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A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

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Summary

A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP+ (K i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP+ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.

Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.

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References

  • ALTMAN, F. P. (1972) An introduction to the use of tetrazolium salts in quantitative enzyme cytochemistry. Koch-Light Laboratories, Colnbrook.

  • ALTMAN, F. P. & CHAYEN, J. (1970) Evidence for two types of hydrogen atom in reduced nicotinamide—adenine dinucleotide phosphate arising from glucose-6-phosphate oxidation, based on the inhibitory action of certain steroids.Biochem. J. 118, 6–7.

    Google Scholar 

  • BUTCHER, R. G. (1971) The effect of phenobarbitone on the production of type I hydrogen from reduced nicotinamide-adenine dinucleotide phosphate in different regions of the liver lobule.Biochem. J. 125, 22–23.

    Google Scholar 

  • BUTCHER, R. G. (1978) The measurement in tissue sections of the two formazans derived from nitroblue tetrazolium in dehydrogenase reactions.Histochem. J. 10, 739–44.

    Google Scholar 

  • BUTCHER, R. G. (1982) The microsomal NADPH oxidation system in lung cancer. InCellular Biology of the Lung (edited by CUMMING, G. and BONSIGNORE, G.), pp. 439–49. New York: Plenum.

    Google Scholar 

  • BUTCHER, R. G. (1984) Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium; the effect of section thickness.Histochemistry 81, 567–72.

    Google Scholar 

  • BUTCHER, R. G. & ALTMAN, F. P. (1973) Studies on the reduction of tetrazolium salts. II. The measurement of the half-reduced and fully reduced formazans of neotetrazolium chloride in tissue sections.Histochemistry 37, 351–63.

    Google Scholar 

  • BUTCHER, R. G. & VAN NOORDEN, C. J. F. (1985) Reaction rate studies of glucose-6-phosphate dehydrogenase activity in sections of rat liver using four tetrazolium salts.Histochem. J. 17, 993–1008.

    Google Scholar 

  • CHAYEN, J., ALTMAN, F. P. & BUTCHER, R. G. (1973) The effect of certain drugs on the production and possible utilization of reducing equivalents outside the mitochondria. InFundamentals of Cell Pharmacology (edited by DIKSTEIN, S.), pp. 196–230. Springfield.

  • DIGNAM, J. D. & STROBEL, H. W. (1977) NADPH-cytochrome P450 reductase from rat liver: purification by affinity chromatography and characterization.Biochemistry 16, 1116–23.

    Google Scholar 

  • HORNBY, W. E., LILLY, M. D. & CROOK, E. M. (1968) Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose.Biochem. J. 107, 669–74.

    Google Scholar 

  • HUANG, M.-T., MIWA, G. T., CRONHEIM, N. & LU, A. Y. H. (1979) Rat liver cytosolic azoreductase. Electron transport properties and the mechanism of dicumarol inhibition of the purified enzyme.J. biol. Chem. 254, 11223–7.

    Google Scholar 

  • HUGGINS, C. & FUKUNISHI, R. (1964) Induced protection of adrenal cortex against 7,12-dimethylbenz(a)anthracene.J. Exp. Med. 119, 923–42.

    Google Scholar 

  • KEYES, S. R., FRACASSO, P. M., HEIMBROOK, D. C., ROCKWELL, S., SLIGAR, S. G. & SARTORELLI, A. C. (1984) Role of NADPH-cytochromec reductase and DT-diaphorase in the biotransformation of Mitomycinc.Cancer Res. 44, 5638–43.

    Google Scholar 

  • KOUDSTAAL, J. & HARDONK, M. J. (1969) Histochemical demonstration of enzymes related to NADPH-dependent hydroxylating systems in rat liver after phenobarbital treatment.Histochemie 20, 68–77.

    Google Scholar 

  • LIND, C. & ERNSTER, L. (1974) A possible relationship between DT diaphorase and the aryl hydrocarbon hydroxylase system.Biochem. Biophys. Res. Commun. 56, 392–400.

    Google Scholar 

  • LIND, C. & ERNSTER, L. (1976) Inhibition of rat liver microsomal D-T diaphorase by benz(a)pyrene metabolite(s). InMicrosomes and Drug Oxidation (edited by ULLRICH, V., ROOTS, I., HILDEBRANT, A., ESTABROOK, R. W. and CONNEY, A. H.), pp. 362–9. New York: Pergamon.

    Google Scholar 

  • LIND, C., HOCHSTEIN, P. & ERNSTER, L. (1982) DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation.Arch. Biochem. Biophys. 216, 178–85.

    Google Scholar 

  • MASTERS, B. S. S. & OKITA, R. T. (1980) The history, properties and function of NADPH-cytochrome P450 reductase.Pharmac. Ther. 9, 227–44.

    Google Scholar 

  • NISHIKIMI, M., RAO, N. A. & YAGI, K. (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen.Biochem. Biophys. Res. Commun. 46, 849–54.

    Google Scholar 

  • ORRENIUS, S. (1965) On the mechanism of drug hydroxylation in rat liver microsomes.J. Cell. Biol. 26, 713–23.

    Google Scholar 

  • PICKER, S. D. & FRIDOVICH, I. (1984) On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium.Arch. Biochem. Biophys. 228, 155–8.

    Google Scholar 

  • PONTI, V., DIANZANI, M. U., CHEESEMAN, K. & SLATER, T. F. (1978) Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.Chem.-Biol. Interact. 23, 281–91.

    Google Scholar 

  • RAAP, A. K. & VAN DUIJN, P. (1983) Studies on the phenazine methosulphate-tetrazolium capture reaction in NAD(P)+-dependent dehydrogenase cytochemistry. II. A novel hypothesis for the mode of action of PMS and the study of the properties of reduced PMS.Histochem. J. 15, 881–93.

    Google Scholar 

  • SCHOR, N. A., BOH, E. & BURKE, V. T. (1978) Carcinogens and dicumarol: opposite effects on rat liver NAD(P)H dehydrogenation.Enzyme 23, 217–24.

    Google Scholar 

  • SCHOR, N. A., MORGAN, K., EPSTEIN, N. & KNIGHT, R. (1982a) The influence of polycyclic hydrocarbons on the activity of NAD(P)H dehydrognating enzymes on rat thymus. A biochemical and histochemical study.Enzyme 29, 167–74.

    Google Scholar 

  • SCHOR, N. A., STEDMAN, R. B., EPSTEIN, N. & SCHALLY, G. (1982b) Rat splenic D-T diaphorase and NAD(P)H-nitroblue tetrazolium reductase: their use to assess the action of polycyclic hydrocarbons in the lymphatic system.Virchows. Archiv. 41, 83–93.

    Google Scholar 

  • SCHOR, N. A. & GILLESPIE, S. (1984) D-T diaphorase and NAD(P)H-NBT reductase activation. A biochemical and histochemical response of the lymph nodes of the rat to the administration of polycyclic hydrocarbons.Virchows. Archiv. 45, 377–84.

    Google Scholar 

  • SHEPHARD, E. A., PHILLIPS, I. R., BAYNEY, R. M., PIKE, S. F. & RABIN, B. R. (1983) Quantification of NADPH: cytochrome P450 reductase in liver microsomes by a specific radioimmunoassay technique.Biochem. J. 211, 333–40.

    Google Scholar 

  • SMITH, M. T., REDICK, J. A. & BARON, J. (1983) Quantitative immunohistochemistry: a comparison of microdensitometric analysis of unlabeled antibody peroxidase—antiperoxidase staining and of microfluorometric analysis of indirect fluorescent antibody staining for nicotinamide adenosine dinucleotide phosphate (NADPH)-cytochromec (P450) reductase in rat liver.J. Histochem. Cytochem. 31, 1183–9.

    Google Scholar 

  • SMITH, M. T. & WILLS, E. D. (1981) The effects of dietary lipid and phenobarbitone on the production and utilization of NADPH in the liver. A combined biochemical and quantitative cytochemical study.Biochem. J. 200, 691–9.

    Google Scholar 

  • STOWARD, P. J. (1980) Criteria for the validation of quantitative histochemical enzyme techniques. InTrends in Enzyme Histochemistry and Cytochemistry (edited by EVERED, D. and O'CONNOR, M.), pp. 11–31. Amsterdam: Excerpta Medica.

    Google Scholar 

  • STROBEL, H. W., DIGNAM, J. D. & GUM, J. R. (1980) NADPH cytochrome P450 reductase and its role in the mixed function oxidase reaction.Pharmac. Ther. 8, 525–37.

    Google Scholar 

  • TAIRA, Y., GREENSPAN, P., KAPKE, G. F., REDICK, J. A. & BARON, J. (1980a) Effects of phenobarbital, pregnenolone-16-carbonitrile, and 3-methylcholanthrene pretreatments on the distribution of NADPH-cytochromec (P450) reductase within the liver lobule.Mol. Pharmacol. 18, 304–12.

    Google Scholar 

  • TAIRA, Y., REDICK, J. A. & BARON, J. (1980b) An immunohistochemical study on the localization and distribution of NADPH-cytochromec (P450) reductase in rat liver.Mol. Pharmacol. 17, 374–81.

    Google Scholar 

  • VAN NOORDEN, C. J. F. & BUTCHER, R. G. (1984) Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.J. Histochem. Cytochem. 32, 998–1004.

    Google Scholar 

  • VAN NOORDEN, C. J. F. & TAS, J. (1982) The role of exogenous electron carriers in NAD(P)-dependent dehydrogenase cytochemistry studiedin vitro and with a model system of polyacrylamide films.J. Histochem. Cytochem. 30, 12–20.

    Google Scholar 

  • VAN NOORDEN, C. J. F., TAS, J. & VOGELS, I. M. C. (1983) Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.Histochem. J. 15, 583–99.

    Google Scholar 

  • WILLIAMS, J. B., WANG, R., LU, A. Y. H. & PICKETT, C. B. (1984) Rat liver DT-diaphorase: regulation of functional mRNA levels by 3-methylcholanthrene, trans-stilbene oxide, and phenobarbital.Arch. Biochem. Biophys. 232, 408–13.

    Google Scholar 

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Authors and Affiliations

  1. Laboratory of Histology and Cell Biology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

    Cornelis J. F. Van Noorden

  2. Department of Lung Pathology, The Cardiothoracic Institute, GU29 0BL, Midhurst, West Sussex, England

    Roger G. Butcher

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  1. Cornelis J. F. Van Noorden
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  2. Roger G. Butcher
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Van Noorden, C.J.F., Butcher, R.G. A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity. Histochem J 18, 364–370 (1986). https://doi.org/10.1007/BF01675217

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