, 162:179 | Cite as

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates

  • Kolliputi V. Narasaiah
  • R. B. Sashidhar
  • C. Subramanyam


The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1–10 μM H2O2 in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [14C]-acetate incorporation into aflatoxin B1 and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.


acetyl CoA carboxylase aflatoxin Aspergillus parasiticus Hydrogen peroxide oxidative stress reactive oxygen species 



Glutathione peroxidase


Glutathione (reduced)


Glutathione (oxidized)


Reactive oxygen species


Superoxide dismutase


Thiobarbituric acid reactive substances


Xanthine oxidase


  1. 1.
    Eaton, DLGroopman, ID eds. 1994The toxicology of aflatoxins: Human health, veterinary and agricultural significanceAcademic PressNew YorkGoogle Scholar
  2. 2.
    International Agency for Research on Cancer (IARC) Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. In: IARC Monographs on the evaluation of carcinogenic risks to humans. Lyon, France: International Agency for Research on Cancer, 1993; Vol. 56: 245–395Google Scholar
  3. 3.
    Hideaki, K, Hajime, H 1999Redox regulation of cellular signalingCell Signal11114CrossRefGoogle Scholar
  4. 4.
    Yu, J, Whitelaw, CA, Nierman, WC, Bhatnagar, D, Cleveland, TE 2004 Aspergillus flavus expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of cropsFEMS Microbiol Lett237333340PubMedGoogle Scholar
  5. 5.
    Fabbri, AA, Feneli, C, Panfili, G, Passi, S, Fasella, P 1983Stimulation of aflatoxin biosynthesis by lipophilic epoxidesJ Gen Microbiol12917211723PubMedGoogle Scholar
  6. 6.
    Shashidhar, J, Sashidhar, RB, Deshpande, V 2005Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production)FEMS Microbiol Lett251113117PubMedCrossRefGoogle Scholar
  7. 7.
    Minto, RE, Townsend, CA 1997Enzymology and molecular biology of aflatoxin biosynthesisChem Rev9725372555PubMedCrossRefGoogle Scholar
  8. 8.
    Yu, J, Bhatnagar, D, Cleveland, TE 2004Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus FEBS Lett564126130PubMedCrossRefGoogle Scholar
  9. 9.
    Shih, CN, Marth, EH 1974Aflatoxin formation, lipid synthesis and glucose metabolism by Aspergillus parasiticus during incubation with and without agitationBiochem Biophys Acta338286296Google Scholar
  10. 10.
    Passi, S, Nazzarro-Poo, M 1986Microsomal and mitocondrial involvement in production of alflatoxins induced by carbon tetrachloride and hydroperoxide cultures of Aspergillus parasiticus Trans Brit Mycol Soc87451456CrossRefGoogle Scholar
  11. 11.
    Buchanan, RL, Federowicz, D, Stahl, HG 1985Activities of tricarboxylic acid cycle enzymes in an aflatoxigenic strain of Aspergillus parasiticus after peptone to glucose carbon shiftTrans Brit Mycol Soc84267275CrossRefGoogle Scholar
  12. 12.
    Bhatnagar, RK, Ahmad, SA, Kohli, KK, Mukerji, KG, Venkitasubramanian, TA 1982Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240Biochem Biophys Res Commun10412871292PubMedCrossRefGoogle Scholar
  13. 13.
    Jayashree, T, Subramanyam, C 2000Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus Free Radic Biol Med29981985PubMedCrossRefGoogle Scholar
  14. 14.
    Cleveland, TE, Bhatnagar, D, Brown, RL 1991Aflatoxin production via cross-feeding intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutantsAppl Environ Microbiol5729072911PubMedGoogle Scholar
  15. 15.
    Umbreit, WW, Burris, RH, Stauffer, JF 1972Manometric and biochemical techniquesBurgess Publishing CompanyMinneapolis, MNGoogle Scholar
  16. 16.
    Jayashree, T, Subramanyam, C 1999Antiaflatoxigenic activity of eugenol is due to inhibition of lipid peroxidationLett Appl Microbiol28179183PubMedCrossRefGoogle Scholar
  17. 17.
    Bradford, MM 1976A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding methodAnal Biochem72248254PubMedCrossRefGoogle Scholar
  18. 18.
    Marklund, S, Marklund, G 1974Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutaseEur J Biochem47469474PubMedCrossRefGoogle Scholar
  19. 19.
    Martinez, JIR, Launay, JM, Dreux, C 1979A sensitive flourimetric microassay for the determination of glutathione peroxidase activity. Application to human blood plateletsAnal Biochem98154159PubMedCrossRefGoogle Scholar
  20. 20.
    Fried, R, Fried, LW 1974Xanthine oxydase(xanthine -dehydrogenase)Bergmeyer, HU eds. Methoden der enzymatischen analyse3Verlag Chemie, Weinheim/BerstrNew York682688Google Scholar
  21. 21.
    Omura, T, Sato, R 1964The carbon monoxide binding pigment of liver microsomesJ Biol Chem23923702378PubMedGoogle Scholar
  22. 22.
    Masters, BSS, Williams, CH, Kamin, H 1967The preparation and properties of microsomal TNPH-cytochrome c reductase from pig liverMeth Enzymol10565573Google Scholar
  23. 23.
    Sumper, M 1981Acetyl CoA carboxylase from yeastMeth Enzymol713437PubMedGoogle Scholar
  24. 24.
    Hissin, P, Hilf, RA 1976Flourometric method for determination of oxidized and reduced glutathione in tissuesAnal Biochem74214226PubMedCrossRefGoogle Scholar
  25. 25.
    Ernster, L, Nordenbrand, K 1967Micosomal lipid peroxidationMeth Enzymol10574580CrossRefGoogle Scholar
  26. 26.
    Praveen Rao, J, Sashidhar, RB, Subramanyam, C 1998Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999World J Microbiol Biotechnol147175Google Scholar
  27. 27.
    Davidson, JF, Whyte, B, Bissinger, PH, Schiestl, RH 1996Oxidative stress is involved in heat- induced cell death in Saccharomyces cervevisiae Proc Natl Acad Sci USA9351165121PubMedCrossRefGoogle Scholar
  28. 28.
    Yao, RC, Hsieh, DPH 1974Step of Dichlorvos inhibition in the pathway of aflatoxin biosynthesisAppl Microbiol285257PubMedGoogle Scholar
  29. 29.
    Snedecor, GW, Cochran, WG 1968Short cut and non parametric methodsSnedecor, GWCochran, WG eds. Statistical methodsOxford and IBHNew Delhi120132Google Scholar
  30. 30.
    Davis, ND, Denier, UL 1970Environmental factors affecting the production of aflatoxinHerznberg, M eds. Proceedings of the first U.S. Japan conference on toxic microorganismU.S Department of InteriorWashington, DC4347Google Scholar
  31. 31.
    Feneli, C, Fabbri, AA, Finnoti, E, Fasella, G, Passi, S 1984Free radicals and aflatoxin biosynthesisExperientia40191193CrossRefGoogle Scholar
  32. 32.
    Fenelli C, Fabbri AA, Finnoti E, Passi S. Lipid peroxidation and aflatoxin biosynthesis by Aspergillus parasiticus and Aspergillus flavus. 1983; 129: 3447–3452Google Scholar
  33. 33.
    Gopa, B, Raj, HG, Mukerji, KG 1989Glutathione levels and γ-glutamyl transpeptidase activities in aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus J Toxicol Toxin Rev8329338Google Scholar
  34. 34.
    Fanelli, C, Fabbri, AA 1989Relationship between lipids and aflatoxin biosynthesisMycopathology107115120CrossRefGoogle Scholar
  35. 35.
    Izawa, S, Maeda, K, Miki, T, Mano, J, Inoue, Y, Kimura, A 1998Importance of glucose-6 phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cervevisiae Biochem J330811817PubMedGoogle Scholar
  36. 36.
    Lee, JS, Hah, YC, Roe, JH 1993The induction of oxidative enzymes in Streptomyces coelicolor upon hydrogen peroxide treatmentJ Gen Microbiol13910131018Google Scholar
  37. 37.
    Saxena, M, Mukerji, KG, Raj, HG 1988Positive correlation between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus Biochem J54567570Google Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Kolliputi V. Narasaiah
    • 1
  • R. B. Sashidhar
    • 1
  • C. Subramanyam
    • 1
  1. 1.Department of BiochemistryUniversity College of Science, Osmania UniversityHyderabadIndia

Personalised recommendations