Studies on the Mechanism of Stimulation of Microsomal H2O2 Formation and Benzo(a)pyrene Hydroxylaton by Substrates and Flavone

  • A. G. Hildebrandt
  • C. Bergs
  • G. Heinemeyer
  • E. Schlede
  • I. Roots
  • B. Abbas-Ali
  • A. Schmoldt
Part of the Advances in Experimental Medicine and Biology book series (AEMB)


The addition of activators like flavone and hexobarbital to hepatic microsomes markedly stimulates H2O2 formation. The similar increase observed with flavone of microsomal hydroxylation of benzo(a)pyrene and its inhibition by catalase and methanol suggests but does not prove a necessary interaction of microsomal H2O2 production with benzo(a)pyrene hydroxylation.

Hexobarbital and flavone-stimulated H2O2 formation is optimal at a stoichiometric relationship of these activators and NADPH. This implies either their direct participation as electron donors or their indirect involvement in electron transport by facilitation of stoichiometric substrate cytochrome P-450/NADPH flavoprotein interactions. Steady state kinetic data are consistent with a scheme in which the formation in microsomes of a complex of 1 mole of NADPH with NADPH-cytochrome P-450 reductase and 1 mole hexobarbital with cytochrome P-450 regulates H2O2 formation.


Liver Microsome H202 Production Hepatic Microsome Redox Protein Mixed Function Oxidase 


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  1. 1.
    R.W. Estabrook and J. Werringloer, Cytochrome P-450 - its role in oxygen activation for drug metabolism, in: “Drug Metabolism Concepts,” D.M. Jerina, ed., ACS Symposium Series 44:1–28(1977).CrossRefGoogle Scholar
  2. 2.
    B. Bösterling, A. Stier, A.G. Hildebrandt, J.H. Dawson and J.R. Trudell, Reconstitution of cytochrome P-450 and cytochrome P-450 reductase into phosphatidylcholine-phosphatidylethanolamine bilayers: characterisation of structure and metabolic activity, Mol. Pharmacol. 16: 332–342 (1979).PubMedGoogle Scholar
  3. 3.
    A.G. Hildebrandt and I. Roots, NADPH-dependent formation and breakdown of hydrogen peroxide during mixed function oxidation reactions in liver microsomes, Arch. Biochem. Biophys. 171: 385–397 (1975).CrossRefGoogle Scholar
  4. 4.
    J. Werringloer, The formation of hydrogen peroxide during hepatic microsomal electron transport reactions, in: “Microsomes and Drug Oxidations,” V. Ullrich, I. Roots, A.G. Hildebrandt, R.W. Estabrook and A. Conney, eds., Pergamon Press, Oxford, 261–268 (1977).Google Scholar
  5. 5.
    G.D. Nordblom and M.J. Coon, Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450, Arch. Biochem. Biophys. 180: 343–347 (1977).PubMedCrossRefGoogle Scholar
  6. 6.
    H.S. Mason, Mechanisms of oxygen metabolism, Advanc. Enzymol. 19: 79–233 (1957).Google Scholar
  7. 7.
    A.G. Hildebrandt, G. Heinemeyer, S. Nigam and I. Roots, Substrate and oxygen activation during hexobarbital metabolism, in: “Microsomes, Drug Oxidations and Chemical Carcinogenesis”, M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P. L. O’Brien, eds., Academic Press, New York, 335–338 (1980).Google Scholar
  8. 8.
    J.R. Gillette, B.B. Brodie and B.N. La Du, The oxidation of drugs by liver microsomes: on the role of TPNH and oxygen, J. Pharmacol. Exp Ther. 119: 532–540 (1957).PubMedGoogle Scholar
  9. 9.
    A.G. Hildebrandt, M. Speck and I. Roots, Possible control of hydrogen peroxide production and degradation in microsomes during mixed function oxidation reactions, Biochem. Biophys. Res. Comm. 54: 968–975 (1973).PubMedCrossRefGoogle Scholar
  10. 10.
    R.W. Estabrook and J. Werringloer, Active oxygen–fact or fancy? in: “Microsomes and Drug Oxidations”, V. Ullrich, I. Roots, A.G. Hildebrandt, R.W. Estabrook and A.H. Conney, eds., Pergamon Press, Oxford, 748–757 (1977).Google Scholar
  11. 11.
    A.G. Hildebrandt, G. Heinemeyer and I. Roots, Stoichiometric cooperation of NADPH and hexobarbital in hepatic microsomes during the catalysis of hydrogen peroxide formation, (1981), submitted for publication.Google Scholar
  12. 12.
    G. Heinemeyer, A.G. Hildebrandt, I. Roots and S. Nigam, Stoichiometry of mixed function oxidase dependent hydroxylation of hexobarbital, in: “Microsomes, Drug Oxidations and Chemical Carcinogenesis, Vol I.”, M.J. Coon, A.H. Conney, R.W. Estabrook, A.V. Gelboin, J.R. Gillette and P.J. O’Brien, eds., Academic Press, New York, 331–334 (1980).Google Scholar
  13. 13.
    A.G. Hildebrandt and R.W. Estabrook, Regulation of microsomal hepatic oxidase activity by substrate and NADPH, NaunynSchmiedeberg’s Arch. Pharmacol., Suppl. 311: R9 (1980).CrossRefGoogle Scholar
  14. 14.
    V. Ullrich and H. Diehl, Uncoupling of monoxygenation and electron transport by fluorocarbons in liver microsomes, Europ. J. Biochem. 20: 509–512 (1971).PubMedCrossRefGoogle Scholar
  15. 15.
    H. Staudt, F. Lichtenberger and V. Ullrich, The role of NADH in uncoupled microsomal monoxygenation, Europ. J. Biochem. 56: 99–106 (1974).CrossRefGoogle Scholar
  16. 16.
    A. Schmoldt, A.G. Hildebrandt, G. Heinemeyer and I. Roots, Digitoxin oxidation and hydrogen peroxide formation by hepatic microsomal cytochrome P-450 oxidase in rat. VIIth Europ. Workshop on Drug Metabolism, Zürich (1980).Google Scholar
  17. 17.
    A.G. Hildebrandt, G. Heinemeyer, C. Bergs, I. Roots, E. Schlede, J. Gundlach and B. Abbas-Ali, Flavone stimulated hydrogen peroxide formation and benzo(a)pyrene hydroxylation in hepatic microsomes from animal and man. (1980) submitted.Google Scholar
  18. 18.
    A.G. Hildebrandt, M. Matsubara, R.W. Estabrook and G. Heinemeyer, unpublished.Google Scholar
  19. 19.
    G. Heinemeyer, S. Nigam and A.G. Hildebrandt, Hexobarbital-binding, hydroxylation and hexobarbital-dependent hydrogen peroxide production in hepatic microsomes of guinea pig, rat and rabbit, Naunyn-Schmiedeberg’s Arch. Pharmacol. 314: 201–220 (1980).PubMedCrossRefGoogle Scholar
  20. 20.
    G.T. Miwa, S.B. West, M.T. Huang and A.H. Lu, Studies on the association of cytochrome P-450 and NADPH-cytochrome c-reductase during catalysis in a reconstituted hydroxylation system, J. Biol. Chem. 254: 5695–5700 (1979).PubMedGoogle Scholar
  21. 21.
    R.W. Estabrook, A.G. Hildebrandt, H. Remmer, J.B. Schenkman, 0. Rosenthal and D.Y. Cooper, The role of cytochrome P-450 in microsomal mixed function oxidation reactions, in: “Biochemie des Sauerstoffs”, B. Hess and H.J. Staudinger, eds., Springer, Berlin, 142–177 (1968).Google Scholar
  22. 22.
    F.J. Wiebel, J.C. Leutz, L. Diamond and H.V. Gelboin, Aryl hydrocarbon (benzo(a)pyrene) hydroxylase in microsomes from rat tissue: differential inhibition and stimulation by benzoflavones and organic solvents. Arch. Biochem. Biophys. 144: 78 (1971).PubMedCrossRefGoogle Scholar
  23. 23.
    F.J. Wiebel and H.V. Gelboin, Aryl hydrocarbon (benzo(a)pyrene) hydroxylases in liver from rats of different age, sex and nutritional status, Biochem. Pharmacol. 24: 1511–1515 (1975).PubMedCrossRefGoogle Scholar
  24. 24.
    J. Kapitulnik, P.J. Poppers, M.K. Buening, J.G. Fortner and A.H. Conney, Activation of monoxygenases in human liver by 7,8-benzoflavone, Clin. Pharmacol. Ther. 22: 475–484 (1977).PubMedGoogle Scholar
  25. 25.
    M.K. Buening, J.G. Fortner, A. Kappas and A.H. Conney, 7,8-Benzoflavone stimulates the metabolic activation of aflatoxin B1 to mutagens by human liver, Biochem. Biophys. Res. Commun. 82: 248–355 (1978).CrossRefGoogle Scholar
  26. 26.
    D.R. Thakker, W. Levin, A.W. Wood, R.E. Lehr, S. Kumar, A.H. Conney and D.M. Jerina, Metabolism and metabolic activation of benzo(e)pyrene 9,10-dihydrodiol by hepatic microsomes from several species, Proc. Amer. Assoc. Cancer Research 21: 89 (1980).Google Scholar
  27. 27.
    A.H. Conney, Microsomes and drug oxidations: perspectives and challenges, in: “Microsomes, Drug Oxidations, and Chemical Carcinogenesis”, M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien, eds., Academic Press, New York, Vol. 2, 1103–1118 (1980).Google Scholar
  28. 28.
    Y. Imai and R. Sato, Activation and inhibition of microsomal hydroxylation by ethyl-isocyanide, Biochem. Biophys. Res. Commun. 25: 80–86 (1966).CrossRefGoogle Scholar
  29. 29.
    K.C. Leibman, Effects of metyrapone on liver microsomal drug oxidation, Mol. Pharmacol. 5: 1–9 (1969).Google Scholar
  30. 30.
    M.W. Anders, Acetone enhancement of microsomal aniline para-hydroxylase activity, Arch. Biochem. Biophys. 126: 269–275 (1968).CrossRefGoogle Scholar
  31. 31.
    M.-T. Huang, R.L. Chang, and A.H. Conney, Studies on the mechanism of microsomal benzo(a)pyrene hydroxylase activity by 7,8-benzoflavone (BF), Feder. Proceedings 39: 2053 (1980).Google Scholar
  32. 32.
    J. Capdevila, R. Renneberg, R.A. Prough and R.W. Estabrook, Multiple products of polycyclic hydrocarbon metabolism, in: “Genetic and Environmental Factors in Experimental and Human Cancer”, H.V. Gelboin, B. MacMahon, T. Matsushima, T. Sugimura, S. Takayama and H. Takebe, eds., Japan Scientific Societies Press, 43–56 (1980).Google Scholar
  33. 33.
    J. Capdevila, Y. Saiki, R.A. Prough and R.W. Estabrook, Biochemical considerations of the enzymology associated with quinone and tetrol formation during benzo(a)pyrene metabolism, in: “13th Jerusalem Symposium in Chemical Carcinogenesis”, B. Pullman, T.O.P. Ts’o and H.V. Gelboin, eds., (1980) in press.Google Scholar
  34. 34.
    S.B. West and A.Y.H. Lu, Liver microsomal electron transport systems, properties of a reconstituted NADH-mediated benzo(a)pyrene hydroxylation system, Arch. Biochem. Biophys. 182: 369–378 (1977).PubMedCrossRefGoogle Scholar
  35. 35.
    M.J. Rogers and P. Strittmatter, Lipid-protein interactions in the reconstitution of the microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase system, J. Biol. Chem. 248: 800–806 (1973).PubMedGoogle Scholar
  36. 36.
    K. Mihara and R. Sato, Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes, J. Biochem. (Tokyo) 78: 1057–1073 (1975).Google Scholar
  37. 37.
    G.H. Hogeboom, Fractionation of cell components of animal tissue, in: “Methods in Enzymology Vol. I.”, S.P. Colowick, N.O. Kaplan, eds., Academic Press, New York, 16–19 (1955).Google Scholar
  38. 38.
    H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with a Folin phenol reagent, J. Biol. Chem. 193: 265–275 (1951).PubMedGoogle Scholar
  39. 39.
    D. Keilin and J.F. Hartree, Properties of azide catalase, Biochem. J. 39: 148–157 (1945).PubMedGoogle Scholar
  40. 40.
    T. Nash, The colorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J. 55: 416–421 (1953).PubMedGoogle Scholar
  41. 41.
    R.M. Philpot, E. Arine and J.R. Fouts, Reconstitution of the rabbit pulmonary microsomal mixed function oxidase system from solubilized components, Drug Metab. Dispos. 3: 118–126 (1975).PubMedGoogle Scholar
  42. 42.
    D.W. Nebert and H.V. Gelboin, Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme, J. Biol. Chem. 243: 6242–6249 (1968).PubMedGoogle Scholar
  43. 43.
    J.W. Depierre and G. Dallner, Structural aspects of the membrane of the endoplasmic reticulum, Biochim. Biophys. Acta. 415: 411–472 (1975).PubMedCrossRefGoogle Scholar
  44. 44.
    I. Roots, G. Laschinsky, A.G. Hildebrandt, G. Heinemeyer, S. Nigam, Inhibition of hexobarbital-induced microsomal H2O2 production by metyrapone, in: “Microsomes, Drug Oxidations, and Chemical Carcinogenesis”, M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien, eds., Academic Press, New York, Vol. 1, 375–378 (1980).Google Scholar
  45. 45.
    V. Ullrich, H. Kuthan, Autoxidation and uncoupling in microsomal monooxygenations, in: “.Biochemistry, Biophysics and Regulation of Cytochrome P-450”, J.-A. Gustafsson, J. Carlstedt-Duke, A. Mode, J. Rafter, eds., Elsevier, Amsterdam, 267–272 (1980).Google Scholar

Copyright information

© Springer Science+Business Media New York 1982

Authors and Affiliations

  • A. G. Hildebrandt
    • 1
  • C. Bergs
    • 1
  • G. Heinemeyer
    • 1
  • E. Schlede
    • 1
  • I. Roots
    • 1
  • B. Abbas-Ali
    • 2
  • A. Schmoldt
    • 3
  1. 1.Institut für Klinische Pharmakologie der Freien, Klinikum SteglitzUniversität BerlinBerlin 45Germany
  2. 2.College of Medicine Al Mustansiryia UniversityBaghdadIraq
  3. 3.Pharmakologisches InstitutUniversität HamburgGermany

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