Abstract
This retrospective reviews the methodology we have developed over several decades for detecting reactive oxygen species (ROS), using the activated polymorphonuclear leukocyte (PMN) as the paradigm of a cell which vigorously generates ROS through activation of NADPH oxidase. In the seventies, the sites of ROS generation by PMN were not clear from biochemical data, and we sought to develop new methods for the cytochemical localization of O·− 2, H2O2, and the H2O2-myeloperoxidase (MPO)-halide system. The H2O2-MPO-halide system in phagocytosing cells was localized at the fine structural level by our development of 3,3′-diaminobenzidine (DAB) as a cytochemical probe for detecting peroxidase activities. Using DAB and exogenous H2O2, we confirmed that azurophil granules discharged MPO into the phagosome, and using particles coated with DAB and relying on endogenous H2O2 to yield oxidized DAB, H2O2 was localized to phagolysosomes. The subcellular sites of H2O2 generation were shown using cerium ions which react with H2O2 and precipitate electron opaque cerium perhydroxides (Ce(OH)2OOH and Ce(OH)3OOH). The results suggested that NADPH oxidase is associated with the plasmalemma, and that the enzyme enters the phagosome along with the invaginating plasmalemma, accounting for the presence of H2O2 in the phagosome. As O·− 2 is the major product of NADPH oxidase, its detection was of some importance. Based on the concept that O·− 2 oxidizes Mn2+ to Mn3+, and Mn3+ oxidizes DAB, a medium containing DAB-Mn2+ was used to localize sites of O·− 2 production in stimulated PMN. The localizations were, as expected, similar to those for H2O2. These techniques have been of considerable usefulness and in general provide the foundation for cytochemistry of ROS in other systems.
Similar content being viewed by others
References
Adams JC (1977) Technical considerations on the use of horseradish peroxidase as a neuronal marker. Neuroscience 2:141–145
Allen RC (1975) Halide dependence of the myeloperoxidase-mediated antimicrobial system of the polymorphonuclear leukocyte in the phenomenon of electronic excitation. Biochem Biophys Res Commun 63:675–683
Allen RC, Stjernholm RL, Steele RH (1972) Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 47:679–684
Angermüller S, Fahimi HD (1988) Light microscopic visualization of the reaction product of cerium used for localization of peroxisomal oxidases. J Histochem Cytochem 36:23–28
Aust SD, Morehouse LA, Thomas CE (1985) Hypothesis paper — role of metals in oxygen radical reactions. J Free Radicals Biol Med 1:3–25
Babbs CF, Cregor MD, Turek JJ, Badylak SF (1991a) Endothelial superoxide production in buffer perfused rat lungs, demonstrated by a new histochemical technique. Lab Invest 65:484–496
Babbs CF, Cregor MD, Turek JJ, Badylak SF (1991b) Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia. A histochemical study. Am J Pathol 139:1069–1080
Babior BM (1978) Oxygen-dependent microbial killing by phagocytes. N Engl J Med 298:659–668
Babior BM, Curnette JT, McMurrich BJ (1976) The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest 58:989–996
Badwey JA, Karnovsky ML (1980) Active oxygen species and the functions of phagocytic leukocytes. Annu Rev Biochem 49:695–726
Badwey JA, Curnette JT, Robinson JM, Lazdins JK, Briggs RT, Karnovsky MJ, Karnovsky ML (1980) Comparative aspects of oxidative metabolism of neutrophils from human blood and guinea pig peritonea: magnitude of the respiratory burst, dependence upon stimulating agents and localization of the oxidases. J Cell Physiol 105:541–551
Baehner RL, Karnovsky MJ, Karnovsky ML (1969) Degranulation of leukocytes in chronic granulomatous disease. J Clin Invest 48:187–192
Bainton DF, Farquhar MG (1966) Origin of granules in polymorphonuclear leukocytes. Two types derived from opposite faces of the Golgi complex in developing granulocytes. J Cell Biol 28:277–301
Bainton DF, Farquhar MG (1968a) Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I Histochemical staining of bone marrow smears. J Cell Biol 39:286–298
Bainton DF, Farquhar MG (1968b) Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. II. Cytochemistry and electron microscopy of bone marrow cells. J Cell Biol 39:299–317
Bainton DF, Ullyot JL, Farquhar MG (1971) The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. J Exp Med 134:907–934
Beckman JS, Beckman TW, Chen J, Marshall PA (1990) Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acd Sci USA 87:1620–1624
Beckman JS, Yao ZY, Anderson PG, Accavitti MA, Tarpey MM, White CR (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe-Seyler 375:81–88
Blough NV, Zafiriou OC (1985) Reaction of superoxide with nitric oxide to form peroxynitrite in alkaline solution. Inorg Chem 24:3502–3504
Bokoch GM (1994) Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. Curr Opin Cell Biol 6:212–218
Briggs RT, Drath DB, Karnovsky ML, Karnovsky MJ (1975a) Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol 67:566–586
Briggs RT, Karnovsky ML, Karnovsky MJ (1975b) Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes. J Cell Biol 64:254–260
Briggs RT, Karnovsky ML, Karnovsky MJ (1977) Hydrogen peroxide production in chronic granulamatous disease: a cytochemical study of reduced pyridine nucleotide oxidases. J Clin Invest 59:1088–1098
Briggs RT, Robinson JM, Karnovsky ML, Karnovsky MJ (1986) Superoxide production by polymorphonuclear leukocytes. A cytochemical approach. Histochemistry 84:371–378
Cammer W, Moore CL (1973) Oxidation of 3,3′-diaminobenzidine by rat liver mitochondria. Biochemistry 12:2502–2509
Candeias LP, Patel KB, Stratford MRL, Wardman P (1993) Free hydroxyl radicals are formed on reaction between the neutrophil-derived species superoxide anion and hypochlorous acid. FEBS Lett 333:151–153
Cavalieri E, Rogan E (1983) One-electron oxidation of aromatic hydrocarbons in chemical and biological systems. In: Cooke M, Dennis AJ (eds) Polynuclear aromatic hydrocarbons. Battle Press, Columbus, Ohio 1–26
Christie KN, Stoward PJ (1982) The cytochemical reactivity of cerium ions with cardiac muscle. J Histochem Cytochem 15:656–672
Corey EJ, Mehrotra MM, Khan AU (1987) Antiarthritic gold compounds effectively quench electronically excited singlet oxygen. Science 236:68–69
Curnette JT, Karnovsky ML, Babior BM (1976) Manganese-dependent NADPH oxidation by granulocyte particles. The role of superoxide and the nonphysiological nature of the manganese requirement. J Clin Invest 57:1059–1067
Deimann W, Angermüller S, Stoward PJ, Fahimi HD (1991) Peroxidases. In: Stoward PJ, Pearse AGE (eds) Histochemistry, vol 3. Churchill Livingstone, Edinburgh, pp 135–159
Dewald B, Baggiolini M, Curnette JT, Babior BM (1979) Subcellular localization of the superoxide-forming enzyme in human neutrophils. J Clin Invest 63:21–29
Drath DB, Karnovsky ML (1974) Bactericidal activity of metalmediated peroxide-ascorbate systems. Infect Immunol 10:1077–1083
Fridovich I (1978) The biology of oxygen radicals. Science 201:875–880
Goldstein IM, Cerqueira M, Lind S, Kaplan HB (1977) Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J Clin Invest 59:249–254
Gossrau R, Van Noorden CJF, Frederiks WM (1990) Pitfall in the light microscopical detection of NADH oxidase. Histochem J 2:155–161
Graham RC, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:219–302
Guiraud HJ, Foote CS (1976) Chemistry of superoxide ion. III. Quenching of singlet oxygen. J Am Chem Soc 98:1984–1986
Halbhuber KJ, Zimmermann N (1985) Light microscopical localization of enzymes by means of cerium-based methods. II. A new cerium-lead-technique for alkaline phosphatase. Acta Histochem 77:67–73
Herzog V, Fahimi HD (1973) A new sensitive colorimetric assay for peroxidase using 3,3′-diaminobenzidine as hydrogen donor. Anal Biochem 55:554–562
Hirai K-I, Moriguchi K, Wang G-Y (1991) Human neutrophils produce free radicals from the cell-zymosan interface during phagocytosis and from the whole plasma membrane when stimulated with calcium ionophore A23187. Exp Cell Res 194:19–27
Hirsch JG (1962) Cinemicrophotographic observations on granule lysis in polymorphonuclear leukocytes during phagocytosis. J Exp Med 116:827–834
Hirsch JG, Cohn ZA (1960) Degranulation of polymorphonuclear leukocytes following phagocytosis of microorganisms. J Exp Med 112:1005–1014
Hobson J, Wright J, Churg A (1991) Histochemical evidence for generation of active oxygen species on the apical surface of cigarette-smoke-exposed tracheal explants. Am J Pathol 139:573–580
Hoover RL, Robinson JM, Karnovsky MJ (1987) Adhesion of polymorphonuclear leukocytes to endothelium enhances the efficiency of detoxification of oxygen-free radicals. Am J Pathol 126:258–268
Hsu S-M, Soban E (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem 30:1070–1082
Jonges GN, Van Noorden CJF, Gossrau R (1990) Quantitative histochemical analysis of glucose-6-phosphatase activity in rat liver using an optimized cerium-diaminobenzidine method. J Histochem Cytochem 38:1414–1419
Karnovsky ML (1962) Metabolic basis of phagocytic activity. Physiol Rev 42:1422–1432
Karnovsky MJ, Robinson JM, Briggs RT, Karnovsky ML (1981) Oxidative cytochemistry in phagocytosis: the interface between structure and function. Histochem J 13:1–22
Khan AU (1977) Theory of electron transfer generation and quenching of singlet oxygen (1∑+ g and 1Δg) by superoxide anion: the role of water in the dismutation of O.− 2. J Am Chem Soc 99:370–371
Khan AU (1981) Direct spectral evidence of the generation of singlet molecular oxygen in the reaction of potassium superoxide with water. J Am Chem Soc 103:6516–6517
Khan AU, Kasha M (1963) The red chemiluminescence of molecular oxygen in aqueous solution. J Chem Phys 39:2105–2106
Khan AU, Kasha M (1970) Chemiluminescence arising from simultaneous transitions in pairs of singlet oxygen molecules. J Am Chem Soc 92:3293–3300
Klebanoff SJ (1968) Myeloperoxidase-halide-hydrogen peroxide antibacterial system. J Bacteriol 95:2131–2138
Klebanoff SJ (1992) Oxygen metabolites from phagocytes. In: Gallin JI, Goldstein IM, Synderman R (eds) Inflammation: basic principles and clinical correlates, second edition. Raven Press, New York, pp 541–588
Klebanoff SJ, Clark RA (1978) The neutrophil: function and clinical disorder. North Holland Publishing Co., Amsterdam
Kono Y, Takahashi M, Asada K (1976) Oxidation of manganous pyrophosphate by superoxide radicals and illuminated spinach chloroplasts. Arch Biochem Biophys 174:454–462
Krinsky NI (1974) Singlet excited oxygen as a mediator of the antibacterial action of leukocytes. Science 186:363–365
Labato M, Briggs RT (1985) Cytochemical localization of hydrogen peroxide generating sites in the rat thyroid gland. Tiss Cell 17:889–900
Liposits Z, Setalo G, Flerko B (1984) Application of the silvergold intensified DAB chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain. Neurosci Ser 13:513–525
Litwin JA (1982) Transition metal-catalysed oxidation of 3,3′-diaminobenzidine [DAB] in a model system. Acta Histochem 71:111–117
Nathan DG, Bachner RL, Weaver DK (1969) Failure of nitroblue tetrazolium reduction in the phagocytic vacules of leukocytes in chronic granulomatous disease. J Clin Invest 48:1895–1904
Neale TJ, Ullrich R, Ojha P, Verhoeven AJ, Kerjaschki D (1993) Reactive oxygen species and neutrophil respiratory burst cytochrome b558 are produced by kidney glomerular cells in passive Heymann nephritis. Proc Natl Acad Sci USA 90:3645–3649
Newman GR, Jasani B, Williams ED (1983) Metal compound intensification of the electron-density of diaminobenzidine. J Histochem Cytochem 31:1430–1434
Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL (1989) Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86:1372–1376
Patel HRH, Frederiks WM, Marx F, Best AJ, Van Noorden CJF (1991) A quantitative histochemical study of d-amino acid oxidase activity in rat liver in relationship with feeding conditions. J Histochem Cytochem 39:81–86
Patriarca P, Dri P, Kakinuma K, Tedesco F, Rossi F (1975) Studies on the mechanism of metabolic stimulation in polymorphonuclear leukocytes during phagocytosis. I. Evidence for superoxide involvement in the oxidation of NADPH2. Biochem Biophys Acta 385:380–386
Rajagopalan KV, Handler P (1964) Hepatic aldehyde oxidase. II. Differential inhibition of electron transfer to various electron acceptors. J Biol Chem 239:2022–2026
Repine JE, White JG, Clawson CC, Holmes BM (1974) Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease. J Clin Invest 54:83–90
Robinson JM, Batten BE (1990) Localization of cerium-based reaction products by scanning laser reflectance confocal microscopy. J Histochem Cytochem 38:315–318
Robinson JM, Karnovsky MJ, Stoward PJ, Lewis PR (1991) Oxidases. In: Stoward PJ, Pearse AGE (eds) Histochemistry, vol 3. Churchill Livingstone, Edinburgh, pp 95–122
Root RK, Metcalf JA (1977) H2O2 release from human granulocytes during phagocytosis. Relationship to superoxide anion formation and cellular catabolism of H2O2: studies with normal and cytochalasin B-treated cells. J Clin Invest 60:1266–1279
Rosenthal I (1975) Singlet molecular oxygen and superoxide radical anion. Israel J Chem 13:86–90
Saito I, Matsuuta I, Inoue K (1983) Formation of superoxide ion via one-electron transfer from electron donors to singlet oxygen. J Am Chem Soc 105:3200–3206
Salin ML, McCord JM (1974) Superoxide dismutases in polymorphonuclear leukocytes. J Clin Invest 54:1005–1009
Segal AW, Abo A (1993) The biochemical basis of the NAPH oxidase of phagocytes. Trends Biochem Sci 18:43–47
Seligman AM, Karnovsky MJ, Wasserkrug HL, Hanker JS (1968) Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol 38:1–14
Shlafer M, Brosamer K, Froder JR, Simon RH, Ward PA, Grum CM (1990) Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused hearts. J Mol Cell Cardiol 22:83–97
Silverstein SC, Steinman RM, Cohn ZA (1977) Endocytosis. Annu Rev Biochem 46:669–722
Steinbeck MJ, Khan AU, Karnovsky MJ (1992) Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap. J Biol Chem 267:13425–13433
Steinbeck MJ, Appel WH, Karnovsky MJ (1993a) NADPH-oxidase and in situ production of superoxide in osteoclasts actively resorbing bone. Mol Biol Cell 4:S:287a
Steinbeck MJ, Khan AU, Appel WH, Karnovsky MJ (1993b) The DAB-Mn++ cytochemical method revisited: validation of specificity for superoxide. J Histochem Cytochem 41:1659–1667
Steinbeck MJ, Khan AU, Karnovsky MJ (1993c) Extracellular production of singlet oxygen by stimulated macrophages quantified using 9,10-diphenyl-anthracene and perlene in a polystyrene film. J Biol Chem 268:15649–15654
Stossell TP (1977) Phagocytosis. Prog Clin Biol Res 13:87–102
Stoward PJ (1991) Reductases and oxygenases. In: Stoward PJ, Pearse AGE (eds) Histochemistry, vol 3. Churchill Livingstone, Edinburgh, pp 123–133
Stoward PJ, Altman FP, Seidler E (1991) Principles of oxidoreductase histochemistry. In: Stoward PJ, Pearse (eds) Histochemistry, vol 3. Churchill Livingstone, Edinburgh, pp 1–25
Strum JM, Karnovsky MJ (1970) Cytochemical localization of endogenous peroxidase in thyroid follicular cells. J Cell Biol 44:655–666
Test ST, Weiss SJ (1986) The generation and utilization of chlorinated oxidants by human neutrophils. Adv Free Rad Biol Med 2:91–116
Turro NJ, Chow M-F, Rigaudy J (1981) Mechanism of thermolysis of endoperoxides of aromatic compounds. Activation parameters, magnetic field and magnetic isotope effects. J Am Chem Soc 103:7218–7224
Van Noorden CJF, Frederiks WM (1993) Cerium methods for light and electron microscopical histochemistry. J Microsc 171, Pt. 1:3–16
Vanderplassche G, Hermans C, Thone F, Borgers M (1989) Mitochondrial hydrogen peroxide generation by NADH-oxidase activity following regional myocardial ischemia in the dog. J Mol Cell Cardiol 21:383–392
Veenhuis M, Wendelaar Bonga SE (1979) Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes. Histochem J 11:561–572
Vigil EL, Swift H, Arntzen CJ (1991) Cytochemistry of plant haem oxidases. In: Stoward PJ, Pearse AGE (eds) Histochemistry, vol 3. Churchill Livingstone, Edinburgh, pp 515–535
Warren JS, Kunkel RG, Simon RH, Johnson KJ, Ward PA (1989) Ultrastructural cytochemical analysis of oxygen radical-mediated immunoglobulin A immune complex induced lung injury in the rat. Lab Invest 60:651–658
Wasserman HH, Larsen DL (1972) Formation of 1,4-endoperoxides from the dye-sensitized photo-oxygenation of alkyl-naphthalenes. J Chem Soc Chem Commun 5:253–254
Wasserman HH, Sheffer JR, Cooper JL (1972) Singlet oxygen reactions with 9,10-diphenylanthracene peroxide. J Am Chem Soc 94:4991–4996
Zgliczynski JM, Stelmaszynska T, Domanski J, Ostrowski W (1971) Chloramines as intermediates of oxidation reaction of amino acids by myeloperoxidase. Biochem Biophys Acta 235:419–424
Zucker-Franklin D, Hirsch JG (1964) Electron microscope studies on the degranulation of rabbit leucocytes during phagocytosis. J Exp Med 120:569–576
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Karnovsky, M.J. Cytochemistry and reactive oxygen species: a retrospective. Histochemistry 102, 15–27 (1994). https://doi.org/10.1007/BF00271045
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00271045