, Volume 31, Issue 1–2, pp 45–51 | Cite as

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

  • Shiro Yamashoji
  • Kenji Isshiki


Menadione-catalyzed H2O2 production by viable cells was proportional to viable cell number, and the assay of this H2O2 production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD50 test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H2O2 production assay. IC50 of each substance required for 50% inhibition of menadione-catalyzed H2O2 production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD50 and IC50, respectively. These results show that menadione-catalyzed H2O2 production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds.

cytotoxicity food safety H2O2 menadione 


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  1. Borenfreund E and Puerner JA (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J Tissue Culture Methods 9: 7–9.CrossRefGoogle Scholar
  2. British Toxicology Society (1984) Special report: A new approach to the classification of substances and preparations on the basis of their acute toxicity. Hum Toxic 3: 85–92.CrossRefGoogle Scholar
  3. Cook JA and Mitchell JB (1989) Viability measurements in mammalian cell systems. Anal Biochem 179: 1–7.CrossRefGoogle Scholar
  4. Ekwall B (1983) Correlation between cytotoxicity in vitro and LD50-values. Acto Pharmacolocia et Toxicolocia 52: 80–99.Google Scholar
  5. Mizoguchi M, Shiga M and Sakamoto K (1993) Diaminodiphenylmethane derivatives as a new chromogenic hydrogen donor in peroxidase-catalyzed oxidation reaction with hydrogen peroxide. Chem Pharm Bull 41: 620–623.Google Scholar
  6. Mosman T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63.CrossRefGoogle Scholar
  7. OECD (1981) Guidelines for the testing of chemical substances-No. 401: Acute oral toxicity. OECD, Paris.Google Scholar
  8. Yam J, Reer PJ and Bruce RD (1991) Comparison of the up-and down method and the fixed-dose procedure for acute oral toxicity testing. Fd Chem Toxic 29: 259–263.CrossRefGoogle Scholar
  9. Yamashoji S, Ikeda T and Yamashoji K (1991) Extracellular generation of active oxygen species catalyzed by endogeneous menadione in yeast cell suspension. Biochim Biophys Acta 1059: 99–105.CrossRefGoogle Scholar
  10. Yamashoji S, Nishimoto F, Usuda F, Kubota H and Isshiki K (1992) Application of the chemiluminescent assay to cytotoxicity test: Detection of menadione-catalyzed H2O2 production by viable cells. Anal Biochem 207: 255–260.CrossRefGoogle Scholar
  11. Yamashoji S (1998) Characterization of extracellular menadione-catalyzed H2O2 production by NIH/3T3 cells. Biochem Mol Biol Int 44: 555–563.Google Scholar
  12. Van den Heuvel ML, Clark DG, Fielder RJ, Koundakjian PP, Oliver GJA, Pelling D, Tomlinson NJ and Walker AP (1990) The international validation of a fixed-dose procedure as an alternative to the classical LD50 test. Fd Chem Toxic 28: 469–482.CrossRefGoogle Scholar
  13. Whitehead A and Curnow RN (1992) Statistical evaluation of the fixed-dose procedure. Fd Chem Toxic 30: 313–324.CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Shiro Yamashoji
  • Kenji Isshiki

There are no affiliations available

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