[CANCER RESEARCH 50, 4300-4307, July 15, 1990]

Metabolic Activation of the Potent Mutagen, 2-Naphthohydroxamic Acid, inSalmonella typhimurium TA981

Mei-Sie Lee2 and Masakazu Isobe3

Department of Chemical Carcinogenesis, Michigan Cancer Foundation, Detroit, Michigan 48201

ABSTRACT

The objective of the present study was to explore the mechanismsresponsible for the strong, direct-acting mutagenicity of 2-naphthohy-droxamic acid (NHA) for Salmonella typhimurium TA98. NHA wasconverted to its O-acetate (O-Ac-NHA) by acetyl-CoA, in the presenceof competent or heat-treated cell-free bacterial preparations. O-Ac-NHA,which is more mutagenic than NHA, reacted nonenzymatically withtRNA in neutral solutions with retention of both the naphthyl andcarbonyl groups in the products, but NHA did not react. Enzymaticsulfate conjugation was not demonstrated. TA98 cells converted NHA to2-aminonaphthalene, presumably through a Lossen rearrangement following 0-acetylation or conjugation by other metabolic pathways. TA98cells reduced O-Ac-NHA to 2-naphthamide, and NADH and NADPHwere shown to be cofactors for reduction in the presence of a cell-freebacterial preparation. Although horseradish peroxidase and Ilo, catalyzed the binding of these compounds to tRNA, no evidence of oxidationof NHA or O-Ac-NHA was obtained with H2O2 and cell-free preparations of TA98 or the cells themselves, as judged by the lack of formationof the peroxidative product, 2-naphthoic acid. Both NHA and O-Ac-NHA reacted with DNA of TA98 with retention of both naphthyl groupand carbonyl of the naphthoyl moiety in the adduct(s). These resultssuggest that NHA may be activated in TA98 by esterification, and theresulting metabolites may amidate or carbamoylate nucleic acids.

INTRODUCTION

Hydroxamic acids (i.e., ArCONHOH, RCONHOH) are agroup of widely distributed chemicals. Although some occurnaturally in fungi, yeasts, bacteria, and plants (1-4), others have

been manufactured for pharmaceutical, consumer, agricultural,and industrial concerns for possible use as antitumor agents,antibiotics, growth factors, and iron-chelating agents (reviewedin Ref. 5). Recently, it has been found that some hydroxamicacids are inhibitors of 5-lipoxygenase (6-8), and they can inhibitthe biosynthesis of leukotrienes which are important mediatorsin a variety of diseases such as asthma, arthritis, and psoriasis.Thus, new and potentially more effective treatments for theseconditions may be developed from these hydroxamic acids.

Certain hydroxamic acids are mutagenic to bacteria (3, 9-13), and acetohydroxamic acid (14) and NHA4-5 are also mu-

Received 7/28/89; revised 4/3/90.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.

' This work from the A. Alfred Taubman Facility was supported by NIH GrantCA-37885 and an institutional grant from the United Foundation of Detroit.

2To whom requests for reprints should be addressed.3 Present address: Faculty of Pharmaceutical Sciences, Setsunan University,

45-1 Nagaotage-cho. Hirakata, Osaka, Japan.' The abbreviations used are: NHA, 2-naphthohydroxamic acid; [3H)NHA, 2-

(rm#-3H]naphthohydroxamic acid; |'4c]NHA, 2-[carfton>7-''tC]naphthohydrox-amic acid; [3H, "CjNHA, 1-[ring-sH, cai-fton>7-l4C]naphthohydroxamic acid; O-Ac-NHA, O-acetate of NHA; O-Ac-|3H, MC]NHA, the O-acetate of [3H, 14C]-NHA; O-Ac-[3H|NHA, O-acetate of [3H|NHA; O-Ac-[14C]NHA, the O-acetate of['"CJNHA; O-SOjH-NHA-Py, pyridinium salt of O-sulfonate of NHA; AN, 2-aminonaphthalene; NpCO2H, 2-naphthoic acid; NpCONH¡, 2-naphthamide: O-CONp-NHA, O-2-naphlhoylate of NHA; di-Np urea, l,3-di-2-naphthyl urea;NpNCO. 2-naphthyl isocyanate; HRP, horseradish peroxidase; PAPS, 3'-phos-phoadenosine-5'-phosphosulfate; HPLC, high-performance liquid chromatogra-phy; R,, retention time; TLC. thin-layer chromatography; DMSO. dimethylsulfoxide. PCP, pentachlorophenol: RC'ONO, nitrosocarbonyl-alkanes or nitro-socarbonyl-arenes; NpCONO, nitrosocarbonyl-2-naphthaline.

5C. Y. Wang, personal communication.

tagenic to V79 Chinese hamster cells. Some are clastogenic andinduce sister chromatid exchange in cultured mammalian cells(5). However, in the only chronic toxicity study of which weare aware, NHA did not significantly increase the incidence oftumors in male or female Sprague-Dawley rats in a 75-wkobservation period following 8 weekly injections of the maximum tolerated dose started at birth.6 It is generally believed

that the covalent binding of a chemical with DNA is an earlystep in most mutagenic and carcinogenic processes (15, 16).However, very little is known about the chemical reactivity ofthe unsubstituted hydroxamic acids with macromolecules andtheir underlying mechanisms of activation.

It has been shown that NHA was highly mutagenic forSalmonella typhimurium TA98 (9, 13), its O-acetate ester beingmuch more mutagenic than the parent compound (11, 13).Additionally, it was observed by Roga et al. (17) that O-acylderivatives of the structurally analogous A'-hydroxyurethanewere more mutagenic than the parent compound. Therefore, itis possible that O-acetylation, similar to the acetyl-CoA-de-pendent activation of arylhydroxylamines in TA98 (18, 19), isone of the major pathways of activation of the unsubstitutedhydroxamic acids in these cells. It was shown in our previousstudy (11) and those of others (3, 12, 13) that the presence ofthe N-H group of hydroxylamine moiety was essential formutagenic activity. This observation led to the proposal (3, 12,13) that a Lossen rearrangement (Ref. 20; Fig. 1), which transforms hydroxamic acids to reactive isocyanates, may be involved in the activation of hydroxamic acids and results in thecarbamoylation of DNA. Our objective in this study was toexplore the mechanisms by which NHA is activated in bacteria.

MATERIALS AND METHODS

Materials. The following were purchased from the sources indicated:2-[r//i£-3H]naphthoic acid (Amersham Corporation, Inc., ArlingtonHeights, IL); 2-naphth[""C]oic acid (Chemsyn Science Laboratories,Lenexa, KS); 2-naphthoic acid, 2-naphthoyl chloride, AN, and benzal-dehyde (Aldrich Chemical Co., Inc., Milwaukee, Wl); NpNCO (AdamsChemical Company, Round Lake, IL); Oxoid No. 2 nutrient broth(Oxoid USA, Inc., Columbia, MO); lysozyme, proteinase K, acetyl-CoA, HRP, 30% H2O2, NADH, NADPH, and PAPS (Sigma ChemicalCo., St. Louis, MO); yeast tRNA (Calbiochem, La Jolla, CA); andRNase (Pharmacia Fine Chemicals, Piscataway, NJ). Other chemicalswere of reagent grade.

S. typhimurium TA98 and TA98/1,8-DNP6 were obtained from Dr.B. N. Ames, University of California, Berkeley, CA, and Dr. E. C.McCoy, Case Western Reserve University, Cleveland, OH, respectively.

Instrumentation and General Procedures. Reverse-phase HPLC wascarried out using a Waters Associates liquid Chromatograph systemequipped with a Model 440 absorbance detector and an automatedgradient controller (Waters Associates, Inc., Milford, MA) interfacedwith a radioactive flow detector (Flow-One HP; Radiomatic Instruments & Chemical Co., Inc., Tampa, FL) and an LKB 2140 rapidspectral detector (LKB-Produkter AB, Bromma, Sweden) by an IBMcomputer using a data acquisition system (Series 760 interface; NelsonAnalytical, Inc., Cupertino, CA). The following systems were used.System I, a PRP-1 column (10 ¿im,150 x 4.1 mm; Phenomenex,

*Unpublished observations.

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METABOLIC ACTIVATION OF 2-NAPHTHOHYDROXAMIC ACID

OAc XN/OS03H-Py

O-Ac-NHA

\ /O-SO3H-NHA-Py

AN UreaFig. 1. Lossen rearrangement of O-acetate and O-sulfonate of NHA. R denotes

a 2-naphthyl or phenyl group.

Torrance, CA), eluted with a linear gradient of methanol in 10 miviTris-HCl (pH 7.4) at 1 ml/min from 50 to 100% methanol in 20 minand then at 100% methanol for 10 min. System II, a C18-MBondapakcolumn (10 ^m, 300 x 3.9 mm; Phenomenex, Torrance, CA), eluted at1 ml/min linearly from 10 mM H3PO4 to methanol in 30 min and then100% methanol for 5 min. System III, the same C,8 column as above,eluted at 1 ml/min linearly from 40 to 80% methanol in 10 mM H.,PO4in 14 min, 80 to 100% methanol in 2 min, and then 100% methanolfor 5 min. The R, values of the compounds are listed in Table 1. TLCwas carried out on silica gel plates with the following solvent systems:Solvent A, benzene:chloroform:methanol (9:1:1); Solvent B, ben-zene:acetone (2:1); and Solvent C, benzene:methanol (9:1). Rf valuesare listed in Table 1. Radioactivity was determined by liquid scintillationspectroscopy. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. Unless otherwise indicated, 0.1 Msodiumphosphate buffer (pH 7.0) was used.

Chemical Synthesis. NHA, O-Ac-NHA, O-SO3H-NHA Py, and di-Np urea were synthesized according to published procedures (11). Forthe preparation of l-(2-naphthyl)-3-phenyl urea, a solution of 40 mg ofNpNCO in 0.5 ml of DMSO was reacted with 0.1 ml of aniline at roomtemperature for 24 h. Benzene and water were added, and the organicphase was washed with 1 ml of l N HC1 and then water (3x) to givecrystals, m.p. 233-234°C (from methanol) [lit. m.p. 233-234°C (21)].

NpCONH2 was prepared by reaction of 2-naphthoyl chloride with cone.NH4OH at room temperature, m.p. 194-195°C[lit. m.p. 195°C(22)].

O-CONp-NHA was prepared by reacting 3.81 g (20 mmol) of 2-naphthoyl chloride with 3.55 g (19 mmol) of NHA in 20 ml oftetrahydrofuran at 70°Cfor 1 h. The product was precipitated by

addition of ether to the cooled reaction mixture, and recrystallizationsfrom acetone-ether gave an analytical sample, m.p. 171-173°C.

C22H15N03

Calculated: C 77.42, H 4.40, N 4.11Found: C 77.52, H 4.51, N 4.13

IR (KBr): >C=O at 1752 and 1621 cm-'. UV (ethanol): Xmax242.5 nm

(( = 124,940), 281.5 nm (<= 21,480), sh, 292 nm (<= 15,280), sh, 269nm (i = 18,050). Mass spectrum (m/e): 170 (14.6%), 169 (100%), 155(26.0%), 141 (28.4%), 127 (32.4%). 'H-NMR (300 MHz, CDC13), a:10.03 (s, N-H), 7.34 to 8.87 (m, 14 aromatic protons).

Radiochemical Synthesis. [3H]NHA was prepared by reaction of 2-[/•m£-3H]naphthoylchloride (prepared by treating a benzene solution of

the acid with 2.4 equivalents of SOC12 in the presence of a catalyticamount of dimethylformamide at 40°Cfor 2 h) and 10 equivalents of

NH2OH (prepared by treating NH2OH-HC1 with an equimolar quantity of KOH in methanol, filtering off the solid KC1 and evaporatingmethanol) in benzene at room temperature for 20 min. Evaporation ofsolvent gave a residue which was triturated with H2O, acidified to pH2 with HC1, and recrystallized from acetone-ether to give [3H]NHA (70

to 75% yield) with a radiochemical purity greater than 98% based onTLC using Solvent C and a specific activity of 443 to 760 mCi/mmol.

[MC[NHA was similarly prepared from 2-naphth[14C]oic acid in 60 to

65% yield with a radiochemical purity greater than 98% and a specificactivity of 43 to 53 mCi/mmol.

O-Ac-pHjNHA was prepared by treating an ether solution of ['H[-

NHA with 1.2 equivalents of acetic anhydride in the presence of acatalytic amount of triethylamine at room temperature for 2 h. Thesolvent was evaporated, and the product was isolated by triturationwith acetone-ether-hexanes (80 to 85% yield) which had a radiochemical purity greater than 98% based on TLC using Solvent C. O-Ac-[14C]NHA was similarly prepared in 65 to 70% yield from [I4C]NHA

and had a radiochemical purity greater than 96%.Decomposition of Esters of NHA. The potential for undergoing

Lossen rearrangement was examined by incubating O-Ac-NHA at 37°C

in a neutral aqueous solution. A mixture of 0.6 ml of pH 7.0 buffercontaining 10 nmol of O-Ac-[3H]NHA (760 mCi/mmol)/10 n\ ofDMSO was incubated at 37°Cand at 0, 2, 4, and 24 h, 50 n\ of the

reaction mixture were mixed with 0.1 ml of methanol and centrifuged,and the supernatant was analyzed by HPLC using System II. The half-life of this compound was also determined from its content at eachtime point.

In order to detect the production of NpNCO from the Lossenrearrangement of O-Ac-NHA, aniline was used to trap it. A solution of1 ml of aniline in 50 ml of pH 7.0 buffer was adjusted to pH 7.0 with1 M H2SO4 and then reacted with 20 mg of O-Ac-NHA/1 ml of DMSOat room temperature for 18 h. It was extracted with 50 ml of ether, andthe extract was washed with an equal volume of l N HC1 and then l NNaOH. Evaporation of solvent and crystallization from methanol gavesome solid which was analyzed by HPLC using System I and TLCusing Solvent C for the detection of ureas. See Table 1 for the R, andRf of l-(2-naphthyl)-3-phenyl urea and di-Np urea.

The potential for O-SOjH-NHA •Py to undergo Lossen rearrangement in pH 7.0 buffer at room temperature was established inour previous study (11). In this study, the half-life of this compound atroom temperature was determined by measuring the decrease with timeof the absorbance at 220 nm of a mixture of 1 ng/10 p\ of ethanol in 1ml of pH 7.0 buffer.

Oxidation of NHA and O-Ac-NHA with H2O2/HRP. Reaction mixtures consisted of 6 ^mol of compound in 150 p\ of DMSO in a totalvolume of 6 ml containing pH 7.0 buffer, 60 pmol of H2O2, and 30units of HRP. Control incubations were carried out in the absence ofHRP or H2O2/HRP. They were incubated at 37°C,and after 10 min, l

h, and 24 h, 2-ml aliquots were extracted with ethyl acetate, and theorganic phase was dried and redissolved in methanol for analysis ofproducts by HPLC using System HI.

Metabolism of NHA and O-Ac-NHA by Cell-free Preparations of 5.typhimurium TA98. Cells from 7 ml of an overnight culture were washedtwice with 7 ml of 0.1 M phosphate/0.9% NaCl buffer, pH 7.0, and

Table 1 HPLC R, and TLC Rf values of compoundsR,(min)"Systems'CompoundNHAO-Ac-NHAO-SOjH-NHA

PyNpCONH2ANNpCO;HO-CONp-NHAdi-Np

ureal-(2-naphthyI)-3-phenylureaI10.814.914.821.36.427.223.2II21.023.622.316.326.131.733.71116.89.28.25.011.815.917.2p

*•VSolventsA0.100.360.050.230.490.400.610.440.43B0.150.540.020.210.490.540.670.610.60aC0.160.390.130.410.510.500.610.450.51

" R,s of the radioactive counterparts were 0.6 min longer than those listed inthe table which were detected by a 254-nm detector.

b The compounds were detected by viewing the TLC plates under 254-nm or366-nm UV light.

' For System I; a PRP-1 column was used and eluted with a linear gradient ofmethanol in 10 HIMTris-HCl (pH 7.4) at 1 ml/min from 50 to 100% methanolin 20 min and at 100% methanol for 10 min; for System II, a C18 column wasused and eluted at 1 ml/min linearly from 10 mM H,PO< to methanol in 30 minand then at methanol for 5 min; for System 111.a C¡8column was used and elutedat 1 ml/min linearly from 40 to 80% methanol in 10 mM H3PO4 in 14 min. 80to 100% methanol in 2 min, and at 100% methanol for 5 min.

d Solvent A, benzene:chloroform:methanol (9:1:1); Solvent B. benzene:acetone

(2:1); Solvent C, benzene:methanol (9:1).

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METABOLIC ACTIVATION OF 2-NAPHTHOHVDROXAMIC ACID

then resuspended in 7 mg of the same buffer. The suspension wasincubated with 1 mg of lysozyme at 37°Cfor 1 h, followed by freezing

and thawing with liquid nitrogen (2x) and then centrifuging at 2000rpm. The supernatant which contained 0.54 mg of protein/ml ofpreparation as determined by the published procedure (23) was storedat -70°Cuntil use.

Incubation mixtures consisted of 135 pg of protein, 2.5 ^mol ofcofactor in 0.5 ml of pH 7.0 buffer, and 10 nmol of [3H]NHA (364mCi/mmol) or O-Ac-[3H]NHA (760 mCi/mmol) in 10 Mlof DMSO.

Control experiments were carried out in the absence of cofactors or inthe presence of heat-treated (100°C,10 min) cell-free preparations.They were incubated at 37°Cfor l h and then mixed with 1 ml of

methanol, and the extracts were analyzed by HPLC using System III.The cofactors used and the atmospheres under which the experimentswere carried out are indicated in Table 2. In the case of PAPS, thebuffer used was 0.1 M bicine, pH 8.0, containing 1.5 ^mol of Mg(OAc)2and 0.5 /¿molof dithiothreitol. The quantitation of the products formedwas based on the percentage of the peak area after correction forappropriate control experiments. The limit of detection was 1% of theadded substrate, i.e., 0.1 nmol.

In Vivo Metabolism of NHA and O-Ac-NHA by TA98 Cell Suspensions. Cells from an overnight culture (8 ml) were washed twice withbuffer (0.1 M sodium phosphate/0.9% NaCl solution, pH 7.0) andresuspended in 8 ml of the same buffer. To 0.5 ml of pH 7.0 buffercontaining 10 nmol of [3H]NHA (364 mCi/mmol) or O-Ac-[3H]NHA

(760 mCi/mmol) in 10 fil of DMSO was added 0.1 ml of the TA98suspension or 0.1 ml of the same buffer as control. At 0 time and afterincubation at 37°Cfor 2 or 4 h, l ml of methanol was added, and the

extracts were analyzed for product formation by HPLC using SystemII. The quantitation of the products formed was based on the percentageof the peak area after correction for the 0 time control experiments.The limit of detection was 1% of the added substrate, i.e., 0.1 nmol.

Similar analyses of sonicates of TA98 or TA98/1,8-DNP6 cells thathad been treated with [3H]NHA were also carried out.

Reaction of NHA and O-Ac-NHA to tRNA. One mg of tRNA in 90M!of 0.14 M NaCl/0.015 M citrate, pH 7, was incubated at 37°Cfor upto 4 h with 10 M!of DMSO containing 0.1 ^mol of O-Ac-[3H, 14C]NHA(39 mCi/mmol of 3H; 6.2 mCi/mmol of I4C) or [3H, 14C]NHA (24.8mCi/mmol of 3H; 3.25 mCi/mmol of 14C).The tRNA was extracted

with organic solvents, recovered by precipitation with ethanol, andassayed for radioisotope content (24). The experiments were carriedout in duplicate.

Reaction of NHA and O-Ac-NHA with tRNA in the Presence ofH2O2/HRP. In order to examine the effect of peroxidation on thereactivity of these compounds toward tRNA, a mixture of 190 n\ of pH

Table 2 In vitro metabolism of NHA and O-Ac-NHA by a cell-free preparation ofSalmonella typhimurium TA98

The incubation mixture consisted of 135 /ig of bacterial protein and 2.5 ^molof cofactors in 0.5 ml of pH 7.0 buffer and 10 nmol of |3H]NHA or O-Ac-[3H]NHA in 10 M!of DMSO. After incubation at 37°Cfor I h, l ml of methanol was

added, and the extracts were analyzed by HPLC using a C18 column, elutedlinearly at 1 ml/min from 40 to 80% methanol in 10 HIMH3PO4 in 14 min, 80to 100% in 2 min, and at 100% for 5 min. The limit of detection was 1% of theadded substrate, i.e., 0.1 nmol.

Substrate[3H]NHAO-Ac-[3H]NHACofactorAcetyl-CoAAcetyl-CoAPAPS*NADHHypoxanthineBenzaldehydeH202NADHNADHNADPHHypoxanthineBenzaldehydeH202AtmosphereAirAirAirArgonArgonArgonAirAirArgonAirArgonArgonAirProductformed

(nmol)O-Ac-NHA(1.2)O-Ac-NHA(2.1)°NoneNoneNoneNoneNoneNpCONH2(1.2)NpCONHj

(4.5)NpCONH;(0.4)NoneNoneNone

" A heat-treated cell-free bacterial preparation was used.* Incubation was carried out in a pH 8.0 bicine buffer containing 1.5 nmol of

Mg(OAc)2 and 0.5 nmol of dithiothreitol.

7.0 buffer, containing 1 mg of tRNA, 1 ¿imolof H2O2, and 5 units ofHRP, was incubated at 37°Cfor 1 min with 0.1 Mmolof [3H, 14C]NHA(18.5 mCi/mmol of 3H, 2.38 mCi/mmol of I4C)or O-Ac-[3H, I4C]NHA(16.2 mCi/mmol of 3H, 2.28 mCi/mmol of I4C) in 10 n\ of DMSO.

Control experiments received no H2O2. Experiments were carried outin duplicate, and the analysis of the extent of reaction with tRNA wasas described (24).

Bacterial DNA Binding with NHA and O-Ac-NHA. Exponentiallygrowing cultures of TA98 were centrifuged, washed with a buffer of0.85% NaCl/50 mM sodium phosphate, pH 7.2, and resuspended inthe same buffer. Separate cultures were used for Experiments 1 and 2.For the reaction with [3H, 14C]NHA (150 mCi/mmol of'H, 26.3 mCi/mmol of 14C),i.e.. Experiment 1, 3.75 ml of a mixture containing 1.13x 10" cells and 1.5 umo\ of compound in 75 u\ of DMSO (0.4 mM incompound, 3 x 10'°cells/ml) were incubated at 37°Cfor up to 60 min.For the reaction with O-Ac-[3H, 14C]NHA (151 mCi/mmol of 3H, 26.3mCi/mmol of 14C;0.4 mM in compound and 4 x 10'°cells/ml), i.e.,

Experiment 2, incubation was for 60 min. Control samples received thetest compound after a 60-min incubation just prior to work-up. Experiments were carried out in triplicate. After incubation, the cells werecollected and washed 4 times with citrate-buffered saline, pH 7.2.Treatment of the cell pellets was as described by Beranek et al. (16).After the cells had been treated with sodium dodecyl sulfate at a finalconcentration of 0.5% at 37°Cfor 30 min (16), the solution was treated

with proteinase K (0.25 mg/ml) for 1 h and then extracted with phenol/8-hydroxyquinoline/m-cresol/H2O (500/0.5/70/55 by weight), chloro-form/isoamyl alcohol (24/1), and ether. The solution was made 0.3mM in sodium acetate, and the DNA was precipitated with 95% alcohol,washed successively with 70% alcohol, 95% alcohol, acetone, and ether,and then dissolved in 1 ml of 100 mM NaCI/10 mM Tris/10 mM EDTA,pH 7.2. Final purification of DNA included sequential incubations at37°Cfor 30 min with heat-treated RNase (0.1 mg/ml) and proteinase

K (0.25 mg/ml) before recovery by extraction and precipitation asdescribed above. The DNA was dissolved in water for determination ofA26onmand tritium content (24). The estimation of DNA contentassumed that a solution of 1 mg/ml would have an absorption of 20.

RESULTS

Decomposition of Esters of NHA. The incubation of O-Ac-[3H]NHA at 3TC in pH 7.0 buffer resulted mainly in the

formation of AN through a Lossen rearrangement. Hydrolysisof the acetate to NHA was a minor pathway. The 2-h incubationmixture showed the presence of both products. At the end of24-h incubation, little of the ester remained, and AN was themajor product as shown in the HPLC profiles (Fig. 2, A andB). The half-life was found to be 9.4 h as judged by the quantitiesof O-Ac-[3H]NHA in the incubation mixture at 0, 2, 4, and 24

h relative to the eluted label (Fig. 3).At higher concentrations, the NpNCO intermediate could be

trapped by reaction with aniline. At the end of an 18-h incubation, both l-(2-naphthyl)-3-phenyl urea and di-Np urea (Fig.1) were present. Their structures were established from theirHPLC R, (Table 1, System I), TLC Rf values (Table 1, SolventC), and UV spectra which were all identical with those ofauthentic samples.

O-SOiH-NHA-Py was previously found to undergo Lossenrearrangement at pH 7.0 and room temperature within 30 minto yield AN and di-Np urea (11). We have now determined thatthe half-life of the sulfate ester in pH 7.0 buffer was 1.4 minbased on the change in absorbance at 220 nm (Fig. 4).

These results showed that these esters of NHA can undergoLossen rearrangement to form NpNCO which can react withH2O to yield AN or with amines to give ureas; the O-sulfonateis much more reactive than the O-acetate.

Oxidation of NHA and O-Ac-NHA with H2O2/HRP. Fig. 5summarizes the results of the oxidation of these compounds.

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METABOLIC ACTIVATION OF 2-NAPHTHOHYDROXAMIC ACID

12

10

8

6

4

2

O-Ac-NHA

0 TIME

10 15 20 25

RETENTION TIME (MIN)

30

O-Ac-NHA

RETENTION TIME (MIN)

Fig. 2. Reverse-phase HPLC profile of the incubation mixture of O-Ac-['H|-NHA at pH 7.0 and 37°Cat 0 time (A) and 24 h (B). The mixture consisted of10 nmol of O-Ac-['H]NHA (760 mCi/mmol)/10 „1of DMSO in 0.6 ml of pH7.0 buffer. At 0 and 24 h, 50 n\ were mixed with 0.1 ml of methanol, and theextracts were analyzed by HPLC using System II as described in Table 1.

100

= 9.4 HzLU

Oü

Iz

<ó

5 10 15 20 25

INCUBATION TIME (H)Fig. 3. The half-life of O-Ac-NHA in pH 7.0 buffer at 37'C. The same

incubation mixture and method of analysis as described for Fig. 2 were used toobtain the quantities of O-Ac-[3H]NHA relative to the eluted label in the mixture

at 0, 2, 4, and 24 h.

In the presence of H2O2/HRP, NHA was almost completelyconverted in 10 min at 37°C,pH 7.0, to the 2 major products,

NpCOjH and O-CONp-NHA, and 3 minor unknown productsas evidenced from the HPLC profile using System III (data notshown). At the end of 1 h (Fig. 6), the product distributionremained similar to that of the 10-min reaction mixture. At theend of 24 h, only the 2 major products remained; there wereapproximately 5 times more of the acid (data not shown).

t,/2=1'4 MIN

TIME (MIN)

Fig. 4. The half-life of O-SO3H-NHA •Py in pH 7.0 buffer at room temperature. The mixture consisted of 1 Mgof compound/10 /il of ethanol in 1 ml ofbuffer. The change of absorbance at 220 nm was obtained by subtracting anarbitrarily chosen steady-state value from the absorbance at 220 nm at each timepoint.

PAC

.OCONp

OTO)O-CONp-NHA

Fig. 5. Peroxidation of NHA and its O-acetate.

Control experiments were carried out in the presence of H2O2alone or in the absence of H2O2/HRP. At the end of a 24-hincubation at 37°C,no NpCO2H was detected, and NHA was

recovered unchanged from these control experiments (data notshown). Presumably, as proposed for the oxidation of hydrox-amic acids (25), in the presence of H2O2/HRP, the reactiveintermediate, NpCONO, was generated which reacted withH2O or NHA to give NpCO2H and O-CONp-NHA, respectively (Fig. 5).

At short intervals, e.g., 10 min, the peroxidation of O-Ac-NHA gave only a small amount of NpCO2H (Fig. 1A). Subsequently, hydrolysis of the acetate to NHA became the majorpathway. At the end of a 24-h incubation, NHA became themajor component. Very little AN was produced (Fig. IB).Control incubations of O-Ac-NHA and H2O2 underwent mainlyhydrolysis of the acetate to NHA; very little Lossen rearrangement occurred. At the end of 24 h, the major productwas NHA with very little AN or O-Ac-NHA present (<1%).These results indicate that only in the presence of H2O2/HRPdid oxidation of O-Ac-NHA take place to form NpCO2H. H2O2suppressed the Lossen rearrangement and facilitated hydrolysisto form NHA. Thus, the formation of NpCO2H is an indicationof peroxidation of NHA or O-Ac-NHA.

Metabolism of NHA and O-Ac-NHA by Cell-free Preparations of TA98. In order to better understand the activation

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METABOLIC ACTIVATION OF 2-NAPHTHOHYDROXAMIC ACID

0.2

UJO

mEO(Om

0.1

0.0

NpCO2H

O-CONp-NHA

NHA

5 10 15

RETENTION TIME (MIN)

20

Fig. 6. Reverse-phase HPLC profile of products from peroxidation of NHAat 1 h. The reaction mixture consisted of 2 »¿molof compound/50 nl of DMSOand 1.95 ml of pH 7.0 buffer, containing 20 ^mol of H2O2 and 10 units of HRP.After 1-h incubation at 37'C, its ethyl acetate extract was analyzed by HPLC

using System III as described in Table 1.

mechanisms of NHA and its O-acetate with TA98, these compounds were treated with cell-free bacterial preparations andvarious cofactors to determine the potentials for conjugations,oxidation, or reduction. The results are summarized in Table2. NHA was acetylated to O-Ac-NHA by acetyl-CoA in thepresence of either a freshly prepared cell-free protein preparation or one that had been heat treated. In the presence of PAPS,NADH, hypoxanthine, benzaldehyde, or H2O2, no product wasformed from NHA and cell-free preparations. Thus, under theseconditions, TA98 can not sulfonate, reduce, or oxidize NHAwith these cofactors. As in the case of NHA, the TA98 preparations cannot oxidize O-Ac-NHA, since the oxidation product,NpCO2H, was not detected when it was treated with H2O2 anda cell-free preparation. On the other hand, TA98 reduced O-Ac-NHA to NpCONH2 in the presence of NADH or NADPH;the reduction was more efficient using NADH and was sensitiveto oxygen. However, hypoxanthine and benzaldehyde could notserve as electron donors in the reduction of O-Ac-NHA.

In Vivo Metabolism of NHA and O-Ac-NHA by Intact TA98Cells. The activation mechanisms of NHA and O-Ac-NHA byTA98 were further studied using intact cells. The metabolismby intact cells converted NHA to AN and 2-naphthamide in atime-dependent fashion (Table 3), presumably through the formation of a conjugate. The conversion of NHA to O-Ac-NHAwas detected only in experiments in which the cells were ruptured by sonication prior to extraction with methanol. Furthermore, a similar experiment using TA98/1,8-DNP6, the N-arylhydroxylamine 0-acetyltransferase-deficient strain of Salmonella (26, 27), also showed the formation of O-Ac-NHA.These results suggest that the conjugation of NHA does notrequire the O-acetyltransferase that can utilize yV-arylhydroxyl-amines as substrates. Small amounts of NpCO2H were detectedat each time point from NHA (Table 3); however, the formationwas not dependent on the presence of bacteria. Reduction ofO-Ac-NHA to the amide, NpCONH2, occurred in the presenceof bacteria. In contrast, formation of AN and NHA from O-Ac-NHA did not require bacteria. No production of NpCO2Hwas observed from O-Ac-NHA in the presence or absence ofbacteria; thus, no peroxidation was detected.

Reaction of NHA and O-Ac-NHA with tRNA. The reactivitiesof these compounds toward macromolecules were studied witha tRNA binding assay (24) using both ring-3H- and carbonyl-

14C-labeled materials. The results are shown in Fig. 8. O-Ac-

NHA bound to tRNA, and the adduci retained both ring andcarbonyl labels. On the other hand, NHA had very little reactivity toward tRNA. These results suggest that NHA needsactivation in order to react with macromolecules and thatadduct formation occurs with retention of both the ring andnaphthoyl carbonyl moieties.

Reaction of NHA and O-Ac-NHA with tRNA in the Presenceof H2O2/HRP. The effect of peroxidation of these compoundson the reactivities towards macromolecules was studied with atRNA binding assay (24) using both ring-'H- and carbonyl->4C-

labeled materials. The results are shown in Table 4. Adductformation was greatly enhanced in the presence of the peroxidation system. NHA was more reactive than O-Ac-NHA, andthe adducts retained both labels, presumably through naphtho-ylation by the reactive intermediate NpCONO (Fig. 5) as proposed by others for the oxidation of hydroxamic acids (25). Thebinding levels of these compounds (Table 4) reflect the extentof formation of NpCO2H from them in the presence of H2O2/HRP as mentioned above in the section of oxidation of NHAand O-Ac-NHA with H2O2/HRP.

Reaction of NHA and O-Ac-NHA with DNA in TA98. Binding of these compounds to DNA in bacteria was studied usingboth ring-'H- and carAo/i>>/-'4C-labeledcompounds. The results

0.2 r

UJu

Å“EOOTm

0.1

0.0

O-AC-NHA

10 MIN

NpC02H

U—K,

5 10 15

RETENTION TIME (MIN)

20

0.2

sz«

111o

mEocom

0.1

0.0

B

24 H

NHA

AN

V

O-Ac-NHA

NpC02H

205 10 15RETENTION TIME (MIN)

Fig. 7. Reverse-phase HPLC profile of the peroxidation of O-Ac-NHA at 10min (A) and 24 h (B). The reaction mixture consisted of 2 i¡mo\of compound/50íílof DMSO and 1.95 ml of pH 7.0 buffer containing 20 ¿unolof H2O2 and 10units of HRP. After 10-min and 24-h incubation at 37"C, their ethyl acetate

extracts were analyzed by HPLC using System III as described in Table 1.

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METABOLIC ACTIVATION OF 2-NAPHTHOHYDROXAMIC ACID

Table 3 Metabolism ofNHA and O-Ac-NHA by intact Salmonella typhimurium TA98 cellsMixtures of 10 nmol of [JH]NHA or O-Ac-[3H]NHA/10 ^1 of DMSO in 0.5 ml of pH 7.0 buffer were incubated at 37'C with or without 0.1 ml of an overnight

culture. After incubation, 1 ml of methanol was added, and the extracts were analyzed by HPLC using a C1Bcolumn, eluted at 1 ml/min linearly from 10 mM H3PO4to methanol in 30 min. The limit of detection was 1% of the added substrate, i.e., 0.1 nmol.

IncubationSubstrate time(h)[3H)NHA

24O-Ac-[3H]NHA

24Metabolites

formed(nmol)Bacteria

AN+

1.40.1+

2.30.2+

0.60.4+

1.21.1NHA0.30.20.30.4NpCONH;1.1N

D1.6ND1.7ND2.20.1O-Ac-NHAND"NDNDNDNpCO2H0.20.30.30.1ND0.1NDND

' ND, not detected.

[3HJNHA

[14CJNHAO-Ac-[3H]NHA

0-Ac-[14C]NHA

INCUBATION TIME (H)

Fig. 8. Binding of [3H, 14C)NHA and O-Ac-[3H, MC]NHA to tRNA at pH 7,37'C. Incubation mixture consisted of 0.1 /imol of compounds in 10 n\ of DMSO

and 1 mg of tRNA in 90 n\ of 0.14 M NaCl/0.015 M citrate buffer. The assayprocedure was as described by King (24). Points, means; bars, SD from duplicateexperiments.

showed that the reaction of NHA with TA98 DNA was timedependent and that binding levels increased linearly up to 60min (graph not shown). The adducts obtained from both compounds (60-min incubation) retained both labels (Table 5).These results suggest that NHA might be activated by forminga conjugate, such as O-Ac-NHA, and the DNA was modifiedby NpCONH- or the Lossen rearrangement product, NpNCO.AN, which could be formed from reaction of NpNCO withwater, was not involved in adduci formation; otherwise, it wouldhave lost the carbonyl label.

DISCUSSION

NHA is stable in neutral aqueous medium, and its O-acelaleand O-sulfonale undergo Lossen rearrangements to form areactive isocyanate which reacts with H2O to give AN. At highconcentrations, the isocyanate can react with AN to form di-Np urea (Fig. 1). Thus, the production of AN is an indicationof the presence of some type of NHA ester. Both NHA and O-Ac-NHA can be peroxidized to form NpCO2H, although to a

much lesser extent with the acetate (Figs. 6 and 7, A and B),presumably through the reaction of H2O with the reactiveintermediate, NpCONO, (Fig. 5). The formation of RCONO(/? = 2-naphthyl in the present study), which has been proposedas a strong acylating agent (25), from the oxidation of hydrox-amic acids is further confirmed by the formation of O-CONp-NHA in the peroxidation mixture of NHA. Thus, the formationof NpCO2H from these compounds is an indication of peroxidation.

In order to better understand the activation pathways of NHAin TA98, in vitro and in vivo metabolism of NHA and O-Ac-NHA with TA98 was investigated. Since there was no evidenceof formation of NpCO2H from NHA or O-Ac-NHA in thepresence of H2O2/TA98 cell-free preparations (Table 2) or inthe presence of bacteria themselves (Table 3), it is unlikely thatTA98 can catalyze the oxidation of these compounds, althougha peroxidative system (H2O2/HRP) increased the reactivity ofNHA and O-Ac-NHA toward tRNA (Table 4).

O-Sulfonalion of NHA by PAPS in the presence of TA98cell-free preparations was not demonstrated (Table 2); otherwise the Lossen rearrangement product, AN, would have beendetected. A previous study also failed to detect the formationof O-sulfonates from /V-arylhydroxylamines by Salmonella (28),although mammalian liver cytosols can O-sulfonate ./V-hydroxy-2-acetylaminofluorene (29-31) as well as A^-hydroxy-2-amino-

fluorene (31).TA98 cell-free preparations can reduce O-Ac-NHA but not

NHA to NpCONH2 in the presence of NADH or NADPH,and the reduction is sensitive to oxygen (Table 2). On the otherhand, TA98 cells can reduce NHA to its amide, thus providingadditional support for the conclusion that NHA was activatedto the O-acetate or other conjugate(s) which can be similarlyreduced as is the O-acetate. It has been reported that liveraldehyde oxidase can reduce hydroxamic acids to amides (32),and that NADH, NADPH, or hypoxanthine is not a cofactorin this reaction. It is also known that yV-hydroxy-2-acetylami-nofluorene reducíasefrom rabbit liver cytosol required NADHor NADPH as an eleclron donor and lhal Ihe reduclion is noi

Table 4 Reactions of NHA and O-Ac-NHA with tRNA in the presence ofH^OJHRP at 37°C for 1 min

The incubation mixtures consisted of 0.1 /¿molof compound in 10^1 of DMSO and 190>il of pH 7 buffer, containing 1 mg of tRNA, 1 ^mol of H2O¡,and 5 unitsof HRP. The procedure for quantitation of adducts was as described (24). The results are from duplicate experiments.

Binding level(nmol/mgCompound[3H,

"C]NHAO-Ac-[3H,

MC]NHAH2O2

Ring+

1.000.03+

0.100.00of

tRNA)Carbonyl0.990.040.080.00Netbinding level(nmol/mg

oftRNA)Ring

Carbonyl0.97

0.950.10

0.08Ring/carbonyltRNA1.021.25

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METABOLIC ACTIVATION OF 2-NAPHTHOHYDROXAMIC ACID

Table 5 Reaction off'H, "CJNHA and O-Ac-f'ff, "CJNHA with DNA of Salmonella typhimurium TA98

Mixtures of 3.75 ml containing 1.5 fjitiol of compound/75 /jl of DMSO and bacterial cells/0.85% NaCl solution, 50 m\i phosphate, pH 7.2, were incubated at37*C for 1 h. Bacteria used for NHA and O-Ac-NHA were 1.1 x 10" cells and 1.5 X 10" cells in Experiments 1 and 2, respectively. For the 0-h experiments,compounds were added at the end of 1-h incubation. See "Materials and Methods" for isolation of DNA. The results from triplicate analyses are shown.

IncubationCompoundtime(h)NHA

01O-Ac-NHA

01Binding

levelRing4.5

±1.1*17.0±

2.03.2

±0.69.0±3.5(/jmol/mol)0CarbonylExperiment

15.0

±0.8918.3±3.1Experiment

22.9

±0.98.9±3.6Net

binding level(^mol/mol)"King

Carbonyl12.5

13.35.8

6.0Ring/carbonylDNA0.940.97" jimol of adduct/mol of nucleotide.*Mean ±SD.

affected by oxygen (33). Thus, the TA98 enzyme which catalyzes the reduction of O-Ac-NHA has characteristics that aredifferent from those of the mammalian enzymes.

In neutral solution, O-Ac-NHA reacted with tRNA, but NHAhad very limited reactivity (Fig. 8). However, incubation ofNHA or O-Ac-NHA with TA98 cell suspensions resulted intheir binding to the bacterial DNA (Table 5) with the retentionof both naphthyl and carbonyl (of naphthoyl) moieties. Theseexperiments were carried out with different bacterial culturesand under slightly different conditions and cannot, therefore,provide for quantitative comparison of the binding of these twocompounds. However, these data do distinguish qualitativelybetween adduct formation from the activated AN as comparedwith reactions with NpNCO or metabolites that are capable ofnaphthoylation or naphthamidation. The activation of NHAthrough the formation of reactive metabolites is further supported by the demonstration that PCP inhibits the mutagenicityof NHA, but not that of O-Ac-NHA, in TA98 in a dose-dependent fashion.6 Thus, the use of PCP as an inhibitor of

metabolic activation may facilitate the search for the enzymaticpathway(s) involved in the activation.

In conclusion, our present results show that NHA requiresmetabolic activation by TA98 to bind to its DNA and that themetabolites responsible for adduct formation may be the O-acetate and/or other metabolites with similar reactivity. Thesedata further suggest that DNA modification pathways mayinvolve naphthamidation or carbamoylation. The characterization of the structure of this adduct(s) is in progress.

ACKNOWLEDGMENTS

We are grateful for the gifts of TA98 and TA98/1,8-DNP6 from Dr.N. B. Ames, University of California, Berkeley, CA, and from Dr. E.C. McCoy, Case Western Reserve University, Cleveland, OH, respectively. We thank A. Meszaros for technical assistance and Dr. C. M.King and Dr. C. Y. Wang for helpful discussions. We also would liketo thank Dr. M. B. Ksebati and M. B. Kempff from the InstrumentationFacility of the Comprehensive Cancer Center of Metropolitan Detroitfor measuring the NMR spectra and mass spectra, respectively. Assistance from E. Thomas-Weber in the preparation of this manuscript ismuch appreciated.

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1990;50:4300-4307. Cancer Res   Mei-Sie Lee and Masakazu Isobe 

TA98Salmonella typhimurium2-Naphthohydroxamic Acid, in Metabolic Activation of the Potent Mutagen,

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