340
Chem. Res. Toxicol. 1992, 5, 340-345
(12) Roselius, W., Vitzthum, O., and Hubert, P. (1979) Process for
the extraction of nicotine from tobacco. US.Patent 4,153,063, 9 PP. (13) Hawthorne, S. B. (1990) Analytical-scale supercritical fluid extraction. Anal. Chem. 62, 633A-642A. (14) Tricker, A. R., Haubner, R., Spiegelhalder,B., and Preussmann, R. (1988) The occurrence of tobacco-specificnitrosamines in oral tobacco products and their potential formation under simulated gastric conditions. Food Chem. Toxicol. 26, 861-865. (15) Chortyk, 0. T., and Chamberlain, W. J. (1991) The application of solid phase extraction to the analysis of tobacco-specific ni-
trosamines (TSNA). J. Chromatogr. Sci. 29, 522-527. (16) Hecht, S. S., Chen, C. B., Dong, M., Ornaf, R. M., and Hoff-
mann, D. (1977)Chemical studies on tobacco smoke. LI. Studies on non-volatile nitrosamines in tobacco. Beitr. Tabakforsch. 9, 1-6. (17) Christensen, S. B., and Krogsgaard-Lamen, P. (1977) Preparation of deuterium labeled guvacine and isoguvacine. J. Labelled Compd. 17, 191-202. (18) Hu, W., Bondinell, W. E., and Hoffman, D. (1974) Synthesis of carbon-14 labelled myosmine, nornicotine, and "-nitrosonornicotine. J. Labelled Compd. 10, 79-88.
The Oxidation of 4-Aminobiphenyl by Horseradish Peroxidase Michael F. Hughes,? Bill J. Smith,$ and Thomas E. Eling* Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, Post Office Box 12233, Research Triangle Park, North Carolina 27709 Received December 3, 1991 The oxidation of the carcinogen 4-aminobiphenyl(I-ABP) catalyzed by the model peroxidase enzyme horseradish peroxidase (HRP) was investigated. 4-ABP served as a reducing cosubstrate for HRP during the enzyme-catalyzed reduction of the synthetic hydroperoxide, 5-phenyl-4penten-1-yl hydroperoxide, to its corresponding alcohol. Spectral analysis during the incubation of HRP, 4-ABP, and H202showed an increase in absorbance a t 230 and 325 nm and decrease at 270 nm, suggesting metabolite formation. Oxygen consumption was not detected in incubations of HRP, 4-ABP, and H202. However, oxygen uptake was observed after the addition of glutathione, which indicated that a free radical metabolite of 4-ABP was formed by the peroxidase. The 4-ABP free radical reacted with glutathione forming a glutathionyl radical which, in turn, reacted with and consumed oxygen. HPLC analysis of organic extracts of incubations with HRP, [3H]-4-ABP,and H202showed the formation of one major peak identified by mass spectroscopy as 4,4'-azobis(biphenyl). The addition of glutathione to the incubations decreased the formation of 4-ABP metabolites, suggesting a reduction of the 4-ABP free radical and/or the formation of glutathione conjugates. Subsequent HPLC analysis of incubations including [35S]glutathione indicated formation of several unidentified 4-ABP-glutathione conjugates as well as recovery of parent compound. These studies suggest that HRP metabolizes 4-ABP by a one-electron oxidation mechanism, resulting in formation of a free radical. This radical can either react with a second radical to form azobis(biphenyl), be reduced by glutathione back to parent, or react with glutathione to form glutathione conjugates.
Introduction Aromatic amines such as 4-aminobiphenyl (4-ABP)' and benzidine have been intensely studied for a number of years to determine the role metabolism has in their activation and subsequent carcinogenic effect (1). The generally accepted hypothesis is that the metabolism of many chemical carcinogens, including aromatic amines, results in the formation of electrophilic intermediates which react covalently with critical nucleophiles in the cell and initiate the process of carcinogenesis (2,3). Cytochrome P-450 is an important catalyst in the activation of many aromatic amines ( 4 ) , with the pivotal step being N-hydroxylation of these chemicals (5). Aromatic amines induce tumors in many different tissues and organs (6-8). 4-ABP induces tumors in several organs such as the bladder of dogs (7,9) and man (10) and the intestine and liver of rats (11). Several extrahepatic organs, such as the bladder, that are susceptible to aro*To whom correspondence should be addressed at NIEHS, MD 19-04, P.O. Box 12233, Research Triangle Park, NC 27709. +Presentaddress: ManTech Environmental Technology Inc., P.O. Box 12313, Research Triangle Park, NC 27709. *Present address: The Procter & Gamble Company, Health & Personal Care Technology Division, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239-8707.
matic amine induced tumorigenesis are low in cytochrome P-450 activity, yet contain appreciable amounts of the mammalian peroxidase, prostaglandin H synthase (PHS) (12, 13). Several aromatic amines are good peroxidase substrates (14). Studies have shown that microsomal PHS has a role in the metabolism of 4-ABP and its structural analogue benzidine (12, 13, 15). The involvement of peroxidases in the metabolism/activation of benzidine has been extensively studied (12,13,15-20). However, limited studies have been conducted on the peroxidase-mediated metabolism of 4-ABP. In this study, we report on the peroxidative oxidation of 4-ABP using horseradish peroxidase (HRP) as a model enzyme.
Experlmentai Procedures Materials. Ascorbic acid, benzidine (caution: a potential carcinogen), glutathione, and HRP type VI were purchased from Sigma Chemical Co. (St. Louis, MO). 5-Phenyl-4-penten-1-yl hydroperoxide (PPHP) was purchased from Oxford Biomedical Research (Oxford, MI). Unlabeled 4-ABP (caution: a potential carcinogen) was purchased from Aldrich Chemical Co. (Mill Abbreviations: 4-ABP, 4-aminobiphenyl; HRP, horseradish peroxidase; PHS, prostaglandin H synthase;PPHP, 5-phenyl-4-penten-1-yl hydroperoxide; PPA, 5-phenyl-4-pentenylalcohol; UV/vis, ultraviolet/ visible; TLC,thin-layer chromatography.
This article not subject to US.Copyright. Published 1992 by the American Chemical Society
4-ABP One-Electron Oxidation waukee, WI). [3H]-4-ABP (0.056 Ci/mol, nonspecifically ring labeled) was obtained from Amersham (Arlington Heights, IL). The purity of [3H]-4-ABPwas found to be >99% as determined by HPLC. [Y3]Glutathione (30 Ci/mmol) was purchased from New England Nuclear (Boston, MA). All other chemicals used were the highest grade available. Instrumentation. A Hewlett-Packard (Palo Alto, CA) 8450A diode array spectrophotometer equipped with a heating and stirring module was used to obtain ultraviolet/visible (UV/vis) spectra. HPLC analysis was conducted with two Waters (Milford, MA) Model 6000A pumps, a Model 721 system controller, a WISP 710B automatic injector, and either a Cls pBondapak column (3.9-mm i.d. X 30 cm) or a Zorbax Cs column (4.6-mm i.d. X 250 mm, Du Pont Instruments, Wilmington, DE). HPLC effluent was monitored with a Waters Model 990 diode array spectrophotometer. Radioactive HPLC effluent was monitored with a Radiomatic Flo-one Beta detector (Radiomatic Instruments and Chemical Co., Meriden, CT). Oxygen consumption was measured with a Clark oxygen electrode and monitor (Yellow Springs Instruments, Yellow Springs, OH). Peroxidase Assay. The ability of chemicals to serve as reducing cosubstrates for peroxidases can be determined by quantitating the amount of the peroxide PPHP reduced to its corresponding alcohol, 5-phenyl-4-pentenyl alcohol (PPA) in the presence of test chemical and enzyme (21). HRP (1g / m L ) was preincubated (3 min, 37 "C) in 0.1 M phosphate buffer, pH 7.6, in the absence or presence of reducing cosubstrate (benzidine or 4-ABP, 100 pM). Benzidine was used as a positive control in the incubations. The incubation was initiated by the addition of PPHP (100 pM). After a 3-min incubation, the incubate was passed through a CISsolid-phase extraction column (PrepSep, Fisher Scientific, Fair Lawn, NJ). The column was washed with HzO,and the metabolites were eluted with methanol (2 X 0.5 mL). Eluates were centifuged, and p-nitrobenzyl alcohol (150 pM) was added as an internal standard. The methanol eluate from the benzidine incubations was then analyzed by HPLC [HzO/ methanol (42/58), 1.8 mL/min] on a Zorbax Cs column, and the column effluent was monitored at 254 nm. A modified solvent system was devised for the methanol incubations since 4-ABP coeluted with PPA in the HzO/methanol system. The new system consisted of 10 mM citrate, pH 6/methanol(42/58) with a flow rate of 1.8 mL/min. A reducing index was determined for 4-ABP and benzidine. This was calculated from the following equation after quantitating the concentrations of PPHP and PPA reducing index = [PPA]/([PPA] [PPHP]). In initial experiments the HRP concentration was varied with 100 pM phenol and 100 pM PPHP to determine the concentration that yielded an index of approximately 0.50. An HRP concentration of 1pg/mL was used in the incubations since at this concentration in the presence of phenol approximately 60% of the PPHP was reduced. Oxygen Consumption Studies. HRP (5 pg/mL) and 4-ABP (100 pM) were preincubated (37 "C) in 1.5 mL of 0.1 M phosphate, pH 7.6, for 2 min. Consumption of oxygen in the absence or presence of glutathione (0.25-1 mM) was then monitored with an oxygen electrode for 2 min after addition of HzO (200 pM). Spectral Studies. HRP (5 pg/mL) and 4-ABP (50 pM) were preincubated (37 "C) for 2 min in 0.1 M phosphate buffer, pH 7.6,2.0 mL, in a reference and sample cuvette. Difference spectra of these incubations were obtained after addition of HzOz (100 pM) into the sample cuvette. Difference spectra (230, 325 nm) were also taken with HRP, 4-ABP, and HzOzand either glutathione (50-500 pM) or ascorbic acid (50-250 pM). Spectra were recorded every 1.5 s with a scan time of 1.0 s. Metabolism of 4-ABP. [3H]-4-ABP(50 pM, 0.45 pCi) was preincubated with HRP (5 pg/mL) at 37 "C for 3 min in 2.0 mL of 0.1 M phosphate buffer, pH 7.6. These incubations were initiated by the addition of HzOz (100 pM)and 1 min later were extracted with 4 volumes of water-saturated ethyl acetate/ether (l/l).The organic phases were taken to dryness in vacuo and reconstituted in 0.25 mL of methanol. Aliquota were then analyzed by reverse-phase HPLC on the ClS pBondapak column. Solvent A was 10 mM citrate, pH 6, and solvent B was methanol. A solvent mixture (2 mL/min) of 50% B for 5 min was followed by a linear gradient to 100% B for 20 min. This was held for 10 min followed by return to initial conditions. 4-ABP has a retention time of 7 min in this system.
+
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 341 Identification of the Major 4-ABP Metabolite. The major metabolite formed during the oxidation of 4-ABP was isolated from a 200-mL incubation containing HRP (5 pg/mL), [3H]-4ABP (50 pM), and H202(100 pM). The mixture was preincubated a t 37 "C for 10 min and the reaction initiated with peroxide addition. Following a 5-min incubation the entire incubate was passed through a preparative-scale Cls solid-phase extraction column and washed with water, and the retained material was eluted with methanol (2 x 5 mL). The 4-ABP oxidation products were separated by preparativescale silica TLC (Whatman PLK5F, 20 cm x 20 cm x 1000 pm, Clifton, NJ) using benzene as the developing solvent. Under these conditions the R, of 4-ABP was 0.29. A pale yellow band (Rf0.95) was scraped from the ?zC plate and eluted with absolute ethanol (10 mL). The ethanol was evaporated under reduced pressure. The residue was reconstituted in benzene and analyzed by mass spectrometry. The mass spectral analysis was performed on a Concept I SQ hybrid mass spectrometer (Kratos Analytical, Manchester, England). The sample was analyzed in the electron impact (70-eV) low-resolution mode using a direct probe. The data were proceased on a SUN 360 workstation utilizing Mach 3 software. Assessment of 4-ABPGllutathione Conjugate Formation. The formation of 4-ABP-glutathione conjugates during HRPcatalyzed 4-ABP oxidation in the presence of glutathione was assessed by reverse-phase HPLC. [3H]-4-ABP (50 pM),[36S]glutathione (500 pM), and HRP (1 pg/mL) in 1 mL of 0.1 M phosphate buffer (pH 7.4) were preincubated at 37 "C for 3 min, and the reaction was initiated with HzOz (100 pM). Following a 5-min incubation, the entire reaction was analyzed directly by HPLC using the system described above with a modified solvent gradient. The initial conditions were 95% citrate (pH 6.0)/5% methanol with a 30-min linear gradient to 50% citrate buffer/50% methanol, held at this composition for 10 min, followed by a 15-min linear gradient to 100% methanol. The elution of the reaction products was monitored using [3H]/[35S]dual-window radioactive detection on the Radiomatic Flo-one Beta detector. The parameters for the radioflow detector channels were set for minimal crwover of %S into the 3H channel. The retention times for glutathione, 4-ABP, and azobis(bipheny1) were 2.7, 37, and 58 min, respectively.
Results Experiments were conducted to determine if the carcinogen 4-ABP could be oxidized by the model peroxidase enzyme HFtP. The initial experiment examined the ability of 4-ABP to serve as a peroxidase-reducing cosubstrate promoting the reduction of the hydroperoxide PPHP to the alcohol PPA. In the presence of 4-ABP a reducing index of 0.55 f 0.02 (mean f SD, N = 3) was determined compared to 0.08 f 0.01 in the absence of 4-ABP. The reducing index represents the fraction of alcohol (PPA) formed from the reduction of hydroperoxide (PPHP). Benzidine had a reducing index of 1.0, indicating all of the hydroperoxide was reduced to alcohol in the incubation. This is characteristic of an excellent peroxidase-reducing cosubstrate. Compared to benzidine, 4-ABP is a moderate HRP-reducing cosubstrate. However, the data indicate that 4-ABP is oxidized by HRP. Oxidation of 4-ABPto a Free Radical Metabolite. Peroxidases catalyze the one-electron oxidation of aromatic amines, resulting in the generation of nitrogen-centered radicals. The ability to detect aromatic amine free radicals by electron spin resonance is difficult. However, aromatic amine free radicals can be indirectly detected because they are known to react with glutathione, forming parent compound and a glutathionyl free radical. The latter radical can react with oxygen, resulting in oxygen consumption (22, 23). Consumption of oxygen in solution can be detected with an oxygen electrode. We used this method to detect the 4-ABP free radical metabolite formed during HRP-catalyzed oxidation of 4-ABP. Oxygen consumption was detected after addition of HzOzonly in the presence
342 Chem. Res. Toxicol., Vol. 5, No. 3, 1992 A
Hughes et al.
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Figure 2. Time course of spectral changes occurring during the oxidation of 4-ABP by HRP. Difference spectra were measured from an incubation containing 4-ABP (50 pM) and HRP (5 pg/mL) in 2.0 mL of 0.1 M phosphate buffer, pH 7.6, after addition of H202(100 pM) to the sample cuvette. The spectra were recorded every 1.5 s with a scan time of 1s. Shown in the figure are spectra at 1.5,4.5,7.5, 10.5, 13.5,16.5,19.5,and 22.5 s. The inset shows the decrease of absorbance at 271 nm with respect to time. of glutathione (Figure 1A). The amount of oxygen consumed was dependent on the concentration of glutathione (Figure 1B). Inspection of Figure 1B indicates a linear relationship between the nanomoles of oxygen consumed and the concentration of glutathione. Approximately 1 nmol of oxygen was consumed for every 10 nmol of glutathione present in the incubation. These observations indicate that HRP oxidizes 4-ABP to a free radical metabolite($.
Spectral Characterization of 4-ABP Oxidation. Oxidation of 4-ABP catalyzed by HRP was monitored by observing changes in the UV/vis absorption spectrum of 4-ABP as displayed in Figure 2. The difference spectra between incubation containing 4-ABP and HRP without and with HzOz showed an absorbance decrease at a wavelength range of 240-300 nm, with the greatest decrease at 270 nm, the absorption maximum of 4-ABP, after addition of H20z. The decrease in absorption maximum is indicativeof oxidation of parent compound. Absorbance increased at a wavelength range of 210-239 nm and 301 to greater than 700 nm, suggesting formation of metabolite(s). The decrease in absorbance at 271 nm, as shown in the inset of Figure 2, was very rapid, and the reaction was almost complete by 1min. No change in absorbance
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Chem. Res. Toxicol., Vol. 5, No. 3, 1992 343
4-ABP One-Electron Oxidation Scheme I
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cleavage of the nitrogen phenyl bond produces fragments at m/z 181 and 153. Cleavage of the azo double bond produces two fragments at m/z 167. Also consistent with the azo structure was the marked UV/vis absorbance (Figure 3) of the product at 360 nm, which is within the azo structure absorbance range of 350-370 nm (24). The formation of azobis(bipheny1) would be an expected product from the coupling of 4-ABP free radicals formed during oxidation by HRP. Inhibition of 4-ABP Metabolite Formation. The ability of glutathione and ascorbic acid to reduce free radical metabolites of 4-ABP and thus inhibit metabolite formation was examined by UV/vis absorption spectroscopy. Inhibition of change in absorbance at 325 nm in incubations with HRP and 4-ABP was dependent on the glutathione concentration (Figure 5). Similar results with glutathione were observed by measuring at 230 nm (data not shown). The lag period for the absorbance increase at 325 nm as shown in Figure 5 was dependent on the glutathione concentration. This suggests that the formation of 4-ABP metabolites are observed only after the glutathione was essentially consumed by reducing the CABP free radical metabolite. Ascorbic acid also inhibited
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increase of absorbance at 325 nm (data not shown) in the presence of HRP and 4-ABP. Maximal inhibition by ascorbic acid occurred at a concentration of 250 FM. Characterization of 4-ABP-Glutathione Conjugates. The spectrophotometric studies with glutathione indicate that this thiol reduces the free radical metabolites of 4-ABP. However, an additional mechanism is the formation of glutathione conjugates. This possibility was further examined by analyzing the products of reaction mixtures containing [3H]-4-ABP, [35S]glutathione,HRP, and Hz02 by reverse-phase HPLC. A typical HPLC chromatogram obtained from the analysis of this reaction is shown in Figure 6. In the absence of glutathione, CABP was completely metabolized to azobis(bipheny1)and several minor unidentified products. However, in the reactions with glutathione, there were significant amounts of 4-ABP remaining and reduced amounts of azobis(biphenyl), indicating that glutathione was inhibiting 4-ABP metabolite formation. These products were only detected in the 3H channel. Most interesting was the appearance of three early eluting polar compounds which coeluted in the 3H and 35Schannels of the radioactive flow detector. This is convincing evidence of 4-ABP-glutathione conjugate formation by the reaction of glutathione with the 4-ABP free radical metabolite.
Dlscusslon We have investigated the oxidation of the chemical carcinogen CABP by the model peroxidase enzyme HRP. A proposed scheme of the reactions that can occur is shown in Scheme I. 4-ABP is a reducing cosubstrate for HRP and is oxidized to a free radical metabolite. This oxidized product can couple with a second free radical metabolite, to form azobis(biphenyl), which we identified by mass spectrometry. Glutathione can inhibit formation of azobis(bipheny1) by either reducing the free radical back to parent or reacting with the free radical to form a glutathione conjugate. The HRP-catalyzed oxidation of 4-ABP in the presence of glutathione resulted in the consumption of oxygen, indicating that a free radical metabolite of 4-ABP is formed. Oxygen consumption in the presence of glutathione and peroxidases has been reported with 2-naphthylamine (25) and p-phenetidine (26). The proposed mechanism (Scheme I) of oxygen consumption first involves the peroxidase-catalyzed one-electron oxidation of an aromatic
Hughes et al.
344 Chem. Res. Toxicol., Vol. 5, No. 3, 1992 ABP-GSH Conjugates
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Figure 6. HPLC radiochromatogram obtained from the HRP-catalyzed oxidation of [3H]-4-ABPin the presence of [35S]glutathione. The incubation contained HRP (1 pg/mL), [3H]-4-ABP(50 pM), and [3sS]glutathione(500 pM) in 1.0 mL of phosphate buffer, pH 7.4. The reaction was initiated by the addition of 100 pM HzO2 After 5 min the entire incubation waa directly analyzed by HPLC. Glutathione disulfide and glutathione sulfonic acid eluted in the void volume with glutathione.
amine to a free radical metabolite. This is followed by a nonenzymatic one-electron reduction of the free radical metabolite by glutathione, resulting in the formation of parent aromatic amine and a glutathionyl free radical. The thiyl radical reacts with molecular oxygen to form a glutathionyl peroxyl radical (22,23),which accounts for the consumption of oxygen. Our observations indicate that 4-ABP is oxidized by HRP to a free radical metabolite. Oxidation of 4-ABP by HRP was also examined by monitoring the change in the W/vis absorbance spectrum of 4-ABP. A rapid decrease in absorbance at the absorption maximum of 4-ABP was observed after addition of H202,and concomitant increases in absorbance at several wavelengths were observed. The changes in absorption after addition of H202suggested that 4-ABP was oxidized by HRP. Oxidation of other aromatic amines such as benzidine (17) and 2-aminofluorene (27) by HRP and PHS peroxidase also results in changes in the W/vis absorption spectrum. Additional evidence that a free radical metabolite of 4-ABP was formed by HRP was the temporal inhibition of change of absorbance at 325 nm by glutathione and ascorbic acid. These chemicals nonenzymatically reduced the free radical metabolite back to 4-ABP. However, ascorbic acid is also a peroxidase substrate (28)and thus may also act by competing with 4-ABP. Glutathione has previously been shown to nonenzymatically reduce the aminopyrine cation radical formed by PHS peroxidase back to aminopyrine (29). Presumably, once all of the glutathione was consumed in our incubations and provided remaining, the oxidation of 4-ABP conthere was H202 tinued with formation of products. This was observed in all of our incubations, as evidenced by changes in absorbance at 325 nm with a time lag, the length of which was related to the concentration of glutathione. Also, in the metabolism studies, 4-ABP was detected by HPLC in the incubations that contained glutathione, but not in those lacking this thiol. 4-ABP is extensively metabolized by HRP as shown by HPLC to one major product which was identified by mass spectrometry as azobidbiphenyl), a coupling product formed from the reaction of two 4-ABP free radical metabolites. The one-electron oxidation of benzidine by peroxidases results in formation of a free radical, which has been detected by electron spin resonance (19,20). The coupling of two benzidine free radicals forms azobenzidine,
which has been isolated and identified in incubations of benzidine and peroxidases (17). Since 4-ABP is similar in structure to benzidine, it is expected that the oneelectron oxidation product would be a free radical. The coupling of two 4-ABP free radicals to form azobis(biphenyl) (Scheme I) is consistent with this mechanism. 2-Aminofluorene (27) and 2-naphthylamine (2.5) also form coupling products from the reaction of two of their respective peroxidase-catalyzed one-electron-oxidized free radical metabolites. We were able to detect by dual-label radioactive detection products we believe are glutathione conjugates formed by the reaction of glutathione with the 4-ABP free radical metabolite. Wise et al. (30) and Josephy and Iwaniw (31) have reported the formation of benzidineglutathione or benzidine-N-acetylcysteine conjugates during the peroxidase-catalyzed oxidation of benzidine to free radical intermediates in the presence of glutathione or N-acetylcysteine, respectively. The formation of 4ABP-glutathione conjugates during HRP-catalyzed oxidation is strong evidence for 4-ABP free radical formation. Thus, glutathione can interact with the 4-ABP free radical by reducing the free radical back to the parent compound and apparently inhibiting oxidation or by nonenzymatic formation of glutathione conjugates (Scheme I). This is important since either reaction will result in detoxication of reactive 4-ABP free radical intermediates. Thus our studies show that 4-ABP is a reducing cosubstrate for HRP and is oxidized by this enzyme to a free radical metabolite. This metabolite can couple with a second free radical metabolite, forming azobis(bipheny1). Glutathione can reduce the radical back to 4-ABP or react with it to form 4-ABP-glutathione conjugates. Additional studies with peroxidases are required to elucidate the actual mechanism of 4-ABP-induced tumorigenesis. Acknowledgment. We thank Mr. Steve McGown for the mass spectral analysis of our samples. Registry No. 4-PhC6H4N=NC6H4-4-Ph,5326-53-4;4-ABP, 92-67-1; 4-ABP free radical, 139655-33-7; 4-ABP-glutathione, 139631-55-3;peroxidase, 9003-99-0;glutathione, 70-18-8.
References (1) Beland, F. A., and Kadlubar, F. F. (1990) Metabolic activation
and DNA adducts of aromatic amines and nitroaromatic hydrocarbons. In Chemical Carcinogenesis and Mutagenesis Z (Cooper, C. S., and Grover, P. L., as. pp )267-325, Springer-Verlag,Berlin.
4-ABP One-Electron Oxidation (2) Miller, J. A. (1970) Carcinogenesis by chemicals-An
overview. Cancer Res. 30, 559-576. (3) Miller, E. C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential address. Cancer Res. 38, 789-804. (4) Kadlubar, F. F., and Hammons, G. J. (1987) The role of cytochrome P-450 in the metabolism of chemical carcinogens. In Mammalian Cytochromes P-450 (Guengerich, F. P., Ed.) Vol. 2, pp 81-130, CRC Press, Boca Raton, FL. (5) Cramer, J. W., Miller, J. A., and Miller, E. C. (1960) NHydroxylation: A new metabolic reaction observed in the rat with the carcinogen 2-acetylaminofluorene. J. Biol. Chem. 235, 885-888. (6) Spitz, S., Maguigan, W. H., and Dobriner, K. (1954) The carcinogenic action of benzidine. Cancer 3, 789-804. (7) Walpole, A. L., Williams, M. H. C., and Roberts, D. C. (1954) Tumours of the urinary bladder in dogs after ingestion of 4aminobiphenyl. Br. J. Ind. Med. 11, 105-109. (8) IARC (1982) Some industrial chemicals and dyestuffs. Benzidine and ita sulphate, hydrochloride and dihydrochloride. IARC monographs of the eualuation of carcinogenic risk of chemicals t o man, Vol. 29, pp 149-183, International Agency for Research of Cancer, World Health Organization, Lyon, France. (9) Deichmann, W. B., Radomski, J. L., Anderson, W. A. D., Coplan, M. M., and Woods, R. M. (1958) The carcinogenic action of pamino-biphenyl in the dog. Ind. Med. Surg. 27, 25-26. (10) IARC (1972) 4-Aminobiphenyl. IARC Monographs of the eualuation of carcinogenic risk of chemicals to man, Vol. 1, pp 74-76, International Agency for Research on Cancer, World Health Organization, Lyon, France. (11) Walpole, A. L., and Williams, M. H. C. (1958)Aromatic amines as carcinogens in industry. Br. Med. Bull. 14, 141-145. (12) Wise, R. W., Zenser, T. V., Kadlubar, F. F., and Davis, B. B. (1984) Metabolic activation of carcinogenic aromatic amines by dog bladder and kidney prostaglandin H synthase. Cancer Res. 44, 1893-1897. (13) Flammang, T. J., Yamazoe, Y., Benson, R. W., Roberts, D. W., Potter, D. W., Chu, D. Z. J., Lang, N. P., and Kadlubar, F. F. (1989) Arachidonic acid-dependent peroxidation activation of carcinogenic arylamines by extrahepatic human tissue microsomes. Cancer Res. 49, 1977-1982. (14) Bull, A. W. (1987)Reducing substrate activity of some aromatic amines for prostaglandin H synthase. Carcinogenesis 8,387-390. (15) Kadlubar, F. F., Frederick, C. B., Weis, C. C., and Zenser, T. V. (1982) Prostaglandin endoperoxide synthetase-mediated metabolism of carcinogenic aromatic amines and their binding to DNA and protein. Biochem. Biophys. Res. Commun. 108, 253-258. (16) Zenser, T. V., Mattammal, M. B., and Davis, B. B. (1979) Cooxidation of benzidine by renal medullary prostaglandin cyclooxygenase. J. Pharmacol. Exp. Ther. 21 1, 460-464. (17) Josephy, P. D., Eling, T. E., and Mason, R. P. (1983) Co-oxidation of benzidine by prostaglandin synthase and comparison
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 345 with the action of horseradish peroxidase. J. Biol. Chem. 258, 5561-5569. (18) Rice, J. R., and Kissinger,P. T. (1982) Cooxidation of benzidine by horseradish peroxidase and subsequent formation of possible thioether conjugates of benzidine. Biochem. Biophys. Res. Commun. 104, 1312-1318. (19) Josephy, P. D., Eling, T. E., and Mason, R. P. (1983) An electron spin resonance study of the activation of benzidine by peroxidases. Mol. Pharmacol. 23, 766-770. (20) Wise, R. W., Zenser, T. V., and Davis, B. B. (1983) Prostaglandin H synthase metabolism of the urinary bladder carcinogens benzidine and ANFT. Carcinogenesis 4, 285-289. (21) Weller, P. E., Markey, C. M., and Marnett, L. J. (1985) Enzydetection matic reduction of 5-phenyl-4-pentenyl-hydroperoxide: of peroxidases and identification of peroxidase reducing substrates. Arch. Biochem. Biophys. 243, 633-643. (22) Moldeus, P., O'Brien, P. J., Thor, J., Berggren, M., and Orrenius, S. (1983) Oxidation of glutathione by free radical intermediates formed during peroxidase-catalyzed N-demethylation reactions. FEBS Lett. 162, 411-415. (23) Ross, D., Albano, E., Nilsson, U., and Moldeus, P. (1984) Thiyl radicals: formation during peroxidase-catalyzed metabolism of acetaminophen in the presence of thiols. Biochem. Biophys. Res. Commun. 125, 109-115. (24) Gordon, A. J., and Ford, R. A. (1972) Electronic absorption and emission spectra: Ultraviolet and visible. In The Chemist's Companion, pp 211-220, Wiley, New York. (25) Boyd, J. A,, and Eling, T. E. (1987) Prostaglandin H synthase-catalyzed metabolism and DNA binding of 2-naphthylamine. Cancer Res. 47, 4007-4014. (26) Ross, D., Larsson, R., Andenson, B., Nilsson, U., Lindquist, T., Lindeke, B., and Moldeus, P. (1985) The oxidation of pphenetidine by horseradish peroxidase and prostaglandin synthase and the fate of glutathione during such oxidations. Biochem. Pharmacol. 34, 343-351. (27) Boyd, J. A., and Eling, T. E. (1984) Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase. J. Biol. Chem. 259, 13885-13896. (28) Markey, C. M., Alward, A., Weller, P. E., and Marnett, L. J. (1987) Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. J. Biol. Chem. 262, 6266-6279. (29) Eling, T. E., Mason, R. P., and Sivarajah, K. (1985) The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical. J. Biol. Chem. 260, 1601-1607. (30) Wise, R. W., Zenser, T. V., and Davis, B. B. (1985) Prostaglandin H synthase oxidation of benzidine and o-dianisidine: reduction and conjugation of activated amines by thiols. Carcinogenesis 6, 579-583. (31) Josephy, P. D., and Iwaniw, D. C. (1985) Identification of the N-acetylcysteine conjugate of benzidine formed in the peroxidase activation system. Carcinogenesis 6,155-158.
