Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Failure of benzene and phenol to serve as substrates for the peroxidatic action of catalase S. Paul Sichak , Oipak Basu & Craig Turner To cite this article: S. Paul Sichak , Oipak Basu & Craig Turner (1990) Failure of benzene and phenol to serve as substrates for the peroxidatic action of catalase, Journal of Toxicology and Environmental Health, 31:3, 227-233, DOI: 10.1080/15287399009531451 To link to this article: http://dx.doi.org/10.1080/15287399009531451
Published online: 20 Oct 2009.
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Date: 06 November 2015, At: 17:15
FAILURE OF BENZENE AND PHENOL TO SERVE AS SUBSTRATES FOR THE PEROXIDATIC ACTION OF CATALASE S. Paul Sichak
Downloaded by [University of Florida] at 17:15 06 November 2015
Environmental Health Sciences Center, University of Rochester School of Medicine, Rochester, New York Oipak Basu, Craig Turner Syracuse Research Corporation, Syracuse, New York
Evidence from several reports in the literature indicates a possible role for catatase in the metabolism of benzene. To investigate possible peroxidatic activity of catalase on benzene or its major metabolite, phenol, we employed an in vitro assay system that had been used previously to study the peroxidation of ethanol by catalase. Under conditions identical to those used to demonstrate catalase-mediated ethanol peroxidation we observed no peroxidase activity of catalase toward either benzene or phenol. We conclude that catalase is not involved in benzene metabolism and make the observation that, to date, no aromatic compounds have been demonstrated to be substrates for the peroxidatic mode of action of catalase.
INTRODUCTION The enzyme catalase, in the presence of hydrogen peroxide, is known to possess peroxidatic activity toward a number of small substrate molecules (i.e., methanol, ethanol, mercury vapor, and others). Several aromatic compounds (i.e., L-dopa, guaiacol, and pyrogallol) are also claimed to be substrates for the peroxidatic mode of action of catalase but only after the enzyme has been structurally altered in some manner (Marklund, 1973; Inada et al., 1961; Caravaca and May, 1964; Sichak and Dounce, 1986). Because these aromatic compounds do not serve as substrates for unaltered catalase, the peroxidase activity of the structurally altered enzyme toward these compounds should be considered as a catalytic but nonenzymatic type of reaction mechanism. Several reports have been found in the literature indicating that catalase in the presence of hydrogen peroxide might show some peroxidatic activity toward benzene, some of its aromatic metabolites, or both. EviWe wish to thank Dr. Kenneth Kun for financial support of this project and also Professors Alexander Dounce and Thomas Clarkson for helpful suggestions and advice in preparing this manuscript. We also wish to acknowledge financial assistance from EHSC center grant ESO1247. Requests for reprints should be sent to S. Paul Sichak, Box EHSC, University of Rochester School of Medicine, Rochester, NY 14642. 227 (ournal of Toxicology and Environmental Health, 31:227-233, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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dence suggesting this possibility is that 3-amino-1,2,4-triazole (an inhibitor of catalase compound I) (Tephly et al., 1961) has been found to inhibit benzene oxidation both in vitro and in vivo (Nomiyama, 1962,1964). Ethanol, a competitive inhibitor of catalase-mediated mercury vapor oxidation (Ogata and Aikoh, 1983, 1984), has been reported to competitively inhibit benzene metabolism in vitro (Sato et al., 1981). In vivo experiments indicate that ethanol-treated mice eliminate benzene from the blood much more rapidly than water-treated control mice following exposure to benzene by inhalation (Driscoll and Snyder, 1984). This may be analogous to the enhanced rate of mercury vapor exhalation in human subjects exposed to the vapor following ethanol consumption (Hursh et al., 1980). That is, ethanol may function to enhance the exhalation of both compounds (i.e., benzene and mercury vapor) by inhibiting a catalasemediated oxidation reaction and therefore making more of the parent compound available for exhalation. A final piece of evidence that suggests a possible role for catalase in benzene metabolism is the finding that horseradish peroxidase, in the presence of hydrogen peroxide, is capable of oxidizing phenol (a major metabolite of benzene) to o,o'- and p,p'-biphenol (Sawahata and Neal, 1982). Horseradish peroxidase and catalase are known to have some substrate overlap in their peroxidase activities. In view of the results just presented, the experiments reported below were carried out to determine whether catalase possesses a significant peroxidatic activity toward benzene or its major metabolite, phenol. MATERIALS AND METHODS
Catalase Two beef liver catalase preparations were used in the present study. One sample had been purchased from Sigma Chemical Company (St. Louis, Mo.); the other had been isolated from bovine liver by the acetone fractionation method (Dounce and Mourtzikos, 1981). Both catalase preparations had been recrystallized at least twice by dialysis of a solution of the enzyme in 10% NaCI (w/v). The resultant suspensions of crystals were examined microscopically. In previous determinations of the activities of these two catalase samples (both catalatic and peroxidatic activities), it was found that both samples possessed similar quantitative activities (Sichak and Dounce, 1987). Although the crystal suspensions were not reassayed quantitatively in the present study, qualitative tests of one drop of the crystal suspensions treated with 3% H2O2 showed extremely high qualitative activities of both samples. No bacteria were noted by phasecontrast microscopic observation. Dry weight was used to estimate the catalase protein concentrations in each of the two preparations as previously (Sichak and Dounce, 1987).
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Besides the catalase sample referred to, the following enzymes and reagents were purchased from Sigma Chemical Company: horseradish peroxidase (type II); glucose oxidase (type X, from Aspergillus niger); guaiacol; ß-D( + )-glucose; and gum arabic (from acacia tree). Prior to the start of the experiment, solutions of catalase and horseradish peroxidase were prepared by dissolving the enzymes in 0.1 M sodium phosphate buffer (pH 7.0) containing 1% (w/v) gum arabic at final enzyme concentrations between 1.95 x 10~3 and 3.23 x 10~3 mM. Glucose oxidase was dissolved in distilled water at a final enzyme concentration of 30 Sigma Company units/ml. The method used to investigate the possible peroxidatic activity of catalase toward benzene or its major metabolite, phenol, involved the use of a slow steady-state hydrogen peroxide generating system that depends upon the reaction of glucose with glucose oxidase. This system has previously been used to measure the peroxidatic oxidation of ethanol by catalase and lyophilized catalase (Oshino et al., 1973; Sichak and Dounce, 1986,1987) and to study the oxidation of L-dopa and guaiacol by horseradish peroxidase (Sichak and Dounce, 1986). The assay used to measure the peroxidatic activity of catalase in this study was similar to the peroxidatic assays used in previous studies (Oshino et al., 1973; Sichak and Dounce, 1986), with the exception that in the present study the reaction volume was increased to 90 ml. Upscaling of the reaction volume to 90 ml was necessary in order to enable extraction of any possible reaction products from the incubation mixture. The initial reaction mixture in this study (total volume of 90 ml) consisted of an oxygen-saturated 50 mM KH2PO4 buffer of pH 7.4, to which was added glucose (10 mM) and one of the two postulated substrates for the peroxidatic mode of action of catalase (i.e., benzene or phenol). Benzene was dissolved in the reaction mixture at its solubility limit (22.5 mM at 20°C) (Verschueren, 1977). Phenol was tested as a potential substrate for the peroxidase action of catalase at a 50 mM concentration. A sufficient quantity of the catalase solutions that had been prepared earlier (usually about 1 ml) was added to the appropriate reaction vessels to give catalase protein concentrations of approximately 2.15 x 10~8 M in the incubation mixture. A catalase concentration of 2.15 x 10~8 M was a sufficient enzyme concentration to enable several investigators (i.e., Sichak and Dounce, 1986; Oshino et al., 1973) to observe the peroxidation of ethanol by catalase. Furthermore, we did not wish to raise the catalase concentration high enough to risk the possibility of occurrence of a catalytic but nonenzymatic reaction mechanism. To start the reaction, 1.0 ml of the glucose oxidase solution that had been prepared earlier was added. This addition provided 30 Sigma Company units of the enzyme to the reaction mixture. This concentration of glucose oxidase had previously been shown to be associated with an H2O2 generation rate of 66 /¿mol
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H2O2/l/min, which maximized the in vitro peroxidation of ethanol to acetaldehyde by catatase (Sichak and Dounce, 1986). The reaction was allowed to run for 20 min. This reaction time was thought to be sufficient because in previous publications (Sichak and Dounce, 1986; Oshino et al., 1973) concerning the peroxidase activities of catalase and horseradish peroxidase, peroxidatic activity of these enzymes toward their respective substrates at comparable substrate concentrations was observed within a matter of minutes. To show that the glucose-glucose oxidase H2O2 generating system would function in the presence of a known toxic agent such as benzene, a separate experiment was done that employed the H2O2 generating system to study the oxidation of guaiacol to tetraguaiacolquinone by horseradish peroxidase. At concentrations of the glucose-glucose oxidase system comparable to those used in attempts to achieve oxidation of benzene by catalase (i.e., H2O2 generation rate of 66 /¿mol H2O2/l/min), the oxidation of guaiacol (50 mlW) to the brown-colored tetraguaiacolquinone by horseradish peroxidase (2.15 x 10~8 M) in the presence of benzene (22.5 mM) was readily observable. The peroxidation reactions involving the enzyme catalase and either of the two substrates examined (i.e., phenol or benzene) were run in 200ml erlenmeyer flasks at room temperature for approximately 20 min. At the end of this time, the contents of the flasks were transferred to 250-ml separatory funnels. The reaction solutions were then acidified to a pH of less than 2 by dropwise addition of a 50% sulfuric acid solution. The samples were then extracted by adding to each funnel 10 ml of méthylène chloride (glass distilled) and shaking about 2 min. After the layers had separated, the méthylène chloride layers were transferred to 250-ml K-D evaporative flasks, fit with 2-ml concentrator tubes, via 10-cm anhydrous sodium sulfate drying columns. The extraction procedure was repeated twice, and after both extractions, the organic layers were transferred to the K-D flasks. The concentrator tubes were partially immersed in hot water, and the méthylène chloride layers were concentrated to 1.0 ml with three ball Snyder columns. Of these concentrated solutions, 2 ¿il was injected into a capillary gas chromatograph/mass spectrometer (GCMS). Possible reaction products were then qualitatively identified by a computer library search initially, and later by trying to match retention times with those of authentic compounds that were injected into the capillary GC-MS. Failure to detect oxidation products of the aromatic substrates being tested either because of a faulty extraction procedure or because of failure of the GC-MS analysis was also investigated. Several possible oxidation products of benzene, phenol, or both, were extracted from an aqueous solution and analyzed by GC-MS using the same procedures that had been employed to examine the original reaction mixture after possible catalase peroxidase action. Phenol, catechol (1,2-benzenediol), hydro-
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quinone (1,4-benzenediol), 1,1'-biphenol-4-ol, and 1,1'-biphenyl-4,4'-diol {p,p '-biphenol) were investigated in this regard. These compounds were made up in solution in concentrations ranging from approximately 50 to 150 ppm. The successful extraction and detection of these compounds using the procedures employed previously for the enzymatic assays indicate that the procedures in question were adequate for determining possible oxidation products of benzene and phenol in the assay mixture. The limits of detection for the various metabolites tested were between 20 and 30 /¿g/l (ppb). This indicated that the conversion of approximately 0.001% of the benzene initially present or 0.0005% of the phenol initially present in the assay mixture to one of the metabolites investigated could have been readily detected. RESULTS AND DISCUSSION The experiments just outlined indicate that benzene and phenol are not substrates for the peroxidase action of catalase. Following a 20-min incubation of these compounds (separately) with catalase in the presence of enzymatically generated H2O2, no oxidation products of benzene or phenol were detected in méthylène chloride extracts of the reaction mixture. Poisoning of the enzyme system responsible for H2O2 generation (glucose oxidase) by the aromatic compounds being tested seems unlikely. Horseradish peroxidase in the presence of the glucose-glucose oxidase H2O2 generating system was able to oxidize guaiacol to tetraguaiacolquinone equally well in the presence or absence of benzene. It is concluded that benzene and phenol are not substrates for the peroxidatic mode of action of catalase, and we have found no evidence in the literature that any aromatic compounds serve as substrates for the peroxidase action of catalase. There are several possible reasons for this. The aromatic compounds that have been tested to date for substrate activity may not, for physicochemical reasons, be able to pass through the relatively small subunit access channels of catalase (described by Reid et al., 1981) that lead to the catalytically active hematin prosthetic groups of the enzyme. It is also possible that the aromatic compounds tested as substrates for the peroxidatic mode of action of catalase may not be able to participate in the stepwise, two-electron transfers postulated for the peroxidatic action of catalase (Sichak and Dounce, 1986). In view of the negative results just reported, it seems necessary to seek an explanation for the inhibitory effects of ethanol and aminotriazole on benzene metabolism that does not involve inhibition of catalase. Aminotriazole has been thought to inhibit benzene metabolism by acting as a mixed-function oxidase inhibitor (Bolcsak and Nerland, 1983), and ethanol has been found to inhibit the metabolism of a number of hydrocarbons (i.e., benzene, toluene, styrene, chloroform, 1,2-
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dichloroethylene, 1,1-dichloroethylene, trichloroethylene, and xylene) by inhibiting an as-yet unspecified liver enzyme(s) (Sato et al., 1981; Driscoll and Snyder, 1984). CONCLUSION
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On the basis of studies in vitro, it is concluded that neither benzene nor phenol can act as substrates for the peroxidatic action of catalase. REFERENCES Bolcsak, L. E., and Nerland, D. E. 1983. Inhibition of erythropoiesis by benzene and benzene metabolites. Toxicol. Appl. Pharmacol. 659:363-368. Caravaca, J., and May, M. D. 1964. The isolation and properties of an active peroxidase from hepatocatalase. Biochem. Biophys. Res. Commun. 16:528-534. Dounce, A. L, and Mourtzikos, C. 1981. Isolation of three crystalline proteins from beef liver after partial purification by acetone fractions. Prep. Biochem. 11:501-523. Driscoll, K. E., and Snyder, C. A. 1984. The effects of ethanol ingestion and repeated benzene exposures on benzene pharmacokinetics. Toxicol. Appl. Pharmacol. 73:525-532. Hursh, J. B., Greenwood, M. R., Clarkson, T. W., Allen, J., and Demuth, S. 1980. The effect of ethanol on the fate of mercury vapor inhaled by man. J. Pharmacol. Exp. Ther. 214:520-527. Inada, Y, Kurozumi, T., and Shibata, K. 1961. Peroxidase activity of hemoproteins. I. Generation of activity by acid or alkali denaturation of methemoglobin and catalase. Arch. Biochem. Biophys. 93:30-36. Marklund, S. 1973. Tryptic digestion and alkaline denaturation of catalase: The influence on catalatic activity, peroxidatic activity towards phenolic compounds, and the reactivity with methyland ethyl-hydroperoxide. Biochim. Biophys. Acta 321:90-97. Nomiyama, K. 1962. Effect of 3-amino-1,2,4-triazole on the catalase activity and the oxidation rate of benzene in the rat liver. Med. J. Shinshu Univ. 7:27-28. Nomiyama, K. 1964. Experimental studies on benzene poisoning. Bull. Tokyo Med. Dent. Univ. 11:297-313. Ogata, M., and Aikoh, H. 1983. The oxidation mechanism of metallic mercury in vitro by catalase. Physiol. Chem. Phys. Med. NMR 15:89-91. Ogata, M., and Aikoh, H. 1984. Mechanism of metallic mercury oxidation in vitro by catalase and peroxidase. Biochem. Pharmacol. 33:490-493. Oshino, N., Oshino, R., and Chance, B. 1973. The characteristics of the 'peroxidatic' reaction of catalase in ethanol oxidation. Biochem. J. 131:555-567. Reid, T. J., III, Murthy, M., Sicignano, A., Tanaka, N., Music, D., and Rossman, M. 1981. Structure and heme environment of beef liver catalase at 2.5 Å resolution. Proc. Natl. Acad. Sci. USA 78:4767-4771. Sato, A., Nakajim, T., and Koyama, Y. 1981. Dose-related effects of a single dose of ethanol on the metabolism in rat liver of some aromatic and chlorinated hydrocarbons. Toxicol. Appl. Pharmacol. 60:8-15. Sawahata, T., and Neal, R. A. 1982. Horseradish peroxidase-mediated oxidation of phenol. Biochem. Biophys. Res. Commun. 109:988-994. Sichak, S. P., and Dounce, A. L. 1986. Analysis of the peroxidatic mode of action of catalase. Arch. Biochem. Biophys. 249:286-295. Sichak, S. P., and Dounce, A. L. 1987. A study of the catalase monomer produced by lyophilization. Biochim. Biophys. Acta 925:282-289. Tephly, T. R., Mannering, G. J., and Parks, R. E. 1961. Studies on the mechanism of inhibition of liver
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and erythrocyte catalase activity by 3-amino-1,2,4-triazole (AT). J. Pharmacol. Exp. Ther. 134:7782. Verschueren, K. 1977. Handbook of Environmental Data on Organic Chemicals, p. 113. New York: Van Nostrand Reinhold.
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Received November 8, 1989 Accepted May 6, 1990
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Failure of benzene and phenol to serve as substrates for the peroxidatic action of catalase S. Paul Sichak , Oipak Basu & Craig Turner To cite this article: S. Paul Sichak , Oipak Basu & Craig Turner (1990) Failure of benzene and phenol to serve as substrates for the peroxidatic action of catalase, Journal of Toxicology and Environmental Health, 31:3, 227-233, DOI: 10.1080/15287399009531451 To link to this article: http://dx.doi.org/10.1080/15287399009531451
Published online: 20 Oct 2009.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 17:15
FAILURE OF BENZENE AND PHENOL TO SERVE AS SUBSTRATES FOR THE PEROXIDATIC ACTION OF CATALASE S. Paul Sichak
Downloaded by [University of Florida] at 17:15 06 November 2015
Environmental Health Sciences Center, University of Rochester School of Medicine, Rochester, New York Oipak Basu, Craig Turner Syracuse Research Corporation, Syracuse, New York
Evidence from several reports in the literature indicates a possible role for catatase in the metabolism of benzene. To investigate possible peroxidatic activity of catalase on benzene or its major metabolite, phenol, we employed an in vitro assay system that had been used previously to study the peroxidation of ethanol by catalase. Under conditions identical to those used to demonstrate catalase-mediated ethanol peroxidation we observed no peroxidase activity of catalase toward either benzene or phenol. We conclude that catalase is not involved in benzene metabolism and make the observation that, to date, no aromatic compounds have been demonstrated to be substrates for the peroxidatic mode of action of catalase.
INTRODUCTION The enzyme catalase, in the presence of hydrogen peroxide, is known to possess peroxidatic activity toward a number of small substrate molecules (i.e., methanol, ethanol, mercury vapor, and others). Several aromatic compounds (i.e., L-dopa, guaiacol, and pyrogallol) are also claimed to be substrates for the peroxidatic mode of action of catalase but only after the enzyme has been structurally altered in some manner (Marklund, 1973; Inada et al., 1961; Caravaca and May, 1964; Sichak and Dounce, 1986). Because these aromatic compounds do not serve as substrates for unaltered catalase, the peroxidase activity of the structurally altered enzyme toward these compounds should be considered as a catalytic but nonenzymatic type of reaction mechanism. Several reports have been found in the literature indicating that catalase in the presence of hydrogen peroxide might show some peroxidatic activity toward benzene, some of its aromatic metabolites, or both. EviWe wish to thank Dr. Kenneth Kun for financial support of this project and also Professors Alexander Dounce and Thomas Clarkson for helpful suggestions and advice in preparing this manuscript. We also wish to acknowledge financial assistance from EHSC center grant ESO1247. Requests for reprints should be sent to S. Paul Sichak, Box EHSC, University of Rochester School of Medicine, Rochester, NY 14642. 227 (ournal of Toxicology and Environmental Health, 31:227-233, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
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dence suggesting this possibility is that 3-amino-1,2,4-triazole (an inhibitor of catalase compound I) (Tephly et al., 1961) has been found to inhibit benzene oxidation both in vitro and in vivo (Nomiyama, 1962,1964). Ethanol, a competitive inhibitor of catalase-mediated mercury vapor oxidation (Ogata and Aikoh, 1983, 1984), has been reported to competitively inhibit benzene metabolism in vitro (Sato et al., 1981). In vivo experiments indicate that ethanol-treated mice eliminate benzene from the blood much more rapidly than water-treated control mice following exposure to benzene by inhalation (Driscoll and Snyder, 1984). This may be analogous to the enhanced rate of mercury vapor exhalation in human subjects exposed to the vapor following ethanol consumption (Hursh et al., 1980). That is, ethanol may function to enhance the exhalation of both compounds (i.e., benzene and mercury vapor) by inhibiting a catalasemediated oxidation reaction and therefore making more of the parent compound available for exhalation. A final piece of evidence that suggests a possible role for catalase in benzene metabolism is the finding that horseradish peroxidase, in the presence of hydrogen peroxide, is capable of oxidizing phenol (a major metabolite of benzene) to o,o'- and p,p'-biphenol (Sawahata and Neal, 1982). Horseradish peroxidase and catalase are known to have some substrate overlap in their peroxidase activities. In view of the results just presented, the experiments reported below were carried out to determine whether catalase possesses a significant peroxidatic activity toward benzene or its major metabolite, phenol. MATERIALS AND METHODS
Catalase Two beef liver catalase preparations were used in the present study. One sample had been purchased from Sigma Chemical Company (St. Louis, Mo.); the other had been isolated from bovine liver by the acetone fractionation method (Dounce and Mourtzikos, 1981). Both catalase preparations had been recrystallized at least twice by dialysis of a solution of the enzyme in 10% NaCI (w/v). The resultant suspensions of crystals were examined microscopically. In previous determinations of the activities of these two catalase samples (both catalatic and peroxidatic activities), it was found that both samples possessed similar quantitative activities (Sichak and Dounce, 1987). Although the crystal suspensions were not reassayed quantitatively in the present study, qualitative tests of one drop of the crystal suspensions treated with 3% H2O2 showed extremely high qualitative activities of both samples. No bacteria were noted by phasecontrast microscopic observation. Dry weight was used to estimate the catalase protein concentrations in each of the two preparations as previously (Sichak and Dounce, 1987).
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Besides the catalase sample referred to, the following enzymes and reagents were purchased from Sigma Chemical Company: horseradish peroxidase (type II); glucose oxidase (type X, from Aspergillus niger); guaiacol; ß-D( + )-glucose; and gum arabic (from acacia tree). Prior to the start of the experiment, solutions of catalase and horseradish peroxidase were prepared by dissolving the enzymes in 0.1 M sodium phosphate buffer (pH 7.0) containing 1% (w/v) gum arabic at final enzyme concentrations between 1.95 x 10~3 and 3.23 x 10~3 mM. Glucose oxidase was dissolved in distilled water at a final enzyme concentration of 30 Sigma Company units/ml. The method used to investigate the possible peroxidatic activity of catalase toward benzene or its major metabolite, phenol, involved the use of a slow steady-state hydrogen peroxide generating system that depends upon the reaction of glucose with glucose oxidase. This system has previously been used to measure the peroxidatic oxidation of ethanol by catalase and lyophilized catalase (Oshino et al., 1973; Sichak and Dounce, 1986,1987) and to study the oxidation of L-dopa and guaiacol by horseradish peroxidase (Sichak and Dounce, 1986). The assay used to measure the peroxidatic activity of catalase in this study was similar to the peroxidatic assays used in previous studies (Oshino et al., 1973; Sichak and Dounce, 1986), with the exception that in the present study the reaction volume was increased to 90 ml. Upscaling of the reaction volume to 90 ml was necessary in order to enable extraction of any possible reaction products from the incubation mixture. The initial reaction mixture in this study (total volume of 90 ml) consisted of an oxygen-saturated 50 mM KH2PO4 buffer of pH 7.4, to which was added glucose (10 mM) and one of the two postulated substrates for the peroxidatic mode of action of catalase (i.e., benzene or phenol). Benzene was dissolved in the reaction mixture at its solubility limit (22.5 mM at 20°C) (Verschueren, 1977). Phenol was tested as a potential substrate for the peroxidase action of catalase at a 50 mM concentration. A sufficient quantity of the catalase solutions that had been prepared earlier (usually about 1 ml) was added to the appropriate reaction vessels to give catalase protein concentrations of approximately 2.15 x 10~8 M in the incubation mixture. A catalase concentration of 2.15 x 10~8 M was a sufficient enzyme concentration to enable several investigators (i.e., Sichak and Dounce, 1986; Oshino et al., 1973) to observe the peroxidation of ethanol by catalase. Furthermore, we did not wish to raise the catalase concentration high enough to risk the possibility of occurrence of a catalytic but nonenzymatic reaction mechanism. To start the reaction, 1.0 ml of the glucose oxidase solution that had been prepared earlier was added. This addition provided 30 Sigma Company units of the enzyme to the reaction mixture. This concentration of glucose oxidase had previously been shown to be associated with an H2O2 generation rate of 66 /¿mol
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H2O2/l/min, which maximized the in vitro peroxidation of ethanol to acetaldehyde by catatase (Sichak and Dounce, 1986). The reaction was allowed to run for 20 min. This reaction time was thought to be sufficient because in previous publications (Sichak and Dounce, 1986; Oshino et al., 1973) concerning the peroxidase activities of catalase and horseradish peroxidase, peroxidatic activity of these enzymes toward their respective substrates at comparable substrate concentrations was observed within a matter of minutes. To show that the glucose-glucose oxidase H2O2 generating system would function in the presence of a known toxic agent such as benzene, a separate experiment was done that employed the H2O2 generating system to study the oxidation of guaiacol to tetraguaiacolquinone by horseradish peroxidase. At concentrations of the glucose-glucose oxidase system comparable to those used in attempts to achieve oxidation of benzene by catalase (i.e., H2O2 generation rate of 66 /¿mol H2O2/l/min), the oxidation of guaiacol (50 mlW) to the brown-colored tetraguaiacolquinone by horseradish peroxidase (2.15 x 10~8 M) in the presence of benzene (22.5 mM) was readily observable. The peroxidation reactions involving the enzyme catalase and either of the two substrates examined (i.e., phenol or benzene) were run in 200ml erlenmeyer flasks at room temperature for approximately 20 min. At the end of this time, the contents of the flasks were transferred to 250-ml separatory funnels. The reaction solutions were then acidified to a pH of less than 2 by dropwise addition of a 50% sulfuric acid solution. The samples were then extracted by adding to each funnel 10 ml of méthylène chloride (glass distilled) and shaking about 2 min. After the layers had separated, the méthylène chloride layers were transferred to 250-ml K-D evaporative flasks, fit with 2-ml concentrator tubes, via 10-cm anhydrous sodium sulfate drying columns. The extraction procedure was repeated twice, and after both extractions, the organic layers were transferred to the K-D flasks. The concentrator tubes were partially immersed in hot water, and the méthylène chloride layers were concentrated to 1.0 ml with three ball Snyder columns. Of these concentrated solutions, 2 ¿il was injected into a capillary gas chromatograph/mass spectrometer (GCMS). Possible reaction products were then qualitatively identified by a computer library search initially, and later by trying to match retention times with those of authentic compounds that were injected into the capillary GC-MS. Failure to detect oxidation products of the aromatic substrates being tested either because of a faulty extraction procedure or because of failure of the GC-MS analysis was also investigated. Several possible oxidation products of benzene, phenol, or both, were extracted from an aqueous solution and analyzed by GC-MS using the same procedures that had been employed to examine the original reaction mixture after possible catalase peroxidase action. Phenol, catechol (1,2-benzenediol), hydro-
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quinone (1,4-benzenediol), 1,1'-biphenol-4-ol, and 1,1'-biphenyl-4,4'-diol {p,p '-biphenol) were investigated in this regard. These compounds were made up in solution in concentrations ranging from approximately 50 to 150 ppm. The successful extraction and detection of these compounds using the procedures employed previously for the enzymatic assays indicate that the procedures in question were adequate for determining possible oxidation products of benzene and phenol in the assay mixture. The limits of detection for the various metabolites tested were between 20 and 30 /¿g/l (ppb). This indicated that the conversion of approximately 0.001% of the benzene initially present or 0.0005% of the phenol initially present in the assay mixture to one of the metabolites investigated could have been readily detected. RESULTS AND DISCUSSION The experiments just outlined indicate that benzene and phenol are not substrates for the peroxidase action of catalase. Following a 20-min incubation of these compounds (separately) with catalase in the presence of enzymatically generated H2O2, no oxidation products of benzene or phenol were detected in méthylène chloride extracts of the reaction mixture. Poisoning of the enzyme system responsible for H2O2 generation (glucose oxidase) by the aromatic compounds being tested seems unlikely. Horseradish peroxidase in the presence of the glucose-glucose oxidase H2O2 generating system was able to oxidize guaiacol to tetraguaiacolquinone equally well in the presence or absence of benzene. It is concluded that benzene and phenol are not substrates for the peroxidatic mode of action of catalase, and we have found no evidence in the literature that any aromatic compounds serve as substrates for the peroxidase action of catalase. There are several possible reasons for this. The aromatic compounds that have been tested to date for substrate activity may not, for physicochemical reasons, be able to pass through the relatively small subunit access channels of catalase (described by Reid et al., 1981) that lead to the catalytically active hematin prosthetic groups of the enzyme. It is also possible that the aromatic compounds tested as substrates for the peroxidatic mode of action of catalase may not be able to participate in the stepwise, two-electron transfers postulated for the peroxidatic action of catalase (Sichak and Dounce, 1986). In view of the negative results just reported, it seems necessary to seek an explanation for the inhibitory effects of ethanol and aminotriazole on benzene metabolism that does not involve inhibition of catalase. Aminotriazole has been thought to inhibit benzene metabolism by acting as a mixed-function oxidase inhibitor (Bolcsak and Nerland, 1983), and ethanol has been found to inhibit the metabolism of a number of hydrocarbons (i.e., benzene, toluene, styrene, chloroform, 1,2-
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dichloroethylene, 1,1-dichloroethylene, trichloroethylene, and xylene) by inhibiting an as-yet unspecified liver enzyme(s) (Sato et al., 1981; Driscoll and Snyder, 1984). CONCLUSION
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On the basis of studies in vitro, it is concluded that neither benzene nor phenol can act as substrates for the peroxidatic action of catalase. REFERENCES Bolcsak, L. E., and Nerland, D. E. 1983. Inhibition of erythropoiesis by benzene and benzene metabolites. Toxicol. Appl. Pharmacol. 659:363-368. Caravaca, J., and May, M. D. 1964. The isolation and properties of an active peroxidase from hepatocatalase. Biochem. Biophys. Res. Commun. 16:528-534. Dounce, A. L, and Mourtzikos, C. 1981. Isolation of three crystalline proteins from beef liver after partial purification by acetone fractions. Prep. Biochem. 11:501-523. Driscoll, K. E., and Snyder, C. A. 1984. The effects of ethanol ingestion and repeated benzene exposures on benzene pharmacokinetics. Toxicol. Appl. Pharmacol. 73:525-532. Hursh, J. B., Greenwood, M. R., Clarkson, T. W., Allen, J., and Demuth, S. 1980. The effect of ethanol on the fate of mercury vapor inhaled by man. J. Pharmacol. Exp. Ther. 214:520-527. Inada, Y, Kurozumi, T., and Shibata, K. 1961. Peroxidase activity of hemoproteins. I. Generation of activity by acid or alkali denaturation of methemoglobin and catalase. Arch. Biochem. Biophys. 93:30-36. Marklund, S. 1973. Tryptic digestion and alkaline denaturation of catalase: The influence on catalatic activity, peroxidatic activity towards phenolic compounds, and the reactivity with methyland ethyl-hydroperoxide. Biochim. Biophys. Acta 321:90-97. Nomiyama, K. 1962. Effect of 3-amino-1,2,4-triazole on the catalase activity and the oxidation rate of benzene in the rat liver. Med. J. Shinshu Univ. 7:27-28. Nomiyama, K. 1964. Experimental studies on benzene poisoning. Bull. Tokyo Med. Dent. Univ. 11:297-313. Ogata, M., and Aikoh, H. 1983. The oxidation mechanism of metallic mercury in vitro by catalase. Physiol. Chem. Phys. Med. NMR 15:89-91. Ogata, M., and Aikoh, H. 1984. Mechanism of metallic mercury oxidation in vitro by catalase and peroxidase. Biochem. Pharmacol. 33:490-493. Oshino, N., Oshino, R., and Chance, B. 1973. The characteristics of the 'peroxidatic' reaction of catalase in ethanol oxidation. Biochem. J. 131:555-567. Reid, T. J., III, Murthy, M., Sicignano, A., Tanaka, N., Music, D., and Rossman, M. 1981. Structure and heme environment of beef liver catalase at 2.5 Å resolution. Proc. Natl. Acad. Sci. USA 78:4767-4771. Sato, A., Nakajim, T., and Koyama, Y. 1981. Dose-related effects of a single dose of ethanol on the metabolism in rat liver of some aromatic and chlorinated hydrocarbons. Toxicol. Appl. Pharmacol. 60:8-15. Sawahata, T., and Neal, R. A. 1982. Horseradish peroxidase-mediated oxidation of phenol. Biochem. Biophys. Res. Commun. 109:988-994. Sichak, S. P., and Dounce, A. L. 1986. Analysis of the peroxidatic mode of action of catalase. Arch. Biochem. Biophys. 249:286-295. Sichak, S. P., and Dounce, A. L. 1987. A study of the catalase monomer produced by lyophilization. Biochim. Biophys. Acta 925:282-289. Tephly, T. R., Mannering, G. J., and Parks, R. E. 1961. Studies on the mechanism of inhibition of liver
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and erythrocyte catalase activity by 3-amino-1,2,4-triazole (AT). J. Pharmacol. Exp. Ther. 134:7782. Verschueren, K. 1977. Handbook of Environmental Data on Organic Chemicals, p. 113. New York: Van Nostrand Reinhold.
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Received November 8, 1989 Accepted May 6, 1990
