n2 atom of guanine and n6 atom of adenine residues as sites for

[CANCER RESEARCH 41, 2664-2671, July 1981]0008-5472/81 /0041-OOOOS02.00

N2 Atom of Guanine and N6 Atom of Adenine Residues as Sites for

Covalent Binding of Metabolically Activated 1'-Hydroxysafroleto Mouse Liver DMA in V/vo1

David H. Phillips,2 James A. Miller,3 Elizabeth C. Miller, and Bruce Adams

McArdle Laboratory for Cancer Research [D. H. P., J. A. M., E. C. M.Â¡and Department of Chemistry Â¡B.A.], University of Wisconsin, Madison. Wisconsin 53706

ABSTRACT

Administration of 1'-[2',3'-3H]hydroxysafrole to adult female

mice resulted in the formation of DMA-, ribosomal RNA-, andprotein-bound adducts in the liver that reached maximum levelswithin 24 hr. The levels of all three macromolecule-boundadducts decreased rapidly between 1 and 3 days after injection, at which time the amounts of the DMA-bound adducts

essentially plateaued at approximately 15% of the maximumlevel. The amounts of the protein and ribosomal RNA adductswere very low by 20 days.

Comparison by high-performance liquid chromatography of

the deoxyribonucleoside adducts obtained from the hepaticDMA with those formed by reaction of deoxyguanosine anddeoxyadenosine with 1'-acetoxysafrole, 1'-hydroxysafrole-2',3'-oxide, and 1'-oxosafrole indicated that the four in vivoadducts studied were derived from an ester of 1'-hydroxysaf-

role. Three of the four in vivo adducts comigrated with adductsformed by reaction of 1'-acetoxysafrole with deoxyguanosine;

the fourth adduct comigrated with the major product of thereaction of this ester with deoxyadenosine. Adduct formationin vivo at low levels by the other two electrophilic metaboliteswas not excluded. The three adducts obtained by reaction of1'-acetoxysafrole with deoxyguanosine appeared to be substituted on the 2-amino group of the guanine residue on the basisof their partitions between aqueous buffer solutions and 1-butanohethyl ether as a function of pH and their retention of 3Hfrom [8-3H]deoxyguanosine. The corresponding three adductsderived from the hepatic DNA of mice given 1'-[2',3'-3H]hy-

droxysafrole had pH partition patterns not significantly differentfrom the three adducts formed in vitro. Adduct II was furthercharacterized from its nuclear magnetic resonance spectrumas /V2-(frans-isosafrol-3'-yl)deoxyguanosine. Adduct IV, derived from the reaction of 1'-acetoxysafrole with deoxyadenosine 5'-phosphate, was characterized in the same manner asiVXfrans-isosafrol-S'-yOdeoxyadenosine.

INTRODUCTION

Safrole [1 -allyl-3,4(methylenedioxy)benzene], a naturally oc

curring flavoring agent that is the major constituent of oil ofsassafras (12, 13, 20), possesses weak hepatocarcinogenicactivity when fed to adult rats or mice (1, 5, 14, 15, 21) andmoderate hepatocarcinogenic activity when injected into miceduring the first few weeks after birth (5, 11, 35). Although the

' This work was supported by Grants CA-07175 and CA-22484 from the

National Cancer Institute. USPHS.2 Present address: Department of Biological Sciences, Stanford University.

Stanford, Calif. 94305.3 To whom requests for reprints should be addressed.

Received December 5. 1980; accepted April 1, 1981.

use of safrole and sassafras oil (containing safrole) as foodadditives was banned in the United States in 1960, safrolecontinues to be ingested in small amounts by humans since itis a minor component of a number of other essential oils and ofsome herbs and spices, including anise, basil, nutmeg, mace,and pepper (12, 13, 20).

Current evidence (reviewed in Refs. 22 and 27) suggeststhat a property common to most chemical carcinogens is thattheir biologically active forms are electrophilic. The majority ofcarcinogens are not electrophilic as such and must undergometabolic activation in vivo to reactive species of this type.Studies on safrole (6, 7, 24, 28-30, 34, 35) indicated themetabolic formation of several electrophilic metabolites. Borch-ert ef al. (5, 6) showed that 1'-hydroxysafrole is a major

metabolite of safrole in the rat and mouse and that it possessesgreater carcinogenic activity than does the parent compoundin these species. 1'-Hydroxysafrole undergoes further metabolism by rat and mouse liver preparations to 1'-sulfonoxysafroleand 1'-hydroxysafrole-2',3'-oxide (30, 34) (Chart 1). Both thelatter metabolite and a model analog of the former, 1'-acetox

ysafrole, possess electrophilic, carcinogenic, and mutagenicactivity (5, 6, 29, 34, 35). In addition, 1'-oxosafrole, small

amounts of which are found as Mannich base derivatives in theurine of rats administered safrole (24), exhibits electrophilicactivity (34). However, 1'-oxosafrole has not shown mutagenic

or carcinogenic activity (34, 35). We have reported recently(26) that 1'-hydroxyestragole, a compound structurally relatedto 1'-hydroxysafrole and a proximate carcinogenic metabolite

of the natural flavoring agent estragÃ³le (1-allyl-4-methoxyben-

zene) (9), is metabolically activated in mouse liver in vivo to aderivative, presumably a 1'-ester, that reacts covalently with

the exocyclic amino groups of guanine and adenine residuesin DNA. The present paper reports that the DNA adductsformed in mouse liver after administration of 1'-hydroxysafroleare analogous to the 1'-hydroxyestragole:DNA adducts (26),

and evidence for their structures is presented. The earlierassignment of the structure of the major product of the reactionof 1'-acetoxysafrole with GMP as O6-(isosafrol-3'-yl)guanylic

acid (6, 34) was found to be incorrect.

MATERIALS AND METHODS

Safrole Derivatives. The syntheses of 1'-hydroxysafrole and1'-acetoxysafrole (6) and of 1'-oxosafrole, 1'-hydroxysafrole-2',3'-oxide, 1'-hydroxy-2',3'-dehydrosafrole, and 1'-{2',3'-3H]hydroxysafrole (34) have been described previously.

Hepatic DNA, rRNA, and Protein from Mice Treated with1'-{3H]Hydroxysafrole. Female CD-1 mice (Charles River

Breeding Laboratory, Wilmington, Mass.), 8 to 10 weeks old(mean weight, 30 g) were given i.p. injections of 1'-[2',3'-

2664 CANCER RESEARCH VOL. 41

Research. on February 17, 2018. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from

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1'-Hydroxysafrole-DNA Adducts in Mouse Liver

1-Ã‡H-

H-C-CH =CH2OH

I-HYDROXYSAFROLE

-,-CH iâ€”CHo -CH,

C-CH=CH2 H-Ã‡-CH=CH2 H-Ã‡-CH-CH20 0-S03H OH MI'-OXO- I'-SULFONOXY- I-HYDROXY

SAFROLE SAFROLE SAFROLE-2',y- OXIDE

Chart 1. Pathways of metabolism of 1'-hydroxysafrole, a proximate carcino

genic metabolite of safrole, to electrophilic species.

3H]hydroxysafrole (404 mCi/mmol, 12 jumol/mouse in 0.1 ml

trioctanoin). At the times indicated, the animals were killed bycervical dislocation, and the livers from groups of 5 mice werepooled for isolation of DMA, rRNA, and protein by the methodof Irving and Veazey (16). DNA and rRNA were dissolved inTris buffer (0.01 M, pH 7.0) and hydrolyzed enzymatically tonucleosides by the method of Baird and Brookes (2). Proteinwas dissolved in Soluene (Packard Instrument Co., Inc., Downers Grove, III.). Aliquots of digests of the 3 macromoleculeswere added to Aquassure scintillation fluid (New England Nuclear, Boston, Mass.) and assayed for radioactivity in Isocap/300 (Nuclear Chicago, Inc., Chicago, III.) or Mark Ml/6880(Searle Analytic, Inc., Des Plaines, III.) scintillation spectrometers. External standardization was used to convert all of thedata to dpm.

Preparation of Nucleoside Adducts. 14C-Labeled nucleo-

side adduct markers were prepared by reacting the appropriate1'-hydroxysafrole derivative (10 mg in 0.5 ml of ethanol) with[14C]dGuo4 or [14C]dAdo (2 mg containing 0.5 jiCi in 0.5 ml

0.01 M Tris, pH 7.0). Unlabeled nucleosides were obtainedfrom Sigma Chemical Co., St. Louis, Mo.; [8-'"C]dGuo and [8-14C]dAdo were purchased from Schwarz/Mann, Orangeburg,

N. Y., and New England Nuclear, respectively. Aliquots (50 jxl)of the reaction mixtures were coinjected on HPLC with 3H-

labeled DNA hydrolysates from mouse liver. In some experiments, 1'-acetoxysafrole (10 mg in 0.5 ml ethanol) was reacted

with dGuo (2 mg in 0.5 ml 0.01 M Tris, pH 7.0) containing 5juCi [8-3H]dGuo (Schwarz/Mann) and 0.5 jtiCi [8-14C]dGuo.

For large-scale preparation of 1'-acetoxysafroleinucleosideadducts, 1'-acetoxysafrole (2.3 g) in ethanol (100 ml) was

reacted with either dGMP or dAMP (1 g) in 75 ml 0.01 M Trisbuffer (pH 7.0) at 37Â° for 17 hr with gentle shaking. The

reaction mixture was then extracted with ethyl ether (4 x 200ml). The aqueous phase was reduced in volume to 5 ml andapplied to a Sephadex LH-20 column (50 x 3 cm) that was

eluted with water, and fractions (approximately 8 ml) werecollected. Pools of 5 to 10 fractions were freeze dried, and theresidues were dissolved in 3 ml 0.1 M Tris buffer, (pH 9.0) and

4 The abbreviations used are: dGuo, deoxyguanosine; dAdo. deoxyadenosine;

HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance.

incubated with 4 units of alkaline phosphatase (Sigma) at 37Â°

for 48 hr. The nucleosides which precipitated were washedwith water and dried in a vacuum. HPLC analysis revealed thatthe material (18 mg) obtained from Fractions 51 to 55 from thedGMP reaction consisted of a single product. Earlier fractionscontained a mixture of this product and unreacted dGuo, andlater fractions contained 2 minor products in addition to themajor product found in Fractions 51 to 55. Similarly, thematerial (28 mg) from Fractions 61 to 70 of the dAMP reactionmixture contained a single product; earlier fractions containedadditional unreacted dAdo, and subsequent fractions contained additional adducts.

NMR spectra were determined on solutions of the adducts indimethyl sulfoxide-d6 (5 mg in 0.4 ml) by use of a 270-MHzBruker WH 270 spectrometer equipped with a B-NC 12 Nicolet

computer.HPLC Chromatography. Chromatography of DNA hydroly

sates and marker nucleoside adducts was performed on anALC/GPC 294 liquid Chromatograph (Waters Associates, Mil-

ford, Mass.) equipped with a Model U6K injector, a Model 660solvent programmer, a Model 440 absorbance detector, anOmniscribe B-5000 strip chart recorder (Houston Instruments,Austin, Texas), and a Spherisorb ODS 5 /im reverse-phase

column (Altex Scientific Inc., Berkeley, Calif.). The solventsystems used were: System A, 100% water for 5 min followedby a linear (Program 6) gradient of 15 to 40% acetonitrile:waterfor 35 min; System B, 25% acetonitrile:water; System C, 45%methanol:water. For each system, the flow rate used was 2 ml/min. As noted previously (26), the retention times of adductsmay be altered by 1 to 2 min depending on the quantity ofother materials, such as unmodified nucleosides, necessarilycoinjected with them. Thus, the retention times quoted foradducts are not absolute, and it was essential for determiningthe identical natures of in vivo- and in wfro-derived adducts

that they be eluted simultaneously after coinjection on HPLC.pH Partition Coefficient Patterns of Adducts. Aliquots (50

to 100 ÃÃl)of the adduct-containing fractions eluted from HPLC

columns were partitioned by the procedure of Moore andKoreeda (23) between 1 ml of 0.05 M buffer solutions (pHrange, 1 to 13) and 1 ml of 1-butanol:ethyl ether (20:80), each

of which had been presaturated with the other. After vigorousshaking, the phases were separated, and portions (0.7 ml) ofeach phase were assayed for radioactivity.

RESULTS

Binding of 1-Hydroxysafrole to Hepatic Macromolecules.The levels to which 1'-hydroxysafrole was bound to liver mac

romolecules after administration of a single dose i.p. to adultfemale CD-1 mice are shown in Chart 2. Maximum binding toprotein, rRNA, and DNA occurred at 5 hr, and by 3 days aftertreatment, the amount of radioactivity bound to all 3 macro-

molecules had fallen by 85%. The levels of binding to proteinand rRNA continued to fall up to 20 days after treatment, butthe level of binding to DNA remained relatively constant between 3 and 20 days after treatment.

HPLC Analysis of the Hepatic DNA Hydrolysates. The elu-tion profiles from reverse-phase HPLC for hepatic DNA hydrolysates from mice killed 23 hr after a single i.p. injection of 1'-[2',3'-3H]hydroxysafrole are shown in Charts 3 to 5. A significant amount of radioactivity eluted in the region of the chro-

JULY1981 2665



D. H. Phillips et al.

300,

l 2 3 4 5 IODAYS AFTER TREATMENT

Chart 2. Binding of 1'-hydroxysafrole to the liver protein, rRNA, and DMA ofgroups of 5 adult female CD-1 mice following treatment i.p. with 12 /imol/30-gmouse. Two experiments per time point were carried out. Bars, S.D.

matographs where unmodified nucleosides eluted (9 to 12min). In the intermediate regions of the chromatographs, from15 to 22 min, low levels of radioactivity eluted, but no well-

defined peaks were discernible. In the later regions of thechromatographs, 4 well-defined peaks of radioactivity eluted;

these were designated I to IV in order of elution. HPLC analysisof the DNA hydrolysates showed essentially the same profilesat all time points. The radioactivity in the later regions of thechromatographs was distributed between the 4 adducts in thefollowing proportions: I, 33 Â±6% (S.D.); II, 62 Â±5%, III, 3 Â±1%, and IV, 2 Â± 1%. Although some variation in the relativeamounts of Adducts I to IV was observed, the differences wererandom with respect to the time at which the mice were killed.

Coinjection on HPLC of hepatic DNA hydrolysates and analiquot of the reaction mixture of 1'-acetoxysafrole with[14C]dGuo (resulted in 14C-labeled products that coeluted inSolvent System A with the 3H-labeled in vivo Adducts I, II, andIII (Chart 3A). The in vivo Adduci IV coeluted with a '4C-labeledproduct from the reaction of 1'-acetoxysafrole with [14C]dAdo(Chart 3B). Comigration of Adducts I, II, and III with 1'-acelox-ysafrole:dGuo adducts and of Adduct IV with a 1'-aceloxysaf-

role:dAdo adduci was also observed on HPLC with SolventSystems B and C. Representalive relenlion limes were asfollows: Solvent Syslem B: I, 9.0 min; II, 11.0 min; III, 13.5 min;and IV, 21.5 min; Syslem C: I, 9.5 min; II, 12.5 min; III, 15 min;and IV, 23.5 min.

When 1'-hydroxysafrole-2',3'-oxide was reacled wilh [14C]-

dGuo, a single major adduci was formed thai eluted al 13.5min on HPLC wilh Solvenl System A. Low levels of 3H-labeled

malerial from liver DNA hydrolysales also eluled in Ihis generalregion (Chart 4/\). Some of Ihe producÃs of Ihe reaclion of 1'-hydroxysafrole-2',3'-oxide with [14C]dAdo also eluted with in

termediate relenlion times lhal were less lhan those of the invivo Adducts I to IV (Chart 48). The reaclion of 1'-oxosafrolewith [14C]dGuo gave a complex pattern of producÃs, none ofwhich coeluted exaclly wilh any of Ine 3H-labeled in vivo

Adducts I to IV. However, some of these products eluted in theintermediate region (20 lo 24 min) of Ihe chromalograph lhalconlained low levels of 3H-labeled material (Chart 5A). The

oxosafrole produci al 23.5 min consislenlly moved about 0.5min faster than did Adduct I in Solvent A. Reaction of 1'-oxosafrole wilh [14C]dAdo gave a major adduci which eluled

close lo, bul noi simullaneously wilh, the in vivo Adduci II inbolh Solvenl Systems A (Chart 5B) and C (dala noi shown).

The coelution from HPLC columns of the in vivo producÃswilh those formed in vitro from 1'-aceloxysafrole indicated lhal1'-hydroxysafrole binds lo mouse liver DNA principally via an

eleclrophilic ester. This cochromalography also indicated thaiadducts were formed wilh bolh Ihe guanine (Adducts I, II, andIII) and adenine (Adduct IV) bases in DNA. However, the possible formation in vivo of minor adducls from reaction of DNAwith 1'-hydroxysafrole-2',3'-oxide or 1'-oxosafrole was noiexcluded. The 3H in mouse liver DNA hydrolysales lhal eluted

from HPLC columns at 15 lo 22 min may be accounted for bysuch adducts. Alternately, Ihese low levels of 3H may denote

polar degradation producÃs of Adducls I lo IV, which resultedfrom chemical or enzymalic modification of the methylenedioxyring of Ihe safrole moiety.

pH Partition Coefficient Patterns of Adducts Obtained inVitro and in Vivo. To further investigate Ihe nalure of Ihe dGuoAdducls I to III, both the adducls obtained in vivo and thosefrom the reaction of 1'-acetoxysafrole wilh [14C]dGuo were

collected from HPLC columns and partilioned betweenaqueous buffer solutions and 1-bulanol:elhyl elher (20:80)

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Chart 3. HPLC profiles of DNA hydrolysates and marker nucleosides. DNAhydrolysates from the livers of mice given injections of 1'-{3H]hydroxysafrolewere cochromatographed with aliquots of the reaction mixture of 1'-acetoxysafrole reacted with ["CJdGuo W and ("CJdAdo (B). The eluting solvent was: 0 to

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Chart 4. HPLC profiles of DNA hydrolysates and marker nucleosides. DNAhydrolysates from the livers of mice given injections of 1'-[3H]hydroxysafrolewere cochromatographed with aliquots of the reaction mixture of 1'-hydroxysaf-role-2',3'-oxide reacted with [14C]dGuo (A) and ["CJdAdo (ÃŸ).The eluting solvent

was as described in the legend to Chart 2.

(Chart 6). For Adducts I and II, the close agreement betweenthe partition coefficients of the in vivo- and in w'fro-derived

samples of each adduci provided further evidence for theirbeing identical. The greater variation in the patterns of AdductIII formed in vivo or in vitro was probably a consequence of themuch smaller amounts of radioactive material (approximately100 dpm/experimental point) with which the experiment wasconducted. However, the data clearly indicated pK0 values atboth acidic and basic pH for each of Adducts I to III. Thesefindings excluded the possibility that any of the adducts wereO6- or N-1-substituted dGuo derivatives, since those adductswould lack an ionizable proton at the N-1 position of guanine

and hence not show a pKa at basic pH. The patterns shown inChart 6 indicate that substitution occurred at the C-8, N-7, orN! position of guanine.

Reaction of 1'-Acetoxysafrole with [8-14C, 8-3H]dGuo.When 1'-acetoxysafrole was reacted with dGuo labeled atposition 8 of guanine with 14C and 3H and the products were

separated by HPLC, Adduct II retained 97% of the tritium(Table 1). Since loss of tritium from position 8 of guanine wouldbe expected for both C-8 and N-7-substituted dGuo derivatives

(32), this and the above results indicate that Adduct II containsa guanine residue substituted at the 2-amino group. Adducts I

and III also showed a high percentage of tritium retention, butthe levels were consistently lower than those for dGuo orAdduct II (Table 1, 86 and 82%, respectively). It is thereforepossible that these in w'fro-derived adduci peaks contain minor

amounts (<20%) of C-8 and/or N-7 adducts, which were notseparable from the major components (i.e., A/2-substituted


dGuo derivatives) of each peak by HPLC with the solventsystems used.

Structure of the dGuo Adduct II. The major nucleosideadduct derived from the large-scale reaction of 1'-acetoxysafrole with dGMP (see "Materials and Methods") comigrated onHPLC in Solvent Systems B and C (see "Materials and Methods") with the major in vivo Adduct II. Its proton NMR spectrum,

shown in Chart 7, was not that of a safrole derivative. Instead,the NMR spectrum is indicative of a 3'-substituted Â¡sosafrole

[1 -propenyl-3,4(methylenedioxy)benzene] derivative with fransconfiguration (Jr,2. = 16.0 Hz) at the C-1', C-2' double bond.The signal for the A/2 proton appeared as a triplet at 6.67 ppmand integrated as a single proton. Its coupling to the C-3'

protons (4.04 ppm) was demonstrated by its changing to asinglet upon irradiation of the C-3' signal. The N7 signal dis

appeared entirely when the sample was exchanged with D20,and at the same time, the C-3' signal changed to a doublet.

The C-8 proton of guanine appeared at 7.90 ppm, which was

similar to its position in the spectrum of dGuo; it did notexchange with D2O. On the basis of these findings, Adduct II isunequivocally assigned the structure A/2-(frans-isosafrol-3'-

yl)deoxyguanosine. The N-1 proton signal was not observed.This result is at variance with the assignment reported pre

viously of the major product of the reaction of 1'-acetoxysafrolewith GMP as O6-(isosafrol-3'-yl)guanylic acid on the basis of

its instability under mildly acidic conditions (34) and a 60-MHz

proton NMR spectrum of a major acetylated nucleoside adductderived from this material (6). Therefore, we have reexaminedsome of the original material (6). Since none of the acetylated

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Chart 5. HPLC profiles of DNA hydrolysates and marker nucleosides. DNAhydrolysates from the livers of mice given injections of 1'-pH]hydroxysafrolewere cochromatographed with aliquots of the reaction mixture of 1'-oxosafrolereacted with ["CJdGuo (A) and ['4C)dAdo (8). The eluting solvent was as

described in the legend to Chart 2.

JULY 1981 2667




adduci was available, the 1'-acetoxysafrole:GMP adduct from

the latter study was converted to the nucleoside adduct, asdescribed for the dGMP and dAMP adducts in "Materials andMethods." HPLC analysis of the material indicated that 85% of

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ADDUCT I

2 4 6 8 10 12pH

Chart 6. Partition of dGuo adducts between 0.05 M aqueous buffers, pH 1 to13 (23) and 1-butanohethyl ether (20:80). â€¢¿�,3H-labeled adducts derived in vivo;O, "C-labeled adducts derived in vitro from the reaction of 1'-acetoxysafrole with("C]dGuo.

the absorbance at 254 nm was eluted in a single peak; 2 lesspolar minor peaks accounted for the remaining 15%. The pHpartition coefficient pattern of the adduct, determined as described in "Materials and Methods" except that absorbance at

254 nm was used to determine the amount of adduct in eachphase, showed pKa values at both acidic (2.2) and basic (9.8)pH. This finding excludes the possibility of the adduct being anO6-substituted derivative, since such an adduct would lack an

ionizable proton at the N-1 position and thus not show a basic

pKa.The 270-MHz proton NMR spectrum of the 1'-acetoxysaf-

role:Guo adduct was similar to that of Adduct II (Chart 7) exceptthat the former was characteristic of a ribonucleoside, ratherthan of a deoxyribonucleoside. Thus, the salient features werethat the N2 proton signal was a triplet at 6.71 ppm and inte

grated as a single proton and that the N-1 proton signal was

seen as an extremely broad signal at 9.37 ppm. The parts ofthe spectrum due to the safrole substituent were indicative ofa 3'-substituted frans-isosafrole derivative. On the basis of thisreexamination of the 1'-acetoxysafrole:Guo adduct with improved techniques, it appears that it is N2-(frans-isosafrol-3'-

yOguanosine.We have also reexamined the original mass and NMR spectra

of the acetylated adduct that was analyzed previously (6). Themass spectrum clearly shows a peak at m/e = 611, which is

consistent with a tetraacetate derivative and which is the highest mass peak observed. However, the integral of the signal

Table 13H:"C ratios of products of the reaction of 1'-acetoxysafrole with

[8-"C; 8-3HldGuo

1'-Acetoxysafrole (10 mg in 0.5 ml of ethanol) was reacted with dGuo (2 mgin 0.5 ml Tris, 0.01 M, pH 7.0) containing 5 /iCi of [8-3H]dGuo and 0.5 fiCi of [8-MC]dGuo. A 0.1-ml aliquot was chromatographed on HPLC, and the fractionswere assayed for 3H and "C.

dGuoAdduct IAdduct IIAdduct III3H:'4C9.2

7.98.9

7.4%

of retention of3H100

869782

DMSO

ADDUCT

Chart 7. 'H NMR spectrum (270 MHz) of Adduct II in

dimethyl sulfoxide (DMSO)-de (5 mg in 0.4 ml). The material was obtained by treatment with alkaline phosphataseof the major product of the reaction of 1'-acetoxysafrolewith dGMP (see "Materials and Methods").





for the acetyl protons in the 60-MHz NMR spectrum accounts

for about 10 protons, i.e., intermediate between that expectedfor triacetate (9 protons) and tetraacetate (12 protons) derivatives. A broad signal at 7.9 ppm (the C-8 signal appeared at

7.65 ppm), which was exchangeable with D2O, was not assigned in the original study (6). It is possible that this signal isdue to the N2 proton and that the signal at 12.35 ppm, originallyassigned to the A/2 proton, is due to the N-1 proton. In view ofthe fact that several A/2-substituted dGuo or guanosine adducts

derived from other chemical carcinogens are hydrolyzed tovarious extents under acidic conditions (17, 18, 26), it is nowevident that ease of hydrolysis by acid is not a reliable indicatorof an O6-substituted guanine derivative (34).

Structure of the dAdo Adduci IV. The nucleoside adductderived from the large-scale reaction of 1'-acetoxysafrole withdAMP (see "Materials and Methods") comigrated on HPLC in

Solvent Systems B and C with the minor in vivo Adduct IV. ItsNMR spectrum is shown in Chart 8. As in the case of AdductII (Chart 7), the C-1 ', C-2', and C-3' signals are indicative of a

frans-isosafrole derivative (Jr.2. = 16.0 Hz) substituted at theC-3' position. The signal at 8.06 ppm was exchangeable withD2O and is assigned to the 6-amino group. The signal integrated as a single proton and exhibited pronounced broadening, as did the Â¡sosafrole C-3' signal (4.23 ppm) and theadenine C-2 signal (8.21 ppm). The reason for these broad-

enings is not clear, but, as discussed earlier (26), similarbroadenings have been observed in the spectrum of the reference compound A/6-methyladenosine and of other A/6-substi-

tuted dAdo adducts (1 7, 26). Thus, Adduct IV is assigned thestructure A/6-(frans-isosafrol-3'-yl)deoxyadenosine.

Structures of Adducts I and III. As noted above, substitutionof Adducts I and III apparently occurred on the N2 atom of the

guanine residues. An attempt was made to determine theposition of attachment to the safrole residue in Adduct I byreduction with H2:platinum, acid hydrolysis of the reducedadduct, and identification of the resulting dihydrosafrole derivative; this procedure was successful in the previous study ona DMA adduct formed from 1'-hydroxyestragole (26). However,

this approach was not practical in the present study becauseof our inability to resolve adequately on HPLC the referencecompounds 1'-hydroxy-2',3'-dihydrosafrole and 3'-hydroxy-

1',2'-dihydroisosafrole. Adduct III was available in such small

amounts that further studies were not undertaken.

DISCUSSION

The pattern of DMA adducts formed in the livers of miceadministered 1'-hydroxysafrole is similar to that which we havedescribed recently for the closely related compound 1'-hydrox

yestragole (26). In each case, 3 dGuo and one dAdo adductswere formed, and all involved covalent attachment of the carcinogen moiety to the exocyclic amino groups of the guanineand adenine residues. The major adduct (II) formed by eachcompound contains the 2-amino group of guanine attached toposition 3' of frans-isoestragole or frans-isosafrole. Althoughthe 2 other dGuo adducts (I and III) formed by 1'-hydroxysafrole

have not been fully characterized, it is probable from theirretention times on HPLC relative to that of Adduct II that theyare analogous to the 1'-hydroxyestragole Adducts I and III.

Evidence was presented that the structures of the latter 2adducts were A/2-(estragol-1 '-yOdeoxyguanosine and A/2-(c/s-isoestragol-3'-yl)deoxyguanosine, respectively (26). A minoradduct (IV) was also formed from both 1'-hydroxysafrole and1'-hydroxyestragole; these adducts involved conjugation of the6-amino group of adenine residues to position 3' of frans-isosafrole or frans-isoestragole. The 3'-substituted isosafrole

adducts (II and IV) could arise by an SN1 mechanism, in whichthe purine bases of DNA substituted position 3' of the carbo-nium ion generated by the loss of the 1'-ester group. Alternatively, they could arise by an SN2' mechanism, whereby the2',3'-allylic double bond shifted to become a 1',2'-p,">penylic

double bond with loss of the ester group. These pocsiblereaction mechanisms were discussed in more detail in >:""earlier paper on the adducts formed from 1'-acetoxyestragole

(26).Although the major share of the 3H in the hydrolysates of

hepatic DNA from mice given either 1'-[3H]hydroxysafrole or1'-[3H]hydroxyestragole migrates with Adducts I to IV formedfrom either 1'-acetoxysafrole or 1'-acetoxyestragole and dGuoor dAdo, some 3H products eluted prior to these adducts(Charts 3 to 5; Ref. 26). Furthermore, the amounts of these 3H

products, which were eluted between 7 and 20 min, appeared

Chart 8. 'H NMR spectrum (270 MHz) of Adduct

IV in dimethyl sulfoxide (DMSOWe (5 mg in 0.4 ml).The material was obtained by treatment with alkalinephosphatase of the major product of the reaction of1'-acetoxysafrole with dAMP (see "Materials andMethods").

ADDUCI 12

-OCH20-

C:Ã

UvJ

->-H C-8H20

3-OH S'-OHta

M

JULY 1981 2669




to be greater when 1'-[3H]hydroxysafrole was administered.

The reasons for this quantitative difference are not known.Some products from the reaction of 1'-hydroxysafrole-2',3'-oxide and 1'-oxosafrole with dGuo or dAdo eluted in this area,

and it is possible that these electrophiles, but not the corresponding electrophilic derivatives from 1'-hydroxyestragole,

contributed small but significant amounts to the DMA adductsformed in vivo. Some radioactivity from tritiated polycyclicaromatic hydrocarbons has been found to elute very early onchromatography of cellular DNA hydrolysates (2, 3, 10, 25).Some of this radioactivity has been attributed to incorporationof tritium into normal deoxyribonucleosides (2, 3, 25). However, in one case, its dependence on the enzymatic methodused for the hydrolysis of the DNA suggested that there mightalso be carcinogen-modified fragments of DNA resistant to

complete hydrolysis (8). Whether or not such circumstancesare involved in the substantial amounts of 3H that eluted be

tween 7 and 12 min on chromatography of hydrolysates ofhepatic DNA from 1'-hydroxysafrole-treated mice is not known.

Although 1'-hydroxysafrole appeared to be bound to mouseliver DNA a little more rapidly (Chart 2) than was 1'-hydroxyes

tragole at the same dose (26), the total amounts bound and theshapes of the curves for the formation and loss of the DNA-,rRNA-, and protein-bound derivatives of 1'-hydroxyestragoleand 1'-hydroxysafrole were quite similar. Moreover, with both

compounds, there were no significant differences in the relativeproportions of the DNA Adducts I to IV at each of the timepoints studied. Thus, although most of the adducts involvingA/2-guanine and /V6-adenine linkage were apparently readily

excised from DNA in vivo, a small but significant proportionwas not removed by 20 days after treatment. Adducts involvingreaction at N2 of guanine and derived from other types of

chemical carcinogens appear to be retained in rodent liverDNA without appreciable loss for several weeks after treatment(4, 19, 31, 33). The type of binding curves found for 1'-hydroxysafrole (Chart 2) and 1'-hydroxyestragole (26), inwhich only a minor proportion of the A/2-guanine (and N6-

adenine) residues persists in the DNA, has not been observedwith other classes of chemical carcinogens.

Safrole and estragÃ³le belong to a large class of ring-substituted allylbenzenes and propenylbenzenes which occur inmany higher plants that are used in the cosmetic and foodflavoring industries (12, 13, 20). Studies are continuing in ourlaboratory on the biological properties and mechanism of actionof various compounds of this class.

ACKNOWLEDGMENTS

The authors are grateful to Grant Grothman for excellent technical assistance.

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JULY 1981 2671



1981;41:2664-2671. Cancer Res David H. Phillips, James A. Miller, Elizabeth C. Miller, et al.

in Vivo-Hydroxysafrole to Mouse Liver DNA ′for Covalent Binding of Metabolically Activated 1

Atom of Adenine Residues as Sites6N Atom of Guanine and 2N

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