bromide) permeabilization treatment without disruption of the E. coli.~5 In the case of GS, the availability of a overproducing strain facilitates these studies. 4 Because relatively small numbers of bacterial cells are used in these experiments, the best method for measurement of oxidized proteins is labeling with tritiated sodium borohydride 14followed by acid hydrolysis and Dowex chromatography. Similar studies are designed for the determination of oxidized proteins in other target cells such as mammalian cells and, in particular, adherent cells which could be treated in culture dishes. 15 R. Backman, Y.-M. Chen, and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 78, 3743 (1981).

[61] X e n o b i o t i c A c t i v a t i o n b y S t i m u l a t e d H u m a n Polymorphonuclear Leukocytes and Myeloperoxidase By DAVID A. EASTMOND and MARTYN T. SMITH

Introduction Numerous pharmacological and environmental chemicals (xenobiotics) are enzymatically converted in the body to reactive intermediates which may be toxic and/or carcinogenic. Although a significant proportion of this metabolic activation occurs via the cytochrome P-450 monooxygenases, numerous studies indicate that other enzymes and cellular components are capable of bioactivation. For example, prostaglandin synthase has been implicated in the nephrotoxicity of phenacetin 1and acetaminophen2 and in the induction of bladder cancer by nitrofurans 3 and aromatic amines. 4 Hydroperoxide-dependent bioactivation may also be involved in the induction of skin cancer by polycyclic aromatic hydrocarbons. 5 Recent studies have demonstrated that the metabolic activation of xenobiotics can occur during the oxidative burst of polymorphonuclear t B. Andersson, M. Nordenskjold, A. Rahimtula, and P. Moldeus, Mol. Pharmacol. 22, 479 (1982).

2p. Moldeus, B. Andersson, A. Rahimtula,and M. Berggren,Biochem. Pharmacol. 31, 1363 (1982). S. M. Cohen, T. V. Zenser, G. Murasaki, S. Fukushima,M. B. Mattammal,N. S. Rapp, and B. B. Davis, Cancer Res. 41, 3355 (1981). 4R. W. Wise,T. V. Zenser, F. F. Kadlubar,and B. B. Davis, CancerRes. 44, 1893(1984). L. J. Marnett, Carcinogenesis 8, 1365(1987). METHODS IN ENZYMOLOGY,VOL. 186

Copyright © 1990by Academic Press, Inc. All rightsof reproduction in any form reserved.




leukocytes (PMNs). 6-~4 The oxidative burst of PMNs can be triggered by various chemicals and physical agents and is part of the inflammatory response) 5 Numerous lysosomal and peroxidative enzymes, as well as large quantities of active oxygen species, are released during this process.16 Chemical bioactivation occurs primarily through peroxidase-mediated oxidations although some direct oxygen radical effects have been reported. 6,7 This bioactivation mechanism may be involved in the myelotoxicity induced by benzene, 7,8 the agranulocytosis caused by procainamide and other d r u g s , 9 and the genotoxicity and carcinogenicity of polycyclic aromatic hydrocarbons,~° diethylstilbestrol, H and azo dyes 12as well as the antiinflammatory action of numerous drugs) 3,~4 Conversion of xenobiotics to reactive and genotoxic species can be studied through the use of various in vitro cellular and enzymatic systems. These methods generally rely on the conversion of the chemical of interest to a reactive species and the subsequent quantitation of protein or DNA binding, mutagenicity, or identification of the reaction products. In this chapter, we focus primarily on the techniques that have been used in our research to follow this process of bioactivation. Detailed protocols and specific information on various aspects of neutrophil isolation and function can be obtained from Ref. 17. Studies with Human Polymorphonuclear Leukocytes Isolation Procedure. Venous blood is obtained from healthy volunteers using acid-citrate-dextrose (ACD)-containing Vacutainers. PMNs 6 M. A. Trush, Toxicol. Left. 20, 297 (1984). 7 D. A. Eastmond, R. C. French, D. Ross, and M. T. Smith, Chem.-Biol. Interact. 63, 47 (1987). 8 D. A. Eastmond, M. T. Smith, and R. D. Irons, Toxicol. Appl. Pharmacol. 91, 85 (1987). 9 j. Uetrecht, N. Zahid, and R. Rubin, Chem. Res. Toxicol. 1, 74 (1988). to M. A. Trush, J. L. Seed, and T. W. Kensler, Proc. Natl. Acad. Sci. U.S.A. 82, 5194 (1985). u D. A. Eastmond, R. C. French, D. Ross, and M. T. Smith, Cancer Lett. 35, 79 (1987). ~2 K. Takanaka, P. J. O'Brien, Y. Tsuruta, and A. D. Rahimtula, Cancer Lett. 15, 311

(1982). 13 S. Ichihara, H. Tomisawa, H. Fukazawa, and M. Tateishi, Biochem. Pharmacol. 34, 1337 (1985). 14 M. Wasil, B. Haliwell, C. P. Moorhouse, D. C. S. Hutchinson, and H. Baum, Biochem. Pharmacol. 36, 3847 (1987). 15 S. T. Test and S. J. Weiss, Ado. Free Radical Biol. Med. 2, 91 (1986). ~6M. J. Karnovsky and J. M. Robinson, in "Histochemistry: The Widening Horizons" (P. J. Stoward and J. M. Polak, eds.), p. 46. Wiley, New York, 1981. ~7j. A. Metcalf, J. I. Gallin, W. M. Nauseef, and R. K. Root, "Laboratory Manual of Neutrophil Function." Raven, New York, 1986.




are isolated by dextran sedimentation following standard protocols. ~8Cell counts are performed using a hemocytometer or a cell counter, and viability is determined by trypan blue exclusion, is Following isolation, the capacity of each batch of PMNs to undergo the oxidative burst should be determined by measuring the production of 02 = from the cells when stimulated by phorbol 12-myristate 13-acetate (PMA) or another stimulating agent. Typical values for neutrophils isolated by this protocol are 13 nmol O2-:/min/106 cells. Standard Incubations. PMNs (10 7 cells/ml) and the chemical (radiolabeled for binding studies) at nontoxic concentrations are incubated in Dulbecco's phosphate-buffered saline (PBS; pH 7.1; 1 ml final volume) at 37° in a shaking water bath. PMA (0.1-1/zg/ml; prepared as described by Markert et al. ~s) is added to initiate the oxidative burst. Under these conditions, the enhanced effect of the stimulated PMNs on the end point of interest is determined and compared with the appropriate controls. Appropriate controls may include incubations without PMA or without the chemical, incubations performed under anaerobic conditions or with heat-killed cells, or incubations containing superoxide dismutase (SOD, 10/zg/ml; from frozen stock 1 mg/ml in phosphate buffer; 3000 U/mg), catalase (650 U/ml), sodium azide (I0 mM), or combinations of the above treatments. The direct measurement of chemical disappearance and metabolite formation l°,19-2l is preferred. Since this is often impossible for reactive metabolites, other indirect measurements of xenobiotic activation have been employed, such as macromolecular binding (Table I). A useful technique for measuring xenobiotic binding to cellular macromolecules (primarily proteins) is described below. PMN-Mediated Macromolecular Binding Assay. The conversion of the xenobiotic to reactive species can be determined by measuring the covalent binding of the activated radiolabeled compound to cellular proteins or other macromolecules. The use of SOD, catalase, and azide treatments can give valuable insights into the mechanism by which metabolic activation is occurring. The standard incubation employs a radiolabeled compound (-0.01-0.1/~Ci), and the reaction is terminated at 20 min by the addition of trichloroacetic acid (TCA; 50/zl of a 100% solution; 5% final concentration). The samples are maintained on ice and centrifuged (550 g for 5 min) prior to the determination of binding. ~8 M. Markert, P. C. Andrews, and B. M. Babior, this series, Vol. 105, p. 358. ~9M. S. Alexander, R. M. Husney, and A. L. Sagone, Jr., Biochem. Pharmacol. 35, 3649 (1986). 20 B. Kalyanaraman and P. G. Sohnle, J. Clin. Invest. 75, 1618 (1985). 21 j. Uetrecht, N. Zahir, N. H. Shear, and W. D. Biggar, J. Pharmacol. Exp. Therapeut. 245, 274 (1988).





Cytochrome c reduction Chemiluminescence DNA breakage Mutagenicity in Salmonella typhimurium Sister chromatid exchange in V-79 cells



Phenols, estrogens, phenytoin Polycyclic aromatic hydrocarbons (PAH), N-methylaminoazobenzene, phenol, aromatic amines Phenols, estrogens PAH, phenols, estrogens, imipramine Bleomycin A2 PAH

a, b a, b, f, I m f



a-e f-k

D. A. Eastmond R. C. French, D. Ross, and M. T. Smith, Chem.-Biol. Interact. 63, 47 (1987). b D. A. Eastmond, R. C. French, D. Ross, and M. T. Smith, CancerLett. 35, 79 (1987). c D. A. Eastmond, M. T. Smith, and R. D. Irons, Toxicol. Appl. Pharmacol. 91, 85 (1987). d S. J. Klebanoff, J. Exp. Med. 145, 983 (1977). e j. Uetrecht and N. Zahid, Chem. Res. Toxicol. 1, 148 (1988). s M. A. Trush, J. L. Seed, and T. W. Kensler, Proc. Natl. Acad. Sci. U.S.A. 82, 5194 (1985). K. Takanaka, P. J. O'Brien, Y. Tsuruta, and A. D. Rahimtula, Cancer Lett. 15, 311 (1982). h p. j. O'Brien, in "Microsomes and Drug Oxidations" (A. R. Boobis, J. Caldwell, F. De Matteis, and C. R. Elcombe, eds.), p. 284. Taylor & Francis, London and Philadelphia, 1985. Y. Tsuruta, V. V. Subrahmanyam, W. Marshall, and P. J. O'Brien, Chem.-Biol. Interact. 53, 25 (1985). M. O. Corbett and B. R. Corbett, Chem. Res. Toxicol. 1, 356 (1988). k M. O. Corbett, B. R. Corbett, M. Hannothiaux, and S. J, Quintana, Chem. Res. Toxicol. 2, 260 (1989). t M. A. Trush, M. J. Reasor, M. E. Wilson, and K. Van Dykes, Biochem. Pharmacol. 33, 1401 (1984). m M. A. Trush, Toxicol. Lett. 20, 297 (1984). n M. A. Trush, T. W. Kensler, and J. L. Seed, Ado. Exp. Med. Biol. 197, 311 (1986). a

Macromolecular binding is determined by liquid scintillation counting using the following modification of the method of Jollow e t a l . 22 The supernatant is discarded, and the pellet is resuspended in 4 ml of 5% TCA by vigorous mixing on a vortex mixer. Occasionally, the use of a wooden applicator stick is necessary to break clumps. The suspension is recentri22 D. J. Jollow, J. R. Mitchell, W. Z. Potter, D. C. Davis, J. R. Gillette, and B. B. Brodie, J. Pharmacol. Exp. Therapeut. 187, 195 (1973).




fuged to form a pellet, and the supernatant is discarded. This procedure is repeated 2 additional times. Similar washes are performed 2-3 times using methanol-water (80: 20) and 2-3 times using ethanol-ether (I : 1; chilled to -20°). By this time, the radioactivity in the supernatant should be similar to background levels. The pellet is then solubilized by incubation at 55 ° for at least 1 hr in a solution containing 1 ml of 0.5% sodium lauryl sulfate in Tris buffer (50 mM; pH 7.4) and 1 ml of 2 N NaOH. The pH is then adjusted to 7.0-7.5 by the addition of 12 N HC1, using 1- to 3-/.d volumes as the pH approaches the neutral range. Aliquots are removed for liquid scintillation counting (1 ml) and for protein determinations (0.2 ml) as described by Lowry et al. 23 Sodium lauryl sulfate can interfere with the Lowry assay so appropriate controls should contain this compound. Additional Considerations With many of these end points, it is prudent to monitor cell viability or cellular respiration (O2 uptake) to ensure that the observed effect is due to metabolism rather than toxicity. In addition, owing to the indirect nature of many of the end points of interest, it is wise to use additional lines of evidence to support the results. The use of metabolic inhibitors should be interpreted with caution. For example, sodium azide, a "specific" inhibitor of myeloperoxidase, may affect other cellular systems and react directly with bioactive metabolites. 24 The use of isolated enzyme systems such as myeloperoxidase (MPO), horseradish peroxidase, xanthine oxidase, and NADPH oxidase or chemicals such as HOCI, H 2 0 2 , and taurine chloramine can be used to more thoroughly investigate and confirm proposed mechanisms of xenobiotic activation by PMNs (see Refs. 7, 10, and 11 for examples). Studies with Myeloperoxidase Initial studies of metabolic activation by neutrophils can be followed by additional experiments to identify the mechanisms of bioactivation and identify reactive metabolites. 7,s,",25-27 A typical sequence of experiments 23 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 24 R. V. Bhat, V. V. Subrahmanyam, A. Sadler, and D. Ross, Toxicol. Appl. Pharmacol. 94, 297 (1988). 25 D. A. Eastmond, M. T. Smith, L. O. Ruzo, and D. Ross, Mol. Pharmacol. 30, 674 (1986). 26 p. j. O'Brien, in "Microsomes and Drug Oxidations" (A. R. Boobis, J. Caldwell, F. De Matteis, and C. R. Elcombe, eds.), p. 284. Taylor & Francis, London and Philadelphia, 1985. 27 j. Uetrecht and N. Zahid, Chem. Res. Toxicol. 1, 148 (1988).




to complement the experiments describing xenobiotic activation and protein binding by neutrophils (see Refs. 7, 8, and 25) is as follows. Incubation Conditions and Analytic Approaches. Protein binding studies are performed by incubating purified MPO (EC; Calbiochem, San Diego, CA) or a PMN lysate (1.5 U/ml) 25,28with the chemical of interest (0.5 mM), an excess of H202 (1 mM), and boiled rat liver protein (0.9 mg). These conditions result in a rapid and complete oxidization of most chemical substrates within 10 min. The reaction is stopped by the addition of 5% TCA (final concentration) and placed on ice. The removal of the chemical from the incubation can be monitored by highperformance liquid chromatography (HPLC), and the recovery of radioactive equivalents bound to protein can be determined as described above. The rat liver protein is prepared from the 9000 g supernatant by previously described standard methods 29 and boiled for 30 min. It is important that the blood be removed from the liver by perfusion prior to microsome preparation because oxyhemoglobin is capable of catalyzing peroxidase reactions. 3° The use of bovine serum albumin is not recommended as it is extensively removed during the washing steps. By omitting the boiled rat liver protein and by stopping the reaction with catalase, the formation of metabolites can be monitored at various time points with subsequent analysis by HPLC or gas chromatographymass spectrometry. The identity of protein-binding species can be determined by the addition of glutathione (5 mM) or other small nucleophiles and the subsequent identification of the chemical-nucleophile conjugate. Glutathione should be added at the completion rather than the beginning of the incubation because gluthatione added at the beginning of peroxidase reactions can act as an antioxidant rather than a nucleophile. The chemical-glutathione conjugates can be isolated by HPLC and identified by the use of fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. In addition, the identity of chemicalglutathione conjugates can also be confirmed by performing the above reaction with the 14C-labeled chemical and [3H] glutathione and recovering both radioisotopes in the isolated conjugate (see Ref. 25 for example). Concluding Comments The use of stimulated polymorphonuclear leukocytes combined with in vitro studies with isolated enzymes and chemicals can provide valuable

s. J. Klebanoff, A. M. Waltersdoff, and H. Rosen, this series, Vol. I05, p. 399. L. Ernster, P. Siekevitz, and G. E. Palade, J. Cell Biol. 15, 541 0962). 3oT. Sawahata, D. E. Rickert, and W. F. Greenlee, in "Toxicologyof the Blood and Bone Marrow" (R. D. Irons, ed.) p. 141. Raven, New York, 1985.


C H E M I L U M I N E S C E N T D E T E R M I N A T I O N OF 0 2 - AND I o 2


insights into the mechanisms of xenobiotic activation of numerous pharmaceutical and environmental agents. Furthermore, this model of bioactivation may be particularly useful in understanding the observed interaction between inflammation and cancer 1°,31 as well as the mechanisms of toxicity and leukemogenesis in leukocyte-rich organs such as the bone marrow and blood, s,32 Since 90% of the immature granulocytes of the body reside in the bone marrow and contain high levels of myeloperoxidase, this mechanism of xenobiotic activation may be particularly important in this organ. Acknowledgments This work was supported by National Institutesof Health Grants P42 ES04705 and P30 ES01896 and the National Foundation for Cancer Research. David A. Eastmond was supported by an appointment to the Alexander Hollaendcr Distinguished Postdoctoral Program administered by the U.S. Department of Energy and Oak Ridge Associated Universities. Work was pcfforrncd in part under the auspices of the U.S. Department of Energy by the Lawrence Livermorc National Laboratory under Contract W-7405-ENG-48. W e thank Dr. David Ross for valuable contributions.

3t T. W. Kensler,P. A. Egner, K. G. Moore,B. G. Taffe,L. E. Twerdok,and M. A. Trush, Toxicol. Appl. Pharmacol. 90, 337 (1987). 32L. E. Twerdok and M. A. Trash, Chem.-BioL Interact. 65, 261 (1988).

[62] Determination of Superoxide Radical and Singlet Oxygen Based on Chemiluminescence of Luciferin Analogs By MINORU NAKANO


2-Methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one or 2methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one reacts with 02- or 102 to emit light, probably via the dioxetanone analog. Superoxide dismutase (SOD, a scavenger of 02-) or NaN3 (a quencher of IO2) can be used for differentiation between 02- and IO2-dependent luminescence. The maximum light intensity or integrated light intensity is detected for the assay of 102 or 02- generation in biological systems. METHODS IN ENZYMOLOGY, VOL. 186

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Xenobiotic activation by stimulated human polymorphonuclear leukocytes and myeloperoxidase.

[61] NEUTROPHIL-MEDIATED METABOLIC ACTIVATION 579 bromide) permeabilization treatment without disruption of the E. coli.~5 In the case of GS, the a...
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