Skip to main content

Cytotoxic effects of methanol, formaldehyde, and formate on dissociated rat thymocytes: A possibility of aspartame toxicity

Abstract

Aspartame is a widely used artificial sweetener added to many soft beverages and its usage is increasing in health-conscious societies. Upon ingestion, this artificial sweetener produces methanol as a metabolite. In order to examine the possibility of aspartame toxicity, the effects of methanol and its metabolites (formaldehyde and formate) on dissociated rat thymocytes were studied by flow cytometry. While methanol and formate did not affect cell viability in the physiological pH range, formaldehyde at 1–3 mmol/L started to induce cell death. Further increase in formaldehyde concentration produced a dose-dependent decrease in cell viability. Formaldehyde at 1 mmol/L or more greatly reduced cellular content of glutathione, possibly increasing cell vulnerability to oxidative stress. Furthermore, formaldehyde at 3 mmol/L or more significantly increased intracellular concentration of Ca2+([Ca2+]i) in a dose-dependent manner. Threshold concentrations of formaldehyde, a metabolite of methanol, that affected the [Ca2+]iand cellular glutathione content were slightly higher than the blood concentrations of methanol previously reported in subjects administered abuse doses of aspartame. It is suggested that aspartame at abuse doses is harmless to humans.

This is a preview of subscription content, access via your institution.

References

  • Becker CE.Methanol poisoning.J Emerg Med.1983:1:51-8.

    PubMed  CAS  Article  Google Scholar 

  • Bray LA, Shao H, Ewald SJ.Effect of ethanol on development of fetal mouse thymocytes in organ culture.Cell Immunol.1993:151:12-23.

    PubMed  Article  Google Scholar 

  • Chikahisa L, Oyama Y.Tri-n-butyltin increases intracellular Ca2+in mouse thymocytes: a flow-cytometric study using fluorescent dyes for membrane potential and intracellular Ca2+PharmacolToxicol.1992:71:190-5.

    CAS  Google Scholar 

  • Chikahisa L, Oyama Y, Okazaki E, Noda K.Fluorescent estimation of H2O2-induced changes in cell viability and cellular nonprotein thiol level of dissociated rat thymocytes.Jpn J Pharmacol.1996:71:299-305.

    PubMed  CAS  Google Scholar 

  • Comment CE, Blaylock BL, Germolec DR, et al.Thymocyte injury after in vitrochemical exposure: potential mechanisms for thymic atrophy.J Pharmacol Exp Ther.1992:262:1267-73.

    PubMed  CAS  Google Scholar 

  • Enan E, Pinkerton KE, Peake J, Matsumura F.Deltamethrin-induced thymus atrophy in male Balb/c mice.Biochem Pharmacol.1996:51:447-54.

    PubMed  CAS  Article  Google Scholar 

  • Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM.Exposure ofphosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.J Immunol.1992:148:2207-16.

    PubMed  CAS  Google Scholar 

  • Farber JL.The role of calcium in toxic cell injury.Environ Health Perspect.1990:84:107-11.

    PubMed  CAS  Google Scholar 

  • Garriga MM, Berkebile C, Metcaife DD.A combined single-blind, double-blind, placebo-controlled study to determine the reproducibility of hypersensitivity reactions to aspartame.J AIIergy Clin Immunol.1991:87:821-7.

    CAS  Article  Google Scholar 

  • Han YC, Lin TL, Pruett SB.Thymic atrophy caused by ethanol in a mouse model for binge drinking: involvement of endogenous glucocorticoids.Toxicol Appi Pharmacol.1993:123:16-25.

    CAS  Article  Google Scholar 

  • Hertelendy Zl, Mendenhall CL, Rouster SD, Marshall L, Weesner R.Biochemical and clinical effects of aspartame in patients with chronic, stable alcoholic liver disease.Am J Gastroenterol.1993:88:737-43.

    PubMed  CAS  Google Scholar 

  • Jain A, Martensson J, Stole E, Auld PAM, Meister A.Glutathione deficiency leads to mitochondrial damage in brain.Proc Natl Acad Sci USA.1991:88:1913-6.

    PubMed  CAS  Article  Google Scholar 

  • Kashiwagi A, Asahina T, Ikebuchi M, et al.Abnormal glutathione metabolism and increased cytotoxicity caused by H2O2in human umbilical vein endothelial cells cultured in high glucose medium.Diabetologia.1994:37:264-9.

    PubMed  CAS  Article  Google Scholar 

  • Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RD, Pals ST, van Oers MH.Annexin V for flow cytometric detection ofphosphatidylserine expression on B cells undergoing apoptosis.Blood.1994:84:1415-20.

    PubMed  CAS  Google Scholar 

  • Kruse JA.Methanol poisoning.Intensive Care Med.1992:18: 391-7.

    PubMed  CAS  Article  Google Scholar 

  • Lai ZW, Fiore NC, Hain PJ, Gasiewicz TA, Silverstone AE.Differential effects of diethylstilbestrol and 2,3,7,8-tetrachlorodibenzo-p-dioxin on thymocyte differentiation, proliferation, and apoptosis in blc-2 transgenic mouse fetal thymus organ culture.Toxicol Appl Pharmacol.2000;168: 15-24.

    PubMed  CAS  Article  Google Scholar 

  • Leon AS, Hunnighake DB, Bell C, Rassin DK, Tephly TR.Safety of long-term large doses of aspartame.Arch Intern Med.1989:149:2318-24.

    PubMed  CAS  Article  Google Scholar 

  • Lutz CT, Browne G, Petzold CR.Methylcholanthrene causes increased thymocyte apoptosis.Toxicology.1998:128:151-67.

    PubMed  CAS  Article  Google Scholar 

  • Matsumoto K, Moriya F, Nanikawa R.The movement of blood formaldehyde in methanoi intoxication.II.The movement of blood formaldehyde and its metabolism in the rabbit.Nippon Hoigaku Zasshi.1990:44:205-11.

    PubMed  CAS  Google Scholar 

  • Meister A, Anderson ME.Glutathione.Annu Rev Biochem.1983:52:711-60.

    PubMed  CAS  Article  Google Scholar 

  • Meyer RJ, Beard ME, Ardagh NW, Henderson S.Methanol poisoning.NZ Med J.2000:113:11-3.

    CAS  Google Scholar 

  • Morris ID, London RG, Waters C, Naylor G, Jones N.Thymic regression and apoptosis in the rat after treatment with the Leydig cell cytotoxin ethylene dimethanesulphonate (EDS).Toxicology 1997:120:19-27.

    PubMed  CAS  Article  Google Scholar 

  • Nakata M, Oyama Y, Okada Y, Yamazaki Y, Chikahisa L, Satoh M.Flow cytometric analysis on tri-n-butyltin-induced increase in annexin V binding to membranes of rat thymocytes.Environ Toxicol Pharmacol.1999:7:267-73.

    CAS  Article  Google Scholar 

  • Nolla-Sala J, Nogue XS, Marruecos SL, Palomar MM, Martinez PJ.Methanol and ethylene glycol poisoning.Study of 18 cases.Med Clin Barc.1995:104:121-5.

    Google Scholar 

  • Okazaki E, Chikahisa L, Kanemaru K, Oyama Y.Flow cytometric analysis of the H2O2-induced increase in intracellular Ca2+concentration of rat thymocytes.Jpn J Pharmacol.1996:71:273-80.

    PubMed  CAS  Google Scholar 

  • Orrenius S, Burkitt MJ, Kass GE, Dypbukt JM, Nicotera P.1992.Calcium ions and oxidative cell injury.Ann Neurol.1992;32:S33-42.

    PubMed  CAS  Article  Google Scholar 

  • Osterloh LD, Pond SM, Grady S, Becker CE.Serum formate concentrations in methanol intoxication as a criterion for hemodialysis.Ann Intern Med.1986:104:200-3.

    PubMed  CAS  Google Scholar 

  • Oyama Y, Noguchi S, Nakata M, et al.Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin.Eur J Pharmacol.1999:384:47-52.

    PubMed  CAS  Article  Google Scholar 

  • Pellicciari C, Bottone MG, Biggioera M.Detection ofapoptotic cells by annexin V labeling at electron microscopy.Eur J Histochem.1997:41:211-6.

    PubMed  CAS  Google Scholar 

  • Quaglio D and Ronchetti IP.Cell death in the rat thymus: a minireview.Apoptosis.2001:5:389-401.

    Article  Google Scholar 

  • Raffray M, McCarthy D, Snowden RT, Cohen GM.Apoptosis as a mechanism of tributyltin cytotoxicity to thymocytes: relationship of apoptotic markers to biochemical and cellular effects.Toxicol Appl Pharmacol.1993:119:122-30.

    PubMed  CAS  Article  Google Scholar 

  • Ranney RE, Oppermann JA, Muldoon E, McMahon FG.1976.Comparative metabolism of aspartame in experimental animals and humans.J Toxicol Environ Health.1976:2: 441-51.

    PubMed  CAS  Google Scholar 

  • Ranney RE. Oppermann JA.A review of the metabolism of the aspartyl moiety of aspartame in experimental animals and man.J Environ Pathol Toxicol.1979;2:979-85.

    PubMed  CAS  Google Scholar 

  • Savabieasfahani M, Lochmiller RL, Janz DM.Elevated ovarian and thymic cell apoptosis in wild cotton rats inhabiting petrochemical-contaminated terrestrial ecosystems.J Toxicol Environ Health A.1999:57:521-7.

    PubMed  CAS  Article  Google Scholar 

  • Shahangian S, Ash KO.Formic and lactic acidosis in a fatal case of methanol intoxication.Clin Chem.1986:32:395-7.

    PubMed  CAS  Google Scholar 

  • Shao H, Zhou J, Ewald SJ.Regulation of signal transduction and DNA fragmentation in thymocytes by ethanol.Cell Immunol.1995:164:11-9.

    PubMed  CAS  Article  Google Scholar 

  • Siesjo BK, Bengtsson F, Grampp W, Theander S.Calcium, excitotoxins, and neuronal death in the brain.Ann NY Acad Sci.1989:568:234-51.

    PubMed  CAS  Google Scholar 

  • Silverstone AE, Frazier DE Jr, Fiore NC, Soults JA, Gasiewicz TA.Dexamethasone, β-estradiol, and 2,3,7,8-tetrachlorodibenzo-p-dioxin elicit thymic atrophy through different cellular targets.Toxicol Appl Pharmacol.1994:126:248-59.

    PubMed  CAS  Article  Google Scholar 

  • Skrzydiewska E, Farbiszewski R.Glutathione consumption and inactivation of glutathione-related enzymes in liver, erythrocytes and serum of rats after methanol intoxication.ArchToxicol.1997:71:741-5.

    Google Scholar 

  • Stegink LD, Brummel MC, McMartin K, et al.Blood concentrations in normal adult subjects administered abuse doses ofaspartame.J Toxicol Environ Health.1981:7:281-90.

    PubMed  CAS  Article  Google Scholar 

  • Stuart MC, Damoiseaux JG, Frederik PM, Arends JW, Reutelingsperger CP.Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes.Eur J Cell Biol.1998:76:77-83.

    PubMed  CAS  Google Scholar 

  • Szondy Z.Adenosine stimulates DAN fragmentation in human thymocytes by Ca2+-mediated mechanisms.Biochem J.1994:304:877-85.

    PubMed  CAS  Google Scholar 

  • Teo SK, Lo KL, Tey BH.Mass methanol poisoning: a clinico-biochemical analysis of 10 cases.Singapore Med J.1996:37: 485-7.

    PubMed  CAS  Google Scholar 

  • Tephly TR.Comments on the purported generation of formal-dehyde and adduct formation from the sweetener aspartame.Life Sci.1999:65: PL157-60.

    PubMed  CAS  Article  Google Scholar 

  • Trefz F, de Sonneville L, Matthis P, Benninger C, Lanz-Englert B, Bickel H.Neuropsychological and biochemical investigations in heterozygotes for phenyiketonuria during ingestion of high dose aspartame (a sweetener containing phenylalanine).Hum Genet.1994:93:369-74.

    PubMed  CAS  Article  Google Scholar 

  • Trocho C, Pardo R, Rafecas I, et al.Formaldehyde derived from dietary aspartame binds to tissue components n vivo.Life Sci.1998:63:337-49.

    PubMed  CAS  Article  Google Scholar 

  • Trump BF, Berezesky IK.Calcium-mediated cell injury and cell death.FASEB J.1995:18:219-28.

    Google Scholar 

  • van den Eeden SK, Koepsell TD, Longstreth WT, van Belle G, Daling JR, McKnight B.Aspartame ingestion and headaches: a randomized crossover trial.Neurology.1994:44: 1787-93.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Oyama, Y., Sakai, H., Arata, T. et al. Cytotoxic effects of methanol, formaldehyde, and formate on dissociated rat thymocytes: A possibility of aspartame toxicity. Cell Biol Toxicol 18, 43 (2002). https://doi.org/10.1023/A:1014419229301

Download citation

  • DOI: https://doi.org/10.1023/A:1014419229301

  • aspartame
  • formaldehyde
  • methanol
  • toxicity
  • thymocytes