metabolic activation of n-hydroxy-n,n'-diacetylbenzidine by … · sulfonation of n-ho-dabz...

[CANCER RESEARCH 40, 751-757, March 1980]

benzidine as a potent human urinary bladder carcinogen (8,14, 44, 57). However,in mice(4, 45, 52), rats (6, 42, 50), andhamsters (48), the liver is a major target organ, while benzidineis at best a weak carcinogen in dogs (i 0, i 2, 44, 50) andrabbits (44, 50) and was shown not to be carcinogenic inmonkeys (50). In an attempt to understand these speciesvariations, we have conducted in vitro metabolic studies onbenzidine and have previously reported on mechanisms for itsmetabolic activation in rodents (4i ). In an acetyb-GoA-dependent reaction, hepatic cytosol catalyzed the conversion of benzidine to DABZ.3 Subsequent N-oxidation of this metabobitetoN-HO-DABZ was also demonstrated with fortified liver microsomes. N-HO-DABZ was then converted to mutagenic andelectrophibic species by the action of hepatic N,O-acybtransfenase, a cytosolic enyme which is involved in the metabolicactivation of several carcinogenic arylhydroxamic acids including N-HO-AAF (i , 27, 30).

Since hepatic sulfotransferase has also been strongly implicated in the metabolic activation of aromatic amine hepatocancinogens, namely, N-HO-AAF (9, 30) and N-hydroxy-N-methyl4-aminoazobenzene (24), and in the conversion of i â€˜-hydroxyalkybarenes(55), purina N-oxides (Si ), and fl-amino alcohols(3) to ultimate carcinogenic electrophiles, the metabolic 0-sulfonation of N-HO-DABZ was investigated. Additionally, themechanism of electrophilic substitution by methionine wasstudied since both carbocations (7, 40) and quinone-imides (7,i 5, i 7, 29) have been proposed as reactive intermediates ofaromatic amine carcinogens.

MATERIALS AND METHODS

Materials. L-[methy!-3H]Methionine (5 mCi/mmol; radiochemical purity, >98%) was purchased from Midwest Research Institute (Kansas Gity, Mo.) and was diluted with Lmethionine to a specific activity of 0.16 mGi/mmol. Bis(2-hydroxyethyl)immnotnis(hydroxymethyl)methanebase and L-methionine were obtained from the Sigma Chemical Co. (St. Louis,Mo.). DABZ (38), N-HO-DABZ (41), N-HO-AAF (43), and PAPS(i 8) were prepared by published methods. Acetanilide and 2-and 3-methylmercaptoanibine were purchased from AldrichChemical Co. (Milwaukee, Wis.). 2-CH3S-acetanilide and 3-CH3S-acetanilide were prepared from the correspondingamines by acetybation in 10 volumes of acetic anhydnide:pyri

3 The abbreviations used are: DABZ, N,N'-diacetylbenzidine; N-HO-DABZ, N-hydroxy-N,N'-diacetylbenzidine; N-HO-AAF, N-hydroxy-2-acetylaminofluorene;PAPS, 3'-phosphoadenosine 5'-phosphosulfate; 2-CH3S-acetanilide, 2-methylmercaptoacetanilide; 3-CH3S-acetanilide, 3-methylmercaptoacetanilide; CH3S-DABZ, methylmercapto-N,N'-diacetylbenzidine; N-acetoxy-DABZ, N-acetoxyN,N'-diacetylbenzidine; 3-CH3S-AAF,3-methyimercapto-2-acetylaminofluorene;HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance;o-CH3S-acetanilide,o-methylmercaptoacetanilide; m-CH3S-acetanilide,m-methylmercaptoacetanilide; o-CH3S-AAF,mixture of 1- and 3-methylmercapto-2-acetylaminofluorene; N-sulfonyloxy-DABZ, N-sulfonyloxy-N,N'-diacetylbenzidine; 2-CH3S-DABZ,2-methylmercapto-N,N'-diacetylbenzidine; 3-CH3S-DABZ,3-methylmercapto-N,N'-diacetylbenzidine.

MARCH1980 751

Metabolic Activation of N-Hydroxy-N,N'-diacetylbenzidine by HepaticSuitotransferase

Kenneth c. Morton,1 Frederick A. Beband, Frederick E. Evans, Nancy F. Fullerton, and Fred F. Kadlubar2

Division of Carcinogenesis Research, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079

ABSTRACT

N-Hydroxy-N,N'-diacetybbenzidmne(N-HO-DABZ) has beenshown to be an in vitro metabobiteof benzidine in several rodentspecies and may represent the proximate form of the carcinogen. Like other arybhydroxamic acids, N-HO-DABZ may beconverted to an ultimate carcinogenic electrophibeby metabolicO-sulfonation in hepatic cytosob.To investigate this possibility,liver cytosols from rats, mice, and hamsters were assayed fortheir ability to catalyze the 3'-phosphoadenosine 5'-phosphosulfate-dependent metabolism of N-HO-DABZ and the fonmalion of an adduct with methionine. For comparative purposes,sulfotmansferaseactivity for N-hydnoxy-2-acetylaminofluorene(N-HO-AF) was also measured. In the rat, N-HO-DABZ and N-HO-AAF were metabolized at rates of 2.5 and 4.3 nmob ofamylhydroxamic acid lost per mm per mg of protein, respectively. In the mouse, these rates were 0.5 nmol for N-HO-DABZand <0.05 nmobfor N-HO-AAF. Sulfotransferase activity forthese substrates in hamster liver cytosobcould not be detected(<0.05 nmol/mmn/mg).

The inclusion of methionine in sulfotransferase incubationmixtures and subsequent heating resulted in the formation ofmethylmercapto arylamides from both N-HO-DABZ and N-HOAAF. From 20 to 40% of the N-HO-DABZ metabolized wastrapped and recovered as an adduct, while 80 to i 00% of theN-HO-AAF metabolized was similarly obtained. A methybmercapto-N,N'-diacetybbenzidine derivative was also obtained byreaction of N-acetoxy-N,N'-diacetylbenzidmne with methionine.Its identity to the adduct formed in the sulfotransferase incubation mixture was established by high-pressure liquid chromatography, ultraviolet light, and mass spectroscopic analyses. By comparing the 13Cnuclear magnetic resonance spectraof the synthetic methylmencapto derivative with several modelcompounds and using chemical shift additivity relationships,the adduct was identified as 3-methylmercapto-N,N'-diacetylbenzidine. Since the yield of the product from N-acetoxy-N,N'diacetybbenzidmneand methionine did not vary appreciably withpH (4 to 8), a reaction mechanism involving an electrophiliccambocationat position 3 is proposed.

These studies demonstrate that N-HO-DABZ can be estenified to an electrophibic reactant by hepatic sulfotransfenases inthe matand the mouse and suggest the involvement of thismetabolite in the hepatocarcmnogenicityof benzidine.

INTRODUCTION

Epidemiobogicalstudies of industrial workers have implicated

1 Present address: Department of Chemical Carcinogenesis, Michigan Cancer

Foundation, 110 E. Warren, Detroit, Mich. 4820i . Supported by InteragencyAgreement 224-75-0002 between the Veterans Administration Hospital, LittleRock, Ark. 72206, and the United States Food and Drug Administration, Jefferson, Ark. 72079.

2 To whom requests for reprints should be addressed.

Received August 30, 1979; accepted December 5, 1979.

on May 15, 2020. © 1980 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

K. C. Morton et a!.

dine (9:1 ) at room temperature for 16 hr. Twenty volumes ofcold water were then added; the mixture was then stirred for 1hr and evaporated to dryness under reduced pressure to yieldcrystalline products.

CH3S-DABZ was synthesized from N-HO-DABZ by the fobbowingprocedure. N-HO-DABZ (0.54 mmob)was converted toN-acetoxy-DABZ by treatment with 10 ml of acetic anhydride:pyridine (9:1 ). After 16 hr at 5Â°,the mixture was filtered, andthe product was precipitated from the filtrate by addition of 20volumes of cold water and stirring for 15 mm. The product wascollected by filtration, washed with cold water, and dried underreduced pressure. The yield was 48%, and mass spectralanalysis (molecular ion at m/e 326; fragments at 284, 266,242, and 200) was consistent with its identity as N-acetoxyDABZ. This compound was reacted with L-methionine in 30%acetone at pH 7 to yield CH3S-DABZby the method previouslydescribed (36) for the synthesis of 3-CH3-S-AAF. The crudeproduct was dissolved in chloroform, filtered, and applied to aneutral alumina (BioRad AG 7) column (2.5 x 27 cm). Thecolumn was washed with 200 ml of chloroform, and the productwas ebutedwith 200 ml of chboroform:ethyl acetate (9:1 ). Onevaporation of the solvent under reduced pressure, white crystalbineCH3S-DABZ(m.p. 258Â°)was obtained (yield, 22% fromN-acetoxy-DABZ). The purity was determined to be >97% by2 HPLC systems. System 1: BioRad BiosibODS-10 column (4x150mm);SolventA,0.1MTnis-HGIbuffer,pH8.5;SolventB, absolute methanol (Fisher HPLC grade); program, 20 to100% B, 30 mm, linear gradient, 1 mI/mm; CH3S-DABZretention time, 18.5 mm. System 2: Waters Associates @BondapakC18 column (3.9 x 300 mm); Solvent A, water; Solvent B,absolute methanol; program, 30% to 90% B, 15 mm, lineargradient, 2 mI/mm; CH3S-DABZ retention time, 13 mm. Electron impact mass spectrometnic analysis of the product gavethe correct molecular ion at m/e 314, with major fragments at272, 267, 257, 230, 226, 215, 199, and 184.

Structural identification of this product as a 3-substitutedderivative required analysis by 13C NMR spectroscopy andcomparison with 13Cspectra of DABZ, acetanibide,and o- andm-CH3S-acetanilide (â€˜â€˜Resultsand Discussionâ€•).

Adult male animals were used. Syrian Golden hamsters andSprague-Dawley rats were from ARS/Sprague-Dawley Division(Madison, Wis.), and inbred mice (BALB/cStCrbfC3Hf/Nctr)were from our breeding stock. Animals were killed by decapitation, and the livers were perfused with 0.9% NaCI solution.Hepatic cytosols were prepared by differential centrifugationof homogenates according to procedures outlined by Hogeboom et a!. (16), and protein concentrations were determinedby the biuret reaction (13).

Enzyme Assays. N-HO-DABZ and N-HO-AAF sulfotransferase assays containing [3H]methionine were carried out as previousbydescribed for N-HO-AAF (24), except that the concentration of the arybhydroxamic acids was only 0.2 m@due to thelimited solubibityof N-HO-DABZ. Enzyme activity was measuredas the PAPS-dependent loss of N-HO-DABZ and N-HO-AAF,which were recovered by ether extraction, converted to theirfernic chelates, and estimated cobonimetnicably(5). The extinction coefficients for N-HO-AAF and N-HO-DABZ femricchebateswere 1.7 x 1o@and 1.9 x 1O@M1, respectively, at 535 nm.The rates of reaction were first order with respect to proteinconcentration and linear with time for 20 mm (rats) or 40 mm(mice).

The concomitant formation of reactive sulfuric acid esterswas estimated from their reaction with the [methy!-3H]methionine. The aqueous portion ofthe ether-extracted assay mixture,which contained enzymaticabby generated (methionin-Syl)arybamideadducts of N-HO-DABZ or N-HO-AAF, was thenanalyzed for 3H-babebedmethylmercaptoarenes as previouslydescribed (9) except that CH3S-DABZwas extracted with ethylacetate instead of benzene:hexane.

Instrumentation. HPLCanalysesand purificationswere carned out on a Waters Associates Model 204 chromatographequipped with a Model 660 programmer, an additional Model6000A pump, a Model 440 dual channel (254 and 280 nm)absorbance monitor, and a Model 3380A Hewlett-Packardrecording Integrator. UV, NMR, and mass spectral analyseswere obtained with Beckman Model 25, Bruken WH 270, andFmnnigan4000 instruments, respectively. â€˜3CNMR spectrawere obtained at 67.9 MHz. Chemical shifts were measured inppm. downfield from internal dimethyb subfoxide-d6 and converted to ppm downfield from tetramethylsibane by addition of39.8 ppm. Radioactivity was measured in Scintisob(Isolab, Inc.,Akron, Ohio) with a SearbeMark III scintillation spectrometer.Melting point determinations were made with a EngineeringLtd. electrothermal apparatus.

RESULTS AND DISCUSSION

Sulfotransferase-medlated Metabolism of N-HO-DABZ toa Reactive Electrophile. Hepatic sulfotransferasehad beenshown to catalyze the conversion of N-HO-AAF in matcytosol(9, 30) and N-hydroxy-N-methyb-4-ammnoazobenzenein rat andmouse (24) cytosols to electrophibic sulfuric acid esters whichare probably ultimate forms of these carcinogens. We soughtto establish whether N-HO-DABZ could also serve as a substrate for hepatic sulfotransferase and be converted to anelectrophilic species.

Preliminary experiments with rats indicated that, under invitro incubation conditions described as optimal for N-HO-AAFsulfotransfenase (24), 35 to 65% of the N-HO-DABZ was lostfrom the complete assay mixture after a 20-mm incubation.Only 10 to 20% was lost if PAPS was omitted, and 5 to 15%was lost if the cytosol was heat denatured at 80Â°for 3 mmprior to incubation. Additional studies on the effects of pH andof substrate and protein concentrations indicated that the N-HO-AAF incubation conditions were also optimal for the PAPSand cytosol-dependent bossof N-HO-DABZ.

To determine whether the enzyme-catalyzed loss of N-HODABZ resulted in formation of an ebectrophilic N-sulfonyboxyderivative, [methy!-3H]methionine was included in the assay asa nucleophilic trapping agent. Several studies had shown thatelectrophibic sulfuric onacetic acid esters of N-hydnoxyarylamides and N-hydroxyarylamines react with methionine to formring-substituted (methionin-S-yl)arylamides or (methionine-SyI)arylamines which are decomposed by heat or alkali to theircorresponding methybmercaptoarenes (1 , 7, 24, 39). The sulfotransferase assay mixtures (â€˜â€˜Materials and Methods' â€˜)werethus analyzed for [3H]CH3S-DABZby methods previously usedfor the recovery of [3H]-o-GH3S-AAFfrom [3H]methionine-supplemented N-HO-AAF sulfotransferase incubations (9). From20 to 40% of PAPS-dependent bossof [3HJ-N-HO-DABZcouldbe accounted for as [3H]-GH3S-DABZ.Furthermore, formationof the 3H-Iabebedproduct was dependent on PAPS and native

752 CANCERRESEARCHVOL. 40



N-H0-DABZ Su!fotransferase

cytosob. HPLC analysis of the ethyl acetate extract using Sobvent System 1 indicated that >90% of the 3H cochnomatographed with the synthetic GH3S-DABZ (Chart 1). Additionalpurification with HPLG System 2 yielded a product the UV(Chart 2) and mass spectra (molecular ion at m/e 314; majorfragments at 272, 267, 257, 230, 215, 199, and 184) of whichwere identical to the synthetic standard. Recovery of synthetic

EC

0

0VCa@0

eâ€˜a@0

GH3S-DABZ from the incubation after heating and extractionwas greater than 90%.

These experiments demonstrate that N-HO-DABZ is metabolized in rat hepatic cytosob to a reactive ebectrophilic ester,presumably N-subfonyboxy-DABZ.Like other esters of N-hydroxy compounds (e.g. , N-subfonyboxy-N-acetyb-2-aminofbuorene), reaction with methionine can occur to yield a ring

@03

0

Time (mm)Chart 1. HPLC analysis of synthetic (â€”) and metabolically formed (â€”â€”â€”â€”)CH3S-DABZ.

4)VCa

.00â€˜a

.0

Wavelength (nm)Chart 2. UV spectra of synthetic (â€”) and metabolically formed (â€”â€”â€”â€”)CH3S-DABZ.

MARCH1980 753



The assay mixture contained 100 mM bis(2-hydroxyethyl)iminotria(hydroxymethyl)methane-HCI buffer (pH 6.6 at 37Â°),5 m@MgCI2,0.5 [email protected],0.65

mM PAPS, 1 mg cytosol protein per ml, 20 mML-[methyl-3H)methionine(0.16mCi/mmol), and 0.2 mM N-HO-DABZ or N-HO-AAF. Activity isexpressedas

nmol of arylhydroxamic acid metabolized per mm per mg of cytosolprotein.Thelimit of detection was <0.05 nmol/min/mg.PAPS-dependent

lossofSpecies

N-HO-DABZN-HO-AAFRat

2.5 Â±0.54a (5)b 4.3 Â±1.4(6)Mouse0.5 Â±0.1 (3) <0.05(4)Hamster

<0.05 (3) <0.05 (4)

K. C. Morton et a!.

substituted methylmercaptoarene.Comparison of N-HO-DABZ and N-HO-AAF Sulfotransfer

ase in Hepatic Cytosols from Rats, Mice, and Hamsters. Inaddition to the nat, hepatic cytosols from mice, but not hamstems,catalyzed the PAPS-dependent bossof N-HO-DABZ fromthe assay media and the concomitant formation of a methionmnybadduct which could be recovered in 20 to 40% yield as CH3S-DABZ (Table 1). However, the enzyme activity in the mousewas only one-fifth of that found in the rat.

For comparative purposes, N-HO-AAF sulfotransferase activity was also measured, both as the PAPS-dependent loss ofsubstrate and as the formation of a methioninyl adduct (as[3H]-o-CH3S-AAF).As previously reported (9), significant activity was obtained only in the rat (Table 1). Additionally, N-HOAAF sulfotransferase activity was about 70% higher than N-HO-DABZ activity; and the o-GH3S-AAF recovered accountedfor 80 to 100% of the PAPS-dependent bossof N-HO-AAF.

Recoveries of N-HO-DABZ and N-HO-AAF from incubationmixtures in the absence of PAPS were dependent upon thesource of liver cytosol. While 80 to 100% of the hydroxamicacids could be recovered from the mouse and rat incubations,only 30 to 35% (N-HO-DABZ) and 60 to 70% (N-HO-AAF) wereobtained from similar mixtures containing hamster cytosob.Theloss of both hydroxamic acids from the hamster incubationswas time dependent and was abolished upon prior heat danaturation of the cytosob. These results suggest that, in thehamster, these compounds are rapidly metabolized by anotherpathway, possibly by hepatic N,0-acybtransferase which ispresent in relatively high bevelsin this species (41).

Thus, bikeN-hydroxy-N-methyl-4-aminoazobenzene (24), N-HO-DABZ is estenified by PAPS in both rat and mouse hepaticcytosobs. Both of these tissues are also susceptible to thecarcinogenic action of N-methyl-4-aminoazobenzene (35) andbenzidine (14, 44). These data imply that the action of hepaticsulfotransferase on N-HO-DABZ in mice and rats may contnibute to the hepatocarcinogenicity of benzidine in these species.In hamster hepatic cytosob, however, other activation mechanisms such as N,0-acybtransferase may be responsible for thegeneration of the ultimate carcinogenic metabobite.

Mechanisms of Electrophilic Substitution by Esters of N-HO-DABZ. Several ebectrophilic species may be generated by0-estenification of N-HO-DABZ (Chart 3). The metabolicallyformed N-sulfonyboxy derivative or the synthetic N-acetoxyderivative may decompose to an ebectrophile with charge bocabization at the nitrogen, the 3-carbon, or the 2'-canbon.Alternatively, this electrophilic form may deprotonate to yield a

Table 1

Ac@@@'203@,,AC

6 5 @OH

N-HO-DABZ

YTOSOLc@2_#_#,__. @@SSS4@AC2O@/PYRIDINE

@ H0503N-SULFONYLOXY-DABZ N-ACETOXY-DABZ

1s,e@00S03H5e

[A:)@cfrq.@(2@c1xSCH3

00H@ H20 2-CH3 -S-DABZ

[AcN(j=!(T3NAc]Ac'..

SCH3

DABZ-DIIMIDE 3-CH3-S-DABZ

Chart 3. Possible mechanismsof electrophilic substitution by esters of N-HODABz. Ac20, acetic anhydride.

diimide which may undergo ebectrophilic â€˜â€˜1,4- and/on 1,8-addition' â€˜reactions. One or several of these mechanisms havebeen shown to exist for 0-esters of arylhydnoxamic acids onN-hydroxyanylamines (1, 7, 15, 24, 31 , 39), for oxidizable phenobicarybamides(17) and arylamines (28, 29), and for quinonederivatives of polycyclic hydrocarbons (2). To eliminate someof these possibilities, the site of substitution on DABZ bymethionine was rigorously investigated.

Methionine was known to react with esters of N-hydmoxyanybaminesand N-hydroxyarybamides to yield 0- and rn-methylmercaptoarenes (1, 7, 15, 24, 31 , 39). Structural proof of thesubstitution positions was based primarily on spectroscopicarguments, notably â€˜HNMR (11, 32, 49, 54), and the mostcharacteristic property of the ortho-substituted derivatives wasa downfield shift of the proton adjacent to the amide function(47, 56). While â€˜H NMR spectra of ring-substituted DABZderivatives have not been reported, we anticipated that a 3-substituted CH3S-DABZwould have a similar downfield shift ofH-5. However, in â€˜HNMR studies on the GH3S-DABZ productthat we obtained from reacting N-acetoxy-DABZ with methionine, the anticipated downfield shift was not observed. Thus,the site of substitution on DABZ was still ambiguous.

Analysis of the â€˜3GNMR spectra of the synthetic GH3S-DABZand several model compounds allowed the question of 2- asopposed to 3-substitution to be resolved. The chemical shiftsof GH3S-DABZ, DABZ, 2-GH3S-acetanilide, 3-CH3S-acetanilide, and acetanibide are reported in Table 2. Note that themethyl and canbonybresonances of GH3S-DABZ are nearlyidentical to those of 3-CH3S-acetanibide.The aromatic carbonchemical shifts of each compound could not be directly compared because of the substitutent effect introduced by theprime ring. However, since effects of this type are known to beadditive (53), a substituent effect was computed by subtractingthe acetanilide chemical shifts from those of the correspondingcarbon atoms of DABZ. For example, C-i of DABZ was 11.4

N-HO-DABZ and N-HO-AAF sulfotransferase activity in hepatic cytosol ofvarious rodents

a Mean Â± S.D.

b Numbers in parentheses, number of animals.

CANCERRESEARCHVOL. 40754



â€˜3CNMR chemical shifts of acetanilideand diacetylbenzidinecompoundsObserved@'PredictedCAssignment8Acetanilideo-CH3S-acet-

m-CH3-S-anilide acetanilideDABZx-CH3S- DABZ2-CH3S- DABZ3-CH3S-DABZ1123.1126.3

121.1134.5137.8132.5137.72128.8126.3138.6126.4123.4136.2123.931

19.21 33.6 116.41 19.61 @33.81 16.8134.04139.5135.7140.0138.5134.2139.0134.75119.2125.4115.7119.6125.7116.1125.86128.8127.0129.3126.4124.6126.9124.6N-Ac

168.4168.7168.6168.3168.7N-Ac24.123.324.124.023.3S-CH315.3

14.815.41'134.5134.72'126.4127.13'119.6119.54'138.5139.15'119.6119.56'126.4127.1N'-Ac

168.3168.5N'-Ac24.024.2

Table3Effectof pH on the reaction of N-acetoxy-DABZ withmethionineThe

reaction mixture was prepared by addition of 8 @imolof N-acetoxy-DABZin 3 ml of acetone to 8 @moIof[methyl-3H]methionine in 10 mMbuffer. Incubationswere carried out at 37Â°for 18 hr, and the CH3S-DABZwas recovered (â€˜â€˜MaterialsandMethodsâ€•),purifiedby HPLC (System 2), and estimated by 3Hcontent.Buffer

pH % yield ofCH3S-DABZSodium

citrate 4.05.35.05.16.05.07.0

3.6Tris-HCI

7.07.28.06.39.0

2.1


Table 2

a Substituent effects, proton-carbon coupling constants, and intensity measurements were the basis for theassignments. The predicted chemical shifts of 3-CH3S-DABZwere also used to assign the x-CH3S-DABZresonances;however, some of the closely spaced signals (e.g., C-3 and C-4) may have reversed assignments. See Chart 3 for thering numbering system for DABZ derivatives. To facilitate comparison with DABZ derivatives, the acetanilides were alsonumbered with the amide function in position 4.

b Chemical shifts were measured in dimethyl sulfoxide-d6 at 20Â° and are reported in ppm from tetramethylsilane(â€œMaterialsand Methodsâ€•).

c Chemical shifts were computed by addition of acetanilide substituent effects to the observed chemical shifts of theappropriate model compounds (â€œMaterialsand Methodsâ€•).

ppm downfield from the analogous position in acetanilide. Inaddition, C-2 and G-6 were upfield by 2.4 ppm, and C-4 wasupfiebdby 1.0 ppm, respectively; while C-3 and C-5 were 0.4ppm downfield. These substituent effects were then added tothe observed chemical shifts of 2- and 3-GH3S-acetanilide topredict chemical shifts for both 2- and 3-CH3S-DABZ. Thepredicted values for 3-GH3S-DABZwere in excellent agreementwith the observed chemical shifts of the compound in question(Table 2). These studies clearly establish that position 3 ofDABZ is the site of electrophibic substitution by methionine.This structure has also been recently confirmed by X-raycrystallographic analysis.4

However, the identification of 3-CH3S-DABZ does not distinguish between an ebectrophilic 3-carbocation intermediate ora diimlde which undergoes a â€˜â€˜1,8 addition' â€˜reaction withmethionine. Therefore, the effect of pH on the reaction of N-acetoxy-DABZ with methionine was investigated. Previousstudies (7) with N-acetoxyphenacetin showed that both anelectrophilic o-carbocation and an analogous reactive quinoneimide were formed as intermediates during reaction with nucleophiles. Under neutral conditions, reaction occurred excbusively through the quinone imide; while under acidic conditions,products arising from both intermediates were detected. Whenwe compared the yield of 3-CH3S-DABZ obtained from N-acetoxy-DABZ and methionine at pH 4 to 8, no appreciabledifferences were detected (Table 3). There were differencesbetween the buffers used, and boweryields were obtained atpH 9. The product obtained at each pH was chromatognaphicalby(HPLC System 2) and spectrally (UV) identical to authentic3-GH3S-DABZ. The failure to detect appreciable pH effects onthe reaction of methionine with N-acetoxy-DABZ strongly suggests that this ester and presumably N-sulfonyboxy-DABZ aswell decompose to yield an electrophibic carbocation with

4 D. van der Helm, personal communication.

charge localization at position 3 (Chart 3, heavy arrow).Thus, electrophilic esters of DABZ appear to involve reaction

mechanisms similar to those of other strong hepatocarcinogenssuch as 2-acetylaminofluorene and N-methyl-4-aminoazobenzene (37) and dissimilar to that of phenacetin, a relatively weakcarcinogen which induces primarily carcinomas of the urinarytract and nasal cavity (19â€”22).Therefore, we propose thathepatic 0-sulfonation as well as N,0-acyl transfer representmajor metabolic activation reactions for DABZ which lead tohepatic tumor induction. On the other hand, benzidine-inducedurinary bladder carcinogenesis, which has been demonstratedonly in humans (44), may involve deacetybated metabolites,namely, N-hydroxybenzidine or N-hydroxy-N'-acetybbenzidine.A correlation between the inability of dogs (33) and humans(34) to acetylate aromatic amines and their increased susceptibility to bladder cancer has been reported. Additionally, biochemical studies on the mechanism of 2-naphthylamine-induced bladder cancinogenesis (23, 25, 26, 46) suggest thatthe nonacetylated N-hydnoxy-2-naphthybamine N-gbucuronide,which is hydrolyzed and converted in an acidic urine to anebectrophilic nucleic acid-binding species, may represent the

MARCH 1980 755



K. C. Morton et a!.

and 2-nitronaphthalene-treated rats. In: A. Aitio (ed), Conjugation Reactionsin Drug Biotransformation, pp. 443â€”454.Amsterdam: Elsevier/North-Holland, 1978.

24. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic metabolism of N-hydroxy-N-methyl-4-aminoazobenzene and other N-hydroxy arylamines toreactive sulfuric acid esters. Cancer Res., 36: 2350â€”2359,1976.

25. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic microsomal N-glucuronidation and nucleic acid binding of N-hydroxy arylamines in relationto urinary bladder carcinogenesis. Cancer Res., 37: 805â€”814,1977.

26. Kadlubar, F. F., Oglesby, L. A., and Flammang, T. J. Effect of urinary pH onthe excretion, resorption, macromolecular binding of carcinogenic N-hydroxy arylamines in the urinary bladder. Proc. Am. Assoc. Cancer Res., 20:129, 1979.

27. King, C. M. Mechanism of reaction, tissue distribution, and inhibition ofarylhydroxamic acid acyltransferase. Cancer Res., 34: 1503â€”1515, 1974.

28. King, C. M., Chang, S. F., and Gutmann, H. R. The oxidation of o-aminophenols by cytochrome c and cytochrome oxidase. V. Inactivation of catslass and arginase by 2-imino-1,2-fluorenoquinone. J. Biol. Chem., 238:2206â€”2212,1963.

29. King, C. M., and Kriek, E. The differential reactivity of the oxidation productsof o-aminophenols towards protein and nucleic acid. Biochim. Biophys.Acta,111: 147â€”153,1965.

30. King, C. M., and Phillips, B. N-Hydroxy-2-fluorenylacetamide. Reaction ofthe carcinogen with guanosine, ribonucleic acid, deoxyribonucleic acid, andprotein following enzymatic deacetylation or esterification. J. Biol. Chem.,244: 6209-6216, 1969.

3%,.Kriek, E. Carcinogenesis by aromatic amines. Biochim. Biophys. Acta, 355:177â€”203,1974.

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756 CANCERRESEARCHVOL. 40

proximate form of the carcinogen. Similarly, N-hydroxybenzidine or N-hydroxy-N'-acetylbenzidmne may represent an ultimate carcinogen for the human urinary bladder. Studies arecurrently in progress to test this hypothesis.

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757MARCH1980



1980;40:751-757. Cancer Res Kenneth C. Morton, Frederick A. Beland, Frederick E. Evans, et al. Hepatic Sulfotransferase

-diacetylbenzidine by′N,N-Hydroxy-NMetabolic Activation of

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