyn C. H. M. M. Phototoxicity of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered Culture media for human leukemic cell lines. Cancer Res. 44: 2253–2254; 1984.PubMedGoogle Scholar
  36. Stoien J. D.; Wang R. J. Effect of near-ultraviolet and visible light on mammalian cells in culture. II. Formation of toxic photoproducts in tissue culture medium by blacklight. Proc. Nat. Acad. Sci. U. S. A. 71: 3961–3965; 1974. doi:10.1073/pnas.71.10.3961.CrossRefGoogle Scholar
  37. Ursini F.; Maiorino M.; Brigelius-Flohe R.; Aumann K. D.; Roveri A.; Schomburg D.; Flohe I. Diversity of glutathione peroxidases. Meth. Enzymol. 252: 38–53; 1995. doi:10.1016/0076-6879(95)52007-4.PubMedCrossRefGoogle Scholar
  38. Wang R. J. Effect of room fluorescent light on the deterioration of tissue culture medium. In Vitro 12: 19–22; 1976. doi:10.1007/BF02832788.PubMedCrossRefGoogle Scholar
  39. Wang R. J.; Nixon B. T. Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light. In Vitro 14: 715–722; 1978. doi:10.1007/BF02616168.PubMedCrossRefGoogle Scholar
  40. Wang X.; Perez E.; Liu R.; Yan L.-J.; Mallet R. T.; Yang S.-H. Pyruvate protects mitochondria from oxidative stress in human neuroblastoma SK-N-SH cells. Brain Res. 1132: 1–9; 2007. doi:10.1016/j.brainres.2006.11.032.PubMedCrossRefGoogle Scholar
  41. Weisburg J. H.; Weissman D. B.; Sedaghat T.; Babich H. In vitro cytotoxicity of epigallocatechin gallate and tea extracts to cancerous and normal cells from the human oral cavity. Basic Clin. Pharmacol. Toxicol. 95: 191–200; 2004.PubMedGoogle Scholar
  42. Yamamoto T.; Lewis J.; Wataha J.; Dickinson D.; Singh B.; Bollag W. B.; Ueta E.; Osaki T.; Athar M.; Schuster G.; Hsu S. Roles of catalase and hydrogen peroxide in green tea polyphenol-induced chemopreventive effects. J. Pharmacol. Exp. Ther. 308: 317–323; 2004. doi:10.1124/jpet.103.058891.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2009

Authors and Affiliations

  • H. Babich
    • 1
  • E. J. Liebling
    • 1
  • R. F. Burger
    • 1
  • H. L. Zuckerbraun
    • 1
  • A. G. Schuck
    • 1
  1. 1.Department of Biology, Stern College for WomenYeshiva UniversityNew YorkUSA

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