Peroxidase Metabolism of the Urinary Bladder Carcinogen 2-Amino-4-(5- Nitro-2-Furyl)Thiazole1

[CANCER RESEARCH 43, 1518-1522, April 1983] 0008-5472/83/0043-0000$02.00 Peroxidase Metabolism of the Urinary Bladder Carcinogen 2-Amino-4-(5- nitro-2-furyl)thiazole1

Ronald W. Wise, Terry V. Zenser,2 and Bernard B. Davis

Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, St. Louis, Missouri 63125 [T. V. Z., B. B. D.J, and Departments of Biochemistry [P. W. W., T. V. Z.] and Internal Medicine [T. V. 2., B. B. D.], St. Louis University School of Medicine, St. Louis, Missouri 63706

ABSTRACT c nitroreductase has been reported (42). Anaerobic metabolism of 5-nitrofurans, however, is not inhibited by aspirin, and reduced Metabolism of 2-amino-4-(5-nitro-2-furyl)thÂ¡azole (ANFT) by a products have different Chromatographie properties than do the variety of different peroxidases was examined. Metabolism of cooxidation products. Aspirin has been shown to significantly ANFT was measured by the binding of radiolabeled substrates reduce rat bladder prostaglandin E2 synthesis (4) and prevent to protein and DMA. Prostaglandin hydroperoxidase but not bladder PES-catalyzed metabolism of FANFT and ANFT (4, 22). horseradish peroxidase, lactoperoxidase, or chloroperoxidase Aspirin has also been demonstrated to inhibit transitional carci metabolically activated ANFT. All four peroxidases catalyzed the noma formation in FANFT-fed rats (38). These studies suggest binding of benzidine to protein and DNA. With peroxide sub that the hydroperoxidase activity of bladder PES may be involved strates, peroxidase-catalyzed binding of both carcinogens was in the initiation of 5-nitrofuran-induced bladder carcinogenesis. observed with or without molecular oxygen. Arachidonic acid- Hemeprotein peroxidases, in addition to prostaglandin hydro dependent binding of ANFT and benzidine by prostaglandin peroxidase, are present in mammalian tissues (1, 19, 30) and endoperoxide synthetase was inhibited by anaerobic conditions may activate carcinogens. A/-Hydroxy-2-acetylaminofluorene has and aspirin. Chloroperoxidase activation of benzidine was also been shown to be converted to the carcinogens 2-nitrosofluorene inhibited by aspirin. Vitamin E inhibited activation of both carcin and A/-acetoxy-2-acetylaminofluorene by horseradish peroxi ogens by all enzymes examined. Prostaglandin hydroperoxidase- dase, lactoperoxidase, and myeloperoxidase (3). Not all peroxi catalyzed binding of benzidine to protein was inhibited by the dases metabolize the same substrates, however, and different 5-nitrofurans ANFT and 3-hydroxymethyl-1-|[3-(5-nitro-2-fu- peroxidases may metabolize a particular substrate to different ryl)allydidene]amino|hydantoin and acetaminophen, while only products. Sulindac sulfide is oxidized by PES but not by horse acetaminophen inhibited horseradish peroxidase-catalyzed bind radish peroxidase or lactoperoxidase (11). Chloroperoxidase ing. These results indicate that different peroxidases may exhibit metabolizes p-chloroaniline to only p-chloronitrosobenzene (6). specificity with respect to their activation of carcinogens. Only Horseradish peroxidase (33) and myeloperoxidase metabolize p- prostaglandin hydroperoxidase activated the 5-nitrofuran ANFT, chloroaniline to multiple products not including p-chloronitroso while a number of peroxidases activated the aromatic amine benzene (2), and lactoperoxidase does not metabolize p-chlo benzidine. roaniline (5). To further evluate ANFT metabolism, this report assessed the metabolism of ANFT by prostaglandin hydroperox INTRODUCTION idase, horseradish peroxidase, lactoperoxidase, and chloroper oxidase. Metabolism of benzidine was also evaluated in order to The hydroperoxidase component of PES3 catalyzes the me assess the activity of the different peroxidases, demonstrate the tabolism of structurally diverse carcinogens which include aro use of inhibitors in assessing activity, and examine peroxidase- matic amines (37,40), 5-nitrofurans (22,37), a synthetic estrogen catalyzed binding to DNA. These results indicate that while (8), and polyaromatic hydrocarbons (21,34). Metabolism of these peroxidases may exhibit specificity with respect to the activation carcinogens results in the formation of activated metabolites of certain carcinogens, other carcinogens may be activated by a which bind to nucleic acids. PES-catalyzed metabolism of carcin number of peroxidases. ogens is initiated by specific unsaturated fatty acid substrates or a broad range of hydroperoxides and is prevented by specific MATERIALS AND METHODS inhibitors of prostaglandin synthesis and antioxidants (8, 21, 34, 39). Renal and bladder cytochrome P-450 mixed-function oxi Materials. [14C]Thiourea (47.5 mCi/mmol) and [U-14C]benzidine (25.7 dases have not been shown to metabolize FANFT or other 5- mCi/mmol) were purchased from New England Nuclear, Boston, Mass. nitrofuran urinary tract carcinogens (36, 42). The only reported Scintillation fluid (ACS) was purchased from Amersham/Searle Corp., mechanism of aerobic 5-nitrofuran metabolism by kidney and Arlington Heights, III. Benzidine dihydrochloride, cesium chloride, p- urinary bladder is cooxidation by prostaglandin hydroperoxidase aminosalicylate, bovine albumin (Fraction V powder), vitamin E, DNA (22, 36). Anaerobic metabolism of 5-nitrofurans by cytochrome (calf thymus), Tween 20, aspirin (acetylsalicylic acid), salicylate, horse radish peroxidase (type I), lactoperoxidase (A412:A28o= 0.6), and chloro peroxidase (RZ = 0.6) were purchased from Sigma Chemical Company, 1This work was supported by the Veterans Administration and by the USPHS St. Louis, Mo. 5,8,11,14-Eicosatetraenoic acid was purchased from Nu- Grant CA-28015 from the National Cancer Institute through the National Bladder Chek Prep, Inc., Elysian, Minn. Hydrogen peroxide and all other solvents Cancer Project. 2 To whom requests for reprints should be addressed, at Geriatric Center (111G- and compounds were purchased from Fisher Scientific Co., Pittsburgh, JB), Veterans Administration Medical Center, St. Louis, Mo. 63125. Pa. Thin-layer Chromatographie plates (Silica Gel F254)were obtained 3 The abbreviations used are: PES, prostaglandin endoperoxide synthetase; from EM Laboratories, Inc., Elmsford, N. Y. 15-HPETE and [14C]ANFT FANFT, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide; ANFT, 2-amino-4-{5-nitro-2-furyl)thiazole; 15-HPETE, 15-hydroperoxy-5,8,11,13-eicosatraenoic acid; TCA, tri- were synthesized and purified as reported previously (22,41). Tween 20 chloroacetic acid. (1.5%)-solubilized ram seminal vesicle microsomes were prepared as Received April 1, 1982; accepted December 30.1982. described previously (22, 26).

1518 CANCER RESEARCH VOL. 43

Incubation Conditions for ANFT. The reaction mixture contained tion of DNA as determined by absorbance at 260 nm on a Beckman enzyme, 0.1 M phosphate buffer for pH 7.0 and pH 7.6 or 0.1 M acetate Acta C11 spectrophotometer. Less than 1% of the radioactivity was buffer for pH 5.0, bovine albumin (2 mg/mt), and 0.02 mw ANFT (0.25 found at the top of the gradient, while >80% was recovered in fractions ÃÃCi).Varioustest substances were added as indicated to a final volume corresponding to densities of 1.59 to 1.74 g/ml. In control experiments of 0.25 ml. The amount of each enzyme used was either 3.5 x 10~2 mg (incubations without DNA), neutralized samples of TCA-precipitable pro solubilized seminal vesicle microsomal protein, 2.4 x 10~5 mg horserad tein were applied to identical CsCI gradients, and >80% of the radioac ish peroxidase, 3.6 x 10~3 mg lactoperoxidase, or 1.2 x 10~3 mg protein tivity was found on the top of the gradient, while <3% occurred in the chloroperoxidase. Two- and 10-fold higher concentrations of each en 1.59- to 1.74-g/ml fractions. zyme were also examined (not shown). Reactions were initiated by The following blanks were used to correct for nonspecific binding of addition of 0.13 mw arachidonic acid, 0.05 mw 15-HPETE, or 0.3 HIM radioactive products to nucleic acid and to correct for the possible carry hydrogen peroxide. over of labeled products bound to protein into the aqueous phase. Blank The pH of the reactions for prostaglandin hydroperoxidase, horserad A consisted of a complete reaction mixture including enzyme and DNA ish peroxidase, lactoperoxidase, and chloroperoxidase was 7.6, 7.0, 7.0, but without arachidonic acid or peroxide. Blank B was a complete and 5.0, respectively. Reactions were terminated after 2 min at room reaction including arachidonic acid or peroxide and DNA but without temperature by addition of 1 ml ice-cold ethyl acetate:ethyl ether (1:1, enzyme. The highest blank value was subtracted from experimental v/v). Following six 1-ml extractions, pooled organic phases from each values, and the data were corrected for recovery of DNA (70 to 80%) individual sample were evporated under N2 gas and dissolved in ethyl and expressed as nmol metabolite bound to precipitatale DNA per mg acetate:ethyl ether (1:1, v/v), and aliquots were applied to thin-layer protein per min. chromatography plates. The plates were developed in mÃ©thylÃ¨nechlc- Determination of Anaerobic Metabolism. Tubes containing reaction ride:ethanol:90% formic acid (80:15:2, v/v/v), and radioactive peaks were mixtures were fitted on top with a septum. Arachidonate or peroxide located using a Packard 7230 radiochromatogram scanner. was added as a 1- or 2-/J droplet on the inside of the tube above the Following organic solvent extraction, an equal volume of 0.6 M TCA incubation solution. While the tube was maintained on ice, a vacuum was applied from a needle through the septum. A vacuum of 10~7 ton- was added to the aqueous phase which was then centrifugea at 2500 x g for 10 min. The resulting precipitates were washed repeatedly with was achieved in 2 min and held for 3 min. The needle was removed, and 1.0 ml 0.3 M TCA, followed by centrifugation until the radioactivity of the the reaction was started by mixing the tubes and placing in a 25Â°water supernatant was background (about 6 washes per sample). Pellets were bath. For aerobic reactions, air was readmitted. Similar results were dissolved in 0.1 ml of N NaOH, diluted to 0.5 ml with distilled water, obtained whether samples were incubated directly in air or first had a aliquoted, and analyzed for radioactivity. Metabolism of ANFT is ex vacuum applied and air readmitted. This suggests that the method used pressed as nmol metabolite bound to TCA-precipitable material per mg to achieve anaerobic conditions did not itself interfere with the results. protein per min. All reactions were linear with respect to protein concen The reactions were stopped by injecting 1 ml ice-cold ethyl ace- tration and incubation time. Blank values were obtained from samples tate:ethyl ether (1:1) through the septum and placing on ice. The septums incubated without enzyme and were subtracted from experimental val were removed, and the extent of the reaction was determined as ues. Similar blank values were obtained with heated microsomes (100Â° described previously. Two oxygen-requiring enzymatic assays were used for 5 min) or with incubations performed in the absence of either arachi to insure that the samples were anaerobic. Arachidonic acid metabolism donic acid or peroxide. by fatty acid cyclooxygenase (32) and 15-HPETE-initiated metabolism of To assure that 14Cmetabolites were covalently bound to TCA-precip 1,3-diphenylisobenzofuran by PES (20) were assessed. Incubation mix itable protein, the solubilized pellets were extracted with ethyl acetate tures were in the same size of tubes and same volumes and contained and chlorofomrethyl ether (1:3, v/v) at pH 4, 7, and 11 (40). Greater than essentially the same components described above. Arachidonic acid- 90% of the radioactivity remained in the aqueous phase. initiated metabolism of benzidine was 94% inhibited in evacuated tubes. Incubation Conditions for Benzidine. The incubation conditions for The 80% inhibition in 1,3-diphenylisobenzofuran metabolism was similar benzidine were the same as those described for ANFT, except that 0.06 to that reported by MarneÂ« ef al. (20) using anaerobic conditions. In rriM benzidine was substituted for ANFT. The reactions were stopped by addition, cytochrome c reduction of FANFT and ANFT, reactions inhibited the addition of unlabeled benzidine to a final concentration of 0.75 HIM, by oxygen (23,42), has been observed using these anaerobic conditions. followed by 1 ml ethyl acetate:ethyl ether (1:1, v/v). Following three 1-ml extractions with organic solvent, cold 0.6 M TCA was added to the RESULTS aqueous phase, and the samples were centrifugea at 2500 x g for 10 min. The pellets were treated as described above for analysis of ANFT. ANFT metabolism by PES, horseradish peroxidase, lactoper Metabolism of benzidine is expressed as nmol metabolite bound to TCA- oxidase, and chloroperoxidase is reported in Table 1. The incor precipitable material per mg protein per min. poration of [14C]ANFT into TCA-precipitable material was cata Determination of Binding of [MC]Benzidine Metabolite(s) to DMA. lyzed only by PES. Horseradish peroxidase, lactoperoxidase, Binding of metabolite(s) to DNA was determined using methods reported previously (39). The incubations included DNA (2 mg/ml) in place of and chloroperoxidase did not show any evidence of ANFT me tabolism in aqueous fractions or TCA-precipitable fractions even bovine serum albumin. The reaction was terminated by adding 0.6 ml 12% sodium p-aminosalicylate followed by 1 ml 30 g phenol:4.2 ml m- when the respective enzyme concentrations were 2- to 10-fold cresol:3.3 ml water. The phenokcresol mixture was saturated with an those used for the benzidine metabolism described below. A equal volume of 100 mw phosphate buffer, pH 7.0. Following phenolcre- thin-layer Chromatographie radioscan of the organic phase re sol extraction, DNA was precipitated from the aqueous phase with 2 sulting from incubations of ANFT with PES shows a peak cor volumes of 2-ethoxyethanol and centrifuged at 4200 x g for 30 min. The responding to authentic ANFT (Peak I) and a peak corresponding precipitate was washed successively with ethanol and ether and dried. to cooxidation product(s) (Peak II) (Chart 1, Scan A). Incubation The precipitate was dissolved in 1.0 ml citrate buffer (pH 7.0) (0.15 M of ANFT with horseradish peroxidase shows only Peak I corre NaCl:0.015 M sodium citrate), and an aliquot was taken for determination sponding to the ANFT substrate (Chart 1, Scan 8). Incubations of radioactivity. with lactoperoxidase and chloroperoxidase also did not produce The binding to DNA was further assessed by determining the migration of radioactive product in a CsCI gradient (16). An aliquot was mixed with organic soluble cooxidation products (not shown). A lack of 5 M CsCI to a final volume of 8 ml in citrate buffer (pH 7.0) and centrifuged ANFT metabolism was also observed when these enzymes were at 30,000 rpm in a Beckman SW 40 rotor for 17 hr. The resultant incubated in phosphate buffer (pH 7.8). Metabolism of ANFT by distribution of radioactivity in the gradient corresponded to the distribu PES was achieved with either arachidonic acid, 15-HPETE, or

APRIL 1983 1519

Table 1 Peroxidase-catalyzed binding of [ÃŒ4C]ANFTtoTCA-precipitable material

nmol/mg protein/min

tration(mM)0.130.050.30.30.3Inhibitors3Aerobic5.7AspirinAnaerobic E(2 Vitamin EnzymePESHorseradish mM)1.2 (0.2 mM) (1 .2 mM)4.7mM) (0.05 Â±0.465.6 ND5.4Â±0.2 5.3 Â±0.4 0.65.1Â±0.8 2.3 Â± Â±0.6NDCNDNOCyanideÂ±1.1NONDNDSalicylateÂ±0.5 6.3 Â±1.3 5.9 Â±0.9 2.0 Â±0.2

peroxidaseLactoperoxidaseChloroperoxidaseSubstrateArachidonate15-HPETEH202H202H2O2Concen

a Aerobic. 6 Mean Â±S.D. (n = 3). c ND, no metabolism detected.

â€” I found to inhibit all the peroxidases to some extent (not shown). Vitamin E was an effective inhibitor of benzidine metabolism by all of the peroxidases examined. Chloroperoxidase was the only peroxidase inhibited by 1.2 mM aspirin. The effect of acetaminophen and different carcinogenic 5- nitrofurans on benzidine activation by PES and horseradish peroxidase was examined (Table 3). ANFT (0.025 mM) inhibited PES-catalyzed metabolism of benzidine by 50% but had no effect on horseradish peroxidase at either 0.025 or 0.050 mM con centrations. 3-Hydroxymethyl-1 -)[3-(5-nitro-2-furyl)allydidene]- aminojhydantoin (0.05 mM), another 5-nitrofuran, also inhibited PES-catalyzed metabolism but had no effect on horseradish peroxidase. Acetaminophen (0.1 mM), however, which is a known substrate for both PES (27) and horseradish peroxidase (29), inhibited benzidine metabolism by both enzymes to approxi mately the same extent. Chart 1. Thin-layer Chromatographie radioscan of organic extractable products of ["CJANFT metabolism by PES (Scan A) and horseradish peroxidase (Scan B). All 4 peroxidases were found to activate benzidine to metab Concentrated organic extracts of reaction mixtures were applied to silica gel thin- olite^) which covalently binds to DNA (Table 4) and tRNA (not layer Chromatographie plates and developed as described in "Materials and Meth ods." Peak I corresponds to the Rf of authentic ANFT, and Peak II appears only shown). The relative rates of radioactivity incorporated into DNA are shown. The large apparent differences in the rates between after cooxidation with PES. PES and horseradish peroxidase are largely due to the relative H2O2 (not shown) as substrate. Although essentially the same purities of the enzyme preparations. Compared to the rate of results were achieved with 0.2 mM H2O2as substrate, 15-HPETE incorporation of benzidine into TCA-precipitable material (Table is reported here, because it more closely resembles the natural 1), the relative rate of incorporation of benzidine into DNA was substrate prostaglandin G2 and is more effective. Only the fatty 1.3, 15.3, 9.1, and 3.9% for PES, horseradish peroxidase, lac- acid cyclooxygenase component of PES (arachidonic acid me toperoxidase, and Chloroperoxidase, respectively. PES has al diated) was inhibited by incubating under anaerobic conditions. ready been shown to catalyze the covalent binding of ANFT to Aspirin (1.2 HIM) also inhibited the arachidonic acid-mediated DNA (22). fatty acid cyclooxygenase component of PES but had no effect on 15-HPETE-mediated prostaglandin hydroperoxidase activity. DISCUSSION Vitamin E (0.05 mM) inhibited ANFT metabolism by PES whether This study demonstrates that of the 4 peroxidases examined, the reaction was mediated by either arachidonic acid or 15- only prostaglandin hydroperoxidase metabolizes the urinary HPETE. Salicylic acid (2 mM) and KCN (0.2 mM) had no apparent bladder carcinogen ANFT. This is the first demonstration of effect on ANFT metabolism at these concentrations. specificity in peroxidase-catalyzed metabolism of a carcinogen. A different pattern of metabolism was observed with benzidine Horseradish peroxidase, lactoperoxidase, and Chloroperoxidase (Table 2). All 4 peroxidases readily metabolized benzidine with do not represent all peroxidases; however, their total lack of or without oxygen. Identical amounts of enzymes were used in metabolism of ANFT suggests that PES may be unique in its Tables 1 and 2. The effect of various inhibitors on PES-catalyzed ability to peroxidatically activate 5-nitrofurans. Anaerobic metab metabolism of benzidine was similar to their effect on ANFT olism of ANFT is observed only by PES (with peroxide substrate) metabolism. Anaerobic conditions and aspirin (1.2 mM) inhibited and cytochrome c nitroreductase. To date, the only demon only the arachidonate-mediated fatty acid cyclooxygenase activ strated aerobic metabolism of ANFT is by PES. This information ity, whereas cyanide (0.2 mM) and salicylate (2 mM) had no is important in interpreting and designing experiments to assess apparent effect on PES whether activated with arachidonate or and to prevent ANFT initiation of bladder cancer. 15-HPETE. Horseradish peroxidase was the only peroxidase Activation of ANFT to bind protein under anaerobic conditions inhibited by 0.2 mM KCN, although higher concentrations were demonstrates that molecular oxygen is not required for prosta-

1520 CANCER RESEARCH VOL. 43

Table 2 Substrate and inhibitor specificity for [*4C]benzidine binding to TCA-precipitable material

nmol/mg protein/min

trationIIMMI0.130.050.30.30.3lnhibitorsaAerobic26.9 E(0.05 EnzymePESHorseradish rriM)27.2 mM)21 HIM)14.8 Â±3.4b29.3 Â±0.330.4 Â±3.130.1 Â±0.230.1 .8Â±2.126.8 Â±1.818.1 Â±3.538.5 Â±4.835.8 Â±2.410.4 Â±1.244.7 Â±1.311.4Â±1.4VitaminÂ±0.3217.9 (IO3)0Lactoperoxidaseperoxidase Â±3.549.3 Â±4.452.2 Â±0.451.6 Â±4.548.7 Â±0.928.1

(10')Chloroperoxidase Â±4.412.9 Â±6.711.5 +2.112.0 Â±2.42.1 Â±2.01.6 (102)SubstrateArachidonate15-HPETEH202HZ02H202Concen Â±1.3Anaerobic1.6Â±6.1Cyanide(0.2Â±0.8Aspirin(1.2mM)1.1Â±0.1Salicylate(2 Â±0.2 a Aerobic. 6 Mean Â±S.D.(n = 3). c Actual value obtained by multiplying by the factor of 10 indicated.

Table 3 the access of ANFT to a site near the heme-iron ligand. The 5- Effect of urinary tract carcinogens on f'CJbenzidine activation by PES and nitrofuran, nitrofurantoin, interacts with methemoglobin at a site horseradish peroxidase distinct from, but in proximity to, the heme-iron atom (10). Incubations included 0.05 mM 15-HPETE or 0.3 mM H202, 0.06 mw benzidine, and bovine serum albumin (2 mg/ml). Control rates of PES- and horseradish Therefore, a lack of access by ANFT to a site near the heme- peroxidase-catalyzed binding to TCA-precipitable material were 26.9 Â±3.4 and iron ligand of horseradish and other peroxidases may explain the 38.5 Â±3.5 x 103 nmol per mg protein per min, respectively. Values represented lack of ANFT metabolism by these peroxidases. as 100% of control were not significantly different from corresponding control rates (n = 3 to 6). The peroxidases examined in this study exhibited different controlAdditionNoneANFTHMN3AcetaminophenConcentration% of responses to inhibitors. Cyanide (0.2 mw) was most effective in inhibiting horseradish peroxidase. The relative lack of effect of (mM)0.025 peroxidase100100 cyanide on PES is consistent with the findings of Hemler and Lands (15). Aspirin is well known for its ability to inhibit the fatty acid cyclooxygenase component of PES by acetylation of a serine residue (31). The lack of effect of salicylate, a deacetylated 0.0500.0500.100PES100505454Horseradish10010045 aspirin metabolite, is consistent with acetylation being the mech anism of aspirin inhibition. Aspirin inhibition of only Chloroperoxi dase may indicate differences in the amino acids near the active site of this enzyme. These results suggest that myeloperoxidase, HMN, 3-hydroxymethyl-1-|[3-(5-nitro-2-furyl)allydidene]amino| hydantoin. which is in many ways mechanistically similar to Chloroperoxi

Table 4 dase (28), may also be inhibited by aspirin. The antioxidant Peroxidase-catalyzed binding of [â„¢C]benzidine to DNA vitamin E was the only compound tested which inhibited metab olism initiated by all the peroxidases. ANFT prevention of ben tration (nmol/mg zidine metabolism by PES but not horseradish peroxidase could EnzymePES (mM)0.05 protein/min)0.38 be explained by substrate competition. Accordingly, acetamino Â±0.04a phen, a substrate for both PES and horseradish peroxidase, Horseradish peroxidase H202 0.3 5.96 Â±0.61 x 103 Lactoperoxidase H202 0.3 45.4 Â±6.7 inhibited metabolism of benzidine by both enzymes. ChloroperoxidaseSubstrate15-HPETEH202Concen 0.3DNA 49.9 Â±3.4 The ability of each enzyme to metabolize benzidine insured a Mean Â±S.D. (n = 3). that each enzyme was active. Not only did all the peroxidases activate benzidine to bind protein, but binding to DNA was also glandin hydroperoxidase metabolism of ANFT. Recent studies demonstrated. Previous publications have reported metabolism have demonstrated, however, that oxygen is inserted into the of benzidine by horseradish peroxidase and lactoperoxidase (33, furan ring during PES metabolism of ANFT (7). Prostaglandin 35). This is the first study, however, to demonstrate that covalent hydroperoxidase has been shown to utilize oxygen from 3 binding to nucleic acids can occur. Covalent binding of carcino sources: molecular oxygen in the cooxidation of 1,3-diphenyli- genic electrophiles to nucleic acids is thought to be a necessary sobenzofuran (20); oxygen from water Â¡nthe cooxidation of step in the initiation of chemical carcinogenesis (13,17, 24, 25). aminopyrine (18); and oxygen from the peroxide substrate in the The results of this study indicate that, considering their wide cooxidation of sulindac sulfide (12). The mechanism of prosta- distribution and ability to activate some carcinogens to bind glandin hydroperoxidase-catalyzed metabolism appears to de DNA, peroxidases may play a role in the carcinogenic process. pend upon the cooxidizable substrate. ANFT apparently acquires A model describing the relationship of prostaglandin hydro an oxygen atom from the aqueous solution or from the peroxide peroxidase and other peroxidases in the activation of carcino substrate. Horseradish peroxidase has been shown to oxidize gens is illustrated in Chart 2. This model is consistent with compounds which incorporate molecular oxygen, such as indole- experimental results in this paper and previous studies. Three 3-acetic acid (9) or oxygen from water as with aminopyrine (14). types of peroxidatic reactions are envisioned. Type 1 reactions ANFT, like sulindac sulfide, is not metabolized by horseradish occur with carcinogens that are just metabolized by the hydro peroxidase and might also incorporate oxygen from the peroxide peroxidase component of PES. In view of the peroxidatic nature substrate. Incorporation of an oxygen atom originating from the of this reaction, arachidonic acid is not required, and a variety of peroxide substrate of heme peroxidases would probably require peroxide cosubstrates may be used. PES-catalyzed activation

APRIL 1983 1521

Inactivation by hydrogen peroxide catalyzed by horseradish peroxidase, metmyoglobin, and protohemin. Biochemistry, 77: 2206-2211,1978. IType * 1 ROOM + Carcinogens (ex. 5-mtrofurans) 15. Hemler, M. E., and Lands, W. E. M. Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J. Biol. Chem., 255: 6253- ROH + 6261,1980. Type *2 ROOM + Carcinogens Activated Carcinogen (ex. aromatic amines) 16. Kadlubar, F. F., Anson, J. F., Dooley, K. L, and Beland, F. A. Formation of urothelial and hepatic DNA adducts from the carcinogen 2-naphthylamine. IType *3 ROOM +(hypothetical) Carcinogens covalant Carcinogenesis (Lond.), 2: 467-470, 1981. binding to nucleic acids 17. Kriek, E. Carcinogenesis by aromatic amines. Biochim. Biophys. Acta, 355: 177-203, 1974. 18. Lasker, J. M., Sivarajah, K., Mason, R. P., Kalyanaraman, B., AbouDonia, M. Initiation of B., and Eling, T. E. A free radical mechanism of prostaglandin synthase- Chemical carclnogenesis dependent aminopyrine demethylation. J. Biol. Chem., 256: 7764-7767,1981. 19. Lyttle, C. R., and DeSombre, E. R. Generality of oestrogen stimulation of ROOM = Prottaglandin Gf lipid peroxide, or H2<>2 peroxidase activity in growth responsive tissues. Nature (Lond.), 268: 337- 339,1977. Chart 2. Proposed model for peroxidatic activation of carcinogens. 20. Marnett, L. J., Bienkowski, M. J., and Pageis, W. R. Oxygen 18 investigation of the prostaglandin synthetase-dependent co-oxidation of diphenylisobenzofuran. J. Biol. Chem., 254: 5077-5082, 1979. 21. Marnett, L. J., and Reed, G. A. Peroxidatic oxidation of benzo(a)pyrene and prostaglandin biosynthesis. Biochemistry, 78: 2923-2929,1979. of 5-nitrofurans is a type 1 reaction. Type 2 reactions occur with 22. Mattammal, M. B., Zenser, T. V., and Davis, B. B. Prostaglandin hydroperox- carcinogens that are metabolized by both prostaglandin hydro- idase-mediated 2-amino-4-(5-nitro-2-furyl)-'4C-thiazole metabolism and nucleic acid binding. Cancer Res., 47: 4961-4966,1981. peroxidase and other peroxidases. In contrast to the other 23. Mattammal, M. B., Zenser, T. V., and Davis, B. B. Anaerobic metabolism and peroxidases, prostaglandin hydroperoxidase is part of a complex nuclear binding of the carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). which generates its own hydroperoxide substrate, prostaglandin Carcinogenesis (Lond.), 3: 1339-1344,1982. 24. Miller, J. A. Carcinogenesis by chemicals: an overviewâ€”G. H. A. Clowes G2. Aromatic amine and diethylstilbestrol activation appears to memorial lecture. Cancer Res., 30: 559-576, 1970. occur by a type 2 reaction. One would expect that certain 25. Miller, J. A., and Miller, E. C. The concept of reactive electrophilic metabolites carcinogens would be metabolized by other peroxidases and not in chemical carcinogenesis: recent results with aromatic amines, safrole, and aflatoxin B. In: D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio (eds.), PES. There is not a known type 3 reaction at this time. Biological Reactive Intermediates, Chap. 2, p. 6. New York: Plenum Publishing Corp., 1977. 26. Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, 0. Purification of ACKNOWLEDGMENTS prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem., 257: 2629-2636, 1976. The authors wish to thank Dr. Michael B. Mattammal for synthesis of 15-HPETE 27. Moldeus, P., and Rahimtula, A. Metabolism of paracetamol to a glutathione and ["CjANFT and Sandy Melliere for secretarial assistance. conjugate catalyzed by prostaglandin synthetase. Biochem. Biophys. Res. Commun., 96: 469-475, 1980. 28. Morrison, M., and Schonbaum, G. R. 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1522 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1983 American Association for Cancer Research. Peroxidase Metabolism of the Urinary Bladder Carcinogen 2-Amino-4-(5-nitro-2-furyl)thiazole

Ronald W. Wise, Terry V. Zenser and Bernard B. Davis

Cancer Res 1983;43:1518-1522.

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