[CANCER RESEARCH 40, 114-1 18, January 1980)0008-5472/80/0040-0000$02.00

Metabolism of N-(4-(5-Nitro-2-furyl)-2-thiazolyl]formamide byProstaglandin Endoperoxide Synthetas&

Terry V. Zenser,2 Michael B. Mattammal, and Bernard B. Davis

Geriatric Research, Education and Clinical Center, VA Medical Canter IT. V. Z., M. B. M., B. B. Dl, and Departments of Biochemisfry fT. V. Z.Jand Medicine fT. V.z., B.B.0.1,St.LouisUniversity,St.Louis,Missouri63125

these experiments was to evaluate thi possibility that FANFTcould be metabolized by a different @nzymesystem, prostaglandin endoperoxide synthetase. Th@results indicate cooxidation of FANFT by prostaglandmnel@mdoperoxidesynthetaseprepared from the rabbit inner medu@aand the ram seminalvesicle. I

MATERIALS AND METHODS

Material. Metyrapone, indomethacir@,allopurinol, hemoglobin (type I, bovine), butylated hydro4ytoluene, sodium benzoate, Tween 20, aspirin (acetylsalicyIk@acid), 11,14,1 7-eicosatrienoic acid, and 9,12,15-octadeca*'ienoic acid were purchased from Sigma Chemical Co., St.@Louis,Mo. 5,8,11,14-Eicosatetraenoic acid and 8,1 1,14-ei4osatrienoic acid werepurchased from Nu-Chek Prep, Inc., Elytpian,Minn. FANFTwaspurchased from Saber Laboratories, Morton Grove, Ill. Thepurity of FANFT was greater than 99.@%as judged by previously published criteria (8). 5,8,1 1,14-@icosatetraynolc acid,ethoxyquin, and prostaglandmnE2 wer@the generous gifts ofDr. W. E. Scott, Hoffmann-La Roche ln4., Nutley, N. J.; Dr. G.L. Romoser, Monsanto Chemical Co., §t.Louis, Mo.; and Dr.John Pike, The Upjohn Co., Kalamazo4, Mich., respectively.All other chemicals were purchased ir@the highest possiblegrade from standard sources. Male New ealand rabbits weighing 1.5 to 2.0 kg were obtained fron@Eldridge LaboratoryAnimals, Barnhart, Mo.

Preparation of Microsomes. Rabbits @ereanesthetized withsodium thiopental (20 mg/kg i.v.). The @cidneyswere quicklyremoved and placed in ice-cold 0.9% Na4@lsolution (33). Renalinner medullary tissue was isolated by c@arefuldissection andwashed free of hemoglobin. Ram semir@alvesicles were obtamed fresh from a local packing plant nd dissected free ofadjacent tissue. Microsomes from either issue were preparedas follows. Minced tissue was homogeni ed for 15 sec with aPolytron homogenizer in 3 volumes of 0. M phosphate buffer,pH 7.8, containing 20% glycerol and 0. 1 Mdithiothreitol. Thehomogenate was centrifuged at 10,000 g for 15 mm and thesubsequent supernatant at 100,000 X g r 60 mm. This pelletwas layered with 1.15% KCI and stored t —40°.Before use,the microsomal pellets were suspended i 1.15% KCl by handhomogenization and centrifuged for 60 nun at 105,000 x g.Pellets were then resuspended in 0.1 M @hosphatebuffer, pH7.8, with a Potter-Elvehjem Teflon-glass @omogenizerto givea final concentration of about 10 mg mk*osomal protein perml. Prostaglandin endoperoxide synthet@se was solubilizedwith Tween 20 using previously publishød procedures (21).The final concentration of Tween 20 was 1.5%. Aliquots wereeither used immediately or stored froze@iat —40°.Proteincontent was determined in microsomes by @hemethod of Lowryet a!. (18), and in the solubilized preparatic@sby the method of

CANCER @ESEARCHVOL. 40

ABSTRACT

Cooxidative metabolism of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyljformamide (FANFT) was exammed using solubilized and particulate microsomal preparationsfrom the rabbit renal inner medulla and the ram seminal vesicle.Metabolism was measured by the rate of decrease in absorbance at 400 nm. In these soluble and particulate preparations,FANFT metabolism was observed only in the presence ofspecific fatty acids. These fatty acids are substrates for prostaglandin endoperoxide synthetase. Structurally dissimilar inhibitors of prostaglandin endoperoxide synthetase such asindomethacin, aspirin, 5,8,1 1,14-eicosatetraynoic acid,ethoxyquin, and meclofenamic acid specifically inhibitedFANFT metabolism. Other inhibitor and substrate specificitystudies suggest that FANFT was not metabolized by nitroreductase, xanthine oxidase, lipoxygenase, lipid peroxidation, ormixed-function oxidases. In addition, the lack of detectable 2-amino-4-(5-nitro-2-furyl)thiazole formation suggests that arylfonmamidasewas not participating in FANFT metabolism measured in these experiments. The data indicate that prostaglandmnendoperoxide synthetase can mediate FANFT metabolism bya cooxidative process.

INTRODUCTION

Carcinoma of the urinary bladder is induced by a number ofchemical carcinogens (6). The 5-nitrofuran FANFT3is a potenturinary bladder carcinogen (9). When fed to experimentalanimals, FANFT is associated with a high incidence of carcinoma of the urinary bladder. Virtually 100% of weanling Fischerrats develop bladder cancer when fed a diet containing 0.2%FANFTfor 26 weeks. It has been proposed that FANFTrequiresmetabolic activation to induce carcinoma of the urinary bladder(25). Several enzyme systems have been shown to metabolizeFANFT. These include nitroreductase (3, 27), xanthine oxidase(27), and arylformamidase (28). Metabolism by these systems,however, has been difficult to relate to the formation of carcinoma of the bladder. For example, when the xanthine oxidaseinhibitor allopuninol is administered to rats in conjunction withFANFT, there appears to be enhancement of the carcinogenicpotential of FANFT to induce carcinoma of the bladder (29).ANFT is the product of FANFT deformylation in the kidney andis observed in the urine of animals fed FANFT (30). However,ANFT is not a urinary bladder carcinogen (7). The purpose of

mThis research was supported by the Veterans Administration.

2 To whom requests for reprints should be addressed, at Geriatric Center

(111G JB), VA Medical Center, St. Louis, Mo. 6312g.3 The abbreviations used are: FANFT, N-[4-(5-nitro-2-furyl)-2-thiazolyl)form

amide; ANfl, 2-ammno-4-(5-nitro-2-furyl)thiazole.Received June 11, 1979; accepted October 10, 1979.

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FANFT Metaba!ism by Prastag!andin Endaperoxide Synthetase

Bensadoun and Weinstein (4). In both methods, bovine serumalbumin was used as the standard.

Incubation Conditionsand Analysisof FANFT Metabolism.The rate of oxygenation of FANFT was determined by the rateof decrease in absorbance at 400 nm at 25°.An extinctioncoefficient of 8.1 mM@1cm'1 for FANFT was used. Kineticmeasurements were made using a Beckman Acta VI recordingspectrophotometer, with a scale expander and a temperaturecontrol device. The reaction mixture contained microsomalprotein, 0.1 Mphosphate buffer (pH 7.8), 0.05 mMFANFT, andvarious test substances in a final volume of 0.2 ml. Reactionswere started by the addition of fatty acid. Following addition ofarachidonic acid, the reaction was linear with respect to proteinconcentration and, as shown in Chart 1, with respect to time.Inhibitors were added to both the sample and reference cuvets.

Inhibitors were preincubated with microsomes for 2 mm atroom temperature prior to addition of fatty acid. All inhibitorsand initiators of cooxidation were found to elicit their respectiveeffects in a dose-dependent manner. In most cases, only resultswith maximally effective concentrations of inhibitors are provided for brevity. To investigate the effects of oxygen onarachidonic acid-dependent metabolism of FANFT, a Thunbergcuvet was used to achieve anaerobic conditions. The cuvetcontained the microsomal preparation with FANFT, and theside arm contained the fatty acids. The cuvet was evacuatedwith a water aspirator for 2 to 3 mm to remove air. The enzymereaction was started by mixing together the solutions in theside arm and the cuvet.

Thin-Layer Chromatographlc Analysis. The reaction mixtunewas extracted with an equal volume of methylene chloride.The organic phase was removed following centnifugation,washed with aqueous NaHCO3and water, dried with Na2SO4,evaporated under N2gas, dissolved in dimethylformamide, andsubjected to thin-layer chromatography on 0.1-mm celluloseplates (EM Laboratories, Inc., Elmsford, N. V.). Plates weredeveIope@with an aqueous solvent of 2% formic acid and

visualized under UV light. The RF's for the FANFT and ANFTstandards were 0.20 and 0.35, respectively. These values aresimilar to those previously reported (28). ANFT was synthetizedfrom FANFT using a modification of a previously publishedprocedure (7).

High-Pressure Liquid Chromatographic Analysis. Metabolism of FANFT was analyzed on a 4.6-mm (inside diameter) X25-cm Whatman Partisil-10 ODS (Whatman Inc., Clifton, N. J.).An Instrumentation Specialities Company Model 1440 highpressure liquid chromatograph was used. The column waseluted at room temperature with a 20-mm linear gradient from80% methanol in water to 100% methanol at a flow rate of 0.8mI/mm. Eluent fractions (0.4 ml) were collected and used forUV-visible spectral analysis (Beckman Acta VI). Samples wereprepared as described above.

Results represent the mean ±S.E. of at least 3 separateexperiments. Statistical differences were evaluated by Student's t test for unpaired values.

RESULTS

Metabolism of FANFT was not observed prior to addition ofarachidonic acid (Chart 1). Arachidonic acid-dependent metabolism of FANFT by inner medullary microsomes is illustrated inTable 1. A maximum nateof FANFT metabolism was achievedbetween 0.12 and 0.18 mM arachidonic acid. With 0.1 8 mMarachidonic acid, greater than 30% of the FANFT is metabolized. Metabolism of FANFT was completely inhibited by 0.025mM indomethacin, and there was no measurable metabolism inthe absence of oxygen. Reaction mixtures from samples inTable 1 were subjected to thin-layer chromatographic analysis.In samples containing 0.1 2 mMarachidonic acid, 2 spots wereobserved, one at the origin and the other at an RFof 0.20. Thespot at RF0.20 was identical to the FANFT standard and wasthe only spot observed with reaction mixtures that did notcontain arachidonic acid (no addition). ANfl, the deformylated

O.O4@Abs

ARACHIDONIC400 nmFANFT

Chart 1. Arachidonic acid-dependent oxidation of FANFT(0.05 mM) by rabbit inner medullary microsomes. Oxidationwas measured by a decreased absorbance at 400 nm. Arachidonic acid (0.12 mM)was added as indicated, followed by0.05 m@ethoxyqumn.

2 1 03

JANUARY1980 115

MICROSOME+

ETHOXYGUIN

TIME(Mm)

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Metabolism of FANFT by inner medullarymicrosomesInExperiment 1, dose-dependent arachidonic acid metabolism of 0.05mMFANFT

was demonstrated. Reaction mixtures contained 0.25 mg ofmicrosomalprotein,0.1 M phosphate buffer (pH 7.8), 0.05 m@.iFANFT, and theindicatedconcentration

of fatty acid (N = 3). In Experiment 2, 0.120 m@arachadonicacidwasused as substrate, and the effect of different conditions on arachidonic acid

dependent metabolism was determined (N —3).Activity

(nmol oxiConcentration dized/mgprotein/Conditions

(mM)mm)Experiment

1NoadditionNDa5,8,1

1,14-Elcosatetraenoic acid 0.240 15.3±(arachidonicacid) 0.180 14.9 ±0.50.120

11.8±0.40.0605.7 ±0.20.0302.9 ±0.2Experiment

2Noaddition 11.8 ±0.4+

indomethacin 0.025 NDND

T. V. Zenser et a!.

product of FANFT metabolism, has an RF of 0.35, and acorresponding spot was not observed atthis position. However,previous studies have shown a nonidentified urinary metaboliteof FANFT (Vi ) at the origin, using this same chromatographicsolvent system (13).

Samples were also analyzed by high-pressure liquid chromatography. Shown in Chart 2A is the elution profile of authentic FANFT. A similar elution profile was observed in samplesincubated without arachidonic acid or with both 0.18 mMarachidonic acid and 0.025 m@,iindomethacin. However, in thepresence of 0.18 mM arachidonic acid (Chart 2B), 2 peakswere observed. Peak II corresponds to authentic FANFT, whilePeak I represents the product of FANFT metabolism. Using thissame solvent system, ANFT had an elution time of 4.7 mm.High-pressure liquid chromatographic fractions correspondingto Peak I were collected, and the UV absorbance spectra weredetermined (Chart 3). The UV absorbance spectra for FANFTand the material in Peak I were different. The material in PeakI only exhibited an absorbance peak in the 280 nm region.However, FANFT exhibited absorbance peaks in both the 280nrn and 390 to 400 nm regions.

The fatty acid substrate requirement for FANFT metabolismis illustrated in Table 2, using solubilized inner medullary microsomal preparations. In addition to arachidonic acid,8,1 1,14-eicosatnienoic acid, the substrate for monoenoic prostaglandmns,was also effective in eliciting FANFT metabolism.ProstaglandmnE2,the major product of arachidonic acid metabolism by inner medullany rnicrosomes (32), was not effective.5,8,1 1,14-Eicosatetraynoic acid (the acetylene analog of arachidonic acid), 11,14, 17-eicosatnienoic acid, and 9,12,15-octadecatnienoic acid were not effective in initiating FANFT metabolism. The specific activity of FANFT metabolism with 0.12mM arachidonic acid was higher in the renal solubilized prep

arations (17.5 ±0.4 nmol per mg protein per mm) than in thenonsolubilized microsomal preparations (11.8 ±0.4 nmol permg protein per mm). The metabolism of FANFT using solubilized ram seminal vesicle preparations was 23 ±0.5 nmol permg protein per mm with 0.12 m@arachidonic acid.

Effect of various compounds on arachidonic acid-dependent

E

wUz4

2

B

1.0I I

2.0 3.0@ 4.0 5.0INJECT

RETENTIONTIME (MIN)

@lutionprofile. A, elutlon pro@escontaining the completeails of these determinations

Chart 2. HIgh-pressure liquid chromatographic ifile of authentic FANFT; B, elution profile of sam@reaction mixture plus 0.18 mMarachidonic acid. Deare provided In ‘Materialsand Methods.―Table 1

FANFT metabolism is illustrated in Tawhich inhibited FANFTmetabolism haveto inhibit prostaglandin endoperoxide scompounds include indomethacin, aspi

le 3. All compoundseen shown previouslynthetase (10). Thesein, 5,8,1 1,14-elcosa

tetraynoic acid, ethoxyqumn,meclofenar ic acid, and butylatedhydroxytoluene. Ethoxyquin inhibition o FANFT metabolism isalso illustrated in Chart 1. Allopurmnol, enzoate, and metyrapone did not inhibit FANFT metabolisi I. FANFT metabolismwas not observed when incubated with arachidonic acid andeither heated microsomes (3 mm at 100@)or in the absence ofmicrosomes.

The solubilized prostaglandin endoperoxide synthetase fromram seminal vesicles demonstrated quaOtativelythe same substrate and inhibitor specificity as did the@enzyme isolated fromthe renal inner medulla.

a ND, not detected.

b Mean ±SE.C A Thunberg cuvet was evacuated with a water aspirator for 2 to 3 mm to

remove oxygen.

116

DISCUSSION

The data demonstrate metabolism of EANFT by renal inner

CANCE RESEARCHVOL. 40

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Fatty acid substrate requirements for the cooxygenation of FANFTbysolubilizedinner medullarymicrosomesReaction

mixtures contained 0.5 mg of solubilized microsomal protein, 0.1Mphosphatebuffer (pH 7.8), 0.05 mM FANFT, 0.001 mM hemoglobin, andtheindicated

concentration of fatty acid (N —3 to 6).Activity

(nmol oxiConcentration dized/mgprotein/Additions

(mM)mm)No

addition ND5,8,1 1.14-Elcosatetraenolc acid 0.1 20 17.5 ±O.4@'(arachidonicacid)5,8,

11,14-Eicosatetraynolc acid 0. 120NDProstaglandmnE2 0.120ND8.1

1.14-Elcosatrienoic acid 0.083 10.0 ±0.311,14,17-Elcosatrienoicacid 0.120ND9,1

2,15-Octadecatrienoic acid 0.120NDa

ND, not detected.

b Mean ±SE.Table

3Effectof various compounds on arachidonic acid-dependent FANFToxidationby

solubilized inner medullarymicrosomesAllreaction mixtures contained 0.120 m@.iarachidonic acid, 0.05 mMFANFT,0.1

M phosphate buffer (pH 7.8), 0.5 mg solublllzed microsomal protein,0.001mMhemoglobin, and the indicated concentration of test agents (N —3 to6).Activity

(nmoloxidized!AdditionsConcentration (mM) mgprotein/mm)No

addition 17.5 ±0.4Indomethacin0.01 2 4.8 ±0.30.025NDbAspirin1.00ND5,8,

11,14-Elcosatetraynoic 0. 120NDacidEthoxyquin

0.050NDMeciofenamicacid 0.100NDBenzoate

5.00 17.2 ±0.3Allopurinol0.125 16.9 ±0.5Butylated

hydroxytoluene 0. 100NDMetyrapone5.00 17.8 ±0.5

FANFT Metabo!ism by Prostag!andin Endoperaxide Synthetase

a reaction product. In previous studies examining kidney deformylation of FANFT, the dialyzed 900 x g supernatant fromwhole kidney was used (28). Our studies used an entirelydifferent kidney preparation. Therefore, our studies are notinconsistent with whole kidney deformylation of FANFT. Theproposed mechanism of metabolism of FANFT in inner medulla,cooxidation by prostaglandin endoperoxide synthetase, is consistent with our previous findings with another furan, 1,3-diphenylisobenzofuran (34), and with another known urinarybladder carcinogen, benzidine (35). Other studies have alsoimplicated prostaglandmnsin carcinogenesis. Such studies havedemonstrated increased prostaglandin synthesis with tumorproducing agents (12, 17) and malignant tumors (15, 26) anddecreased prostaglandmn synthesis with agents, such asethoxyqumnand butylated hydroxytoluene, which inhibit thedevelopment of chemically induced tumors (23, 31).

FANFT metabolism by nitroreductase and cooxidation byprostaglandin endoperoxide synthetase are both monitored byfollowing the decrease in absorbance at 400 nm. However, themetabolism of FANFT reported in this paper is distinctly different from that observed with nitroreductase (27). Oxygen is anecessary requirement for both prostaglandin synthesis andcooxidation of FANFT. By contrast, nitroreductase-mediatedmetabolism of FANFT is inhibited by oxygen. In addition, nitroreductase has not been shown to require arachidonic acid for

Table 2

0.5

0.4

0.3

0.2

0.1

Chart 3. UV spectra of authentic FANFTand the eluant containing Peak I fromthe high-pressure liquid chromatographic elution profile described in Chart 2.

medulla. The product of FANFT metabolism has been characterized and shown to be different from FANFT in its thin-layerchromatographic, high-pressure liquid chromatographic andUV spectral properties. FANFT was metabolized by a processof cooxidation by prostaglandmner@Joperoxidesynthetase. Thisconclusion is supported by the substrate specificity of thereaction and inhibitor data. Arachidonic acid and 8,11,14-eicosatrienoic acid, both substrates for prostaglandin endoperoxide synthetase, initiated inner medullary metabolism ofFANFT. Other unsaturated fatty acids which are substrates forlipoxygenase enzymes (11) and susceptible to lipid peroxidation but are not substrates for synthetase (1) did not initiatemetabolism of FANFT. Benzoate, an inhibitor of hepatic microsomal lipid peroxidation (16), had no effect on FANFT metabolism. FANFT metabolism was inhibited by aspirin, meclofenamic acid, and indomethacin, all known inhibitors of prostaglandmnendoperoxide synthetase (10). Conversely, allopurinol,a xanthine oxidase inhibitor (29), and metyrapone, an inhibitorof the microsomal mixed-function oxidase system (14), had noeffect upon FANFT metabolism. In addition, NADPH, a necessary cofacton for mixed-function oxidases, was not required forarachidonic acid-dependent FANFT metabolism. These resultsare consistent with the lack of renal inner medullary cytochrome P-450 content and mixed-function oxidase activity (2,33). Although rodent kidneys have been shown to enzymatically deformylate FANFT to ANfl, ANFT was not identified as

wUz4a,

2

FANFT

WAVELENGTH (nm)

a Mean ± SE.

b ND, not detected.

JANUARY 1980 117

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T. V. Zenser et a!.

activation, nor has it been shown to be inhibited by aspirin andindomethacin.

Prostaglandin endoperoxide synthetase-mediated cooxidation appears due to the peroxidative activity of this enzyme.Purified prostaglandin endoperoxide synthetase has both cyclooxygenase and hydroperoxidase activities (22). The cyclooxygenase activity is responsible for the oxidative conversion of arachidonic acid to prostaglandin G2 (a hydroperoxyprostaglandin endoperoxide), while the hydroperoxidase activity converts prostaglandin G2 to prostaglandin H2 (a prostaglandin endoperoxide). In addition to arachidonic acid, prostaglandin G2 and other organic hydroperoxides but not prostaglandin H2 can elicit cooxidation (19, 20). Therefore, thehydroperoxidase and not the cyclooxygenase activity is necessary for cooxidation. Recently, the cooxidative metabolismof 1,3-diphenylisobenzofuran was reported to occur by a radical chain pathway (19). The similarity in the furan structurebetween 1,3-diphenylisobenzofuran and FANFT suggests acommon mechanism of metabolism of both furans by prostaglandin endoperoxide synthetase.

These data are consistent with the hypothesis that the renalinner medulla is a site for the metabolism of chemicals whichinduce bladder cancer. Prostaglandmnendoperoxide synthetase mediates cooxidative metabolism of the bladder carcinogens FANFT and benzidine (35). The renal inner medulla hasthe highest content of kidney prostaglandmnendoperoxide synthetase (32), with the epithelial cells of the collecting ductcontaining the highest level of synthetase (24). In addition,these epithelial cells are the last cells in contact with urine priorto its entry into the urinary space (5). The renal inner medullais the site of drug and xenobiotic concentration and excretion.Therefore, the unique anatomical, physiological, and biochemical characteristics of the inner medulla, along with the closeproximity of the renal inner medulla to the bladder, make theinner medulla ideally suited for playing a key role in the pathogenic mechanisms involved in the induction of bladder cancer.

M., and Crovetti, A. J. The production of cardnoma of the urinary bladderin rats by feeding N-(4-(5-nitro-2-furyl).2-thiazOlylJformamlde. Cancer Ass.,27:1998—2002,1967.Flower, R. J. Drugs which inhibit prostaglan4in biosynthesis. Pharmacol.Rev., 26: 33-67, 1974.Hamberg, M., and Samuelsson, B. On the spe@lficityof the oxygenation ofunsaturated fatty acids catalyzed by soybeai@lipoxidase. J. Biol. Chem.,242: 5329—5335, 1967.Hassid, A., and Levine, L. Induction of fatty ac@dcyclooxygenase sctlvity Incanine kidney cells (MDCK) by benzo(a)pyrenO.J. Bid. Chem.. 252: 6@91—6593, 1977.Hayashida, S., Wang, C. Y., and Bryan, G. T. fl simple method for detectionand analysis of carcinogenic nitrofuran compo4indsand thelr metabolites bycombining chromatography and spot mutatiot) tests. Gann, 6 7: 617—619.1976.Hildebrandt, A. G. The binding of metyrapone @ocytochrome P-450 and itsinhibitory action on microsomal hepatic mixed function oxidation reactions.Biochem. Soc. Symp., 34: 79-102, 1972.Humes, J. L., and Strausser, H. A. Prostaglan@insand cyclic nucleotides inMaloney sarcoma tumors. Prostaglandins, 5: @83—196, 1974.Lai, C. S., and Platte, L. H. Spin-trapping studies of hydroxyl radicalproduction involved In lipid peroxidatlon. Arøh.Biochem. Biophys., 190:27-38, 1978.Levine,L., and Hassid, A. Effects of phorbol-i 2,13-diesters on prostaglandmnproduction and phospholipase activity in cani@ekidney (MDCK) cells. Blochem. Blophys. Res. Commun., 79: 477-484,@1977.Lowry, 0. H., Rosebrough, N. J., Farr, A. L and Randall, A. J. Proteinmeasurementwith the Folin phenol reagent. J. Blol. Chem., 193: 265—275,1951.Marnett, L. J.. Bienkowskl, M. J., and Pagels,of the prostaglandin synthetase-dependent czofuran. J. Biol. Chem., 254: 5077-5082, 19Marnett, L J., Wlodawer, P., and Samuelsson,substrates by the prostaglandin synthetase ofChem., 250:8510—8517, 1975.Miyamoto, T., Ogino, N., Yamamoto, S., aprostaglandmnendoperoxide synthetase fromsomes. J. Biol. Chem., 251: 2629-2636, 197Ohki, S., Ogino, N., Yamamoto, S.. and Hayperoxidase, an Integral part of prostaglandmnbovine vesicular gland microsomes. J. Blol.Slaga, T. J., and Bracken, W. M. The effectsinitiation and aryl hydrocarbon hydroxylase. Q1977.Smith, W. L., and Wilkin, G. P. Immunochemis$oxide-forming cyclooxygenases: the detectio$rat, rabbit and guinea pig kidneys by immunc@13: 873—892,1977.Swaminathan,S., and Lower, G. N., Jr. Biotrai@nitrofurans. In: G. T. Bryan (ed.), Carcinogene4Vol. 4, pp. 59—97.New York: Raven Press, laTashjian, A. H., Jr., voelkel, E. F., Levine, L.,@that the bone resorption-stimulating factor prcx4cells is prostaglandmnE2.A new model for th@Exp. Med., 136: 1329—1343,1972.Wang, C. Y., Behrens, B. C., Ichikawa, M., a@of 5-nitrofuran derivatives by rat liver xanthmnepamide adenine dinucleotide phosphate-cytocI@Phannacol., 23: 3395-3404, 1974.Wang, C. Y., and Bryan, G. T. Deacylationderivatives by mammaliantissues. Chem.-Biol.@Wang, C. Y., Hayashida, S., Pamukcu, A. M.,)effect of allopurmnolon the induction of blad@nitro-2-furyl)-2-thiazolyl]formamlde. Cancer F4Wang, C. Y., and Lee, L. H. Mutagenic activtt@cinogenic nitrofurans and of urine of rats fed tt@Interact.,15:69—75,1976.Wattenberg, L W. Inhibition of carcinogenic $hydrocarbons by phenolic antioxidants and ettj48:1425—1431,1972.Zenser, T. V., Levitt, M. J., and Davis, B. B. EtPGE and PGF production by rat kidney sIlc@151,1977.Zenser, T. V., Mattammal, M. B., and Davis, B@mixed function oxidase activities in rabbit kid207: 719—725,1978.Zenser, T. V., Mattammal, M. B., and Davis, B.pathways for drug metabolism in rabbit kidn208:418—421, 1979.Zenser, T. V., Mattammal, M. B., and Davis,by renal medullary prostaglandmncyclooxygenin press.

I. A. Oxygen 18 investIgationoxidation of diphenylisoben‘9.B.Co-oxygenation of organicbeep vesicular gland. J. Biol.

Hayaishi, 0. PurIfication oflovIne vesicular gland micro

shi, 0. Prostaglandmnhydro@doperoxidesynthetase from

hem., 254: 829-836, 1979.If antloxidants on skin tumorancer Res., 37: 1631-1635,

lv of prostagiandin endoper@ of the cyclooxygenases in

fluoresence. Prostaglandmns,

sformatlons and excretion ofus,a ComprehensiveSurvey,78.and Goidhaber, P. Evidencelucedby mousefibrosarcomahypercalcemla of cancer. J.

I Bryan, G. T. NitroreductionDxidaseand reduced nicotinrome c reductase. Biochem.

of carcinogenic 5-nitrofuranInteract., 9: 423—428,1974.and Bryan, G. T. Enhancinger cancer in rats by N-(4-(5-es., 36: 1551-1555, 1976.of carcinogenic and noncarass compounds. Chem.-Blol.

rid toxic effects of polycyclic)xyquln. J. NatI.Cancer Inst.,

fect of oxygen and solute onB. Proetaglandmns, 13: 143-

B. Differential distribution of‘y.J. Pharmacol. Exp. Ther.,

B.Demonstration of separateV. J. Pharmacol. Exp. Ther.,

B. Cooxidationof benzldlnese. J. Pharmacol. E.xp.Ther.,

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

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ACKNOWLEDGMENTS

Appreciation Is expressed to Sandy Melliere for secretarial assistance and toHarald Buhle for technical assistance.

REFERENCES

1. Andersen, N. Program notes on structures and nomenclature. Ann. N. Y.Acad. Scm.,180: 14—23,1971.

2. Armbrecht, H. J., Bimbaum, L. S., Zenser, T. V., Mattammal, M. B., andDavis, B. B. Renal cytochrome P450's—electrophoretic and electron paramagnetic studies. Arch. Biochem. Biophys. 197: 277—284,1979.

3. Aufrere, M. B., Hoener, B-A., and Vore, M. Reductive metabolism ofnitrofurantoin In the rat. Drug Metab. Dispos., 6: 403—411, 1978.

4. Bensadoun, A., and Weinstein, D. Assay of proteins in the presence ofinterfering materials. Anal. Biochem., 70: 241-250, 1976.

5. Bonventre. J. V.. Kamovsky, M. J., and Lechene, C. P. Renal papillaryepithelial morphology in antidiuresis and water diuresis. Am. J. Physlol.,235: F69-F76, 1978.

6. Clayson, D. B., and Gamer, R. C. Carcinogenic aromatic amines and relatedcompounds. In: C. E. Searle (ed), Chemical Carcinogens, pp. 366—461.Washington, D. C.: American Chemical Society, 1976.

7. Cohen, S. M., Lower, G. M., Jr., ErtUrk, E., and Bryan, G. T. Comparativecarcmnogenicityin Swiss mice of N-(4-(5-nitro-2-turyl)-2-thiazolyl]acetamideand structurally related 5-nitrofurans and 4-nltrobenzenes. Cancer Res., 33:1593—1597,1973.

8. ErtUrk, E., Atassi, S. A., Yoshida, 0., Cohen, S. M., Price, J. M., and Bryan,G. T. Comparativeurinaryand gallbladdercarcmnogenicityof N.{4-(5-nitro2-furyl)-2-thiazolyl)formamide and N.{4-(5-nifro-2-furyl)-2-thiazolyl)acetamide in the dog. J. NatI. Cancer Inst., 45: 535—542,1970.

9. ErtUrk, E., Price, J. M., Morris, J. E., Cohen, S., Leith, R. S., Von Esch, A.

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