Oxidative stress in cell culture: an under‐appreciated problem?

Cell culture studies have given much valuable information about mechanisms of metabolism and signal transduction and of regulation of gene expression, proliferation, senescence, and death. However, cells in culture may behave differently from cells in vivo in many ways. One of these is that cell culture imposes a state of oxidative stress on cells. I argue that cells that survive and grow in culture might use ROS‐dependent signal transduction pathways that rarely or never operate in vivo. A further problem is that cell culture media can catalyse the oxidation of compounds added to them, resulting in apparent cellular effects that are in fact due to oxidation products such as ROS. Such artefacts may have affected many studies on the effects of ascorbate, thiols, flavonoids and other polyphenolic compounds on cells in culture.

[1]  K. Fukuchi,et al.  Production of hydrogen peroxide and methionine sulfoxide by epigallocatechin gallate and antioxidants. , 2001, Anticancer research.

[2]  B. Halliwell The antioxidant paradox , 2000, The Lancet.

[3]  M. Inoue,et al.  Catalase contents in cells determine sensitivity to the apoptosis inducer gallic acid. , 2001, Biological & pharmaceutical bulletin.

[4]  R Gopalakrishna,et al.  Protein kinase C signaling and oxidative stress. , 2000, Free radical biology & medicine.

[5]  B. Meier Reactive oxygen intermediates involved in cellular regulation , 2005, Protoplasma.

[6]  R. Dashwood,et al.  Inhibition of beta-catenin/Tcf activity by white tea, green tea, and epigallocatechin-3-gallate (EGCG): minor contribution of H(2)O(2) at physiologically relevant EGCG concentrations. , 2002, Biochemical and biophysical research communications.

[7]  M. Leist,et al.  Conventional cell culture media do not adequately supply cells with antioxidants and thus facilitate peroxide-induced genotoxicity. , 1996, Free radical biology & medicine.

[8]  J. Crapo,et al.  Hyperoxia enhances lung and liver nuclear superoxide generation. , 1984, Biochimica et biophysica acta.

[9]  B. Halliwell,et al.  The antioxidants of human extracellular fluids. , 1990, Archives of biochemistry and biophysics.

[10]  J. Lunec,et al.  The behaviour of caeruloplasmin in stored human extracellular fluids in relation to ferroxidase II activity, lipid peroxidation and phenanthroline-detectable copper. , 1985, The Biochemical journal.

[11]  J. Hoidal,et al.  Requirement for Reactive Oxygen Species in Serum-induced and Platelet-derived Growth Factor-induced Growth of Airway Smooth Muscle* , 1999, The Journal of Biological Chemistry.

[12]  R. Burdon Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. , 1995, Free radical biology & medicine.

[13]  Jang-Soo Chun,et al.  Tumor Necrosis Factor-α Generates Reactive Oxygen Species via a Cytosolic Phospholipase A2-linked Cascade* , 2000, The Journal of Biological Chemistry.

[14]  Richard A. Ashmun,et al.  Tumor Suppression at the Mouse INK4a Locus Mediated by the Alternative Reading Frame Product p19 ARF , 1997, Cell.

[15]  J. Kanner,et al.  Can apple antioxidants inhibit tumor cell proliferation? Generation of H(2)O(2) during interaction of phenolic compounds with cell culture media. , 2002, Journal of agricultural and food chemistry.

[16]  H. de Groot,et al.  Hypoxia, reactive oxygen, and cell injury. , 1989, Free radical biology & medicine.

[17]  S. Sanz-González,et al.  Peroxynitrite generated from constitutive nitric oxide synthase mediates the early biochemical injury in short‐term cultured hepatocytes , 2000, FEBS letters.

[18]  Xiangdong Wu,et al.  Hydrogen Peroxide Generated during Cellular Insulin Stimulation Is Integral to Activation of the Distal Insulin Signaling Cascade in 3T3-L1 Adipocytes* , 2001, The Journal of Biological Chemistry.

[19]  C. Laurent,et al.  Hydrogen Peroxide Generation in Caco-2 Cell Culture Medium by Addition of Phenolic Compounds: Effect of Ascorbic Acid , 2002, Free radical research.

[20]  S. Baker,et al.  Selenium deficiency in tissue culture: implications for oxidative metabolism. , 1998, Journal of pediatric gastroenterology and nutrition.

[21]  H. Joenje,et al.  Chromosomal instability and progressive loss of chromosomes in HeLa cells during adaptation to hyperoxic growth conditions. , 1989, Mutation research.

[22]  J. Shay,et al.  Historical claims and current interpretations of replicative aging , 2002, Nature Biotechnology.

[23]  H. Joenje,et al.  Some characteristics of hyperoxia-adapted HeLa cells. A tissue culture model for cellular oxygen tolerance. , 1985, Laboratory investigation; a journal of technical methods and pathology.

[24]  M. E. Perry,et al.  The p53 Tumor Suppressor Protein Does Not Regulate Expression of Its Own Inhibitor, MDM2, Except under Conditions of Stress , 2000, Molecular and Cellular Biology.

[25]  G. Bartosz,et al.  Reactive oxygen species are formed in cell culture media. , 2000, Acta biochimica Polonica.

[26]  H. Nakamura,et al.  Redox regulation by thioredoxin superfamily; protection against oxidative stress and aging , 2000, Free radical research.

[27]  J. Lepock,et al.  Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions. , 1991, Cryobiology.

[28]  O. Aruoma,et al.  Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation? , 1987, The Biochemical journal.

[29]  R. DePinho,et al.  Cellular Senescence Minireview Mitotic Clock or Culture Shock? , 2000, Cell.

[30]  T. Galeotti,et al.  Reactive Oxygen Species as Downstream Mediators of Angiogenic Signaling by Vascular Endothelial Growth Factor Receptor-2/KDR* , 2002, The Journal of Biological Chemistry.

[31]  E. Schiffrin,et al.  Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells. , 1999, Hypertension.

[32]  Q. Chen,et al.  Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts. , 2000, The Biochemical journal.

[33]  B. Halliwell,et al.  Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. , 2000, Biochemical and biophysical research communications.

[34]  S. Cadenas,et al.  Low mitochondrial free radical production per unit O2 consumption can explain the simultaneous presence of high longevity and high aerobic metabolic rate in birds. , 1994, Free radical research.

[35]  S. Pervaiz,et al.  Reactive oxygen intermediates regulate cellular response to apoptotic stimuli: an hypothesis. , 1999, Free radical research.

[36]  J. Labat-Robert,et al.  Inhibition of cell proliferation and fibronectin biosynthesis by Na ascorbate , 2002, European journal of clinical investigation.

[37]  M. Qiu,et al.  Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-α , 2001, Oncogene.

[38]  D. Harrison,et al.  Dual role of reactive oxygen species in vascular growth. , 1999, Circulation research.

[39]  S. Kumazawa,et al.  Role of Lipophilicity and Hydrogen Peroxide Formation in the Cytotoxicity of Flavonols , 2001, Bioscience, biotechnology, and biochemistry.

[40]  K. Davies The Broad Spectrum of Responses to Oxidants in Proliferating Cells: A New Paradigm for Oxidative Stress , 1999, IUBMB Life - A Journal of the International Union of Biochemistry and Molecular Biology.

[41]  P. Moldéus,et al.  Individual, culture-specific alterations in the human endothelial glutathione system: relationships to oxidant toxicity. , 1994, Pharmacology & toxicology.

[42]  H. Jaeschke,et al.  Reactive oxygen as modulator of TNF and fas receptor-mediated apoptosis in vivo: studies with glutathione peroxidase-deficient mice. , 2002, Antioxidants & redox signaling.

[43]  J. Reiners,et al.  Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells. , 2000, Toxicology letters.

[44]  J. Crapo,et al.  The effect of hyperoxia on superoxide production by lung submitochondrial particles. , 1982, Archives of biochemistry and biophysics.

[45]  B. Halliwell,et al.  Hydrogen Peroxide. Ubiquitous in Cell Culture and In vivo? , 2000, IUBMB life.

[46]  B. Halliwell Free radicals, proteins and DNA: oxidative damage versus redox regulation. , 1996, Biochemical Society transactions.

[47]  R. Schnellmann,et al.  L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture. , 1996, The American journal of physiology.

[48]  H. S. Mason,et al.  Are physiological oxygen concentrations mutagenic? , 1978, Nature.

[49]  F. Visioli,et al.  Vitamin C matters: increased oxidative stress in cultured human aortic endothelial cells without supplemental ascorbic acid , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[50]  H. Messer,et al.  Removal of trace metals from culture media and sera for in vitro deficiency studies. , 1982, The Journal of nutrition.

[51]  S. Kakumu,et al.  Extracellular superoxide dismutase and glomerular mesangial cells: its production and regulation , 2002 .

[52]  B. Wallaert,et al.  In vitro effects of hyperoxia on alveolar type II pneumocytes: inhibition of glutathione synthesis increases hyperoxic cell injury. , 1992, Experimental lung research.

[53]  B. Halliwell,et al.  Oxidation and generation of hydrogen peroxide by thiol compounds in commonly used cell culture media. , 2001, Biochemical and biophysical research communications.

[54]  B. Halliwell,et al.  The cytotoxicity of dopamine may be an artefact of cell culture , 2002, Journal of neurochemistry.

[55]  W. Dröge Free radicals in the physiological control of cell function. , 2002, Physiological reviews.

[56]  J. Tredger,et al.  Catalytic metal ions and the loss of reduced glutathione from University of Wisconsin preservation solution. , 1996, Transplantation.

[57]  G. Yang,et al.  Effect of black and green tea polyphenols on c-jun phosphorylation and H(2)O(2) production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction. , 2000, Carcinogenesis.

[58]  B. Halliwell,et al.  The in vitro cytotoxicity of ascorbate depends on the culture medium used to perform the assay and involves hydrogen peroxide. , 2001, Antioxidants & redox signaling.

[59]  A. Bowie,et al.  Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. , 2000, Biochemical pharmacology.

[60]  T. Zglinicki Oxidative stress shortens telomeres , 2002 .

[61]  T. Buttke,et al.  Autocrine production of extracellular catalase prevents apoptosis of the human CEM T-cell line in serum-free medium. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[62]  H. Sies,et al.  Oxidative stress: oxidants and antioxidants , 1997, Experimental physiology.

[63]  L. Gerweck,et al.  Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection. , 1984, Radiation research.

[64]  T. K. Baker,et al.  Temporal gene expression analysis of monolayer cultured rat hepatocytes. , 2001, Chemical research in toxicology.

[65]  J. Shay,et al.  Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. , 2001, Genes & development.

[66]  B. Halliwell,et al.  Free radicals in biology and medicine , 1985 .