Methyl-4-Aminoazobenzene and Other N -Hydroxy- N Hepatic

[CANCER RESEARCH 36, 2350-2359, July 1976] Hepatic Metabolism of N-Hydroxy-N-methyl-4-aminoazobenzene and Other N-Hydroxy Arylamines to Reactive Sulfuric Acid Esters

Fred F. Kadlubar, 2 James A. Miller, and Elizabeth C. Miller

McArd/e Laboratory for Cancer Research, University of Wisconsin Medica/ Center, Madison, Wisconsin 53706

SUMMARY scribed PAPS-dependent reaction, were detected in rat liver. Hepatic cytosols catalyzed a 3'-phosphoadenosine 5'- phosphosulfate (PAPS)-dependent O-sulfonation of N-hydroxy-N-methyl-4-aminoazobenzene (N-HO-MAB) and ot INTRODUCTION several other N-hydroxy arylamines. The presumed product from N-HO-MAB, N-methyl-4-aminoazobenzene-N-sulfate, The metabolic N-oxidation of carcinogenic arylam- reacted with added guanosine to yield N-(guanosin-8-yl)-N- ines and arylamides appears to be a necessary step in their methyl-4-aminoazobenzene, with methionine to form a sul- conversion to ultimate carcinogenic derivatives (48). Two fonium derivative that decomposed to yield 3-methylmer- hepatic microsomal enzymes, a cytochrome P-450-depend- capto-N-methyl-4-aminoazobenzene, and with ribosomal ent oxidase (21, 33, 69, 73) and a flavoprotein amine oxi- RNA to give a bound derivative. N-Methyl-4-aminoazoben- dase (33, 77, 78), have been implicated as catalysts for these zene was converted to N-(guanosin-8-yl)-N-methyl-4-ami- oxidations. The N-hydroxy amines and N-hydroxy amides noazobenzene in concerted N-oxidation and O-sulfonation are then subject to further hepatic metabolism. A NADH-de- reactions conducted aerobically with a fortified 10,000 • g pendent microsomal N-hydroxy amine reductase and a rat liver supernatant. In the absence of an added nucleo- NADPH- and oxygen-dependent microsomal N-hydroxy phile, metabolically formed N-methyl-4-aminoazobenzene- amine oxidase catalyze the reduction and oxidation, re- N-sulfate (or the nitrenium ion from this unstable ester) was spectively, of a number of N-hydroxy amines (32, 34, 53). A reduced by N-HO-MAB to form N-methyl-4-aminoazoben- microsomal deacetylase for N-HO-AAF 3 has been demon- zene; the N-HO-MAB was oxidized, probably through a ni- strated in the livers of a number of species (26). trone intermediate, to yield products that included N-hy- Hepatic cytosols from rats and some other species droxy-4-aminoazobenzene and formaldehyde. An analo- contain N-hydroxy arylacylamide-dependent N-hydroxy gous reaction .was noted between N-benzoyloxy-N-methyl- arylamine acyltransferase (6, 7, 36), ATP-dependent seryl- 4-aminoazobenzene and N-HO-MAB in the absence of transferase (66, 67), and PAPS-dependent N-hydroxy amide cytosol and PAPS. sulfotransferase (16, 37, 64) activities. These enzymes Hepatic N-HO-MAB sulfotransferase activities were in the catalyze the conversion of certain N-hydroxy arylamines order: male rat > female rat, male rabbit, male guinea pig, and N-hydroxy arylamides to their reactive acetic acid, male mouse > male hamster. Male'rat kidney and small serine, and sulfuric acid esters, respectively. The highly intestine cytosols had low activities; the other tissues stud- reactive and mutagenic sulfuric acid ester of N-HO-AAF is ied had little or no activity. Hepatic sulfotransfera'~e activi- strongly implicated as an ultimate carcinogenic metabolite ties for N-HO-MAB and N-hydroxy-N-acetyl-2-aminoflu- of AAF in rat liver (16, 17, 23, 41, 74). Similarly, reactive orene displayed different pH optima and inhibitor and acti- ester metabolites of other N-hydroxy compounds have been vator responses. considered as ultimate carcinogens (48). The NADPH- The rates of PAPS-dependent rat liver cytosol-catalyzed dependent reduction of N-hydroxy-2-naphthylamine (52) esterification of N-hydroxy-N-ethyl-4-aminoazobenzene, N- and an S-adenosylmethionine-dependent O-methylation of hydroxy-4-aminoazobenzene, and N-hydroxy-1- and 2- N-HO-AAF (39) by hepatic cytosols have also been reported. naphthylamine were 20 to 50% of that for N-HO-MAB. Ac- This communication reports the PAPS-dependent esteri- tivities for trans-N-hydroxy-4-aminostilbene, N-hydroxy-2- fication of N-HO-MAB, a metabolite of the hepatocarcino- aminofluorene, N-hydroxyaniline, and N-hydroxy-N-methyl- gen MAB (33), and of several other carcinogenic and non- N-benzylamine were not detected. carcinogenic N-hydroxy arylamines. Some enzymatic prop- No microsomal reduced nicotinamide adenine dinucleotide-dependent reduction or reduced nicotinamide adenine 3The abbreviations used are: N-HO-AAF, N-hydroxy-N-acetyl-2-aminofluorene; PAPS, 3'-phosphoadenosine5'-phosphosulfate; AAF, N-acetyl-2-ami- dinucleotide phosphate-dependent oxidation or cytosolic nofluorene; N-HO-MAB, N-hydroxy-N-methyl-4-aminoazobenzene;MAB, N- transferase reactions for N-HO-MAB, except the above-de- methyl-4-aminoazobenzene;Bis-Tris, bis(2-hydroxyethyl)-Tris; PAP, 3'-phosphoadenosine 5'-phosphate; N-HO-AB, N-hydroxy-4-aminoazobenzene;N- HO-EAB, N-hydroxy-N-ethyl-4-aminoazobenzene;N-(guanosin-8-yl)-MAB, N- 1This research was supported in part by funds from USPHS Grants CA- (guanosin-8-yl)-N-methyl-4-aminoazobenzene; 3-CHsS-MAB, 3-methylmer- 07175 and CA-15785. capto-N-methyl-4-aminoazobenzene; MAB-N-sulfate, N-methyl-4-aminoazo- 2 Postdoctoral Fellow of the Foundation for Cancer Research. Chicago. benzene-N-sulfate; N-benzoyloxy-MAB, N-benzoyloxy-N-methyl-4-amino- Received January 26, 1976; accepted April 7, 1976. azobenzene.

2350 CANCER RESEARCH VOL. 36

erties of the sulfotransferase(s) and the chemical reactivi- or diluted in absolute ethanol to a concentration of 25 mM ties of the metabolically formed esters were examined. and was added to the enzyme incubation mixture at a final concentration of 0.5 mM. The oxidative decomposition of MATERIALS AND METHODS the N-hydroxy arylamines in the reaction media was mini- mized by the inclusion of 0.5 mM EDTA which prevented Materials. NAD § NADP +, glucose 6-pl~osphate, Bis-Tris, metal-catalyzed autoxidation (31) and/or lipid peroxidation ATP, 3',5'-ADP(PAP), p-nitrophenol, o-iodosobenzoate, S- (29). This low level of EDTA stabilized the N-hydroxy deriva- adenosylmethionine iodide, S-acetyI-CoA, catalase (type tives even in assay media that required high concentrations C-40), and serum albumin (type V) were purchased from the of Mg 2+. Assays that did not require oxygen were carried Sigma Chemical Company (St. Louis, Mo.). NADPH and out in an argon atmosphere to minimize further nonenzy- NADH were obtained from P-L Biochemicals, Inc. (Milwau- matic oxidation of these compounds. Unless otherwise indi- kee, Wis.). Glucose-6-phosphate dehydrogenase (Code ZF) cated, hepatic cell fractions from adult male rats (age, 4 to 6 was purchased from Worthington Biochemicals (Freehold, months) were used as enzyme sources. N. J.). rRNA and PAPS were prepared from rat liver by the Microsomal N-hydroxy amine oxidase and reductase methods of Irving and Veazey (28) and Irving et al. (27), assays were carried out as previously reported (32). Unless respectively. Radioactive materials were obtained as fol- otherwise indicated, the assay media for measuring cyto- lows: [8-14C]guanosine, Schwarz/Mann (Orangeburg, N. Y.); solic S-adenosylmethionine-dependent O-methylase (39), [8J4C]adenosine, Amersham/Searle Corp. (Arlington N-hydroxy arylacetamide-dependent acetyltransferase (7), Heights, II1.); [2,6-14C]uridine, Nuclear Dynamics (El Monte, ATP-dependent seryltransferase (66), and NADPH-depend- Calif.); and [2,6-~4C]thymidine, New England Nuclear (Bos- ent N-hydroxy arylamine reductase (52) were used as ton, Mass.). [G-3H]-N-Hydroxy-2-naphthylamine (110 mCi/ described in the references. mmole) was prepared from [G-3H]-2-nitronaphthalene Attempts to detect acetyI-CoA-dependent metabolism in (Amersham/Searle custom tritiation) (75). [G-'3H]-N-HO - N-HO-MAB were carried out in assay media that contained MAB was synthesized as described previously (33). 100 mM Tris-HCI buffer (pH 7.3 at 37~ 0.5 mM EDTA, 1 mM The following amine derivatives were synthesized by pub- acetyI-CoA, 20 to 100 mM methionine, 3 mg cytosol protein lished procedures: N-hydroxy-N-methyI-N-benzylamine per ml, and 0.5 mM N-HO-MAB. (20), N-hydroxyaniline (35), N-HO-AB (56), N-HO-MAB and The assay medium that was found to be optimal for hepa- N-HO-EAB (33), N-hydroxy-2-aminofluorene (40), N-HO- tic N-hydroxy arylamine sulfotransferase activity contained AAF (50), trans-N-hydroxy-4-aminostilbene (1), N-hydroxy- 100 mM Bis-Tris-HCI buffer (pH 6.2 at 37~ 5 mM MgCI.,, 0.5 4-aminobiphe~yl (42), N-hydroxy-1- and 2-naphthylamine mM EDTA, 1 mM PAPS, 0.5 to 5.0 mg cytosol protein per ml, (75), 2-amino-l-naphthylsulfate (13), 2,2'-azoxynaphthalene and the specified N-hydroxy amine (0.5 mM). The assay (43), MAB (46), 3-methylmercapto-4-aminoazobenzene and medium that was optimal for N-HO-AAF sulfotransferase its N-methyl derivative (57), and 3-methylmercapto-4-ami- contained 100 mM Bis-Tris-HCI buffer (pH 6.6 at 37~ 5 mM nobiphenyl (16). 3-Methylmercapto-N-ethyl-4-aminoazo- MgCI2, 0.5 mM EDTA, 0.65 mM PAPS, 0.2 to 1.0 mg cytosol benzene was synthesized by the procedure described for protein per ml, and N-HO-AAF (0.5 mM). When compared the synthesis of its N-methyl analog (57); after purification directly with the assay medium described by DeBaun et by chromatography on alumina and crystallization (m.p. al. (16), the above medium yielded 10 to 20% higher activi- 38~ it eluted as a single peak on gas-liquid chromatogra- ties. L-Methionine (100 mM for N-hydroxy amine assays; 20 phy and had the expected mass spectrum (M + = 271). mM for N-HO-AAF assays), [8-14C]guanosine (1 mM, 4 mCi/ Analyses for each element were within 0.2% of theory (Huff- mmole), or rRNA (6.7 mg/ml) was routinely added to the man Laboratories, Wheatridge, Colo.). 3-Methylmercapto- sulfotransferase assays to trap the enzymatically generated 2-aminofluorene and 3-methylmercapto-N-acetyl-2-amino- sulfuric acid esters. fluorene were generously supplied by Dr. To L. Fletcher (The Each of the above assays was carried out in the presence Fred Hutchinson Cancer Research Center, Seattle, Wash.). or absence of the coenzymes and with intact or heat- Microsomal and cytosolic cell fractions were obtained by denatured (85 ~ for 3 min) cell fractions so that coenzyme- differential centrifugation according to the principles out- and enzyme-dependent metabolism could be ascertained. lined by Hogeboom et al. (25). Livers were perfused in situ Chemical Assays. The concentrations of the N-hydroxy with 0.9% NaCI solution prior to homogenization. Adult amines in aliquots of the incubation mixtures were deter- animals were obtained as follows: CD random-bred rats and mined colorimetrically as n-amyl acetate-extractable Fe 3+- CD-t mice, Charles River Breeding Laboratories (Wilming- reducing equivalents. Aliquots (0.5 ml) were agitated on a ton, Mass); rabbits, Sand Valley Farms (Spring Green, vortex mixer with 3 ml of water-saturated n-amyl acetate, Wis.); guinea pigs, O'Brien Co. (Madison, Wis.); and ham- and the phases were separated by centrifugation. The con- sters (Con Olson Co. (Madison, Wis.). The rats, mice, and centrations of the N-hydroxy secondary amines (N-HO- hamsters were fed Wayne Breeder Blox pellets (Allied Mills, MAB, N-HO-EAB, N-hydroxy-N-methyI-N-benzylamine)in Inc., Chicago, II1.); the rabbits and guinea pigs were given the amyl acetate extracts were determined by a method Teklad Guinea Pig Diet (Teklad Mills, Winfield, Iowa). described for the estimation of "lipid-soluble" N-hydroxy Enzyme Assays. The enzyme-catalyzed reactions of N- amines (32), except that, in order to stabilize the chromo- HO-AAF, N-HO-MAB, and other N-hydroxy arylamines were phore, 0.05 ml of 1.7 M acetic acid was added after the measured by monitoring the loss of these substrates from specified addition of EDTA. the assay media. Each N-hydroxy compound was dissolved The concentrations of N-hydroxy primary arylamines and

JULY 1976 2351

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1976 American Association for Cancer Research. F. F. Kadlubar et al. phenolic amines in the amyl acetate extracts were mea- was measured by the method of Nash (49), except that prior sured by a modification of a method developed for the extraction with chloroform was required to remove dye estimation of tocopherols as Fe3§ equivalents derivatives; semicarbazide (1 mM) was included in the reac- (72), in which 0.2 ml of the amyl acetate extract was mixed tion mixture to obtain quantitative recovery of the formalde- with a solution that consisted of 0.1 ml of 0.4 M sodium hyde. acetate:0.6 M acetic acid, 0.6 ml of 95% ethanol, 0.2 ml of Products from the [G-3H]-N-hydroxy-2-naphthylamine 4,7-diphenyl-l,10-phenanthroline [10 mg/ml in anhydrous (110 mCi/mmole) sulfotransferase reaction were isolated amyl acetate:absolute ethanol (15:1)], and 0.04 ml of 0.01 M similarly by thin-layer chromatography on silica gel (East- Fe(NO~),~ :0 .1 M acetic acid; after 1 min the reaction was man 6060 sheets) with 1-butanol:l-propanol:0.1 M NH:~ terminated by addition of 0.04 ml of 0.02 M H:~PO4. The (2:1:1) and 1-butanol:acetic acid:H.,O (10:1:1) [Solvents (a) absorbance at 535 nm was 39,200 (2 equivalents of Fe 3+ and (m) of Boyland and Manson (11)] and with Solvents A reduced per equivalent of N- or o-hydroxy amine) for each and F (33). of the following compounds: N-HO-AB, N-hydroxyaniline, Instrumentation. Mass spectra were obtained with a Var- N-hydroxy-1- and 2-naphthylamine, N-hydroxy-4-aminobi- ian CH-7 mass spectrometer (Varian Associates, Palo Alto, phenyl, N-hydroxy-2-aminofluorene, N-hydroxy-4-amino- Calif.) equipped with a mass marker and a magnet-driven stilbene, 2-amino-l-naphthol, and o- and p-aminophenol. If direct insertion probe (Variset Corp., Madison, Wis.). Elec- reducing compounds are formed from a N-hydroxy amine tronic spectra were measured with a Zeiss PMQ II or Beck- substrate during an enzyme-catalyzed reaction, this assay man DB spectrophotometer. 3H was determined in Scintisol can provide only a minimum estimate of the amount of N- (Isolab, Inc., Akron, Ohio) with a Packard Tri-Carb scintilla- hydroxy amine metabolized. tion spectrometer. Gas chromatography was performed N-HO-AAF was determined spectrophotometrically as its with a Barber-Colman Model 10 chromatograph equipped ferric chelate as described by Booth and Boyland (9). Pro- with a 0.6- x 120-cm column packed with 3% OV-1 on tein was determined by the biuret reaction (22). Chromsorb W/HP (Pierce Chemical Co., Rockford, III.). Reactivity Studies and Product Identification. When methionine was used to trap reactive metabolites, the methionyl derivatives of the aminoazo dyes were decomposed RESULTS and analyzed as 3-methylmercapto derivatives as described by Poirier et ah (51), except that the yield of the methylmer- Microsomal N-Hydroxy Arylamine Metabolism capto compounds was increased 30 to 50% by decomposition of the sulfonium adduct in alkali at 90 ~ for 45 min. The In view of the metabolic formation of N-hydroxy amines identification of 3-CH:3S-MAB (M + = 257) from N-HO-MAB by the endoplasmic reticulum of liver cells, the ability of sulfotransferase reaction mixtures was confirmed by gas male rat liver microsomes to metabolize certain N-hydroxy chromatography-mass spectrometry. The sulfonium deriva- arylamines was examined. Under the assay conditions used tive of 4-aminobiphenyl was decomposed and analyzed as (cf. "Materials and Methods"), the NADH-dependent reduc- 3-methylmercapto-4-aminobiphenyl by gas-liquid chroma- tion of N-hydroxy-N-methyI-N-benzylamine and N-hydroxy- tography as previously described (7). aniline occurred at rates of 8.6 -+ 0.8 and 2.1 +-- 0.5 nmoles/ The formation of [8-1"C]-N-(guanosin-8-yl)-MAB in N-HO- min/mg microsomal protein (n = 3). On the basis of analy- MAB sulfotransferase assays that contained [8-1"C]guan - ses for extractable reducing equivalents, no NADH-depend- osine was measured by thin-layer chromatography (Ref. ent loss of N-HO-MAB or N-HO-EAB was observed, and 38, Solvents A to D) and liquid scintillation spectrophotom- these compounds were recovered quantitatively from the etry. Similar incubation mixtures that contained other assay medium with incubation periods of up to 6 min. [14C]nucleosides were chromatographed by the procedures Incubation for longer times resulted in significant losses, of Poirier et al. (51). When the N-hydroxy amine sulfo- but similar losses occurred with heat-denatured micro- transferase assays contained rRNA, the recovery and esti- somes. Similarly, 93 to 98% of the N-hydroxy derivatives of mation of RNA-bound substrate were carried out according 4-aminoazobenzene, 4-aminobiphenyl, 2-aminofluorene, 4- to the method of Wislocki et ah (76). aminostilbene, and 1- and 2-naphthylamine were recovered For structural identification the products formed during as extractable reducing equivalents after 6-min incubations the PAPS-dependent esterification of N-HO-MAB were iso- with NADH-fortified microsomes. With fortified heat-dena- lated by extraction 3 times with 2 volumes of ethyl acetate, tured microsomes only 20 to 50% of each N-hydroxy pri- concentration of the extract under reduced pressure, and mary arylamine was recovered under the same conditions. thin-layer chromatography of the concentrate in Solvents A, The much higher recoveries of these compounds from incu- B, and F (33). After elution the products were analyzed by bations with fresh microsomes may be due to a reduced mass spectrometry. Quantitation of these products was pyridine nucleotide-dependent enzymatic reduction of achieved with the use of [3H]-N-HO-MAB (503 mCi/mmole) aerobic oxidation products (nitroso derivatives?) of the as follows. A 0.5-ml aliquot of the incubation medium was N-hydroxy amines (54). mixed with an equal volume of 95% ethanol that contained Under the standard conditions (pH 7.6 in air, cf. "Mate- carrier dyes (0.01 M MAB, N-HO-MAB, and N-HO-AB); after rials and Methods") for measuring microsomal NADPH-de- centrifugation 2 /~1 were analyzed as previously described pendent N-hydroxy amine oxidase activity, the N-hydroxy (33) by thin-layer chromatography in Solvents A, B, and F aminoazo dyes oxidized rapidly (20 to 50 nmoles/min/ml of and liquid scintillation spectrophotometry. Formaldehyde reaction mixture) even in the absence of NADPH and/or

2352 CANCER RESEARCH VOL. 36

microsomes. Thus, if an NADPH-dependent enzymatic oxi- Neither ATP nor PAP could substitute for PAPS, and heat- dation occurred, it could not be detected under these con- denatured cytosol did not support the reaction. A low PAPS- ditions. The rapid air oxidation of N-hydroxy amines under independent loss of N-HO-MAB was achieved only with aerobic conditions (pH > 7) has been studied (31,33). cytosols from 0.9% NaCI solution-perfused livers. The loss of N-HO-MAB was 5 to 10 times greater in cytosols from Cytosolic N-Hydroxy Arylamine Metabolism nonperfused livers, and variable amounts of heme or Fe 2§ in these cytosols may have promoted the rapid decomposition In preliminary experiments with liver cytosol from male of the N-HO-MAB (31). Omission of methionine from the rats no coenzyme-dependent loss of N-HO-MAB or N-HO- assay medium did not affect the PAPS-dependent loss of N- EAB was detected (<0.1 nmole/min/mg cytosol protein) HO-MAB, but addition of methionine was essential to the using assay media described (cf. "Materials and Methods") formation of a sulfonium-dye conjugate. Protein (added for N-hydroxyaryl acetamide- or acetyI-CoA-dependent O- serum albumin) apparently competed with methionine for acetylation, ATP-dependent O-serylation, S-adenosylme- the reactive ester. thionine-dependent O-methylation, or NADPH-dependent re- Experiments with N-HO-MAB and other N-hydroxy arylam- duction of certain N-hydroxy amines or N-hydroxy amides. ines indicated that the optimal conditions for N-hydroxy Furthermore, the formation of a methionyl-dye adduct was amine sulfotransferase activity differed from those for sulfo- not detected (<0.01 nmole/min/mg protein) in methionine- transferase activity for N-HO-AAF or 3-hydroxyxanthine (16, supplemented O-acetylation or O-serylation incubation mix- 64); the major differences were the optimal pH and the tures. Attempts to detect these latter activities at other pH's protein and PAPS concentrations. The optimal assay condi- (6.2, 6.6, 7.0), with substrate concentrations up to 0.5 mM, tions for sulfotransferase activity for N-HO-MAB and N-HO- or with protein concentrations of 1 to 5 mg/ml were unsuc- AAF (cf. "Materials and Methods") were defined after test- cessful. However, PAPS-dependent losses of N-HO-MAB of ing different methods of rat hepatic cytosol preparation greater than 10% occurred under the assay conditions used (with or without liver perfusion, various homogenization to demonstrate the formation of the sulfuric acidQ ester of N- media, gel filtration of the cytosol), incubation times, pH, HO-AAF (16), and 3-CH:}S-MAB was recovered when methio- buffer compositions, and the concentrations of Mg 2+, nine was included in the incubation medium. Similar results EDTA, PAPS, substrate, substrate solvent (ethanol, ace- were obtained with several other N-hydroxy arylamines. tone, or dimethyl sulfoxide), and cytosol protein. Under the These experiments suggested that rat hepatic cytosol con- optimal assay conditions the PAPS-dependent loss of N-HO- tains enzyme(s) that catalyze the PAPS-dependent esterifi- MAB and the formation of 3-(methion-S-yl)-N-methyl-4- cation of N-hydroxy amines, and the properties of this enzy- aminoazobenzene (determined as 3-CH:,S-MAB) were 1st matic activity are described in the following section. order with respect to protein concentration up to 5 mg/ml and linear with time for 10 to 15 min. The loss of N-HO-AAF N-Hydroxy Amine Sulfotransferase Activity and the formation of 1- and 3-(methion-S-yl)-N-acetyl-2-aminofluorene (as o-methylmercapto-N-acetyl-2-aminoflu- Assay Requirements and Comparisons with N-HO-AAF Sul- orene) were 1st order with protein concentration up to 1 fotransferase Activity mg/ml and linear with time for 30 to 60 min. Inhibition by PAPS occurred at concentrations above 1.0 and 0.65 mM, The loss of N-HO-MAB and formation of 3-CH.~S-MAB respectively, for N-HO-MAB and N-HO-AAF sulfotransferase from methionine-supplemented incubation mixtures were activities. dependent on the presence of PAPS and cytosol (Table 1). The pH optima for the cytosol-catalyzed PAPS-dependent losses of N-HO-MAB and N-HO-AAF, as well as for the Table 1 formation of their sulfonium derivatives, were approxi- Rat hepatic N-HO-MAB sulfotransferase: assay requirements mately 6.2 and 6.6, respectively (Chart 1). The pH versus Relative rates"

Formation Loss of N- of 3-CH:~S- LOSS ? Incubation mixture HO-MAB MAB 9 FORMATION .... %0 Complete system 100 100 With heat-denatured cytosol 5 <1 - PAPS 8 <1 - PAPS + PAP (1 mM) 10 <1 - PAPS + ATP (1 mM) 12 <1 - Methionine 101 <1 + Serum albumin (5 mg/ml) 101 59 " Assays were carried out as described in "Materials and Meth- ods" with 5 to 10 different preparations of hepatic cytosol from 0 |0 LO male rats; the rates are expressed relative to that of the complete , 4.5 ~.o ~.5- 6'.o 1.5 7'.o 7'.~ 8.o system. The mean N-HO-MAB sulfotransferase activity of the com- z plete system was 4.7 + 1.6 nmoles N-HO-MAB metabolized per min pH 01 37 ~ per mg protein (n = 10). The formation of 3-CH:~S-MAB by the Chart 1. N-HO-MAB and N-HO-AAF sulfotransferase activities in rat he- complete system averaged 0.61 - 0.05 nmole/min/mg protein (n = patic cytosol, pH vs. rate profile. Rates are expressed as nmoles/min/mg 10). cytosol protein, o-CH3S-AAF, o-methylmercapto-N-acetyl-2-aminofluorene.

JULY 1976 2353

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1976 American Association for Cancer Research. F. F. Kadlubar et al. rate profile for N-HO-AAF sulfotransferase activity is similar Table 2 to that described by DeBaun et al. (16). Effects of various agents on rat hepatic N-HO-MAB and N-HO-AAF Previous studies on the sulfonation of phenols, amines, sulfotransferase activities and steroids have demonstrated that several cytosolic pro- Relative rates of forma- teins catalyze PAPS-dependent sulfonation reactions (19). tion ~ These sulfotransferases have multiple and overlapping sub- o-Methyl- strate affinities and differ in their pH optima and sensitivity mercapto- to inhibitors and activators. A "phenol" sulfotransferase N-acetyl-2- that reportedly esterifies only phenols is unaffected by 3-CH3S- aminoflu- added Mg 2§ has optimal activity at pH 5.8, and, depending Reaction conditions" MAB orene on the thiol content of the enzyme preparation, is activated Complete system 100 100 or inhibited by o-iodosobenzoate (3, 4, 15, 45). Another - Mg =+ 78 86 sulfotransferase that acts on androgens, cholesterol, 2- + o-lodosobenzoate (0.25 mM) 42 4 + 2-Naphthylamine (2.0 mM) 23 33 naphthylamine, p-nitrophenol, and L-tyrosine methyl ester + p-Nitrophenol (1.0 mM) 1 2 is stimulated by Mg 2+, has optimal activity at pH 7.0 to 7.5, + Dehydroepiandrosterone (0.1 mM) 98 128 and is inhibited by o-iodosobenzoate (3, 5, 15). A 3rd sulfo- + Estrone (0.1 mM) 113 120 transferase that acts on estrogens, 2-naphthylamine, and + Deoxycorticosterone (0.1 mM) 132 138 + L-Tyr0sine methyl ester (3 mM) 104 108 p-nitrophenol is stimulated by Mg2+; it has optima! activity + N-HO-AAF (0.5 mM.) 139 at pH 6.2 for estrogens and 2-naphthylamine and at pH 7.0 + N-HO-MAB (0.5 mM) 13 for p-nitrophenol (3, 19). Sulfonation of 2-naphthylamine is " The incubation medium was a compromise between the opti- stimulated by addition of steroids while steroid sulfonation mal systems for N-HO-MAB and N-HO-AAF and contained 100 mM is inhibited by added 2-naphthylamine (3, 55). Another Bis-Tris-HCI buffer (pH 6.4 at 37~ 100 mM methionine, 5 mM MgCI._,, steroid sulfotransferase is thought to catalyze the esterifi- 0.5 mM EDTA, 0.65 mM PAPS, rat liver cytosol protein (1 mg/ml), cation of deoxycorticosterone, but no further characteriza- and 0.5 mM N-HO-MAB or N-HO-AAF. Incubation time was 15 min. "Assays were carried out as described in "Materials and Meth- tion has been made (3). An o-iodosobenzoate-sensitive ods" using 4 to 6 different cytosol preparations; the rates are ex- sulfotransferase with activity toward L-tyrosine methyl pressed relative to the control. The mean rates of methylmercapto- ester, tyramine, and p-nitrophenol has also been described arylamine formation in the complete system were 0.31 +- 0.03 and (5, 44). 6.1 - 0.5 nmoles/min/mg (n = 6) for N-HO-MAB and N-HO-AAF, respectively. Similar relative rates were obtained under the various As an aid in evaluating the roles of the various sulfotrans- reaction conditions by measuring the PAPS-dependent loss of ferases in the esterification of N-HO-AAF and N-HO-MAB, N-HO-MAB or N-HO-AAF. the effects of several inhibitors and activators on these reactions were investigated. The assays were carried out "phenol" sulfotransferase, while the activity for N-HO-AAF either under optimal conditions for N-HO-MAB (1 mM PAPS, exhibits properties similar to those described for both pH 6.2) and for N-HO-AAF (0.65 mM PAPS, pH 6.6) or under phenol and steroid sulfotransferases. intermediate conditions (0.65 mM PAPS, pH 6.4), so that both activities could be measured on a comparable basis Reactivity of MAB-N-sulfate (Table 2). At pH6.4, omission of Mg 2+ decreased the activity toward both N-HO-MAB and N-HO-AAF by 10 to 20%. At pH A relatively high concentration of methionine (100 mM) 7.0, the effect on N-HO-AAF activity was more pronounced; was required to trap appreciable amounts of the sulfuric the rate in the absence of added Mg 2+ was only 50 to 60% of acid ester of N-HO-MAB, and under these conditions the the control level. These differences may reflect the catalytic formation of a sulfonium dye-conjugate accounted for only participation of several sulfotransferases and/or the inhibi- 5 to 15% of the PAPS-dependent loss of N-HO-MAB (Chart tory effect of Mg 2+ on PAPS-sulfohydrolase (18). o-lodoso- 2). With N-HO-AAF as substrate 20 mM methionine trapped benzoate inhibited both N-HO-MAB and N-HO-AAF sulfo- 65 to 85% of the reactive ester as a methionyl derivative. transferase activity, but it was a much more effective inhibi- Addition of [8-1"C]guanosine (optimal concentration, 0.8 tor of the latter activity. Both 2-naphthylamine and p-nitro- to 1.2 mM) to the sulfotransferase incubation medium phenol inhibited the esterification of N-HO-MAB and N-HO- yielded a [8-14C]guanosinyl adduct that accounted for 6 to AAF. Dehydroepiandrosterone, estrone, and deoxycorticos- 12% of the N-HO-MAB metabolized. The adduct was identi- terone increased sulfotransferase activity for N-HO-AAF by fied as [8-14C]-N-(guanosin-8-yl)-MAB on the basis of its 20 to 40%, while only estrone and deoxycorticosterone ap- chromatographic properties in 4 thin-layer systems (38). peared to increase sulfotransferase activity for N-HO-MAB. Similar sulfotransferase incubation mixtures that contained L-Tyrosine methyl ester did not significantly alter either N- 1 mM [~4C]adenosine, [14C]cytidine, [~4C]uridine, or [~4C]- HO-MAB. or N-HO-AAF sulfotransferase activity. The addi- thymidine yielded no significant amounts of nucleoside-dye tion of N-HO-AAF to assays for N-HO-MAB sulfotransferase adducts (<0.5% of the N-HO-MAB metabolized). This result activity caused a 40% stimulation, while N-HO-MAB was was consistent with the lack of detectable reactivity of these strongly inhibitory to N-HO-AAF sulfotransferase activity. nucleosides with the synthetic ester N-benzoyloxy-MAB Although several sulfotransferases may catalyze the esterifi- (51). The sulfuric acid ester of N-HO-MAB also reacted with cation of both N-HO-MAB and N-HO-AAF, the activity for N- added rRNA (6.7 mg/ml), but only 1 to 3% of the N-hydroxy- HO-MAB appears to be similar to that described for amine metabolized was recovered as an rRNA adduct.

2354 CANCER RESEARCH VOL. 36

Tissue, Sex, and Species Distribution 1 and 5). Thus, the lower N-HO-MAB activities shown in Tables 3 and 4 reflect this age variation. Of the extrahepatic tissues studied only the cytosol fractions of rat kidney and small intestine contained detectable Substrate Specificity sulfotransferase activity for N-HO-MAB (Table 3). These activities are no more than 20% of that found in hepatic N-HO-EAB, N-HO-AB, N-hydroxy-1-and 2-naphthyla- cytosol, but a comparable level of sulfotransferase activity mine, and N-hydroxy-4-aminobiphenyl also appeared to be for N-HO-AAF was not detected in rat kidney or small intes- converted to reactive esters by rat hepatic cytosol and tine (16). No activity for N-HO-MAB was detected in rat PAPS (Table 5). The PAPS-dependent metabolism of each muscle or lung cytosols. of these N-hydroxy amines was optimal under the assay The highest hepatic sulfotransferase activity for N-HO- conditions.for N-HO-MAB sulfotransferase rather than those MAB was found in male rats. Female rat liver and the livers for N-HO-AAF esterification. In the absence of PAPS, 80 to from males of the other species studied, except the ham- Table 4 ster, had at least 25% of the activity of male rat liver (Table N-HO-MAB sulfotransferase activity in hepatic cytosols from 4). N-HO-AAF sulfotransferase activity has been detected various species only in rat and rabbit liver cytosols (16). PAPS-dependent Young adult animals were used in these comparative loss of N-HO-MAB studies. In young rats hepatic N-HO-MAB sulfotransferase No. of ani- (nmoles/min/mg activity was consistently lower than in older rats (cf. Tables Species Sex mals protein)" Rat M 10 3.6 - 1.8~' I.O- -5.0 Rat F 5 i.1 _+ 0.5 Rabbit M 3 1.9 -+ 0.5 Guinea pig M 3 1.3 + 0.3 z o Mouse M 3 1.1 _ 0.1 o o.8- ~4.0 Hamster M 3 0.2 - 0.03 I-- -r ,~ oJ " See "Materials and Methods" for assay procedure. For the livers from each species at least 2 reactions were carried out in the o 0.6- -3.0 la.. -3'i presence of methionine. In each case the amount of 3-CH:~S-MAB A that could be isolated at the end of the incubation period was 0 [] equivalent to 5 to 10% of the PAPS-dependent loss of N-HO-MAB. m 0.4- -2.0 -,1 t, Mean _+ S.D. < O m Table 5 Substrate specificity of rat hepatic N-hydroxy amine sulfotransferase(s) PAPS-de- ! " 0 pendent loss % isolable o 2'0 40 eo ,oo of substrate as methyl- METHIONINE (raM) (nmoles/min/ mercapto- Substrate mg protein)" arylamine b Chart 2. Effect of methionine on methylmercaptoarene formation from N- HO-MAB and N-HO-AAF. Rates are expressed as nmoles methylmercaptoar- N-HO-MAB 4.8 _+ 1.8" 5-20 erie isolated per rain per mg cytosol protein, o-CH~-S-AAF, o-methylmer- N-HO-EAB 2.2 -+ 0.6 8-12 capto-N-acetyl-2-aminoftuorene. N-HO-AB 1.9 _+ 0.6 1-3 N-Hydroxy-l-naphthylamine 2.4 _ 1.0 -' Table 3 N-Hydroxy-2-naphthylamine 1.4 __+ 0.3 -" N-HO-MAB sulfotransferase activity in various tissues from male N-Hydroxy-4-aminobiphenyl 1.1 + 0.4 25-40 rats trans-N-Hyd roxy-4-am i n osti I be n e, <0.1 -' N-hyd roxy-2-aminofluorene, N- PAPS-dependent loss of N- hydroxyaniline, and N-hydroxy- HO-MAB (nmoles/min/mg N-methyI-N-benzylamine Tissue protein)" " Assays were carried out as described in "Materials and Meth- Liver 2.7 _+ 0.5~,' ods." Kidney 0.5 - 0.3b b The trapping efficiencies differed between liver cytosols, possi- Small intestine mucosa 0.5 _ 0.3~ bly because of variations in the amounts of endogenous nucleo- Muscle <0.2" philes. Lung <0.2' e Mean - S.D. for analyses of 5 to 10 different cytosol prepara- " Assays were carried out with the cytosol fraction of each tissue tions. The wide standard deviations reflect the variations in activity homogenate, and the values reported represent determinations on from one liver preparation to another. tissues from 3 animals. ' Not studied. b For each tissue at least 2 reactions were also carried out jn the The methylmercaptoarylamine was too labile to air oxidation to presence of methionine. In each case the amount of 3-CH3S-MAB permit its isolation and estimation by chromatography. Therefore, that could be isolated at the end of the incubation period was rRNA was used as a trapping agent; 0.2 to 0.3% of the PAPS- and equivalent to 5 to 10% of the PAPS-dependent loss of N-HO-MAB. cytosol-dependent loss of [:~H]-N-hydroxy-2-naphthylamine was ac- " Mean _ S.D. counted for as a rRNA-bound adduct. By comparison, 1 to 3% of a These values were judged to be the lower limit of detection. the N-HO-MAB metabolized was recovered as rRNA-bound dye.

JULY 1976 2355

100% of each N-hydroxy amine added could be recovered amines such as N-HO-MAB are strong reducing agents. The after the 10-min incubation period; the low PAPS-independ- latter property may account for the apparent reduction of ent losses were presumed to be nonenzymatic. When me- the aminoazo nitrenium ion to MAB and the concomitant thionine or rRNA was added to the complete assay media, formation of oxidation products (N-HO-AB and nitrone-de- methionyl or rRNA adducts were formed. rived adducts) of N-HO-MAB. Reduction of acylaryl nitrenium ions by several reducing agents has been demonstrated Products Formed in the Sulfotransferase Incubation Me- (61, 65). This reaction sequence may account for the low dium yields of arylamine-nucleophile adducts formed from N- hydroxy amines, as compared to the high yields of such From N-HO-MAB. Incubation of N-HO-MAB in the sulfo- adducts obtained from esters of N-HO-AAF. transferase assay in the absence of added guanosine, Further support for this mechanism was obtained by incu- methionine, or RNA yielded the following products (ex- bation of 0.5/.Lmole of N-benzoyloxy-MAB with 0.5/~mole of pressed as the percentage of the N-HO-MAB metabolized): N-HO-MAB under the sulfotransferase assay conditions, ex- MAB (50 to 60%), N-HO-AB (25 to 35%), formaldehyde cept for the omission of PAPS and cytosol. Both of the (20 to 30%), and 2 unidentified products (15 to 25%). added dyes were consumed within 10 min, and 0.16/~mole MAB and N-HO-AB were identified by thin-layer chro- of MAB, 0.28 /~mole of N-HO-AB, 0.14 /~mole of formalde- matography, their UV absorption maxima in 95% ethanol hyde, and 5 to 6 other unidentified dye-containing products (402 and 375 nm, respectively), and their mass spectra (M + were obtained. Incubation of N-HO-MAB in the absence of = 211 and 213, respectively). Formaldehyde was character- N-benzoyloxy-MAB under these conditions yielded only the ized spectrally as the 2,4-pentanedione-ammonia adduct starting material, while incubation of N-benzoyloxy-MAB (~ ...... = 412 nm). The unidentified products exhibited ab- alone yielded several of the above unidentified dye prod- sorption maxima at 390 to 400 nm in 95% ethanol and at 520 ucts. to 530 nm in 40% ethanol:0.5 M HCI; these products had low From N-Hydroxy-2-naphthylamine. A similar reaction se- volatilities and did not yield molecular ions on electron quence appeared to occur when [3H]-N-hydroxy-2-naphthyl- impact mass spectrometry. These properties are consistent amine was incubated with PAPS and hepatic cytosol (Chart with those expected for a dinitrone or a nitrone-N-hydroxy 4), but the identification of the naphthalene derivatives was amine addition product. All of the above reaction products hampered by the relatively low rate of esterification of this were also obtained in assays supplemented with methionine N-hydroxy amine. The tentative identifications of the prod- or guanosine, but the yields were proportionately de- ucts are based on their chromatographic behaviors in 2 creased. thin-layer systems and the characteristic fluorescence of A reaction mechanism that is consistent with the above each product under 254 nm light (10). The formation of each results is presented in Chart 3. In view of the great lability of product was dependent on the presence of PAPS in the the N-sulfate of N-HO-AAF and some other esters of N- incubation mixture. 2-Amino-l-naphthyl sulfate accounted hydroxy metabolites (7, 41,48, 51, 60), the apparent initial for 30 to 35% of the substrate metabolized. 2-Naphthyla- product from N-HO-MAB, MAB-N-sulfate, is presumed to be rapidly converted to a reactive nitrenium ion. This ion could OH then bind to cellular nucleophiles (cf. Ref. 59). However, ~NH 2 * H(~+ HS04e unlike N-hydroxy amides (e.g., N-HO-AAF), N-hydroxy 2-AMINO-I- NAPHTHOL ~ - L ~ ~OS%H j\ '0'"I , so:. ~N:oH J 2-AMINO-I-NAPHTHYL- P"PSTcY'~176 SULFATET ~:'H~AB TM ~ TLREN~II U M ~ON N~H N'. \OH cytosol ) .-\'OSO3 r %N "CH2 ] A ~,-N "CH3 / | ~ ~o I ~ ",4 ~-~ "H 2"~MINE: ~ "~ ] N-(p-PHENYLAZOPHENYL)[ ...... L NITRONE j ...... - ..... + HSO~ HCHO ,~ H20 "%'"" (20-30 %)~ ~'~ OTHER DYE PRODUCTS H (15-25 %) N~OH y>; >, N-HO-AB (25- 35%)

%= YIELD FROM N-HOMAB Chart 3. Reaction scheme for the formation of the products obtained from Chart 4. Reaction scheme for the formation of the prod ucts obtained from the PAPS-dependent cytosol-catalyzed esterification of N-HO-MAB in the the PAPS-dependent cytosol-catalyzed esterification of N-hydroxy-2-naph- absence of added nucleophiles. thylamine in the absence of added nucleophiles.

2356 CANCER RESEARCH VOL. 36

mine and 2,2'-azoxynaphthalene, which were only partially DISCUSSION resolved by chromatography, accounted for another 35 to 40%. While the 5 to 10% yield of 2-amino-l-naphthol was The hepatocarcinogen MAB is N-hydroxylated by a dependent on PAPS, 2-amino-l-naphthol was converted to mixed-function amine oxidase in the hepatic endoplasmic 2-amino-l-naphthyl sulfate in the sulfotransferase reaction reticulum (33). As shown in this report the product of this medium at 5 to 10 times the rate of conversion of N-hydroxy- oxidation, N-HO-MAB, is sulfonated by 1 or more sulfo- 2-naphthylamine to this product. The reaction scheme, transferases in the hepatic cytosol in a PAPS-dependent while only tentative, allows for both sulfamate and ester reaction to form a reactive electrophile, presumably the formation and is consistent with the reported chemical ester MAB-N-sulfate. This electrophile forms products of properties of N- and O-sulfonates of N-hydroxy arylamines known structure with nucleophiles such as methionine and (12-14). guanosine that are identical to products derived from the protein- and nucleic acid-bound dyes formed in vivo in the Conversion of MAB to N-(Guanosin-8-yl)-MAB by the livers of rats fed MAB (38, 58). MAB-N-sulfate is thus a 10,000 x g Supernatant of Rat Liver possible ultimate carcinogenic metabolite of MAB. Evi- dence supporting this concept has come from the recent By appropriate modifications of the MAB N-oxidase (33) observations of Blunck and Crowther (8) that the hepatocar- and N-HO-MAB sulfotransferase assay procedures, it was cinogenicity of the closely related dye, 3'-methyI-N,N-di- possible to demonstrate the conversion of MAB to a nucleo- methyl-4-aminoazobenzene, is greatly enhanced in the rat side adduct on incubation with the 10,000 x g supernatant by dietary administration of a high level of sodium sulfate. fraction of rat liver. For this purpose [3H]MAB and guano- These data on the metabolism and carcinogenicity of MAB sine were used as substrate and trapping agent, respec- thus closely parallel data previously obtained for the metab- tively. Demonstration of this overall reaction required the olism and carcinogenicity of AAF in the rat liver (16, 17, 23, selection of an intermediate pH and elimination of potas- 48, 69, 74), although the N-oxidase and sulfotransferase sium phosphate buffer, which inhibited sulfotransferase ac- activities for MAB and N-HO-MAB, respectively, differ from tivity,, and Tris-HCI buffer, which inhibited N-oxidation. Me- those for AAF and N-HO-AAF. The microsomal N-oxidase thionine (100 raM) could not be used as the trapping agent, for MAB does not depend on cytochrome P-450 and pre- since it completely inhibited the N-oxidase. PAPS was also sumably is a flavoprotein amine oxidase similar to that somewhat inhibitory (40%) to the N-oxidase. The incubation described by Ziegler et al. (33, 78). The microsomal oxidase medium selected for demonstration of the reaction con- for AAF involves cytochrome P-450 (69). Similarly, as shown tained 100 mM Bis-Tris-HCI buffer (pH 7.0 at 37~ 1 mM in the present report (cf. "Results"), the hepatic sulfotrans- NAN3, 5 mM MgCI=, 0.5 mM EDTA, 1 mM PAPS, an NADPH- ferase activities for N-HO-AAF and N-HO-MAB appear to be and NADH-generating system (33), 1 mM guanosine, 5 mg different. The former activity appears to have properties protein from 10,000 x g supernatant per ml, and 0.5 mM noted for both phenol and steroid sulfotransferases, while [3H]MAB (1.5 Ci/mmole). The isolation, separation, and the latter activity appears to be more similar to that of the estimation of [3H]-N-(guanosin-8-yl)-MAB were carried out phenol sulfotransferase. as described above for the 1"C-labeled derivative. Under The inability to detect either O-acetylation or O-serylation these conditions the formation of [3H]-N-(guanosin-8-yl)- of N-HO-MAB, as has been demonstrated for other carcino- MAB from [3H]MAB was demonstrated and shown to be genic N-hydroxy arylamines (6, 7, 36, 66, 67), emphasizes enhanced by the addition of both PAPS and a NADPH, the probable importance of O-sulfonation in the metabolism NADH-generating system (Chart 5). of this N-hydroxy dye. The failure of hepatic cytosols (NADPH-dependent) and of hepatic microsomes (NADH-dependent) to catalyze the reduction of certain N-hydroxy MAB ~ PAPS ; N-HO-MAB l, MAB-N-SULFATE NADPH arylamines further conserves these substrates for this met- 3.0- GUANOSINL~ N" (GUANOSIN- 8 -YL ) MAB abolic pathway. Y The tissue, sex, and species distributions of microsomal COMPLETE SYSTEM~ N-oxidase activity for MAB (33) and cytosolic sulfotransferase activity for N-HO-MAB (Tables 3 and 4) are consistent 2.0- with the observation that MAB and related aminoazo dyes ? are strongly carcinogenic only in the liver of the male rat d, (47). Likewise, the low hepatocarcinogenicities of EAB and i z AB (2, 47, 68), 1-naphthylamine (Ref. 24, pp. 100-101; Ref. 1.0- - PAPS /'k 30, p. 168; Ref. 62, p. 183; Ref. 63, p. 126; Ref. 70, p. 559; Ref. 71, pp. 271-272), 2-naphthylamine (Ref. 24, pp. 101- 103; Ref. 30, pp. 169-170; Ref. 62, pp. 183-185; Ref. 63, pp. -NADPH -NADH 127-128; Ref. 70, pp. 559-564; Ref. 71, p. 272), and 4- G aminobiphenyl (Ref. 30, p. 104; Ref. 62, p. 102; Ref. 63, p. 71; Ref. 70, pp. 375-376; Ref. 71, p. 200) may result in part O O 5 IO 115 from the lower rates of O-sulfonation of the N-hydroxy MIN Chart 5. N-Oxidation of MAB and O-esterification of N-HO-MAB by the metabolites of these amines in the rat liver as compared to 10,000 x g supernatant of rat liver. those noted for N-HO-MAB (Table 5).

JULY 1976 2357

N-(p-Phenylazo)phenylnitrone also deserves consideration 16. DeBaun, J. R., Miller, E. C., and Miller, J. A. N-Hydroxy-2-acetylaminofluorene Sulfotransferase: Its Probable Role in Carcinogenesis and in as a possible ultimate carcinogen of N-HO-MAB. As noted Protein-(methion-S-yl) Binding in Rat Liver. Cancer Res., 30: 577-595, previously (33), N-HO-MAB is easily oxidized aerobically to a 1970. reactive derivative, presumably a nitrone, that can form 17. DeBaun, J. R., Smith, J. Y. R., Miller, E. C., and Miller, J. A. Reactivity in Vivo of the Carcinogen N-Hydroxy-2-acetylaminofluorene: Increase by adducts in anhydrous media with carbon-carbon and car- Sulfate Ion. Science, 167: 184-186, 1970. bon-nitrogen double bonds in a variety of tissue constitu- 18. Denner, W. H. B., Stokes, A. M., Rose, F. A., and Dodgson, K. S. Separation and Properties of the Soluble 3'-Phosphoadenosine-5'-Phos- ents. From data presented in this report, this nitrone also phosulfate-Degrading Enzymes of Bovine Liver. Biochim. Biophys. Acta, appears to be formed during the oxidation of N-HO-MAB by 315: 394-401, 1973. metabolically formed MAB-N-sulfate to yield products in- 19. Dodgson, K. S., and Rose, F. A. Sulfoconjugation and Sulfohydrolysis. In: W. H. Fishman (ed.), Metabolic Conjugation and Metabolic Hydroly- cluding MAB, N-HO-AB, and formaldehyde. The latter 2 sis, Vol. 1, pp. 239-325. New York: Academic Press, Inc., 1970. products may arise by hydrolysis of N-(p-phenyl- 20. Exner, O. Derivatives of Oximes. I1. Reduction of O- and N-Alkyl Oximes azo)phenylnitrone, an initial product in the oxidation-re- with Lithium Aluminum Hydride. Collection Czech. Chem. Commun., 20: 202-209, 1955. duction reaction. A reaction between N-benzoyloxy-MAB 21. Gillette, J. R., Davis, D. C., and Sasame, H. A. Cytochrome P-450 and Its and N-HO-MAB occurred in the absence of cytosol and Role in Drug Metabolism. Ann. Rev. Pharmacol., 12: 57-84, 1972. 22. Gornall, A. G., Bardawill, C. J., and David, M. M. Determination of Serum PAPS and yielded similar products. In a previous report (33) Proteins by Means of the Biuret Reaction. J. Biol. Chem., t77: 751-766, it was noted that N-HO-EAB did not form a reactive nitrone 1949. upon aerobic oxidation. Thus, these observations are con- 23. Gutmann, H. R., Malejka-Giganti, D., Barry, E. J., and Rydell, R. E. On the Correlation between the Hepatocarcinogenicity of the Carcinogen, sistent with the earlier findings that an N-methyl group was N-2-Fluorenylacetamide, and Its Metabolic Activation by the Rat. Cancer required for the formation of high levels of protein-bound Res., 32: 1554-1561, 1972. dye in vivo and for strong hepatocarcinogenicity by ami- 24. Hartwell, J. L. Survey of Compounds Which Have Been Tested for Carci- nogenic Activity. U. S. Public Health Service Publication No. 149. Wash- noazo dyes in the rat (2, 47, 68). ington, D. C.: U. S. Government Printing Office, 1951. 25. Hogeboom, G. H., Schneider, W. C., and Palade, G. E. Cytochemical Studies of Mammalian Tissues. I. Isolation of Intact Mitochondria From ACKNOWLEDGMENTS Rat Liver: Some Biochemical Properties of Mitochondria and Submi- croscopic Particulate Material. J. Biol. Chem., 172: 619-635, 1948. 26. Irving, C. C. Enzymatic Deacetylation of N-Hydroxy-2-Acetylamino-flu- The authors wish to express their gratitude to Nancy Korda for her expert orene by Liver Microsomes. Cancer Res., 26 (Part 1): 1390-1396, 1966. technical assistance during the course of these studies. 27. Irving, C. C., Janss, D. H., and Russell, L. T. Lack of N-Hydroxy-2- acetylaminofluorene Sulfotransferase Activity in the Mammary Gland and Zymbal's Gland of the Rat. Cancer Res., 31: 387-391, 1971. REFERENCES 28. Irving, C. C., and Veazey, R. A. Isolation of Deoxyribonucleic Acid and Ribosomal Ribonucleic Acid from Rat Liver. Biochim. Biophys. Acta, 166: 246-248, 1968. 1. Andersen, R. A., Enomoto, M., Miller, E. C., and Miller, J. A. Carcino- 29. Jacobson, M., Levin, W., Lu, A. Y. H., Conney, A. H., and Kuntzman, R. genesis and Inhibition of the Walker 256 Tumor in the Rat by trans-4- The Rate of Pentobarbital and Acetanilide Metabolism by Liver Micro- Acetylaminostilbene, Its N-Hydroxy Metabolite, and Related Com- somes: A Function of Lipid Peroxidation and Degradation of Cytochrome pounds. Cancer Res., 24:128-143, 1964. P-450 Heine. Drug. Metab. Disposition, I: 766-774, 1973. 2. Arcos, J. C., and Simon, J. Effect of 4'-Substituents on the Carcinogenic 30. John I. Thompson and Co. Survey of Compounds Which Have Been Activity of 4-Aminoazobenzene Derivatives. ArzneimitteI-Forsch., 12: Tested for Carcinogenic Activity, Vol. 1968-1969, U. S. Public Health 270-275, 1962. Service Publication No. 149. Washington, D. C.: U. S. Government 3. Banerjee, R. K., and Roy, A. B. The Sulfotransferases of Guinea Pig Printing Office, 1973. Liver. Mol. Pharmacol., 2: 56-66, 1966. 31. Johnson, D. H., Rogers, M. A. T., and Trappe, G. Aliphatic Hydroxyla- 4. Banerjee, R. K., and Roy, A. B. Kinetic Studies of the Phenol Sulfotrans- mines. Part I1. Autoxidation. J. Chem. Soc., 1093-1103, 1956. ferase Reaction. Biochim. Biophys. Acta, 151: 573-586, 1968. 32. Kadlubar, F. F., McKee, E. M., and Ziegler, D. M. Reduced Pyridine 5. Barford, D. J., and Jones, J. G. ThioI-Dependent Changes in the Proper- Nucleotide-Dependent H-Hydroxy Amine Oxidase and Reductase Activi- ties of Rat Liver Sulfotransferases. Biochem. J., 123: 427-434, 1971. ties of Hepa tic Microsomes. Arch. Biochem. Biophys., 156: 46-57, 1973. 6. Bartsch, H., Dworkin, C., Miller, E. C., and Miller, J. A. Formation of 33. Kadlubar, F. F., Miller, J. A., and Miller, E. C. Microsomal N-Oxidation of Electrophilic N-Acetoxyarylamines in Cytosols from Rat Mammary Gland the Hepatocarcinogen N-Methyl-4-aminoazobenzene and the Reactivity and Other Tissues By Transacetylation from the Carcinogen N-Hydroxy- of N-Hydroxy-N-methyl-4-aminoazobenzene. Cancer Res., 36: 1196- 4-acetylaminobiphenyl. Biochim. Biophys. Acta, 304: 42-55, 1973. 1206, 1976. 7. Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. Electrophilic N- 34. Kadlubar, F. F., and Ziegler, D. M. Properties of a NADH-Dependent N- Acetoxyaminoarenes Derived from Carcinogenic N-Hydroxy-N-acetyl- Hydroxy Amine Reductase Isolated from Pig Liver Microsomes. Arch. aminoarenes by Enzymatic Deacetylation and Transacetylation in Liver. Biochem. Biophys., 162: 83-92, 1974. Biochim. Biophys. Acta, 286: 272-298, 1972. 35. Kamm, O. /3-Phenylhydroxylamine. In: H. Gilman (ed.), Organic 8. Blunck, J. M., and Crowther, C. E. Enhancement of Azo Dye Carcinogen- Syntheses, Coll. Vol. 1, pp. 445-447. New York: John Wiley and Sons, esis by Dietary Sodium Sulfate. European J. Cancer, 11: 23-32, 1975. Inc., 1941. 9. Booth, J., and Boyland, E. The Biochemistry of Aromatic Amines. 10. 36. King, C. M. Mechanism of Reaction, Tissue Distribution, and Inhibition Enzymic N-Hydroxylation of Arylamines and Conversion of Arylhydroxyl- of Arylhydroxamic Acid Acyltransferase. Cancer Res., 34: 1503-1515, amines into o-Aminophenols. Biochem. J., 91: 362-369, 1964. 1974. 10. Booth, J., Boyland, E., and Manson, D. Metabolism of Polycyclic Com- 37. King, C. M., and Phillips, B. N-Hydroxy-2-fluorenylacetamide. Reaction pounds. 9. Metabolism of 2-Naphthylamine in Rat Tissue Slices. Bio- of the Carcinogen with Guanosine, Ribonucleic Acid, Deoxyribonucleic chem. J., 60: 62-71, 1955. Acid, and Protein following Enzymatic Deacetylation or Esterification. J. 11. Boyland, E., and Manson, D. The Biochemistry of Aromatic Amines. 2- Biol. Chem., 244: 6209-6216, 1969. Formamido-l-naphthyl Hydrogen Sulfate, a Metabolite of 2-Naphthyla- 38. Lin, J. K., Miller, J. A., and Miller, E. C. Structures of Hepatic Nucleic mine. Biochem. J., 99: 189-199, 1966. Acid-Bound Dyes in Rats Given the Carcinogen N-Methyl-4-aminoazo- 12. Boyland, E., and Manson, D. The Biochemistry of Aromatic Amines. The benzene. Cancer Res., 35: 844-850, 1975. Metabolism of 2-Naphthylamine and 2-Naphthylhydroxylamine Deriva- 39. Lotlikar, P. D. Enzymatic N-O-methylation of Hydroxamic Acids. tives. Biochem. J., 101: 84-102, 1966. Biochim. Biophys. Acta, 170: 468-471, 1968. 13. Boyland, E., Manson, D., and Sims, P. The Preparation of o-Amino- 40. Lotlikar, P. D., Miller, E. C., Miller, J. A., and Margreth, A. The Enzymatic phenyl Sulfates. J. Chem. Soc., 3623-3628, 1953. Reduction of the N-Hydroxy Derivatives of 2-Acetylaminofluorene and 14. Boyland, E., and Nery, R. Arylhydroxylamines. Part II. Phenylhydroxyl- Related Carcinogens by Tissue Preparations. Cancer Res., 25: 1743- amine N- and O-Sulphonic Acids. J. Chem. Soc., 5217-5222, 1962. 1752, 1965. 15. Carroll, J. and McEvoy, F. A. The Activation of Liver Phenol Sulfotrans- 41. Maher, V. M., Miller, E. C., Miller, J. A., and Szybalski, W. Mutations and ferase. Biochem. J., 119: 27p, 1970. Decreases in Density of Transforming DNA Produced by Derivatives of

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the Carcinogens 2-Acetylaminofluorene and N-Methyl-4-aminoazoben- of Free Radicals upon Decomposition of N-Acetoxy-N-arylacetamides. zene. Mol. Pharmacol., 4:411-426, 1968. Cancer Res., 33: 1159-1164, 1973. 42. Maher, V. M., Miller, J. A., Miller, E. C., and Summers, W. C. Mutations 61. Scribner, J. D., and Naimy, N. K. Destruction of Triplet Nitrenium Ion by and Loss of Transforming Activity of Bacillus subtillis DNA after Reaction Ascorbic Acid. Experientia, 31: 470-471, 1975. with Esters of Carcinogenic N-Hydroxy Aromatic Amides. Cancer Res., 62. Shubik, P., and Hartwell, J. L. Survey of Compounds Which Have Been 30: 1473-1480, 1970. Tested for Carcinogenic Activity, Suppl. 2, U. S. Public Health Service 43. Manson, D. Oxidation of N-Naphthylhydroxylamines to Nitrosonaphthols Publication No. 149. Washington, D. C.: U. S. Government Printing by Air. J. Chem. Soc. Perkin Trans. I, 192-194, 1974. Office, 1969. 44. Mattock, P., and Jones, J. G. Partial Purification and Properties of an 63. Shubik, P., and Hartwell, J. L. Survey of Compounds Which Have Been Enzyme from Rat Liver That Catalyzes the Sulfation of L-Tyrosyl Deriva- Tested for Carcinogenic Activity, Suppl. 1. U. S. Public Health Service tives. Biochem. J., 116: 797-803, 1970. Publication No. 149. Washington D. C.: U. S. Government Printing 45. McEvoy, F. A., and Carroll, J. The Isolation of Rat Liver Phenol Sulfo- Office, 1957. transferase. Biochem. J., 119: 26p-27p, 1970. 64. StOhrer, G., Corbin, E., and Brown, G. B. Enzymatic Activation of the 46. Miller, J. A., and Baumann, C. A. The Determination ofp-Dimethylamino- Oncogen 3-Hydroxyxanthine. Cancer Res., 32: 637-642, 1972. azobenzene, p-Monomethylaminoazobenzene and p-Aminoazobenzene 65. StOhrer, G., and Salemnick, G. Oxidizing Action of Purine N-Oxide in Tissue. Cancer Res., 5: 157-161, 1945. Esters. Cancer Res., 35: 122-131, 1975. 47. Miller, J. A., and Miller, E. C. The Carcinogenic Aminoazo Dyes. Advan. 66. Tada, M., and Tada, M. Enzymatic Activation of the Carcinogen 4- Cancer Res., 1: 339-396, 1953. Hydroxyaminoquinoline 1-Oxide and Its Interaction with Cellular Macro- 48. Miller, J. A., and Miller, E. C. The Metabolic Activation of Aromatic molecules. Biochem. Biophys. Res. Commun., 46: 1025-1032, 1972. Amines and Amides. Progr. Exptl. Tumor Res., 11: 273-301, 1969. 67. Tada, M., and Tada, M. Seryl-tRNA Synthetase and Activation of the 49. Nash, T. The Colorimetric Estimation of Formaldehyde by Means of the Carcinogen 4-Nitroquinoline 1-Oxide. Nature, 255: 510-512, 1975. Hantzsch Reaction. Biochem. J., 55: 416-421, 1953. 68. Terayama, H. Aminoazo Carcinogenesis-Methods and Biochemical 50. Poirier, L. A., Miller, J. A., and Miller, E. C. The N- and Ring-Hydroxyl- Problems. In: H. Busch (ed.), Methods in Cancer Research, Vol. 1, pp. ation of 2-Acetylaminofluorene and the Failure to Detect N-Acetylation of 399-449. New York: Academic Press, Inc., 1967. 2-Aminofluorene in the Dog. Cancer Res., 23: 790-800, 1963. 69. Thorgeirsson, S. S., Jollow, D. J., Sasame, H. A., Green, I., and Mitchell, 51. Poirier, L. A., Miller, J. A., Miller, E. C., and Sato, K. N-Benzoyloxy-N- J. R. The Role of Cytochrome P-450 in N-Hydroxylation of 2-Acetylami- methyl-4-aminoazobenzene: Its Carcinogenic Activity in the Rat and Its nofluorene. Mol. Pharmacol., 9: 398-404, 1973. Reaction with Proteins and Nucleic Acids and Their Constituents in Vitro. 70. Tracor/Jitco. Survey of Compounds Which Have Been Tested for Carci- Cancer Res., 27: 1600-1613, 1967. nogenic Activity, Vol. 1961-1967. U. S. Public Health Service Publication 52. Poirier, L. A., and Weisburger, J. H. Enzymic Reduction of Carcinogenic No. 149. Washington, D. C.: U. S. Government Printing Office, 1972. Aromatic Nitro Compounds by Rat and Mouse Liver Fractions. Biochem. 71. Tracor/Jitco. Survey of Compounds Which Have Been Tested for Pharmacol., 23: 661-669, 1974 Carcinogenic Activity, Vol. 1970-1971. U. S. Public Health Service Publi- 53. Poulsen, L. L., Kadlubar, F. F., and Ziegler, D. M. Role of the Microsomal cation No. 149. Washington, D. C.: U. S. Government Printing Office, Mixed-Function Amine Oxidase in the Oxidation of N,N-Disubstituted 1974. Hydroxylamines. Arch. Biochem. Biophys., 164: 774-775, 1974. 72. Tsen, C. C. An Improved Spectrophotometric Method for the Determina- 54. Poulsen, L. L., Masters, B. S. S., and Ziegler, D. M. Mechanism of 2- tion of Tocopherols Using 4,7-Diphenyl-l,10-phenanthroline. Anal. Naphthyiamine Oxidation Catalyzed by Pig Liver Microsomes. Xenobi- Chem., 33: 849-851, 1961. otica, in press. 73. Uehleke, H. The Role of Cytochrome P-450 in the N-Oxidation of Individ- 55. Roy, A. B. The Enzymic Synthesis of Aryl Sulphamates. Biochem. J., 74: ual Amines. Drug. Metab. Disposition, I: 299-313, 1973. 49-56, 1960. 74. Weisburger, J. H., Yamamoto, R. S., Williams, G. M. Grantham, P. H., 56. Sato, K., Poirier, L. A., Miller, J. A., and Miller, E. C. Studies on the N- Matsushima, T., and Weisburger, Eo K. On the Sulfate Ester of N-Hy- Hydroxylation and Carcinogenicity of 4-Aminoazobenzene and Related droxy-N-2-fluorenylacetamide as a Key Ultimate Hepatocarcinogen in the Compounds. Cancer Res., 26: 1678-1687, 1966. Rat. Cancer Res., 32: 491-500, 1972. 57. Scribner, J. D., and Miller, J. A. Intramolecular Hydrogen Bonding in o- 75. Willstatter, R., and Kubli, H. Uber die Reduktion von Nitroverbindungen Methylmercapto Derivatives of N-Methylaniline and N-Methyl-4-aminoa- nach der Methode von Zinin. Chem. Ber., 41: 1936-1940, 1908. zobenzene. J. Org. Chem., 32: 2348-2349,-1967. 76. Wislocki, P. G., Borchert, P., Miller, J. A., and Miller, E. C. The Metabolic 58. Scribner, J. D., Miller, J. A., and Miller, E. C. 3-Methylmercapto-N- Activation of the Carcinogen l'-Hydroxysafrole in Vivo and in Vitro and Methyl-4-aminoazobenzene: An Alkaline-Degradation Product of a La- the Electrophilic Reactivities of Possible Ultimate Carcinogens. Cancer bile Protein-Bound Dye in the Livers of Rats Fed N,N-Dimethyl-4-aminoa- Res., 36: 1686-1695, 1976. zobenzene. Biochem. Biophys. Res. Commun., 20: 560-565, 1965. 77. Ziegler, D. M., McKee, E. M., and Poulsen, L. L. Microsomal Flavopro- 59. Scribner, J. D., Miller, J. A., and Miller, E. C. Nucleophilic Substitution tein-Catalyzed N-Oxidation of Arylamines. Drug Metab. Disposition, 1: on Carcinogenic N-Acetoxy-N-arylacetamides. Cancer Res., 30: 1570- 314-321, 1973. 1579, 1970. 78. Ziegler, D. M., and Mitchell, C. H. Microsomal Oxidase: IV. Properties of 60. Scribner, J. D., and Naimy, N. K. Reactions of Esters of N-Hydroxy-2- a Mixed-Function Amine Oxidase Isolated from Pig Liver Microsomes. acetamidophenanthrene with Cellular Nucleophiles and the Formation Arch. Biochem. Biophys., 150: 116-125, 1972.

JULY 1976 2359

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Fred F. Kadlubar, James A. Miller and Elizabeth C. Miller

Cancer Res 1976;36:2350-2359.

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