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Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review

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Abstract

Methylglyoxal is a toxic metabolite unavoidably produced in mammalian systems as a by-product of glycolysis. Detoxification of this compound occurs principally through the glyoxalase pathway, which consists of glyoxalase I and glyoxalase II, and requires reduced glutathione as a co-enzyme. Recently, it has been demonstrated that variations in glucose, glutamine and fetal bovine serum levels can cause significant changes in the intracellular concentration of methylglyoxal. More importantly, comparative studies involving wild-type Chinese hamster ovary cells and clones overexpressing glyoxalase I indicate that glucose and glutamine, within the range normally found in cell culture media, can cause decreased cell viability mediated solely through increased production of methylglyoxal. In addition, endogenously produced methylglyoxal has been shown to cause apoptosis in cultured HL60 cells. While the exact mechanism of the impact of methylglyoxal on cultured cells is unknown, methylglyoxal is a potent protein and nucleic acid modifying agent at physiological concentrations and under physiological conditions. Protein modification occurs mainly at arginine, lysine and cysteine residues and is believed to be an important signal for the degradation of senescent proteins. Modification of arginine and lysine results in the irreversible formation of advanced glycation endproducts, whereas modification of cysteine results in the formation of a highly reversible hemithioacetal. Methylglyoxal also forms adducts with nucleic acids, principally with guanyl residues. At high extracellular concentrations, it is genotoxic to cells grown in culture. Even at physiological concentrations (100 nM free methylglyoxal), methylglyoxal can modify unprotected plasmid DNA and cause gene mutation and abnormal gene expression.

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References

  • Al-Abed Y, Lee A, Reis D, Di Stefano C, Liebach H, Papsoulis A and Bucala R (1996) Methylglyoxal induces DNA modification: A model system of DNA mutagenesis. Biochemistry. 35(28): 9289.

    Google Scholar 

  • Alberts G, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular biology of the cell, 3rd Ed., Garland Publishing. Inc. New York.

    Google Scholar 

  • Anderson DC and Goochee CF (1995) The effect of ammonia on O-linked glycosylation of granulocyte colony-stimulating factor produced by Chinese hamster ovary cells. Biotechnol. Bioeng. 47: 96-105.

    Article  Google Scholar 

  • Argiles JM (1986) Has acetone a role in the conversion of fat to carbohydrate in mammals? TIBS 11: 61-63.

    CAS  Google Scholar 

  • Baum VC (1967) Lack of effect of thiamine deficiency on oxidation of methylglyoxal-14C in the rat. J. Nutrition. 91: 399-406.

    Google Scholar 

  • Bebbington, CR, Renner G, Thompson S, King D, Abrams D and Yarrantonm GT (1990) High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10: 169-175.

    Article  Google Scholar 

  • Bibila TA and Robinson DK (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol. Prog. 11: 1-13.

    Article  PubMed  CAS  Google Scholar 

  • Bonarius HPJ, Hatzimanikatis V, Meesters KPH, de Gooijer CD, Schmid G and Tramper J (1996) Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol. Bioeng. 50: 299-318.

    Article  CAS  Google Scholar 

  • Box EPG, Hunter WG and Hunter JS (1978) Statistics for experimenters, John Wiley & Sons, Inc. New York.

    Google Scholar 

  • Brambilla G, Sciaba L, Faggin P, Finollo R, Bassi AM, Ferro M and Marinari UM (1985) Methylglyoxal induced DNA-protein crosslinks and cytotoxicity in Chemese hamster overy cells. Carcinogenesis 6(5): 683-686.

    PubMed  CAS  Google Scholar 

  • Brown ME, Renner G, Field RP, Hassell T (1992) Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system. Cytotechnology 9: 231-236.

    PubMed  CAS  Google Scholar 

  • Cajelli E, Canonero R, Martelli A and Brambilla G (1987) Methylglyoxal-mutation to 6thioguanine resistance in V79 cells. Mutat. Res. 190: 47-50.

    Article  PubMed  CAS  Google Scholar 

  • Carrington SJ and Douglas KT (1986) The glyoxalase enigma-the biological consequences of this ubiquitous enzyme. IRCS Med. Sci. 11: 762-768.

    Google Scholar 

  • Chaplen FWR (1996) Analysis of Methylglyoxal Metabolism in Mammalian Cell Culture, Ph. D. Thesis, University of Winconsin-Madison, Madison, Wisconsin.

    Google Scholar 

  • Chaplen FWR, Cameron DC and Fahl WE (1996a) Effect of Endogenous Methylglyoxal on Chinese Hamster Ovary Cells Grown in Culture. Cytotechnology 22: 33-42.

    Article  CAS  Google Scholar 

  • Chaplen FWR, Cameron DC and Fahl WE (1996b) Detection of Methylglyoxal as a Degradation Product of DNA and Deoxyribonucleotides Treated with Strong Acid. Anal. Biochem. 236: 262-269.

    Article  PubMed  CAS  Google Scholar 

  • Chaplen FWR, Fahl WE and Cameron DC (1996c)Method for determination of free extracellular and intracellular methylglyoxal in animal cells grown in culture. Anal. Biochem. 238: 171-178.

    Article  PubMed  CAS  Google Scholar 

  • Cheung S-T and Fonda ML (1979) Reaction of phenylglyoxal with Arg. The effect of buffers and pH. Biochem. Biophys. Res. Comm. 90: 940-947.

    Article  PubMed  CAS  Google Scholar 

  • Chaudhary AK, Nokubo M, Reddy GR, Yeola SN, Morrow JD, Blair IA and Marnett LJ (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265: 1580-1582.

    PubMed  CAS  Google Scholar 

  • Cooper RA(1984) Metabolism of methylglyoxal in microorganisms. Annu. Rev. Microbiol. 38: 49-68.

    Google Scholar 

  • Dakin HD and Dudley HW (1913) On glyoxalase. J. Biol. Chem. 14(4): 423-431.

    CAS  Google Scholar 

  • Dorner AJ, Wasley LC and Kaufman RJ (1992) Overexpression of GRP78 mitigates stress induction of glucose regulated proteins and blocks secretion of selective proteins in Chinese hamster overy cells. EMBO 11(4): 1563-1571.

    CAS  Google Scholar 

  • Együd LG and Szent Györgyi A (1966) On the regulation of cell division. Proc. Natl. Acad. Sci. USA 56: 203-207.

    Article  PubMed  Google Scholar 

  • Eigenbrodt E, Fister P and Reinacher M (1985) New perspectives on carbohydrate metabolism in tumor cells, pp. 141-169. In: R. Brietner (ed.), Regulation of Carbohydrate Metabolism, Vol. 2, CRC Press, Boca Raton, Florida.

    Google Scholar 

  • Fujita Y, Wakabuayashi K, Nagao M and Sugimura T (1985) Implication of hydrogen peroxide in the mutagenicity of coffee. Mutat. Res. 144: 227-230.

    Article  PubMed  CAS  Google Scholar 

  • Glacken MW, Fleischacker RJ and Sinskey AJ (1986) Reduction of waste product excretion via nutrient control: Possible strategies for maximizing for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol. Bioeng. 28: 1376-1389.

    Article  CAS  Google Scholar 

  • Hara S, Yamaguchi M, Takemori Y, Yoshitake T and Nakamura M (1988) 1,2Diamino4,5methylenedioxybenzene as a highly sensitive fluorogenic reagent for α-dicarbonyl compounds. Anal. Chim. Acta 215: 267-276.

    Article  CAS  Google Scholar 

  • Hopkins FG and Morgan EJ (1945) On the distribution of glyoxalase and glutathione. Biochem. J. 39: 320-324.

    PubMed  CAS  Google Scholar 

  • Hu WS and Himes VB (1989) Stoichiometric considerations of mammalian cell metabolism in bioreactors. In: Bioproducts and Bioprocesses Feichter A, Okada H, Tanner RD, (eds.), Springer-Verlag, Berlin, pp. 33-45.

    Google Scholar 

  • Inlow D, Maiorella B and Snauger AE (1992) Methods for adapting cells for increased product production through exposure to ammonia. US Patent Number 5,156,964.

  • Jocelyn PC (1977) Biochemistry of the SH Group, Academic Press, New York, pp. 1-46.

    Google Scholar 

  • Krymkiewicz N (1973) Reaction of methylglyoxal with nucleic acids. FEBS Lett. 29: 51-54.

    Article  PubMed  CAS  Google Scholar 

  • Lo TWC, Westwood ME, McLellan AC, Selwood T and Thornalley PJ (1994) Binding and modification of proteins by methylglyoxal under physiological conditions. J. Biol. Chem. 269: 32299-32305.

    PubMed  CAS  Google Scholar 

  • McLellan AC, Phillips SA and Thornalley PJ (1992) The assay of methylglyoxal in biological systems by derivatization with 1,2diamino4,5dimethoxybenzene. Anal. Biochem. 206: 17-23.

    Article  PubMed  CAS  Google Scholar 

  • Migliore L, Barale R, Bosco E, Girogelli F, Minunni M, Scarpato R and Loprieno N (1990) Genotoxicity of methylglyoxal: Cytogenic damage in human lymphocytes in vitroand in the intestinal cells of mice. Carcinogenesis 11(9): 1503-1507.

    PubMed  CAS  Google Scholar 

  • Miller WM and Blanch HW (1991) Regulation of animal cell mettabolism in bioreactors. In: Animal Cell Bioreactors. Ho CS and Wang DIC (eds.), Butterworth-Heinemann, Boston, Ch. 5 pp. 119-157.

    Google Scholar 

  • Miller WM, Wilke CR and Blanch HW (1988) Transient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture. Bioproc. Eng. 3: 113-122.

    Article  CAS  Google Scholar 

  • Nagaraj RH, Shipanova IN and Faust FM (1996) Protein cross-linking by the Maillard reaction. Isolation, characterization, and in vivodetection of a lysine-lysine crosslink derived from methylglyoxal. J. Biol. Chem. 271: 19338-19345.

    Article  PubMed  CAS  Google Scholar 

  • Neuberg C (1912) über die Zerstörung von Milchsäurealdehyd und Methylglyoxal durch tierische organe. Biochem. Z. 49: 502-506.

    Google Scholar 

  • Ohmori S, Mori M, Kawase Mand Tsuboi S (1987a) Determination of methylglyoxal as 2methylquinoxaline by high performance liquid chromatography and its application to biological sample. J. Chromatog. 414: 149-155.

    CAS  Google Scholar 

  • Ohmori S, Kawase M, Mori M and Tsuboi S (1987b) Simple and sensitive determination of methylglyoxal in biological samples by gas chromatography with electron caputure detection. J. Chromatog. 415: 221-229.

    CAS  Google Scholar 

  • Ohmori S, Mori M, Shiraha K and Kawase M (1989) Biosynthesis and degradation of methylglyoxal in animals. In: Enzymology and Molecular Biology of Carbonyl of Carbonyl Metabolism 2, Alan R. Liss, Inc., pp 397-412.

    Google Scholar 

  • Ozturk SS, Riley MR and Palsson BO (1992) Effects of ammonia and lactate on hybridoma growth, metabolism and antibody production. Biotechnol. Bioeng. 39: 418-431.

    Article  CAS  Google Scholar 

  • Papsoulis A, Al-Abed Y and Bucala R (1995) Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end-product. Biochemistry 34: 648-655.

    Article  Google Scholar 

  • Phillips SA and Thornalley PJ 61993) Formation of methylglyoxal and D-lactate in human red blood cells in vitro. Biochem. Soc. Trans 21: 163S.

    PubMed  CAS  Google Scholar 

  • Polgár L (1989) Mechanism of Protease action, CRC Press, Inc., Baton Rouge, FL.

    Google Scholar 

  • Ray M and Ray S (1987) Aminoacetone oxidase from goat liver. J. Biol. Chem. 262: 5974-5977.

    PubMed  CAS  Google Scholar 

  • Ray S and Ray M(1981) Isolation of a methylglyoxal synthase from goat liver. J. Biol. Chem. 256: 6230-6234.

    PubMed  CAS  Google Scholar 

  • Rehemtulla A, Dorner AJ and Kaufman RJ (1992) Regulation of PACE propeptide processing activity: Requirement for a post-endoplasmic reticulum compartment and autocatalytic activation. Proc. Nat. Acad. Sci. USA 89: 8235-8239.

    Article  PubMed  CAS  Google Scholar 

  • Richard JP (1984) Acid-base catalysis of the elimination and isomerization reactions of triose phosphates J. Am. Chem. Soc. 106: 4926-4936.

    Article  CAS  Google Scholar 

  • Richard JP (1991) Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance. Biochemistry 30: 4581-4585.

    Article  PubMed  CAS  Google Scholar 

  • Riordan JF, McElvany KD and Borders CL (1977) Arginyl residues: Anion recognition sites in enzymes. Science 195: 884-886.

    PubMed  CAS  Google Scholar 

  • Sawicki E and Sawicki CR (1975) Aldehydes - Photometric Analysis, Academic Press, New York.

    Google Scholar 

  • Schumpp B and Schlaeger EJ (1992) Growth study of lactate and ammonia double resistant clones of HL60 cells. In: Animal Cell Technology: Developments, Processes and Products. Spier RE, Griffiths JB, MacDonald C (eds.), Butterworth-heinemann: Oxford, UK, pp. 224-229.

    Google Scholar 

  • Shapiro, R., Cohen, BI, Shiuey, SJ. and Maurer, H. (1969) On the reaction of guanine with glyoxal, pyruvaldehyde and kethoxal, and the structure of acylguanines. A new synthesis of N 2-alkylguanines. Biochemistry 8: 238-245.

    CAS  Google Scholar 

  • Shinohara M, Giardino I and Brownlee M(1996) Overexpression of glyoxalase I inhibits intracellular advanced glycation endproduct (AGE) formation. Diabetes 25Supplement 2 126A.

    Google Scholar 

  • Shih MJ, Edinger JW and Creighton DJ (1997) Diffusion-dependent kinetic properties of glyoxalase I and estimates of the steady-state concentrations og glyoxalase-pathway intermediates in glycolyzing erythrocytes. Eur. J. Biochem. 244: 852-857.

    Article  PubMed  CAS  Google Scholar 

  • Siwiora S, Fingberg M, Buntermeyer H and Lehmann J (1994) Inhibitory substances secreted in cell culture media of a recombinant CHO and a hybridoma cell line In: Animal Cell Culture Technology: Products of Today, prospects of Tomorrow. Spier RE, Griffiths JB, Berthold E (eds.). Butterworth-Heinnemann: Oxford, UK, pp. 167-169.

    Google Scholar 

  • Takahashi K (1977a) Further studies on the reactions of phenylglyoxal and related reagent with proteins. J. Biochem. (Tokyo) 81: 403-414.

    PubMed  CAS  Google Scholar 

  • Takahashi K (1977b) The reactions of phenylglyoxal and related reagent with amino acids. J. Biochem. (Tokyo) 81: 395-402.

    PubMed  CAS  Google Scholar 

  • Takahashi K (1968) The reaction of phenylglyoxal with arginine residues in proteins. J. Biochem. (Tokyo) 23: 6171-6179.

    Google Scholar 

  • Thornalley PJ, Edwards LG, Kang Y, Wyatt C, Davies N, Ladan MJ and Double J (1996) Antitumor activity of S-p-bromobenzylglutathione cyclopentyl diester in vitroand in vivo. Inhibition of glyoxalase I and the induction of apoptosis. Biochem. Pharmacol. 51(10): 1365-72.

    Article  PubMed  CAS  Google Scholar 

  • Thornalley PJ (1996) Pharmacology of methylglyoxal: Formation, modification of proteins and nucleic acids, and enzymatic detoxification A role in pathogenesis and anti-proliferative chemotherapy. Gen. Pharmac. 27(4): 565-573.

    CAS  Google Scholar 

  • Thornalley PJ (1995) Advances in glyoxalase research. Glyoxalase expression in malignancy, anti-proliferative effects of methylglyoxal, glyoxalase I inhibitor diesters and S-D-lactoylglutathione, and methylglyoxal-modified protein binding and endocytosis by the advanced glycation endproduct receptor. Crit. Rev. Oncology & Hematol 20: 99-128.

    Article  CAS  Google Scholar 

  • Thornalley PJ (1993) The glyoxalase system in health and disease. Mol. Aspects Med. 14: 287-371.

    Article  PubMed  CAS  Google Scholar 

  • Thornalley PJ (1990) The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life. Biochem. J. 269: 1-11.

    PubMed  CAS  Google Scholar 

  • Thornalley PJ (1988) Modification of the glyoxalase system in human red blood cells by glucose in vitro. Biochem. J. 254: 751-755.

    PubMed  CAS  Google Scholar 

  • Tucker JD, Taylor TT, Christenson ML, Strout CL, Hanna ML and Carrano AV (1989) Cytogenic response of 1,2dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes. Mutat. Res. 224: 269-279.

    Article  PubMed  CAS  Google Scholar 

  • Vaca CE, Fang J-L, Conradi M and Hou SM(1994) development of a 32P-postlabelling method for the analysis of 2‘deoxyguanosine3’ monophosphate and DNA adducts of methylgloxal. Carcinogenesis 15: 1887-1994.

    PubMed  CAS  Google Scholar 

  • Vander Jagt DL, Robinson B, Taylor KT and Hunsaker LA (1992) Reduction of trioses by NADPH-dependent aldo-keto reductase. J. Biol. Chem. 267: 4364-4369.

    PubMed  CAS  Google Scholar 

  • Wells-Knecht KJ, Zyzak DV, Litchfield JE, Thorpe SR and Baynes JW (1995) Mechanism of autoxidative glycosylation: Identification of glyoxal and arbinose as intermediates in the autoxidative modification of proteins by glucose. Biochemistry 34: 3702-3709.

    Article  PubMed  CAS  Google Scholar 

  • Westwood ME, McLellan AC and Thornalley PJ (1994) Receptor-mediated endocytic uptake of methylglyoxal-modified serum albumin. J. Biol. Chem. 269: 32293-32298.

    PubMed  CAS  Google Scholar 

  • Westwood ME and Thornalley PJ (1995) Molecular characteristics of methylglyoxal-modified serum albumins. J. Protein Chem. 14(5): 359-372.

    Article  PubMed  CAS  Google Scholar 

  • Westwood ME and Thornalley PJ (1996) Induction of synthesis and secretion of interleukin 1β in the human monocyteic THP-1 cells by human serum albumins modified by methylglyoxal and advanced glycation endproducts. Immunol. Lett. 50: 17-21.

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi M, Hara S and Nakamura M (1989) Determination of methylglyoxal in mouse blood by liquid chromatography with fluorescence detection. Anal. Chim. Acta, 221: 163-166.

    Article  CAS  Google Scholar 

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  1. Department of Bioresource Engineering, Oregon State University, 116 Gilmore Hall, Corvallis, OR, 97331-3906, USA

    Frank W.R. Chaplen

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Chaplen, F.W. Incidence and potential implications of the toxic metabolite methylglyoxal in cell culture: A review. Cytotechnology 26, 173–183 (1998). https://doi.org/10.1023/A:1007953628840

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