nitroreductases and glutathione transferases that act on 4 … · rat, and mouse liver cytosols and...

ICANCERRESEARCH52, 58-63, January 1, 19921

ABSTRACT

These studies concern the initial steps in 4-nitroquinoline 1-oxide(4NQO) metabolism in relation to mechanisms of anticarcinogenesis.Butylated hydroxyanisole (BHA) administration by a protocol known toinhibit the pulmonarytumorigenicityof 4NQO in A/HeJ miceenhancedhepatic and pulmonary activities for 4NQO metabolism by two majorpathways, conjugative detoxification and nitroreductive activation. Highperformance liquid chromatography analysis showed approximate doubling of two types of glutathione transferase subunits with 4NQOconjugating activity in livers of BHA-treated mice. Similar increases wereobserved in hepatic 4NQO-conjugating activity and in V.,,,,, while K,, for4NQO was 39 to 43 jiM. Pulmonary 4NQO-glutathione transferaseactivity increased 24 to 29%. DT diaphorase activity toward 4NQO waselevated 3.3-fold in livers and 2.7-fold in lungs of BHA-treated mice.However, the predominant4NQO reductase of liver and lung was dicumarol resistant, had a strong preference for NADH, and showed little ifany response to BHA. This M@200,000 enzyme, partially purified fromlivers of Swiss mice, exhibited the stoichiometry of 2-NADH/4NQOexpected for reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide.Its high affinityfor 4NQO (K.., 15pM)signifieda muchgreater influenceon4NQO metabolismthan DT diaphorase (K.,, 208AM).The dicumarolresistant 4NQO reductase differed from several known cytosolic nitroreductases. The results suggest that protection by BHA may result fromalteration of the balance between4NQO activationand conjugation.

INTRODUCTION

The mechanisms of 4NQO4 activation and carcinogenesishave been studied extensively (1â€”4).The initial metabolism of4NQO may proceed by either nitroreduction or displacementof the nitro group by GSH (2â€”9).Nitroreduction produces aproximate carcinogenic metabolite, 4-hydroxyaminoquinoline1-oxide (2, 3, 10). Observations that most of the 4NQO nitroreductase activity of rat liver was localized in the cytosol, thateither NADH or NADPH could serve as the electron donor,and that the activity could be almost completely inhibited bydicumarol led Sugimura et aL (4) and Kato et aL (5) to concludethat DT diaphorase [NAD(P)H:(quinone acceptor) oxidoreductase; EC 1.6.99.2] was responsible for the reduction of 4NQOto its hydroxyamino metabolite. Also, purified DT diaphorasecatalyzed this reduction (4, 11).

4NQO is also susceptible to reaction with cellular thiols andundergoes both enzymic and spontaneous reaction with GSH(6â€”9).We recently developed a spectrophotometric assay forthe reaction of 4NQO with GSH and demonstrated that, atphysiological pH, this reaction was highly dependent uponcatalysis by GSH transferases (EC 2.5. 1.18) (9). Whereas GSH

Received7/26/91; accepted 10/18/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Grant BE-65B from the American Cancer Society.

2 Present address: Department of Biochemistry, University of Tennessee

Memphis, 800 Madison Avenue, Memphis, TN 38163.3 To whom requests for reprints may be addressed, at Department of Biochem

istry and Molecular Biology,Slot 516, U. A. M. S., 4301 W. Markham Street,Little Rock, AR 72205-7199.

4 The abbreviations used are: 4NQO, 4-nitroquinoline I-oxide; GSH, glutathi

one; BHA, 2(3)-tert-butyl-4-hydroxyanisole(butylated hydroxyanisole);HPLC,high-performance liquid chromatography.

transferases catalyze several types of reactions including conjugations, reductions, and isomerizations (12â€”14),the productof the enzyme-catalyzed reaction of 4NQO with GSH has beenshown to be the conjugate, 4-(glutathion-S-yl)-quinoline-1-oxide (15). 4NQO-GSH transferase activity is present in human,rat, and mouse liver cytosols and is widely distributed amongextrahepatic organs of mice (16). This reaction is catalyzedprimarily by isoenzymes of the @iand ir classes (16, 17). Conjugation by GSH transferases provides an alternate metabolicpathway that obviates nitroreductive activation of4NQO (Fig. 1).

In studies on the chemoprevention of carcinogenesis, Wattenberg (18, 19) demonstrated that a multidose schedule of i.p.BHA inhibited the tumorigenicity of 4NQO in female A/HeJmice by 52%, under conditions where 4NQO alone caused anaverage of 7.7 pulmonary adenomas per mouse. Large increasesin DT diaphorase and GSH transferase activities in responseto BHA have been reported for other animal models, differentprotocols of BHA administration, and substrates other than4NQO (20â€”23).The present investigation focused upon theenzymic nitroreduction and GSH conjugation of 4NQO andthe effects of BHA, administered to female A/HeJ mice i.p.according to Wattenberg's (18) protocol, on the enzymes catalyzing these reactions.

MATERIALS AND METhODS

Chemicals and Proteins. 4NQO, allopurinol, and pyrazole were purchased from Aldrich Chemical Company, Milwaukee,WI. BHA, GSH,NADH, NADPH, flavin adenine dinucleotide, xanthine, 2,6-dichloroindophenol, dicumarol, sesame oil, Triton X-lO0, Sephadex G-150,Sepharose CL-6B, apoferritin, @-amylase,alcohol dehydrogenase, andcarbonic anhydrase were from Sigma Chemical Company, St. Louis,MO. Bovineplasma albumin was purchased from Armour Pharmaceutical Company, Tarrytown, NY. The reagent for the dye-binding proteinassay was from Bio-Rad Laboratories, Richmond, CA.

Treatment of Mice and Organs. Female A/HeJ mice, 9 wk old whenobtained from Jackson Laboratories, Bar Harbor, ME, were acclimatized for 18 days prior to treatment. The mice were housed in stainlesssteel cages with hardwood chip bedding, in an environmentally controlled room on a 12-h light/dark cycle. Purina laboratory chow andtap water were available ad libitum. BHA was administered by theprotocol described by Wattenberg (18). Each mouse received0.2 ml of2% (w/v) BHA in sesame oil, i.p., on Days 1, 2, 4, 5, 8, and 9. Controlgroups received only the vehicle. On the ninth day, starting 3 h afterthe last injection, instead of receiving 4NQO as in the Wattenbergprotocol (18), the mice were sacrificed by cervical dislocation and theirorgans were excised. Cold 0.15 M KCI in 2 m@iEDTA (pH 7.0) wasused to perfuse the livers and to rinse the other organs. Mucosa wascollected from the upper and lower halves of the small intestine andfrom the colon by scraping with a stainless steel spatula on a glass plateon ice. All tissues were frozen in liquid nitrogen and stored at â€”70T.A/HeJ mice were used for all studies except the partial purification ofnitroreductases from liver cytosol. Because a substantial number oflivers were required for development of the purification procedure andbecause of the limited availability of A/HeJ mice, these studies utilizedlivers obtained from Swiss mice and purchased from Pel-Freez Biologicals, Rogers, AR. Tissues were homogenized in 0.25 M sucrose at 0C,and cytosol fractions were prepared as described previously (22). Protein concentrations were measured by the method of Bradford (24).

58

Nitroreductases and Glutathione Transferases That Act on 4-Nitroquinoline 1-Oxide

and Their Differential Induction by Butylated Hydroxyanisole in Mice'

J. Steven Stanley,2 J. Lyndal York, and Ann M. Benson3

Department ofBiochemistry and Molecular Biology, University ofArkansasfor Medical Sciences,Little Rock, Arkansas 72205-7199

on July 20, 2021. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

proteins were precipitated by 90% saturation with ammonium sulfate,redissolved in Buffer A, and chromatographed on a 1.5- x 97-cmcolumn of Sephadex G-150. The column was developed with Buffer Aat a flow rate of 1 1 ml/h and 2.9-mi fractions were collected andassayed for 4NQO nitroreductase activity. Mr values were estimated bythe method of Andrews (29) on a 1.0- x 109-cm column of SepharoseCL-6B in 50 mMTris in 100 mrviKC1,containing 0.1 mrvtdithiothreitol(pH 8.25 at O'C).The column, run at 8 ml/h, wascalibrated as describedin Sigma Chemical Co. Technical Bulletin GF-3 with 2-mi samples ofblue dextran, horse spleen apoferritin (Mr 443,000), sweet potato fiamylase (Mr 200,000), yeast alcohol dehydrogenase (Mr 150,000),bovine plasma albumin (Mr 66,000), and bovine erythrocyte carbonicanhydrase (Mr 29,000). The absorbance of the effluent was monitoredat 280 nm by use of a Pharmacia UV-2 monitor and REC-2 recorder.The nitroreductases were detected in fractions by their enzymic activities, and molecular weight was estimated from the ratio of elutionvolume to void volume, by linear regression analysis using a standardcurve.

RESULTS

4NQO-conjugating GSH Transferase Activities of Mouse Organ Cytosols. Elevated GSH transferase activities toward4NQO were observed in cytosol fractions from the livers(+187%; P < 0.001; n = 6) and the lungs (+29%; P < 0.001;n = 6) of mice that had received BHA i.p. by the multidoseprotocol described above in comparison with mice that hadreceived only the vehicle. These initial observations were confirmed in further studies which also examined the effects ofBHA on three digestive tract organs known to be targets for4NQO tumorigenicity in mice. The results are shown in Fig. 2.In both uninduced and BHA-treated mice, the highest 4NQOconjugating activities were observed in the liver, with esophagusand lung exhibiting relatively low activities. BHA treatmentincreased 4NQO-conjugating activity 2.4-fold in liver (P <0.001) and 24% in lung (P < 0.01). A significant increase inGSH transferase activity toward 4NQO was observed in theforestomach (+36%; P < 0.01) in response to BHA, but not inthe esophagus or in the glandular stomach.

HPLC Analysis of Hepatic GSH Transferases. To determinewhich isoenzymes were induced by BHA in the i.p. protocolused in these studies, the GSH transferases were obtained fromliver cytosols of vehicle-treated and BHA-treated mice by affinity purification and subjected to HPLC subunit analysis asdescribed previously (26). The results are shown in Table 1.The subunit HPLC profiles (not shown) from the livers of the

Hxxxx x xxxxxxxxx x xxxxxxxxxxx,â€”@III

I

4NQO-METABOLIZING ENZYMES AND ANTICARCINOGENESIS

GSNO2

@ N'

+ +0 0

4-HydroxyaminoGSH conjugate

quinoline 1-oxide

Fig. 1. Chemical structures of 4NQO, GSH conjugate, and 4-hydroxyarninoquinoline 1-oxide.

Measurement of GSH Transferase Activities and Kinetic Constants.GSH transferase activity toward 4NQO was measured spectrophotometrically at 25T (9). The assay system contained 0.1 M potassiumphosphate (pH 6.5), 0.1 mrvi 4NQO, 1 mM GSH, and enzyme sourcecontaining 1 to 20 @zgof cytosol protein, in a final volume of 1.0 ml.The reaction was initiated by addition of the 4NQO in 10 @ilof ethanol.Initial velocities were determined from the change in absorbance at 350nm (@E = 7.20 mM@'cm@). For the determination ofkinetic constants,portions of pooled liver cytosols from 5 vehicle-treated mice and from5 BHA-treated mice were used as the enzyme source. Kinetic parameters were determined by linear regression of double reciprocal plots ofsubstrate concentrations and initial velocities.

HPLC Analysis of GSH Transferase Subunits. GSH transferaseswere affinity purified from liver cytosol by use of S-hexylglutathioneSepharose (25) and then subjected to subunit analysis by reverse-phaseHPLC as described previously (26). The individual subunits were identified and quantitated by comparison of their retention times and peakareas with those of purified GSH transferases (26).

Measurement of 4NQO Reductase and DT Diaphorase Activities.4NQO nitroreductase activities were measured spectrophotometricallyat 340 nm as the enzymic oxidation of NADH by 4NQO, using amodification of the method of Sugimura et a!. (4). A 10 mM stocksolution of 4NQO was prepared in ethanol. The assay system, at 25'C,contained 50 mM potassium phosphate (pH 6.5), 5 @Mflavin adeninedinucleotide, 0.1 mM 4NQO, 0 or 15 @Mdicumarol, an appropriateamount of enzyme corresponding to 40 to 200 @gof cytosol protein,and 60 @zMNADH which was added to initiate the reaction. NADHoxidation was linear with time and with enzyme concentration underthe conditions used. Rates were corrected for the oxidation of NADHin the absence of4NQO. The dicumarol-sensitive portion ofthe activitywas taken as the DT diaphorase activity. With 2,6-dichloroindophenolas a substrate, DT diaphorase activity was measured by a modification(22) of the method of Ernster (27).

Reduction of 4NQO by Xanthine Oxidase in Mouse Liver Cytosol.The xanthine oxidase-catalyzed reduction of 4NQO was measuredspectrophotometrically under anaerobic conditions at 25'C as the allopurinol-sensitive, 4NQO-dependent oxidation ofxanthine to uric acid(28). The 3-ml assay system contained 50 mrsi potassium phosphate atpH 6.5, 5 @tMflavin adenine dinucleotide, 0.1 mr@t4NQO, 0.1 to 0.3mg of liver cytosol protein, 0 or 50 @iMallopurinol, and 167 j@Mxanthine. The reactions were carried out in Thunberg cuvets. Oxygenwas removed by repeated evacuation and flushing with nitrogen beforeinitiation of the reaction by addition ofthe xanthine from the side arm.The rate of uric acid formation was measured as the rate of increase inabsorbance at 292 nm (EM1 cm 10,500) (28).

Nitroreductase Purification and M, Determination. All steps werecarried out at 0â€”4'C. Mouse livers (16 g) from Pel-Freez were thawed,minced, and homogenized in 48 ml of 0.25 M sucrose. The cytosolfraction (49 ml), obtained as described previously (22), was made 0.1mM in dithiothreitol and passed through a 2.5- x 12-cm column ofcarboxymethylcellulose (Whatman CM-52) and a 2.5- x 12.5-cm col

umn of DEAE-cellulose (Whatman DE-52). The columns were connected in series and operated in 10 mM potassium phosphate (pH 7.16)containing dithiothreitol (0.1 mM) (Buffer A). NADH-dependent4NQO nitroreductase activity was recovered in 90% yield in 64 ml,being unretained by these adsorbents. After addition of 0.63 ml of 100mM dithiothreitol and 6.4 ml of 1 M potassium phosphate, pH 7.0,

4N00

GI. STOMACH Hx x x xH

FORESTOMACH Hxxxxxxxxx}-t

ESOPHAGUS::@t

LUNG@ mm

LIVER

CONTROLEx@ BHA

0 500 1000 1500 2000

SPECIFIC ACTIVITY (nmol/min/mg protein)

Fig. 2. Effects of BHA administration to A/HeJ mice on 4NQO-GSH transferase activities of organ cytosols. Assays were performed as described in â€œMaterials and Methods.â€•Each column represents the mean specific activity for 6organs; bars, SEM; GI. stomach, glandular stomach.

59

HNOH

â€”@

0



Table 1 Inductionofhepatic GSH transftrase subunitsby administrationof BHAi.p. to A/He) mice

The micereceivedmultipledosesof either BHA in sesameoil, i.p.,or only thevehicle. GSH transferases, obtained from pooled liver cytosol by chromatographyon S-hexylglutathione Sepharose were subjected to HPLC subunit analysis.

TreatmentgroupGT-8.7/8.8 iLGT-9.0 TGT-9.3GT-10.3 aGT-10.6aControl5â€¢[email protected]

GSH transferase subunitsare presentedas rng/gof cytosolprotein.

4NQO-METABOLIZINGENZYMES AND ANTICARCINOGENESIS

quantitation, but rose to measurable levels in response to i.p.BHA.

Kinetics of Cytosolic Conjugation of 4NQO. Examination ofsubstrate kinetics for the conjugation of 4NQO with GSH byliver cytosols showed that BHA treatment led to increased Vmaxwith little effect on Km for either 4NQO or GSH (Fig. 3). With1 mM GSH present in the assay system, pooled liver cytosolfrom vehicle-treated mice exhibited an apparent Km of 43 sMfor 4NQO and a Vmaxof 1.7 @tmol/min per mg of protein.Treatment with BHA increased the Vmaxto 3.8 @mol/minpermg, while the Km for 4NQO was 39 sM. At 0.1 mM 4NQO, nochange was observed in the apparent Km for GSH (94 zM), butthe Vmaxof 1.1 @tmol/minper mg of protein for liver cytosolfrom vehicle-treated mice was increased to 2.6 @mol/minpermg of protein by BHA treatment.

The observed increase in Vmaxas the major factor in theenhanced rate of conjugation of 4NQO by liver cytosols fromBHA-treated mice is in quantitative accord with the results ofHPLC subunit analyses. Previous studies on GSH transferaseisoenzymes purified from livers of CD-i mice showed that thehighest specific activities toward 4NQO were exhibited by GSHtransferases GT-8.8 (85 smol/min/mg), GT-8.7 (44 zmol/min/mg), and GT-9.0 (27 @mol/min/mg) (16), and that the Km ofGT-8.8 for 4NQO was 35 @M(9). In A/HeJ mice administeredBHA i.p., the approximate doubling of liver cytosol activity inthe conjugation of 4NQO was accompanied by parallel increases in the concentrations of GT-8.7/8.8 and GT-9.0 subunits in the liver cytosol as determined directly by HPLC.

Dicumarol-sensitive 4NQO Reductase Activity. The dicumarol-sensitive activity was measured because Kato et a!. (5)had shown that the nitroreduction of 4NQO by rat liver cytosolwas almost completely inhibited by 10 @sMdicumarol, a potentinhibitor of DT diaphorase (27). In nine organs from mice thathad received only the sesame oil vehicle, the rate of dicumarolsensitive, NADH-dependent reduction of 4NQO ranged from3 to 157 nmol/min per mg of cytosolic protein. The specific

activities (nmol/min per mg of protein; n = 6) were 22 Â±4 forthe kidney, 46 Â±8 for the upper small intestine, 25 Â±5 for thelower small intestine, and 35 Â±4 for the colon. Fig. 4 showsdicumarol-sensitive 4NQO reductase activities for liver and fourtarget tissues for 4NQO-induced tumor formation in mice, andthe effects of BHA administered i.p. according to the protocol

6

4

2

0 50 100 150 200

1/ [4NQO] (mM@')

6

4,

2

.@

4J014

II

.@04.)0I40.

EII

â€”10 0 10 20 30 40

l/E GSH] (@_1)Fig. 3. Effects of BHA treatment of mice on the kinetics of cytosolic conjuga

tion of 4NQO with GSH. The data are presented as Lineweaver-Burk analysesof the kinetics of conjugation of 4NQO with GSH as catalyzed by pooled livercytosols from BHA-treated (â€¢)or vehicle-treated (0) mice. Each point representsthe average of at least two determinations.

vehicle-treated A/HCJ mice were qualitatively indistinguishableand quantitatively similar to those obtained previously forhepatic GSH transferases of female CD-i mice (26). The relationships between isoenzyme GT-8.7 and a less abundant form,GT-8.8, are unclear, and the HPLC analysis does not distinguish between the subunits of these very similar transferases,which are therefore designated GT-8.7/8.8 (26). The class @tisoenzymes, GT-8.7 and GT-8.8, and the class ir isoenzyme,GT-9.0, most actively catalyze the conjugation of 4NQO (16).The cytosolic concentrations of GT-8.7/8.8 and GT-9.0 subunits were induced about 2-fold over control levels, while theconcentration of GT-10.6 subunits was increased to a muchlesser extent by BHA. GT-9.3 and GT-i0.3 subunits werepresent in livers of vehicle-treated mice at levels insufficient for

GI. STOMACH

ESOPHAGUS@@@@

c@ CONTROLLUNG@ ExJ BHA

UVER

I I I I

0 50 100 150 200

SPECIFIC ACTIVITY (nmol/min/mg protein)

Fig. 4. Effects of BHA administration to A/HCJ mice on the reduction of4NQO by DT diaphorase. Activities were measured as the cytosolic oxidation ofNADH by 4NQO in the presence of 30 MMdicurnarol and in the absence ofdicumarol. The dicumarol-sensitive portion ofthe activity was taken as the activityof DT diaphorase. Each column represents the mean specific activity for 6 organs;bars, SEM; Gl. stomach,glandular stomach.

60



Table 2 Electron donor and inhibitor specificities of4NQO reductionbydicumarol-resistantreductase(s)and xanthine oxidaseofmouse livercytosol4NQO

reductaseactivitywas measuredin livercytosol fractions fromA/HeJmicethat had received either BHA or only the sesame oilvehicle..

4NQO reductase activity

DicumarolElectron donor (I 5 hiM) Additive Control BHAtreatedNADPH(60@zM)

+ None 0.6Â±0.la1.0Â±0.1NADH(60 MM) + None 19.3 Â±0.4 22.2 Â±0.8NADH(60 @zM) + Allopurinol (50 18.9 Â±0.7 21.4 Â±0.6

@iM)bNADH(6OMM)+ TritonX-l00 18.8Â± 1.021.6Â±0.8(0.0028%)NADH

(60 @zM) + Pyrazole (1 mM) 18.4 Â±0.8 21.4 Â±0.6Xanthine(167 @M)C None 1.7 Â±0.1 2.7 Â±0.5Xanthine(167 @M)c Allopurinol (50 0.270.55@zM)

Table 3 Distinguishing characteristics ofthe two predominant 4NQOnitroreductases,NR-1 and NR-2, partially purifledfrom mouselivercytosolCharacteristic

NR-lNR-2Molecular

weight (M,) 200,000 43,000Specificactivitya

NADH (60 MM) 38 23NADPH (60 MM) I 23NADH (60 @M)and dicurnarol (30 @M) 45 2

Km (hiM)for 4NQOâ€• 15208a

Nitroreductase-specific activities are expressed as nrnol of NADH orNADPH oxidized/mm per rng of protein, with 4NQO as the electron acceptor.

b The Km for 4NQO was determined with NADH (60 MM) as the electron

donor and variable 4NQO concentration in the range of 0 to 30 tiM.


groups, specifically xanthine oxidase (EC 1.2.3.2), aldehydeoxidase (EC 1.2.3.1.), and alcohol dehydrogenase (EC i.i.1.l.)(30). The results are shown in Table 2. Aldehyde oxidase, whichis not known to utilize pyridine nucleotides, and xanthineoxidase are greatly inhibited by Triton X-iOO and allopurinol,respectively (31â€”33).The lack of any substantial inhibition byTriton X-100 or allopurinol suggests that neither of theseenzymes was responsible for the NADH-dependent, dicumarolresistant, 4NQO reductase activity. This activity was also resistant to inhibition by pyrazole, an alcohol dehydrogenaseinhibitor with a I(@of 0.2 sM (34). Interaction of pyrazole withalcohol dehydrogenase requires NAD@ (34, 35), present at verylow concentrations during assay. However, other criteria (below) distinguished the dicumarol-resistant 4NQO reductasefrom alcohol dehydrogenase.

The extent to which xanthine oxidase in liver cytosol wouldcatalyze 4NQO reduction anaerobically with xanthine as theelectron donor was also examined. As shown in Table 2, reduction of 4NQO was observed under these conditions. Its susceptibility to inhibition by allopurinol confirmed the role of xanthine oxidase. The rate of4NQO reduction by xanthine oxidasewas only 7% to 8% as great as the NADH-dependent 4NQOreductase activity.

Distinguishing Characteristics ofthe Two Predominant 4NQOReductases of Mouse Liver Cytosol. The studies on A/HeJ micesuggested that NADH-dependent 4NQO reduction by mouseorgan cytosols must be catalyzed by at least two distinct enzymes. Swiss mouse livers obtained from Pel-Freez were usedfor the further characterization ofthe murine 4NQO reductases.The enzymes were partially purified and then chromatographedon a Sephadex G-150 column, as described in â€œMaterialsandMethods.â€• The cytosolic NADH-dependent 4NQO nitroreductase activity was recovered in 66% yield and was resolvedinto two distinct components, designated NR-i and NR-2, thatdiffered in several characteristics (Table 3). The molecularweights (Mr) of 200,000 for NR-1 and 43,000 for NR-2 wereobtained by chromatography of these enzymes on a calibratedcolumn of Sepharose CL-6B as described in â€œMaterialsandMethods.â€• Thus, NR-1 was further distinguished from liveralcohol dehydrogenase, a dimer having a molecular weight ofapproximately 80,000 (35). Examination of the kinetics of4NQO reduction by NR-i and NR-2 revealed a marked difference in Km,with much greater efficiency of NR-i at low 4NQOconcentrations (Fig. 5). In catalysis of 4NQO reduction, thedual pyridine nucleotide specificity and dicumarol sensitivity ofNR-2 confirmed its identity with DT diaphorase, whereas NRi exhibited a strong preference for NADH and was completelyresistant to inhibition by dicumarol.

Stoichiometry of NADH Utilization in 4NQO Reduction byNR-l. The amount of NADH oxidation accompanying theenzymic reduction of 4NQO by NR-1 was measured in a l-ml

a Mean Â±SEM of the specific activity, nrnol/min/mg of cytosolic protein (n= 5 mice). All values have been corrected for reduction of electron donors in the

absenceof 4NQO.b Additives were dissolved in water. Assay systems contained, per ml, 25 @lof

2 mr@iallopurinol, 20 @lof0.l4% (v/v) Triton X-l00, or 10 @lof100 [email protected] With xanthine as the electron donor, assays were performed under anaerobic

conditions.

described by Wattenberg (18). BHA administration increasedDT diaphorase activity toward 4NQO 3.3-fold in liver (P <0.001) and 2.7-fold in lung (P < 0.01) while not altering thisactivity significantly in the esophagus, forestomach, or glandular stomach.

The induction of hepatic and pulmonary DT diaphorase bythis protocol of BHA administration was confirmed in a separate experiment with 2,6-dichloroindophenol as the substrate.Liver and lung cytosols from six vehicle-treated mice hadactivities of 126 Â±5 nmol/min and 77 Â±6 nmol/min per mgof protein, respectively, in the dicumarol-sensitive, NADHdependent reduction of 2,6-dichloroindophenol. These activities increased 2.5-fold in liver (P < 0.001; n = 6) and 2.2-foldin lung (P < 0.001; n = 6) in response to i.p. BHA.

Dicumarol-resistant 4NQO Reductase Activity. In contrast torat liver cytosol (5), mouse liver cytosol was found to exhibit4NQO reductase activity that was resistant to inhibition bydicumarol and highly selective for NADH rather than NADPHas the electron donor (Table 2). In liver cytosols from femaleA/HeJ mice, 84% of the total activity was dicumarol resistant.Treatment of the mice with BHA yielded only a 15% increase(n = 5; P < 0.02) in the dicumarol-resistant activity. Thus,despite the 3-fold induction of hepatic DT diaphorase activitytoward 4NQO, the total NADH-dependent 4NQO reductasespecific activity ofliver cytosol was increased to only 1.45 timescontrol levels by BHA. Dicumarol-resistant 4NQO reductaseactivities of extrahepatic organ cytosols were measured withNADH and are uncorrected for its oxidation in the absence of4NQO. Glandular stomach, forestomach, and esophagus cytosols exhibited dicumarol-resistant 4NQO reductase activities of.27.8 Â±1.8, 27.8 Â±1.5, and 25.9 Â±2.9 nmol/min per mg ofprotein, respectively (n = 6). Whereas DT diaphorase activity(Fig. 4) predominated in these digestive tract organs, lungcytosol resembled liver cytosol in having relatively low 4NQOreductase activity (13.5 Â±0.6; n = 5), of which 77% wasdicumarol resistant. None of the extrahepatic organs examinedshowed any significant change in dicumarol-resistant 4NQOreductase activity in response to BHA.

Xanthine Oxidase and Other Nitro- and/or Nitrosoreductasesin 4NQO Reduction. Experiments were undertaken to determine whether the dicumarol-resistant 4NQO reductase activitymight be due to the action of cytosolic enzymes known tocatalyze the reduction of xenobiotic nitro and/or nitroso

61




assay system under standard conditions. Each cuvet contained930@ of buffer and 20, 40, or 50 nmol of 4NQO added in 5 @lof ethanol. The enzyme source was 50@ of the most activefraction of an NR-i peak from a Sephadex G-150 column.Reaction was initiated by addition of 150 nmol of NADH, andthe change in absorbance at 340 nm was monitored. Correctionswere made for any change in absorbance in the absence of4NQO. As shown in Fig. 6, 2.0 to 2.1 nmol of NADH wereoxidized per nmol of 4NQO present. This stoichiometry isconsistent with the reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide, without significant reoxidation of this product or of the intermediate, 4-nitrosoquinoline 1-oxide.

DISCUSSION

These studies are aimed at elucidating the enzymology of4NQO metabolism in relation to the mechanisms by whichBHA protects against this carcinogen. The present phase ofthis investigation concerns the balance between the two opposing pathways in the initial metabolism of 4NQO, catalyzed bycytosolic enzymes that promote either the detoxification of4NQO by its conversion to a GSH conjugate (16), or alternatively, the nitroreductive activation of4NQO to form 4-hydroxyaminoquinoline 1-oxide, a proximate carcinogenic metabolite(2, 3). The dose, route, schedule, and vehicle for administrationof BHA and the use of female A/HeJ mice as the animal modelwere selected to match conditions under which BHA has beendemonstrated to confer substantial protection against the pulmonary tumorigenicity of 4NQO (i 8, i 9). This regimen ofBHA administration resulted in the induction of hepatic andpulmonary enzymes in both the detoxification pathway and theactivation pathway for 4NQO.

Studies on rat liver have shown 4NQO nitroreduction to becatalyzed primarily by DT diaphorase, a reductase characterizedby its sensitivity to inhibition by dicumarol (4, 5, 27). Dicumarol-sensitive 4NQO reductase activity increased 3.3-fold inmouse liver and 2.7-fold in lung in response to BHA. However,a dicumarol-resistant enzyme which differed from DT diaphor

120

100

80

40

20

0 20 40 60Minutes

000EC

0I0

z

Fig. 6. Stoichiornetry between NADH oxidation and 4NQO reduction bymurine nitroreductase NR-l. The oxidation of NADH accompanying the reduction of 4NQO by NR-l was monitored at 340 nm. As described in â€œMaterialsand Methods,â€•the 1-mI incubation mixtures contained buffer, enzyme, 150 nmolof NADH, and either 20 nmol (U), 40 nmol (0), or 50 nmol (â€¢)of 4NQO.

ase in catalytic and molecular properties was found to beresponsible for most of the hepatic and pulmonary 4NQOreductase activity. Since DT diaphorase exhibited a relativelylow affinity for 4NQO, having a Km of 208 @sM,catalysis by thedicumarol-resistant 4NQO reductase, with a Km of 15 @iMfor4NQO, would have much greater quantitative significance atcellular 4NQO concentrations achievable in vivo. These resultsimply that the observed induction of DT diaphorase in mouseliver and lung may have little impact upon 4NQO metabolismor carcinogenicity due to the functional predominance of thedicumarol-resistant enzyme. The induction of 4NQO-conjugating GSH transferases by BHA without a corresponding increasein the dicumarol-resistant 4NQO reductase activity would appear to constitute a mechanism by which BHA protects againstthe tumorigenicity of 4NQO.

The results of this study have further elucidated the enzymology of the initial steps in 4NQO metabolism by confirmingthe role of DT diaphorase and revealing the presence of anotherpyridine nucleotide-dependent nitroreductase as well as a minorcontribution of xanthine oxidase. Further characterization ofthe enzymes catalyzing these reactions is in progress. Theresults indicate that protection by BHA against the carcinogenicity of 4NQO may be explained, at least in part, by alterationof the balance between activation and detoxication of 4NQO asa result of BHA administration. Furthermore, increased hepaticmetabolism of 4NQO may result in decreased bioavailability of4NQO in the lung. Whether BHA may also influence furthersteps in the 4NQO metabolic pathway is under investigation.

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1992;52:58-63. Cancer Res J. Steven Stanley, J. Lyndal York and Ann M. Benson Butylated Hydroxyanisole in Mice4-Nitroquinoline 1-Oxide and Their Differential Induction by Nitroreductases and Glutathione Transferases That Act on

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