Hydroxysafrole and the Electrophilic Reactivity of I'-Acetoxysafrolel

Home , Piperonal, Safrole

[CANCER RESEARCH 33,575-589, March 1973] The Metabolism of the Naturally Occurring Hepatocarcinogen Safrole to 1' -Hydroxysafrole and the Electrophilic Reactivity of I'-Acetoxysafrolel

Peter Borchert, Peter G. Wislocki, James A. Miller, and Elizabeth C. Miller McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center, Madison, Wisconsin 53706

reported: l'-acetoxysafrole, 3'-acetoxyisosafrole, 3'- SUMMARY bromoisosafrole, 3'-methylmercaptoisosafrole, and l'-me- A conjugated form of l'-hydroxysafrole was identified asa thoxysafrole. urinary metabolite of safrole; the conjugate was cleaved by commercial 0-glucuronidase preparations. The conjugated l'-hydroxysafrole accounted for 1 to 3% of an i.p. dose of INTRODUCTION safrole administered to rats, hamsters, or guinea pigs. Pretreatment of rats with phÃ©nobarbitalor 3-methyl- Safrole (Chart 1) or 4-allyl-l,2-methylenedioxybenzene is a cholanthrene increased about 10-fold the excretion of l'-hy plant constituent that is a hepatotoxin for animals and man ( 1, droxysafrole conjugate(s) after a dose of safrole; these 11, 13, 16-20, 30, 63). It is a major (70 to 80%) component treatments had little effect on the urinary excretion of of oil of sassafras and certain oils from the Heterotropa genus, conjugated l'-hydroxysafrole after injection of the l'-hydroxy a minor component (1 to 4%) of some other essential oils, and derivative. Pretreatment with phÃ©nobarbitaldidnot increase a very minor (ca. 0.1%) component of oils of nutmeg, star the excretion of conjugated l'-hydroxysafrole by guinea pigs anise, mace, and cinnamon leaf (30, 52â€”55,57). Safrole is or hamsters given safrole injections. Male mice excreted 30% thus a natural ingredient of sassafras tea, and it was used as a or more of a dose of safrole or l'-hydroxysafrole as a flavoring component in soft drinks such as root beer in the conjugate of the latter compound. The l'-hydroxysafrole United States until 1960. In that year the use of safrole as a released by 0-glucuronidase treatment of urine from rats given food additive was banned in the United States (30) after a safrole injections was isolated and characterized by compari study by the United States Food and Drug Administration son of its ultraviolet, nuclear magnetic resonance, and mass indicated that safrole was a weak hepatocarcinogen in the rat. spectra with those of the synthetic compound. Zeitlin (B. Zeitlin, personal communication cited in Ref. 19), The synthetic model ester l'-acetoxysafrole is an electro- Long et al. (30), Homburger et al. (18, 19), and Abbott et al. philic reactant. It reacts at neutrality with methionine to yield (1) in the early 1960's showed that administration of 0.04 to 3'-methylmercaptoisosafrole, which was characterized by the 1.0% of safrole in the diet of male and female rats for 150 identity of its ultraviolet, nuclear magnetic resonance, and days to 2 years produced hepatic adenomas and hepatic mass spectra with those of the compound prepared from carcinomas. Further confirmatory data were published shortly 3'-bromoisosafrole and methylmercaptan. l'-Acetoxysafrole thereafter (13, 17, 63). Subsequently, it was noted that also reacts with guanosine and adenosine and, probably to a hepatomas were also induced in mice by the long-term small extent, with cytidine as evidenced by the formation in administration of safrole p.o. beginning at the 7th day of life reaction mixtures containing 14C-labeled nucleosides of (20). Likewise, Epstein et al. (11) have reported that 58% of l4C-containing products which are less polar than the parent male Swiss mice killed 1 year after 4 s.c. injections of safrole nucleosides. These products did not contain a significant quantity of 3H from l'-acetoxysafrole-acetoxy-3H. A major (total dose of 6.6 mg) in infancy had hepatomas as compared to a 0% incidence in female mice treated with the same dose of nucleotide product was isolated from a large-scale reaction of l'-acetoxysafrole with guanosine monophosphate. This nucleo safrole and a 6% incidence in control male mice. Thus, under appropriate conditions safrole can act as a moderately potent tide was degraded to the nucleoside, which was acetylated. hepatocarcinogen. The ultraviolet, nuclear magnetic resonance, and mass spectra The present work was undertaken to test experimentally of the acetylated nucleoside are consistent with its characteri zation as 0-6-(isosafrol-3'-yl)-jV-2-acetylguanosine-2',3',5'-tri- our prediction of the metabolic activation of a chemical carcinogen. The prediction was based on the generalization acetate. (43, 44) that the ultimate carcinogenic forms of most, if not The syntheses of the following new compounds are all, chemical carcinogens are strong electrophilic reactants. The structure of safrole was judged to possess little, if any, 'This work was supported by Grants CA-07175 and CRTY-5002 of electrophilic reactivity, and its carcinogenicity in a tissue the National Cancer Institute, USPHS, and by a grant from the Jane distant from the sites of administration further implied that Coffin Childs Memorial Fund for Medical Research. safrole requires metabolic activation to act as a carcinogen. Received October 5, 1972; accepted December 11, 1972. The specific nature of this activation was suggested by the

MARCH 1973 575

Instruments, Inc. (Westbury, N. Y.), respectively. With the exception of chromatograms of nucleotide or nucleoside derivatives, the plates were generally developed in mixtures of H-C-C=CH, n-hexane (Skellysolve B) and ethyl ether. Reference spots of 'l1 t 3' "- OH synthetic safrole derivatives were used as markers on each l'-HYDROXYSAFROLE SAFROLE chromatogram. The chromatograms were viewed under a Chart 1. The structural formulae of safrole and its l'-hydroxy 254-nm mercury lamp to locate UV-absorbing materials. For derivative. detection of small amounts of safrole derivatives or other impurities, the plates were sprayed with vanillin-sulfuric acid reagent (35) and heated at 110-120Â° for about 5 min. Safrole characterization of the pyrrole metabolites of the hepatocarcinogenic pyrrolizidine alkaloids as strongly electrophilic allylic and isosafrole derivatives generally yield purple colors with esters (7, 22, 36â€”39). Similarly, the carcinogenic diaryl this reagent and show distinctive shades and rates of color acetylenic carbamates (14, 15) are benzylic esters and contain development. leaving groups attached to an electrophilic carbon atom. From these considerations we postulated that safrole might undergo Chemicals conversion in vivo to an electrophilic allylic and benzylic ester. The metabolism of safrole in the rat and mouse, as well as in Safrole was purchased from Aldrich Chemical Co. (Milwau certain other species, to the allylic alcohol l'-hydroxysafrole is kee, Wis.); its purity was shown to be greater than 99% upon described in this paper. Data are also reported on the thin-layer chromatography on silica and subsequent examina electrophilic reactivity of its synthetic acetic acid ester, tion of the plates under UV or after treatment with the l'-acetoxysafrole, with methionine and with guanyl residues in vanillin-sulfuric acid reagent. The other safrole and isosafrole guanosine and GMP. The accompanying paper (5) presents derivatives were synthesized by the following procedures. data on the much greater carcinogenic activity of l'-hydroxy Syntheses for l'-hydroxysafrole (61, 62) and 3'-hydroxysafrole, as compared to safrole, for the liver of the rat and isosafrole (2, 12, 48) have been reported previously, but our mouse. Sarcomas developed in the interscapular region of mice procedures are preferable because of the greater simplicity of fed l'-hydroxysafrole and in rats at the site of repeated s.c. the reactions and improved yields of product. Each compound injections of l'-acetoxysafrole. Some of these data have been was analyzed for each of its constituent elements (Huffman published in abstract form (4, 68). Laboratories, Inc., Wheatridge, Colo.); except for the small discrepancies noted below, all of the analyses were within 0.4% of theory. In all cases the assigned structures are MATERIALS AND METHODS consistent with the NMR data (Table 1). All of the isosafrole derivatives, the syntheses of which are described in this report, Instrumentation and General Procedures are the trans isomers, as judged from the large coupling constants (about 15 cps) for the olefmic protons in the NMR Corrected melting points were determined from the spectra. Unless otherwise noted, the organic chemicals for the inflections in rapid time-temperature melting curves obtained syntheses were obtained from Aldrich or Eastman Organic with the Accumelt apparatus (American Instrument Co., Silver Chemicals (Rochester, N. Y.). Acidic conditions were avoided Springs, Md.). UV and infrared spectra were measured with a in the syntheses and use of the safrole compounds to prevent Beckman DB spectrophotometer equipped with a Sargent acid-catalyzed rearrangements to isosafrole derivatives. The recorder and a Beckman 1R-10 spectrophotometer, re compounds were routinely stored at â€”¿20Â°. spectively. NMR2 spectra were determined on a 60-MHz l'-Hydroxysafrole. Vinyl bromide (422 g, 3.95 moles), Perkin-Elmer R-12 spectrometer. A Varian CH-7 mass spec dissolved in 1.5 liters of tetrahydrofuran, was added dropwise trometer was used for determination of mass spectra; the (2 hr) to 80 g (3.3 moles) of magnesium turnings in a flask instrument was standardized with perfluorokerosene (Peninsu equipped with a condenser and a drying tube. The reaction lar ChemResearch, Inc., Gainesville, Fla.). Scans for radio mixture was stirred mechanically and cooled, when necessary, activity were made with a Packard Chromatoscanner equipped to maintain a temperature of about 55Â°.Then 248 g (1.65 with a recording ratemeter. 3H and 14C were determined with moles) of piperonal dissolved in 1 liter of tetrahydrofuran Packard Tri-Carb scintillation spectrometers with the use of were added over a 2-hr period with the temperature of the either Scintisol (Isolab, Inc., Akron, Ohio) or RPI Scintillator reaction maintained at 50Â°.A molar ratio of piperonal to PPO-POPOP (Research Products International, Elk Grove Grignard reagent of 0.5 was used, since higher ratios favored Village, 111.) formation of piperonyl alcohol. The reaction mixture was Thin- (0.25- and 0.5-mm) and thick-layer (1.5- to 2-mm) hydrolyzed with a solution of 450 g of NH4C1 in 2 liters of ice plates were prepared on glass from Silica Gel HF254 and water, and the combined organic phase and an ethyl ether PF254, respectively (E. Merck, Darmstadt, Germany). Silica extract of the aqueous phase were dried with 10 g of gel for column chromatography (Merck, 0.05 to 0.2 mm) was anhydrous sodium carbonate and 200 g of anhydrous used for column separations. Aluminum- and plastic-backed magnesium sulfate. After filtration, the solvent was removed silica gel plates were purchased from Merck and Brinkmann under reduced pressure at 40Â°.The residue was stripped of low boiling components at 0.5 mm of Hg and 100Â°,and the !The abbreviation used is: NMR, nuclear magnetic resonance. l'-hydroxysafrole was distilled rapidly at 0.2 mm of Hg and

576 CANCER RESEARCH VOL. 33

Table 1 NMR data for certain safrole and isosafrole derivatives (I1) (31) (5) (6) H 0) (CH,-C=C-) H2C=CH-CH (21)H,

The data for each compound are listed in the following order: signal [ppm downfield from trimethylsilane (internal standard)] ; relative number of protons; multiplicity; coupling constant (J, cycles/sec). The abbreviations used for expressing multiplicity are: S, singlet; D, doublet, and M, multiplet. The relative intensity equals the apparent number of protons in the signal as calculated from the ratio of the integration for the signal to the total integration. All compounds were dissolved in deuterated chloroform.

H-2' H-31 Compound H-3, 5,6 H2-7 H-r OH" Other

l'-Hydroxysafrole3'-Hydroxy ,D,46.88,3,M,*6.85,3,M,*6.85,3,M,*6.8,3 1,M,*6.3, 3.08,C,1,S4.28,2,0,4.8 isosafrolel'-Acetoxysafrole3'-Acetoxy 1,M,*6,1,M,*6.1.1.M,*5.9,1.75,dl,S5.23,2,M,*4.69,2,0,5.55.22,2,M,*4.18,2,0,6.93.2,2,0,6AcetylAcetylMethoxyMethyl-mercapto 1,M*6.6,1,0,15.54.53,1,D,66.62,1,0,15.56.05,2 isosafrolel'-Methoxysafrole3'-Bromoisosafrole3'-Methylmercapto-isosafrole6.74,3 ,M,*6.92,3 1,M,*6.22,1,M,*(Included ,M,*6.82,3,M,*5.82,2,55.96,2,55.88,2,55.94,2,55.95,2,55.98,2,55.9,2,55.05,l,M*b6.46,1,D,156.21, ,M,155.95, withH-iy5.25,2,D(M),10

" The signals of the OH protons were exchangeable with D2O. b *, overlapping J which could not be measured accurately. c 250 mg/ml. d 125 mg/ml. e Signals for H-l' and H-2' protons of 3'-methylmercaptoisosafrole overlap completely.

120Â°to avoid decomposition. The product was judged to have propionitrile) (1 g) was added to initiate the reaction. If the a purity of greater than 99.5% by thin-layer chromatography. reaction mixture did not turn yellow after 8 to 10 min of Yield, 76%; colorless oil; nD23, 1.5610. Xmax 238 nm (e, refluxing, another 1 g of the azonitrile was added. The 4,560), 286 nm (e, 3,930) in methanol. Mass spectrum: m/e = reaction was complete after approximately 40 min of 178 (m+), 161 (m+--OH), 149, 135, 131. The product was refluxing, as indicated by the disappearance of the N- presumably racemic, since no rotation was observed with a bromosuccinimide and the formation of solid succinimide. 10% solution in ethanol and a 10-cm light path. Suga et al. With a reaction time of 70 to 80 min, decomposition products (61) obtained a yield of 51% with a similar synthesis starting were formed and interfered with crystallization of the 3'-bromoisosafrole. The reaction mixture was cooled in an ice with vinyl chloride. 3'-Hydroxyisosafrole. 3'-Hydroxyisosafrole was prepared bath and filtered, the CC14 was removed, and the product was much more readily by rearrangement of l'-hydroxysafrole poured into an evaporating dish to crystallize at room than by any of the literature methods (2, 12, 48). temperature. After 4 to 5 hr on an unglazed plate, the product l'-Hydroxysafrole (50 g, 0.28 mole) was dissolved in 250 ml was dissolved in 450 ml of ethyl ether and boiled with 500 mg of tetrahydrofuran and 50 ml of 0.02 N HC1, and the mixture of Norit A. The solution was evaporated to about 100 ml, and was refluxed until thin-layer chromatography showed com the 3'-bromoisosafrole crystallized as colorless needles at 5Â°. plete disappearance of the l'-hydroxysafrole (about 2 hr). The Yield, 54%; m.p. 69Â°.Xmax 276 nm (e, 13,700), 310 nm (e, solution was then neutralized with solid sodium bicarbonate, 11,200) in ethyl ether. poured into 200 ml of water, and extracted 4 times with 150 l'-Acetoxysafrole. Pyridine (250 ml, Mallinckrodt reagent) ml of ethyl ether. The combined organic extracts were dried was added to the l'-hydroxysafrole (not distilled) obtained with anhydrous magnesium sulfate, and the solvent was from 1.65 moles (248 g) of piperonal, and 204 g (2 moles) of removed under reduced pressure at 40Â°.The liquid residue was acetic anhydride were added dropwise to the magnetically transferred to a 6.8-cm diameter column containing 1 kg of stirred solution at room temperature. When the esterification silica, which was developed with hexane:ethyl ether (1:1) and was complete as determined by thin-layer chromatography (4 monitored by thin-layer chromatography. The 3'-hydroxy- to 5 hr), the reaction mixture was diluted with 600 ml of isosafrole eluted from the column in essentially pure form, as mÃ©thylÃ¨nedichloride and extracted several times with 250-ml judged from the thin-layer chromatography, and was recrystal- aliquots of cold l N HC1 and, as the pH began to fall, with 0.1 lized from ethyl ether. Yield, 60%; m.p. 77Â°;analysis for C, N HC1. As soon as the aqueous extract remained acidic (pH 2 0.45% lower than theory (67.41%). Xmax 265 nm (e, 12,400), to 3), the organic layer was immediately extracted with 400 304 nm (e. 6,400) in methanol. Mass spectrum: m/e = 178 ml of l M sodium carbonate solution. After being dried with (m+), 161(m+-OH), 139, 127. anhydrous MgS04 the solvent was removed under reduced 3'-Bromoisosafrole. Safrole (100 g, 0.62 mole) was dissolved pressure, and the remaining oil was distilled at 102Â°and 2 mm in 600 ml of CCU, 110 g ofTV-bromosuccinimide were added, of Hg. Yield, 84% (based on piperonal); colorless oil;Â«D23, and the suspension was stirred magnetically in a flask equipped 1.5263. Xraax 239 nm (e, 4,970), 285 nm (e, 4,100) in with a condenser and drying tube. 2,2'-Azobis(2-methyl- methanol. Mass spectrum: m/e = 220 (m*), 178 (m*

MARCH 1973 577

- CH2=CO), 160, 148, 131. On the basis of thin-layer sulfate. The ether was removed under reduced pressure, and chromatography, the fresh product or that stored at -20Â° had the residue (22 g) was chromatographed on a column of silica a purity of at least 99%. (1 kg) with hexane:ethyl ether (3:1). The flow rate was l'-Acetoxysafrole-acetoxy-3H. After the acetic anhydride- maintained at 10 ml/hr/sq cm to facilitate the separation of 3H (Amersham/Searle, Inc., Arlington Heights, 111.,26 mg, 100 the l'-methoxysafrole from the byproducts, 3'-hydroxy- and 3'-methoxyisosafrole. The column fractions which contained mCi/mmole) was concentrated at the bottom of the shipping vial by cooling with liquid nitrogen, the vial was opened and the l'methoxysafrole were combined and distilled under 214 mg (1.2 mmoles) of l'-hydroxysafrole in 3 ml of pyridine reduced pressure. Yield, 40%; b.p. 127Â°(3 mm of Hg);nD23, were added. After 2 hr at room temperature, 75 mg (0.72 1.5324. Xmax 239 nm (e, 4,800), 285 nm (e, 4,000) in mmole) of nonradioactive acetic anhydride were added. The methanol. Mass spectrum: m/e =192 (m+), 165 (m* mixture stood overnight and, after addition of 7 ml of CH=CH2), 161(w+CH30), 149,131. mÃ©thylÃ¨nedichloride, was extracted twice with 10 ml of l N Isolation of l'-Hydroxysafrole from Rat Urine HC1 and 5 times with 10 ml of 0.1 N HC1. The organic phase, which still contained some pyridine, was then extracted with 5 ml of 5% sodium carbonate solution, dried with anhydrous Safrole [90 mg in 0.5 ml of trioctanoin (Eastman)] was MgSO4 and evaporated. The residue was chromatographed on injected i.p. into each of 8 male rats (approximately 300 g a thick-layer plate which was developed with hexane:ether body weight), in which the bile ducts had been ligated 24 hr (2:1), and the material with the RF of the reference previously. The urine (170 ml) was collected for 30 hr in tubes 1'-acetoxysafrole was eluted with 250 ml of chloroform. The cooled by Dry Ice and diluted with 200 ml of 0.1 M phosphate yield was 136 mg (specific activity, 10.2 mCi/mmole). buffer, pH 6.9, containing 4.1 g of ÃŸ-glucuronidase (Sigma, 3'-Acetoxyisosafrole. 3'-Hydroxyisosafrole (6 g) was dis Type 1, 42 units/mg) and 5 ml of chloroform. After 5 hr at 37Â°,the mixture was extracted 4 times with 100 ml of ethyl solved in 150 ml of pyridine and 7 g of acetic anhydride and stirred in a closed flask for 4 hr, after which it was diluted ether. The ether was dried with anhydrous MgSO4 and with 300 ml of mÃ©thylÃ¨nedichloride and extracted several evaporated, and the residue (60 mg) was spotted on a silica gel thick-layer plate which was developed with hexane:ethyl ether times with l N HC1 until the extracts remained acidic. The (1:1). The silica at the RF's of I'-hydroxysafrole and organic layer was dried with anhydrous MgSO4, the solvent 3'-hydroxyisosafrole was eluted with ethyl acetate. This was removed under reduced pressure, and the residual oil was preparation yielded 3 mg of l'-hydroxysafrole and 0.5 mg of crystallized from ethyl ether. Yield, 67%; white crystals, m.p. 39Â°;analysis for C, 0.46% greater than theory (65.45%). Xmax 3'-hydroxyisosafrole. 265 nm (e, 13,200), 304 nm (e, 7,410) in methanol. Mass A 2nd isolation was carried out on the urine from 12 male spectrum: m/e = 220 (m+), 177 (m+ - CH3CO), 160, 148. rats (300 g), each of which was given an i.p. injection of 15 mg S'-Methylmercaptoisosafrole. 3'-Bromoisosafrole (900 mg) of 3-methylcholanthrene in 0.5 ml of corn oil 24 hr prior to in 30 ml of acetone was added dropwise with stirring to an the first of 3 daily injections of 150 mg of safrole in 0.5 ml of ice-cold mixture of 60 ml of 0.2 N NaOH, 30 ml of acetone, trioctanoin. The urine from these rats, which was incubated and 4 g of methylmercaptan. Thin-layer chromatography of an for only 3 hr with (3-glucuronidase, yielded 120 mg of aliquot after 15 min indicated that the 3'-bromoisosafrole had l'-hydroxysafrole and only trace amounts of 3'-hydroxy- been completely converted to 3'-methylmercaptoisosafrole isosafrole. with no detectable byproducts. The reaction mixture was then Analysis of Urines for 1'-Hydroxysafrole3 extracted twice with 200 ml of ethyl ether, the ethereal solution was dried over anhydrous MgSO4 and the ether was Rats (CD random bred) and mice (GDI) were obtained from removed in a stream of N2. The residue was spotted on 3 thick-layer plates (20 x 20 cm) which were developed with the Charles River Breeding Laboratory, Wilmington, Mass.; golden Syrian hamsters (Con Olson Co.) and random-bred hexanetethyl ether (4:1). The silica containing the product guinea pigs (O'Brien Co.) were obtained in Madison, Wis. All was eluted with 125 ml of CC14. The product was distilled at 92Â°(3 mm of Hg). Yield, 67%; colorless oil;Â«D24, 1.6080;C of the animals were fed a grain diet (50) for at least 1 week prior to the administration of the safrole derivative; the diet analysis was 0.47% less than theory (63.43%) and S analysis for the guinea pigs was supplemented with 1 g of ascorbic acid was 0.48% more than theory (15.39%). The compound had per kg. In some cases sodium phÃ©nobarbital(Merck and Co., absorption maxima at 266 and 306 nm in methanol. Mass spectrum: m/e = 210 (m+ + 2), 208 (m+), 161 (m* - CH3S), Rahway, N. J.) was included in the drinking water for 1 week prior to and after administration of the test compound; the 131.l'-Methoxysafrole. l'-Hydroxysafrole (20 g, 0.11 mole) was weanling rats received 0.5 mg/ml and all other animals received 1 mg/ml of water. In a few experiments 3-methylcholanthrene stirred for 4 hr with 40 g of dry Ag2O (Allied Chemicals and (5 mg per 100 g body weight per 0.25 ml corn oil) was Dye Corp., New York, N. Y.), 60 g of methyl iodide, and 40 ml of dimethyl formamide. When, as determined by thin-layer injected i.p. 24 hr before the safrole. In some animals of each chromatography, all of the l'-hydroxysafrole had been used 3In each case studied, the I'-hydroxysafroie was excreted in the up (about 2.5 hr), the reaction mixture was poured into 200 urine entirely, or nearly so, as a conjugate that was cleaved by the crude ml of water and extracted 3 times with 150 ml of ethyl ether. /3-glucuronidase preparation. However, for convenience, in this paper The combined ethyl ether extracts were filtered, extracted the urinary metabolite(s) that yield I'-hydroxysafrole by the procedure several times with water, and dried with anhydrous magnesium described are referred to collectively as I'-hydroxysafrole.

578 CANCER RESEARCH VOL. 33

species, the common bile duct was ligated with surgical thread 0.30 I TTT I I ! I I III_ 24 hr before injection of the safrole. Safrole (30 mg/100 g body weight) and l'-hydroxysafrole (10 or 20 mg/100 g body *A 6 ,' \ weight) were injected i.p. in solution in trioctanoin (0.1 ml/100 g body weight). Control animals received similar injections of trioctanoin and, where appropriate, 3-methyl- ll o cholanthrene injections or p.o. phÃ©nobarbital. 0.20-> After injections with safrole or l'-hydroxysafrole, the animals were maintained in stainless steel metabolism cages equipped with a screen for the separation of feces and a tube A cooled with Dry Ice for collection of the urine. The animals received water but no food during this period. At the end of the 24-hour collection period, the metabolism funnels were 0.10 washed with 0.1 M phosphate buffer, pH 6.8. The bile from the gallbladders and ligated ducts was collected in a syringe. All samples were stored at -17Â°. \- In another experiment, urine was collected at various times V during the continuous feeding of the grain diet containing 0.5% of safrole or 0.55% of l'-hydroxysafrole. The male rats IIII 230 240 250 260 270 280 290 300 (initial weights, 200 to 250 g) were trained to eat only during three 45-min periods each day (starting at 8 a.m., 12 noon, nm Chart 2. The UV absorption spectra in methanol of synthetic and 4 p.m.) and were fed outside the metabolism cages. One 1'-hydroxysafrole (X) and the metabolite (o) obtained by extraction group fed the safrole-containing diet also received 0.1% of and thick-layer chromatography from the urine of rats given safrole sodium phÃ©nobarbital in the drinking water. In a similar injections. Arrow, point at which the 2 spectra were arbitrarily experiment, male mice were fed 0.50% safrole or 0.55% l'-hydroxysafrole on the same time schedule for 2 weeks, at matched. which time they were transferred to metabolism cages for collection of two 24-hour samples of urine. The urinary excretion data have been corrected for the The urine samples were thawed immediately prior to absorption at 286 nm of equivalent preparations from the analysis and diluted to 100 ml with the phosphate buffer. urine of comparably treated control animals and for the 0-Glucuronidase (1680 units, Sigma type 1) in 1 ml of recovery of l'-hydroxysafrole added to urine of control phosphate buffer and 0.3 ml of chloroform were added to a animals of the same species. The absorption at 286 nm of 10-ml aliquot. After incubation at 37Â°for 4 hr, each sample preparations from the urine of control animals was usually no was immediately extracted 3 times with 10 ml of ethyl acetate more than 10% of the absorption due to l'-hydroxysafrole in (equilibrated with water), the combined extracts were dried extracts from the urine of safrole-treated animals. With the overnight with anhydrous magnesium sulfate and sodium urines from phenobarbital-pretreated guinea pigs and hamsters, carbonate (to ensure basicity), and the extracts were filtered this blank value was higher (up to 25%); with animals and evaporated at 30Â°. Each residue was extracted 3 times excreting high levels of l'-hydroxysafrole (mice and 3-methyl- with 2 ml of ethyl ether. These extracts were concentrated and cholanthrene- or phenobarbital-treated rats), the blank value chromatographed on 0.5-mm silica gel thin layers on glass was less than 5% of the absorption due to l'-hydroxysafrole. plates with hexane:ethyl ether (1:1). The areas corresponding The recovery of l'-hydroxysafrole averaged about 70% unless to l'-hydroxysafrole (as judged from the RF of the a 2nd chromatography was necessary, in which case the simultaneously chromatographed standard and observation recovery was about 50%. under UV) were eluted with ethyl ether. The residues after evaporation were dissolved in methanol for determination of Reaction of 1'-Acetoxysafrole with Methionine and Isolation of 3'-Methylmercaptoisosafrole the absorption from 220 to 300 run. The positions of the absorption maxima (238 and 286 nm) and the correct ratio of the absorbance at these wavelengths (Chart 2) were used as For a typical reaction, a solution of methionine (2.7 g, 18 criteria for the purity of the 1'-hydroxysafrole. In some cases mmoles) in 175 ml of 0.1 M sodium-potassium phosphate (especially with the urine from guinea pigs and hamsters and buffer, pH 7 or 8, was mixed with a solution of 4 g (18 from rats given phÃ©nobarbital),2 successive thin-layer chroma- mmoles) of l'-acetoxysafrole in 175 ml of acetone. After 24 hr at 37Â°,l'-acetoxysafrole could no longer be detected. The tograms were necessary to obtain spectroscopically pure 1'-hydroxysafrole. incubation mixture was then extracted twice with 300 ml of The yields of l'-hydroxysafrole were not increased by ethyl ether, and the ethereal solution was dried with longer incubations with 0-glucuronidase or by doubling the anhydrous MgS04 and evaporated to dryness. The entire amount of /3-glucuronidase with the usual incubation time residue (3.1 g) was streaked on 3 thick-layer silica plates (20 x of 4 hr. However, with longer incubations more 3'-hydroxy- 20 cm) which were developed in hexane:ethyl ether (4:1). The silica from the area with the RF of 3'-methylmercaptoisosafrole was found; this product presumably arose from isomerization of the l'-hydroxysafrole. isosafrole was scraped from each plate (although no product

MARCH 1973 579

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Borchert, Wislocki, Miller, and Miller band was detected on examination under UV light) and eluted to 8 sections, and the silica from each section was transferred with ethyl acetate. This solution was concentrated and to Scintisol for determination of radioactivity. The percentage streaked on a thin-layer plate developed in the same solvent. of reaction was calculated as the percentage of the I4C from The product (3.5 mg, approximately 0.1% yield) was eluted the nucleoside that moved as a band with RF higher than that from the silica at the RF of 3'-methylmercaptoisosafrole and of the parent nucleoside. The RF of the guanosine product(s) characterized by comparison of its mass, UV, and NMR was about 1.3 times that of guanosine, and the RF of the spectra with those of S'-methylmercaptoisosafrole. adenosine produces) was about 1.4 times that of adenosine. Before being mixed with the safrole derivative, the appropriate labeled nucleoside [guanosine-8-'4C, adenosine-8- Quantitation of the Reaction of Safrole Derivatives I4C, cytidine-2-I4C, thymidine-2-I4C, or uridine-2-'4C with Methionine (Schwarz/Mann, Orangeburg, N. Y.)] was added to the The reaction of 1'-acetoxysafrole with methionine was unlabeled nucleoside to give a final specific activity of about 0.33 Â¿iCi/Aimole.In some cases l'acetoxysafrole-acetoxy-3H quantitated by conversion of radioactivity from methionine- methyl-3H to 3'-methylmercaptoisosafrole, by analogy with (10.2 nd/fimok) was used in the assay. the procedure used to quantitate the reactions of esters of the Reaction of 1'-Acetoxysafrole with GMP aromatic hydroxamic acids and hydroxylamines with methio nine (8, 31, 41, 49, 56). For this purpose 1 ml of 50% acetone:water containing 45 AmÃ³lesof 1'-acetoxysafrole (10 GMP (4 g, 9.8 mmoles), dissolved in 1070 ml of 0.1 M Tris-chloride buffer, pH 8, was mixed with a solution of 17.7 g mg) or another safrole or isosafrole derivative, 0.13 mg (0.9 (80 mmoles) of 1'-acetoxysafrole dissolved in 940 ml of /Â¿mole)of methionine-methyl-3H (Amersham/Searle) [specific acetone. After 3.5 hr at 37Â°, the solution was extracted 3 activity, 0.08 piCi//imole after dilution and preextracted with benzene:hexane (3:7) to reduce the amount of 3H in the times with 450 ml of ethyl ether. The aqueous phase was freeze-dried, and the residue (19 g) was dissolved in 30 ml of blank], and 50 AmÃ³les of sodium phosphate buffer, pH 7.4, was incubated at 37Â°for 2, 5, or 20 hr. After addition of 2 ml water and applied to a Sephadex LH-20 column (1.5 m x 3 of 0.55 N NaOH, each sample was extracted twice with 3 ml cm; bed volume of 1.2 liters). The initial flow rate was 3.7 of benzene:hexane (3:7), and the combined organic extracts ml/hr/sq cm. GMP (1.8 g) was eluted after about 650 ml of were washed twice with 2 ml of water and dried with water had passed through the column; at this time the flow anhydrous magnesium sulfate. Aliquots (2.0 ml) were evapo rate was 1 ml/hr/sq cm. After 1.22 liters of water had passed through the column, 2 overlapping UV-absorbant fractions rated to dryness, redissolved, and chromatographed as 2 to 3 cm bands on aluminum-backed silica gel thin-layer plates with (Products 2 and 3) were eluted. The elution of the major UV-absorbing product (Product 1) began after 1.43 liters of hexane:ethyl ether (3:1) after addition of 75 /ig of synthetic 3'-methylmercaptoisosafrole. In the absence of this carrier, a eluant had been collected. An aliquot of each fraction was major share of the radioactive 3'-methylmercaptoisosafrole chromatographed on a silica thin-layer plate, and the fractions apparently oxidized on the plates, since the radioactivity with similar products were pooled. moved with a RF less than that of 3'-methylmercapto- Product 1. The pooled fractions containing this product isosafrole. The silica in the UV-absorbing bands was trans (which was assumed to be a nucleotide) were pooled and ferred to scintillation vials that contained PPO-POPOP freeze-dried to a volume of 2 ml, which from the absorption at scintillator. With reaction mixtures containing 1'-acetoxy 260 nm and the extinction coefficient of GMP was calculated safrole and methionine-methyl-3H and with the addition of to contain about 250 mg of product. After addition of 18 ml carrier 3'-methylmercaptoisosafrole before chromatography, of 0.1 M Tris buffer, pH 8, and 4 mg of alkaline phosphatase (Sigma, 33 units/mg), the solution was incubated at 25Â°for 24 60 to 80% of the ex tractable tritium cochromatographed with 3'-methylmercaptoisosafrole. hr. The precipitate that formed was collected by centrifuga- tion, washed 3 times by suspension in 7 ml of water and recentrifugation, and freeze-dried to yield 174 mg of dry white Quantitation of the Reaction of Safrole Derivatives powder. with Nucleosides For acetylation, 150 mg of the above residue (assumed to be a nucleoside) were dissolved in 5 ml of dry pyridine, 2 ml The safrole or isosafrole derivative (4.5 /Â¿molesin 0.1 ml of of acetic anhydride were added, and the mixture was stirred acetone) was added to 0.9 /Â¿moleof nucleoside in 0.9 ml of for 24 hr at room temperature. After the residual acetic 0.1 M buffer (sodium phosphate or Tris-HCl), pH 7.4 (37Â°). anhydride was hydrolyzed with 8 ml of water, the solution After 2 hr at 37Â°,the samples were extracted 3 times with 3 was extracted twice with 10 ml of chloroform. The combined ml of benzene:hexane (3:7). The residual organic solvent was extracts were washed 10 times with 5 ml of water, once with 5 evaporated under a stream of nitrogen, and 25 fi\ of the ml of 5% NaHCOs solution, and 10 times with 10% NH4C1 aqueous phase were chromatographed as a 2.5-cm band on solution. After being dried over anhydrous MgS04, the plastic-backed silica gel thin-layer plates with 1-butano!: chloroform was evaporated, and the residue was applied to a wateracetic acid (100:50:22). The UV-absorbing areas (usu silica thick-layer plate. The thick-layer plate was developed ally only the parent nucleosides) were located on the air-dried first with ethyl ether to separate the residual pyridine (RF 0.5) plates and, in some experiments, the radioactive areas were from the reaction products (RF 0.0). After the band located with a scanner. Each chromatogram was divided into 6 containing the pyridine was scraped from the plate, it was

580 CANCER RESEARCH VOL. 33

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Metabolism and Reactivity of Safrole Derivatives redeveloped with chloroform:methanol (8:1) 6 times until the optical rotation within the error of our instrumentation was major acetylated derivative had separated from the smaller noted. A small amount (approximately 0.5 mg) of 3'-hydroxyiso- amount of another product which, from its Rp, was probably derived from Product 2. The silica containing the major safrole was also isolated from the thick-layer chromatograms acetylated product was eluted with two 50-ml aliquots of in the 1st experiment. The identity of this product with chloroform and two 50-ml aliquots of methanol. The 3'-hydroxyisosafrole was established through comparison of its combined eluants yielded 145 mg of a product which gave RF on silica gel thin-layer plates and its mass, NMR, and UV only 1 spot (RF about 0.3) on thin-layer chromatography on absorption spectra with those of the synthetic compound. However, further studies indicated that the 3'-hydroxyiso- silica with chloroform:methanol (4:1) and which has been safrole was formed by rearrangement of l'-hydroxysafrole. tentatively characterized. Product 2. The pooled freeze-dried column fractions Thus, only traces of 3'-hydroxyisosafrole were observed on containing Product 2 showed on thin-layer chromatography 2 isolation of 120 mg of l'-hydroxysafrole from urine when a UV-absorbing materials in addition to Products 1 and 2. 3-hr incubation at 37Â°was used instead of the 5-hr incubation. Treatment with alkaline phosphatase yielded about 90 mg of a Furthermore, the analyses on the urine from various species revealed only traces, if any, of 3'-hydroxyisosafrole. A 4-hr new product, assumed to be a nucleoside. Because of a major UV-absorbing band which moved slightly above and partially incubation at 37Â°was used, the urine samples were frozen overlapping the acetylated product, thick-layer chromatog until they were analyzed, and the l'-hydroxysafrole was raphy of 90 mg of the crude product acetylated as described extracted immediately after the incubation with (3-glucuroni- for Product 1 yielded only 5 mg of apparently pure product. dase. This product gave only 1 spot (RF about 0.4) on thin-layer The l'-hydroxysafrole is probably excreted in the urine as a glucuronide, since less than 3% of the l'-hydroxysafrole in the chromatography on silica with chloroform:methanol (4:1), but the NMR and mass spectra were not adequate for urine of rats given safrole injections could be extracted prior characterization of the compound. to treatment with 0-glucuronidase. However, the possible activity of other hydrolases in the bacterial (3-glucuronidase preparation has not been excluded. Since 1,4-saccharolactone RESULTS is reported to be a relatively specific inhibitor of ÃŸ-glucuronidase (27, 28), its ability to inhibit the liberation of Isolation and Identification of 1'-Hydroxysafrole from the 1'-hydroxysafrole by j3-glucuronidase preparations was studied. Urine of Rats Given Injections of Safrole. 1'-Hydroxysafrole At 10~4 M, l ,4-saccharolactone inhibited the release of was isolated by thick-layer chromatography of extracts of ether-extractable l'-hydroxysafrole from urine incubated for /3-glucuronidase-treated urine from safrole-treated rats. Three 0.5 hr with 150 units of ÃŸ-glucuronidaseper ml by 50%; the mg of the compound were isolated from the urine of 8 rats, |3-glucuronidase was limiting under these conditions. This each of wlÃ¼chreceived an i.p. injection of 30 mg of safrole per degree of inhibition was less than was anticipated from the 100 g body weight, and 120 mg were obtained from the urine literature (27, 28), but other studies in our laboratory have of 12 rats given 3-methylcholanthrene injections prior to the shown that 10~4 M l ,4-saccharolactone inhibits the release of i.p. injection of 50 mg of safrole per 100 g body weight. The TV-hydroxy-2-acetylaminofluorene from its pure O-glucuro- identity of the metabolite as l'-hydroxysafrole was suggested nide by only 70% (B. W. Butler, E. C. Miller, and J. A. Miller, by the fact that the RF for both the synthetic compound and unpublished data). the metabolite was about 0.5 on chromatography on silica gel Urinary Excretion of 1'-Hydroxysafrole after Administra thin-layer plates with hexane:ethyl ether (1:1) and both tion of Safrole or 1'-Hydroxysafrole.3 Rats excreted about 2% products showed a characteristic purple color when the plates of an i.p. dose of 30 mg of safrole per 100 g of body weight in were sprayed with the vanillin-sulfuric acid reagent. The UV the urine as conjugate(s) of l'-hydroxysafrole (Table 2). The absorption specta of the synthetic and metabolic compounds urinary excretion was similar for 4-week-old or adult male and for adult female rats. The urinary excretion of l'-hydroxy were essentially identical (Chart 2). The structure of the product from urine was confirmed by mass spectrum analysis safrole after a dose of safrole increased about 10-fold if young (Chart 3); both the synthetic and metabolic compounds or adult male rats were pretreated by administration of showed the expected mole peak of 178, and they had very phÃ©nobarbital in the drinking water or by i.p. injection of 3-methylcholantlirene. When l'-hydroxysafrole (10 mg/100 g similar fragmentation patterns. The NMR of the metabolite was also very similar to that of synthetic l'-hydroxysafrole body weight) was injected i.p. into 4-week-old male rats, about 40% was excreted in the urine as l'-hydroxysafrole, and the (Chart 4). The doublet from the aromatic protons at 6.74 ppm, the singlet of the methylenedioxy protons at 5.82 ppm, amount excreted was not markedly altered by pretreatment and the multiple peaks for the l'-, 2'-, and 3'-protons centered with phÃ©nobarbitalor 3-methylcholanthrene. With rats, the percentage of p.o. safrole or l'-hydroxy at 5.05, 5.95, and 5.25 ppm, respectively, are the same for the safrole excreted in the urine as l'-hydroxysafrole conjugates metabolite and the synthetic compound. The proton of the l'-hydroxy group is exchangeable with D20. The signal for was similar to that observed after i.p. injection. When the this proton moves from 1.9 ppm at a concentration ofSOmg compounds were fed continuously in the diet to male rats, the of l'-hydroxysafrole per ml to 3.2 ppm at 500 mg/ml. An excretion of l'-hydroxysafrole was 5 to 10% of the daily attempt to determine whether the racemic compound or 1 intake of safrole during the 1st 18 days and thereafter optical isomer was excreted was inconclusive; only a very low averaged 3 to 4% (Chart 5). With continuous administration of

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Borchen, Wislocki, Miller, and Miller The urinary excretion of 1'-hydroxysafrole by adult male 0.1% sodium phÃ©nobarbitalin the drinking water, the urinary excretion of 1'-hydroxysafrole averaged 7% of the safrole hamsters or guinea pigs was similar to that of rats (average intake over an 11-week period. The urinary excretion of p.o. values of 2.5 and 3.5%, respectively), but with these species l'-hydroxysafrole averaged 40% over the 7-month period. the administration of phÃ©nobarbital in the drinking water did not increase it. Male mice, on the other hand, excreted in the urine about 33% of an i.p. dose of safrole as conjugate(s) of 100,1 1'-hydroxysafrole; this level was similar to that found after administration of l'-hydroxysafrole. PhÃ©nobarbital pretreat ment did not increase the excretion of l'-hydroxysafrole by 50'.I-METABOLITE1li mice, but the dose used (0.1% in the drinking water) was somewhat toxic. Male mice fed 0.5% of safrole or 0.55% of I li 11ilII 1L1IIIIII II-1 1'-hydroxysafrole in the diet for 2 weeks excreted 17 and 6%, III respectively, of the ingested compound as l'-hydroxysafrole. The species differences in the excretion of l'-hydroxy 0050SYNTHETIC l'-HYDROXY-SAFROLE1. safrole were apparently not due to differences in the relative amounts of excretion via the biliary and urinary systems. Thus, with rats, guinea pigs, and hamsters, the amounts excreted were not increased if their bile ducts were ligated 24 1iItili 11II i.lllili 11 hr prior to the injection of safrole. Furthermore, the bile that could be recovered from the gallbladders and distended bile 180 160 140 120 IOC m/ei ducts of these animals contained very little, if any, free or Chart 3. The mass spectra of synthetic 1'-hydroxysafrole and the conjugated l'-hydroxysafrole. Reaction of l'-Acetoxysafrole with Methionine and Charac metabolite obtained by extraction and thick-layer chromatography from terization of the Products as 3'-Methylmercaptoisosafrole and the urine of rats given safrole injections.

TMS

I'-HYDROXYSAFROLE

Chart 4. The NMR spectra in CDC13of synthetic 1'-hydroxysafrole and the metabolite obtained by extraction and thick-layer chromatography from the urine of rats given safrole injections. This sample of the metabolite was obtained from rats pretreated with 3-methylcholanthrene prior to injection of safrole. The concentrations of the synthetic and metabolic compounds were 150 and 50 mg/ml, respectively; inset, peak for the hydroxyl proton of synthetic l'-hydroxysafrole at 50 mg/ml. As indicated by the shading, the hydroxyl proton signal was lost on addition of D,O. Different instrument settings were used for the 2 spectra to obtain signals of comparable intensities. The crossed out peaks appeared to be derived from solvents used in the isolation and purification of the compound.

582 CANCER RESEARCH VOL. 33

Table 2 Urinary excretion of I'-hydroxysafrole after administration ofsafrole or l'-hydroxysafrote to various animals

l'-hydroxysafrole SpeciesRatMouseGuinea injected0SafroleSafroleSafroleSafroleSafroleSafroleSafroleSafrolel'-Hydroxysafrolel'-Hydroxysafrole1(%ofdose)c1.62.81.115,17,2.129224358293339,13.2.12.72.33.50.91.1Â±

0.7(4)Â± tedNone3-MethylcholanthrenePhÃ©nobarbitalNone3-MethylcholanthrenePhÃ©nobarbitalNone3-MethylcholanthrenePhÃ©nobarbitalNoneNoneNoneNoneBileduct liga 0.6(5), 1.32020Â±

wk4 0.8(4)8.2(4)10(4)11(3)13(4)10(4)2.7 wk4 wk4 wk4 wk4 wkAdultAdultAdultAdultAdultAdultAdultAdultAdultCompound'-HydroxysafroleSafrolel'-HydroxysafroleSafroleSafroleSafroleSafroleSafroleSafroleSafrolePretreatmentbNoneBile (4)4022,2.5,2.7,4.2Â±

pigHamsterSexMMFMMMMMMMMMMFMMMMMMAgeAdultAdultAdultAdultAdult4 tedPhÃ©nobarbitalNoneBileduct liga

0.3(4), ductligatedPhÃ©nobarbitalUrinary 1.0Â± 0.7 (3) " Safrole (30 mg/100 g body weight) and I'-hydroxysafrole (10 and 20 mg/100 g body weight for rats and mice, respectively) were injected i.p. in trioctanoin (0.1 ml total volume per 100 g body weight). b Bile duct ligations were performed 24 hr prior to injection of safrole. 3-Methylcholanthrene (5 mg/100 g body weight) was injected i.p. 24 hr before the safrole or I'-hydroxysafrole. Sodium phÃ©nobarbital(0.05% for weanling rats and 0.1% for all other animals) was administered in the drinking water for 1 week prior to the injection ofsafrole or I'-hydroxysafrole. e The urine from individual guinea pigs or the pooled urine from 2 hamsters or adult rats, three 4-week-old rats, or 5 mice was used for each analysis. Each value has been corrected by subtraction of the average absorbance value at 286 nm obtained for identical preparations from control urine from the same species and pretreatment and for the average recovery values for comparable amounts of I'-hydroxysafrole added to the control urine. See "Materials and Methods" for more details. If 3 or more samples were analyzed, the average value Â±1S.D. is given with the number of samples indicated by the number in parentheses. If only 2 samples were analyzed, each value is listed. l'-Methoxysafrole. By analogy with the reactivity of esters isotope) of about 4%. The peak at m/e =161 (m* - SCH3) such as 7V-acetoxy-2-acetylaminofluorene with methionine to confirmed the presence of a methylmercapto group. yield o-methylmercaptoamides (8, 31, 41, 49, 56), it was The NMR spectrum of the product was typical for an reasoned that 1'-acetoxysafrole might react with methionine isosafrole derivative and essentially identical to that of to yield a thioether (Chart 6). Evidence for this reaction was 3'-methylmercaptoisosafrole synthesized from 3'-bromoiso- obtained from the formation of benzene-soluble tritium on safrole. The doublet of the allylic protons on the 3'-carbon incubation of 1'-acetoxysafrole with methionine-methyl-3H was found at 3.2 ppm and the signal for the -SCH3 protons and the cochromatography on silica thin layers of most of the appeared at 2 ppm. tritiated product with 3'-methylmercaptoisosafrole. Small amounts (approximately 10% of the amount of Proof came with the isolation of 3'-methylmercaptoiso- 3'-methylmercaptoisosafrole) of another product were also safrole from large-scale preparations. Thus, thick-layer chroma- observed on chromatography of some reaction mixtures. This tography of an extract from the incubation of 1'-acetoxy product had a Rp slightly lower than that of 3'-methylmercaptoisosafrole and was identified as l'-methoxysafrole on the safrole and methionine (molar ratio of 1:1) yielded a product with the RF's on silica gel thin-layer plates and the basis of the identity of its RF on silica thin-layer plates with characteristic yellow color with the vanillin-sulfuric acid that of synthetic l'-methoxysafrole. This identification was reagent of 3'-methylmercaptoisosafrole. In methanol solution, confirmed by its mass and NMR spectra. The mass spectrum of the spectrum of the product was very similar to that of the minor product yielded a molecular ion with m/e = 192, an 3'-methylmercaptoisosafrole, with maxima at 266 and 306 m* â€”¿27(vinyl) fragment, which is characteristic of safrole nm. The mass spectrum was identical to that of 3'-methyl- derivatives, an m+ â€”¿15(methyl) fragment, and an m* â€”¿31 mercaptoisosafrole prepared from 3'-bromoisosafrole and (methoxy) fragment. The entire fragmentation pattern was methylmercaptan (Chart 7). It showed the expected molecular identical with that of synthetic l'-methoxysafrole. ion at m/e = 208 and a m* + 1 peak =210 (due to the sulfur Quantitation of the Reaction of 1'-Acetoxysafrole and

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Borchen, WisloctÃ¤,Miller, and Miller When a 50-fold molar excess of l'-hydroxysafrole, 3'-hydroxyisosafrole, or 3'-acetoxyisosafrole was incubated with #60 o I -HYDROXYSAFROLE methionine-methyl-3H for 20 hr, no reaction was detected. o Reaction of 1'-Acetoxysafrole with GMP and the Tentative 40 Identification of the Major Reaction Product. Preliminary experiments indicated that the incubation of l'-acetoxysafrole with GMP resulted in a marked loss of GMP and the formation 20- of substantial amounts of products (Chart 6). Thus, on chromatography on silica gel thin-layer plates with 1-butanol: watenacetic acid (100:50:22), 3 new UV-absorbing products with Rp's of about 0.3, 0.4, and 0.5 were noted; the RF of GMP was 0.1. The purple colors that these products gave with -l 10 the vanillin-sulfuric acid reagent and their UV absorption SAFROLE + PB spectra (maxima at 262 nm with shoulders at 302 nm) suggested that each might be an adduct of an isosafrole ? 5 derivative with guanylic acid. When the reaction was carried er out on a large scale (see "Materials and Methods"), substantial i amounts of the major product (Product 1) and lesser amounts II 8 18 78 88 2ÃŒO of a 2nd product (Product 2) were isolated. Incubation of the DAYS products with alkaline phosphatase converted these water- Chart 5. The urinary excretion of l'-hydroxysafrole by rats fed 0.5% safrole or 0.55% l'-hydroxysafrole in the diet for up to 7 months. 100 One group of rats received 0.1% sodium phÃ©nobarbital(PB) in the drinking water during the entire period. A

50-

SULFONIUM -HOMOSERINE

S-CH3 100 3-METHYLMEPCAPTOISOSAFROLE B.1

lllllÂ¡i .11 II i200i i i i i i i i i i 5'-GUANYLIC 180 160 140 120 100 m/e ACID Chart 7. The mass spectra of 3'-methylmercaptoisosafrole (A) synthesized from 3'-bromoisosafrole and methylmercaptan and the compound obtained by reaction of methionine with l'-acetoxysafrole (B). Chart 6. The reactions of l'-acetoxysafrole with methionine and Table 3 GMP. The structure of the GMP-isosafrole adduci has been placed in The reaction of methionine with safrole and isosafrale derivatives brackets since the characterization is not unequivocal, although the The ratio of safrole or isosafrole derivative to methionine was 50:1. NMR and mass spectrum data strongly favor the structure shown. In See "Material and Methods" for experimental conditions. addition to the compound depicted, the reaction with GMP yielded 2 other adducts the structures of which have not been determined. Nu, nucleophile. 3'-Methylrnercaptoisosafrole formed (% of theory0)at Other Safrole Derivatives with Methionine. When a 50-fold molar excess of l'-acetoxysafrole was incubated with methio- Compound 2hr 5hr 20 hr nine-methyl-3H for 2 lir at neutrality (50% acetone), 4% of the 1'-Acetoxysafrolel'-Hydroxy 3H was converted to a benzene :hexane-soluble product with safrole3'-Acetoxyisosafrole3 the same mobility on silica gel plates as 3'-methylmercaptoiso- '-Hydroxy isosafrole4.0bbb4.7bbb4.8<0.05<0.05<0.05 safrole (Table 3). Incubation times of 5 or 20 hr increased the yield of 3'-methylmercaptoisosafrole by only 20%. No evidence for 3H-labeled l'-methoxysafrole was obtained on 0 Based on the % of the radioactivity from methionine-methyl-3 H, which migrated on thin-layer chromatograms with the Rp of cochromatography of the 2-hr reaction mixture with synthetic 3'-methy Imereap toisosafrole. l'-methoxysafrole. b Not studied.

584 CANCER RESEARCH VOL. 33

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Metabolism and Reactivity ofSafrole Derivatives soluble derivatives to water-insoluble compounds (presumably Table 4 nucleosides) and thus facilitated the removal of water-soluble NMR data on the acetylated nucleoside derivative of Product 1 obtained by reaction of CMP with I'-acetoxysafrole impurities. However, the low solubilities of the nucleosides in most solvents and their low volatilities handicapped NMR and )-CH2 (7) mass spectra determinations. These problems were reduced by acetylation of the nucleosides. The following characterization data refer only to the derivative obtained from Product 1; adequate studies have not been carried out on the lesser amounts of the derivative of Product 2. The NMR spectrum of the acetylated nucleoside from Product 1 was consistent with its characterization as O-6-{isosafrol-3'-yl)-7V-2-acetylguanosine-2',3',5'-triacetate (Ta ble 4). The presence of the C-8 proton at 7.65 ppm and the fact that this proton did not exchange with D20 appeared to exclude C-8 or N-7 substitution; Tomasz (64) has demonstra ted the lability of the proton in the C-8 position of N-7-substituted guanine derivatives. The signal at 12.3 ppm, AcO AcO which integrated for 1 proton and was exchangeable with The resolution of this NMR spectrum was insufficient to measure D20, was assigned to the proton of the acetylated NH group coupling constants. attached to C-2 of guanine. The same signal was observed in the NMR spectrum of 7V-2-acetylguanosine-2',3',5'-triacetate, Signal ofprotons11352512Multi which was synthesized as a model compound (29). In the (ppm)12.35b7.656.85.6-6.75.94.322.1No.plicity09CSMMSMSAssignment-NH-2 NMR spectrum of guanosine, the signal for the 2 protons of (guanine)H-8 the amino group was found at 6.6 ppm. These data exclude (guanine)Hj-3,5,6 the possibility that the safrole derivative was attached to the (isosafrole)H-l',H-2',H-3' guanosine through substitution of the amino group, since, in andH-l',H-2' (ribose) order to form the yV-2-acetyltriacetate derivative, the amino (isosafrole)H2 -7(isosafrole)H4',H2-5' group in the nucleoside must have been unsubstituted prior to andHÂ¡-3'(ribose) acetylation. The signal of the acetyl protons at 2.1 ppm (isosafrole)H, integrated for 12 protons, the expected value for a ./V-acetyltri- , (acetyl) acetate derivative. No signal was detected; this was consistent a See Table 1 for abbreviations used in expressing multiplicity. with the result expected for the proton attached to N-l of b The signal at 12.35 ppm was exchangeable with D2O. guanine. From the NMR spectra of jV-2-acetylguanosine- c Not clear. 2',3',5'-triacetate and guanosine-2',3',5'-triacetate, this signal was expected at 10.9 ppm. The characterization of the product as an isosafrole The mass spectrum of the acetylated nucleoside was consistent with a structure of 0-6-(isosafrol-3'-yI)-7V-2-acetyl- derivative is consistent witli the lack of the signals between 5 2',3',5'-guanosine triacetate. The expected molecular ion at and 5.5 ppm, which are characteristic for the side-chain protons of safrole. The signal at 4.3 ppm can be assigned to m/e = 611 was observed. Other observed ions and their the 2 allylic protons on the 3'-carbon of isosafrole. The signal postulated structures were 569 (isosafrolylguanosine triace for the protons on the 3'-carbon of 3'-methoxyisosafrole tate), 451 (isosafrolylguanosine monoacetate), 353 (acetyliso- appeared at 4.00 ppm (5). The similarity in the positions of safrolylguanine), 310 (isosafrolylguanine -H), 259 ribose these signals is evidence that substitution on the guanine ring triacetate -OH), 194, 176(acetylguanine -OH or hydroxyiso- occurred at the O-6 position, rather than on N-l or N-3. safrole -2 H), 161 (isosafrole -H), 151, 130 (ribose because the oxygen at C-6 of guanine and the methoxy group monoacetate â€”¿OH).Nometastable ion peaks were observed. of 3'-methoxyisosafrole would be expected to have similar The UV spectrum of the acetylated nucleoside in methanol deshielding effects on the allylic protons. For a N-l- or had a maximum at 261 nm with an extinction coefficient of N-3-substituted guanine derivative, the signal of the 2 allylic 29,300 and a shoulder at 302 nm. While the spectrum protons on the 3'-carbon of isosafrole would be expected at a approached the addition spectrum of TV-2-acetylguanosine triacetate and 3'-hydroxyisosafrole, the lack of appropriate higher field. The typical doublet of the allylic protons of the isosafrole derivatives could not be seen, since the 2 protons on reference compounds and the instability of the isosafrole the 5'-carbon and the proton on the 4'-carbon of the ribose portion in acidic solutions did not permit a structural absorbed in this region. The signals for the 3 aromatic protons interpretation of the UV spectra. of isosafrole appeared at the usual region (6.8 ppm), and the Attempts were made to compare the infrared spectrum of characteristic marker signal for the 2 protons of the the nucleoside from Product 1 with that of guanosine (65), methylenedioxy group was at 5.9 ppm. The signals for the 2 especially for the probable absence of a 6-carbonyl function in olefinic protons at the 1'- and 2'-positions of isosafrole were the nucleoside. However, these efforts failed since the buried under the signals for the protons at the l'-, 2'-, and nucleoside was much less soluble in D20 or H20, especially at 3'-positions of guanosine. acid pH, than guanosine and since the spectra taken in the

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Borchert, Wislocki, Miller, and Miller or 3'-acetoxyisosafrole with guanosine-8-'4C yielded much solid state were not interpretable, presumably for the reasons discussed by Shapiro (58). less, if any, product (Table 5). Quantitation of the Reaction of l'-Acetoxysafrole and of In preliminary studies, the reactions of the nucleotides with l'-acetoxysafrole were determined. With a 50-fold excess of Other Safrole Derivatives with Nucleosides. For quantitation 1'-acetoxysafrole the relative amounts of GMP, AMP, CMP, of the reaction with nucleosides, the safrole derivative was incubated with the appropriate l4C-labeled nucleoside, and UMP, and TMP converted to less polar forms were 50, 33, 18, the amount of less polar nucleoside derivative(s) was deter 13, and 12%, respectively. No reaction was observed between GMP and l'-hydroxysafrole or 3'-hydroxyisosafrole, while mined by chromatography of the reaction mixture on silica gel thin-layer plates and scintillation counting of the gel fractions. 3'-acetoxyisosafrole appeared to yield 1 to 2% of product on Incubation of 1'-acetoxysafrole with guanosine-8-14C or reaction with GMP. On incubation of l'-acetoxysafrole-2',3'- adenosine-8-14C converted 12 or 4%, respectively, of the 3H (3) with a phosphate buffer, 80% of the tritium was radioactivity to a less polar form (Table 5). Similar incubations converted to a water-soluble form while only 8% became with cytidine-2-14C yielded a low level (1.3%) of presumed water-soluble in a similar incubation mixture containing Tris product, while incubation with uridine-2-14C or thymidine- buffer. The yield of 14C-labeled product from GMP-8-14C was 2-14C did not yield a significant amount of radioactive prod the same with phosphate or Tris buffer. This finding suggested that l'-acetoxysafrole may be transesterified to yield l'-hy uct. On rechromatography of the guanosine and adenosine droxysafrole phosphate or 3'-hydroxyisosafrole phosphate. derivatives under the same conditions, 70 and 50% of the radioactivity moved with the original RF, and the remaining While the reaction with phosphate has not been established, radioactivity had RF's equal to or less than that of the parent the findings suggest that reaction of l'-acetoxysafrole with nucleoside. This finding and the apparent decomposition of nucleotides may involve not only the bases but also the some of the derivatives formed from GMP and l'-acetoxy- phosphate group. This formulation is consistent with the safrole during the large-scale isolations (see above) suggested apparent reaction of UMP and TMP but not uridine or thymidine with l'-acetoxysafrole and with the greater reactiv that the adducts are somewhat labile. When the reaction mixtures contained l'-acetoxysafrole- ity of GMP, AMP, and CMP as compared to the corresponding acetoxy-3H, low levels of tritium were distributed over much nucleosides. of the chromatogram with no definite bands of radioactivity. When the tritium contents of corresponding areas of chroma- DISCUSSION tograms of reaction mixtures without a nucleoside were subtracted from those of the product areas for reaction The data in this and the following paper (5) establish mixtures containing guanosine or adenosine, it was concluded l'-hydroxysafrole as a proximate carcinogenic metabolite of that no more than 5% of the reaction products from either of safrole. The urinary excretion of a conjugate of l'-hydroxy these nucleosides could have contained the acetyl group from safrole by rats, mice, hamsters, and guinea pigs given safrole the 1'-acetoxysafrole. These data appear to rule out the clearly establishes the l'-hydroxy derivative as a new presence of appreciable amounts of acetylated nucleosides in metabolite. That l'-hydroxysafrole is a proximate carcinogen the product areas. Under the conditions used for reaction with 1'-acetoxy (i.e., closer to the ultimate carcinogenic form than safrole) safrole incubation of l'-hydroxysafrole, 3'-hydroxyisosafrole, appears evident from the much greater hepatocarcinogenicity of l'-hydroxysafrole, as compared to safrole, for rats and mice (5). l'-Hydroxysafrole also has some carcinogenic activity at

Table 5 sites where safrole showed little or no activity. Thus, mice fed 1'-hydroxysafrole developed angiosarcomas in the interscapu- The reaction of nucleosides with safrole and isosafrole derivatives The ratio of safrole or isosafrole derivative to the nucleoside was lar region after 1 year, and 2 rats developed sarcomas at the 5:1. The incubation time was 2 hr; see "Materials and Methods" for sites of repeated s.c. injections of this metabolite. Isolated other experimental conditions. small papillomas developed in the forestomachs of rats fed l'-hydroxysafrole. % reaction0 with The metabolic hydroxylation of safrole to l'-hydroxy or3'hydroxyisosafrole<0.1bbbbsafrole is similar to earlier observations on the oxidation in vivo of ethyl-, isopropyl-, and /i-propylbenzene to benzylic safrole11.84.11.3<1.0<1.03'-Acetoxy-isosafrole0.2bbbbl'-Hydroxysafrole alcohols (10, 51, 59, 60). On the basis of its allylic structure, the oxidation of safrole to l'-hydroxysafrole is also analogous GuanosineAdenosineCytidineThymidineUridinel'-Acetoxy to the oxidation in the rat of the allylic carbon of allethrin to its l'-hydroxyprop-2'-enyl derivative (9). Like the other benzylic alcohols, l'-hydroxysafrole appears to be excreted primarily as a glucuronide in the urine. With the 4 species " Based on the percentage of the radioactivity from the nucleoside studied, the conjugate of l'-hydroxysafrole appeared to be that migrated as a band on thin-layer chromatograms with an Rp excreted primarily, if not entirely, in the urine. Thus, unlike greater than that of the nucleoside. The following blanks, obtained for the excretion of ./V-hydroxy-2-acetylaminofluorene by rats (21, identical samples except for the omission of the safrole derivatives, have 42), there was no significant increase in the urinary excretion been subtracted: guanosine, 0.6%; adenosine, 0.7%; cytidine, 0.8%; of l'-hydroxysafrole conjugates in animals in which the bile thymidine, 1.0%; and undine, 1.0%. 6 Not studied. ducts had been ligated 24 hr before injection of safrole.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. Metabolism and Reactivity ofSafrole Derivatives The excretion of conjugated l'-hydroxysafrole by rats given (25), the reaction of l'-acetoxysafrole was most facile with safrole was markedly increased by pretreatment of the rats guanine derivatives,. Some reaction was also observed with with either 3-methylcholanthrene or phÃ©nobarbital. The adenosine and, possibly, with cytidine; these nucleosides are increased amount of conjugated l'-hydroxysafrole appears to also substituted by other alkylating agents. Reaction of be due primarily to an increased level of l'-hydroxylation, l'-acetoxysafrole with GMP appeared to yield 3 products. The since pretreatment with 3-methylcholanthrene or phÃ©nobarbi product which has been provisionally characterized is not tal did not similarly augment the total excretion of l'-hy substituted in either the N-7 or the C-8 position of the guanine droxysafrole when 1'-hydroxysafrole was injected. This find portion, the major sites of reaction with certain alkylating and ing suggests that coadministration of 3-methylcholanthrene or arylamidating agents, respectively (24, 25). The major reaction product from l'-acetoxysafrole appears to be substituted on phÃ©nobarbital with safrole might increase the hepatic damage and tumor yield with the latter compound, but in a study still the oxygen at C-6 {i.e., 0-6) of guanine. Loveless (33) and in progress no differences have been found by 14 months for Lawley and Thatcher (26) have presented correlative evidence rats given 0.5% safrole in the diet with or without 0.1% which suggests that the O-6 substitution of guanine in nucleic phÃ©nobarbitalin the drinking water. acids may be associated with the carcinogenic and mutagenic The inducibility of the mixed-function oxygenases of many action of some alkylating agents. tissues with phÃ©nobarbital and 3-methylcholanthrene suggests Alternative routes of activation of l'-hydroxysafrole might that the l'-hydroxylation is mediated by a mixed-function develop from its oxidation to reactive epoxides or from its oxidation to l'-ketosafrole. The latter electrophilic a,(3-unsatu- oxygenase system (6), and this is under study. Parke and Rahman (46, 47), Wagstaff and Short (66), and Lotlikar and rated ketone has recently been synthesized in good yield in Wasserman (32) have shown that safrole can also induce our laboratory. McKinney et al. (40) and we (unpublished) mixed-function oxygenase systems as well as an increased level have found that it readily alkylates dimethylamine, piperidine, of microsomal heme proteins in rat liver. While in vitro studies and pyrrolidine to yield Mannich bases. These bases have been are needed for more definitive evidence, the 50% decrease in isolated and identified as urinary metabolites of safrole in rats the urinary excretion of l'-hydroxysafrole conjugates by rats and guinea pigs by Oswald et al. (45). These data, as well as fed safrole for 2 weeks or more, as compared to those fed the confirmatory data from our laboratory (5), thus provide compound for shorter times, provides no evidence that safrole tentative evidence for the formation of l'-ketosafrole in vivo. induces its l'-hydroxylation system. Further studies on the reactions of l'-ketosafrole, esters of The elucidation of the metabolic steps leading to the l'-hydroxysafrole, and other possible model compounds with ultimate carcinogenic derivative(s) of l'-hydroxysafrole is in cellular nucleophiles are in progress. The comparison of these progress. The activation of yV-hydroxy-2-acetylaminofluorene products with the radioactive derivatives formed in vivo on by sulfuric acid esterification in rat liver and the major role of administration to rats of l'-hydroxysafrole-2',3'-3H of high this ester in the hepatocarcinogenicity of 7V-hydroxy-2-acetyl- specific activity will hopefully provide evidence on the nature aminofluorene (8, 23, 67) suggest esterification of l'-hydroxy of the ultimate carcinogenic and reactive metabolites of safrole safrole as a metabolic activation mechanism. This mechanism in vivo (3). also seems plausible in view of the findings that the active forms of the hepatocarcinogenic pyrrolizidine alkaloids are ac tivated allylic esters (7, 22, 36â€”39) and that the carcinogenic REFERENCES diaryl acetylenic carbamates are benzylic esters (14, 15). The synthetic acetic acid ester of l'-hydroxysafrole also reacts with 1. Abbott, D. D., Packman, E. W., Wagner, B. M., and Harrisson, J. W. E. 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Chem. Ber., 57: 1327-1330, 1924. Chromatography of "Puxuri" Essential Oils. Anais Assoc. Brasil. 49. Poirier, L. A., Miller, J. A., Miller, E. C, am Quim., 26: 73-78, 1967. Abstracted in Chem. Abstr., 68: 11309, Af-Benzoyloxy-JV-methyl-4-aminoazobenzene: Its Carcinogenic 1968. Activity in the Rat and Its Reactions with Proteins and Nucleic 58. Shapiro, R. Chemistry of Guanine and Its Biologically Significant Acids and Their Constitutents in Vitro. Cancer Res., 27: Derivatives. Progr. Nucleic Acid Res. Mol. Biol.,S: 73-112, 1968. 1600-1613, 1967. 59. Smith, J. N., Smithies, R. H., and Williams, R. T. Studies in 50. Poirier, M. M., Miller, J. A., and Miller, E. C. The Carcinogenic Detoxification 55. Metabolism of Alkylbcnzene: a) Glucuronic Activities of Ar-Hydroxy-2-acetylaminonuorene and Its Metal Acid Excretion Following the Administration of Alkylbenzene, b) Chelates as a Function of Retention at the Injection Site. Cancer Elimination of Toluene in the Respired Air of Rabbits. Biochem. Res., 25: 527-533, 1965. J.,56: 317-320, 1954. 51. Robinson, D., Smith, J. N., and Williams, R. T. Studies in 60. Smith, J. N., Smithies, R. H., and Williams, R. T. Studies in Detoxication. 60. The Metabolism of Alkylbenzenes. Isopropyl- Detoxification. 56. The Metabolism of Alkylbenzenes. Stereo- benzene (Cumene) and Derivatives of Hydratropic Acid. chemical Aspects of the Biological Hydroxylation of Ethylbenzene Biochem. J., 59: 153-159, 1955. to Methylphenylcarbinol. Biochem. J., 56: 320-324, 1954. 52. Saiki, Y., Akahori, Y., Morinaga, K., Taira, T., Noro, T., 61. Suga, K., Watanabe, S., and Suematsu, M. Grignard Reaction of Fukushima, S., and Harada, T. Gas Chromatography of Natural Vinylchloride with oi,(3-Unsaturated Carbonyl Compounds. Yuki Volatile Oils. III. Gas Chromatography of the Volatile Oils of Gosei Kagaku Kyokai Shi, 24: 213-215, 1966. Plants Belonging to the Heterotropa Genus. 1. Yakugaku Zasshi, 62. Tamayo, M. L., and Ossorio, R. P.-A. La SÃntesisDe Ã•cidos 87: 1535-1538, 1967. Abstracted in Chem. Abstr., 68: 11301, Fenilalilsuccinicos. Anales Real Soc. Espan. Fis. Quim., 44B: 1968. 981-998, 1948. 53. Saiki, Y., Akahori, Y., Morinaga, K., Taira, T., Noro, T., 63. Taylor, J. M., Jenner, P. M., and Jones, W. I. A Comparison of the Fukushima, S., and Harada, T. Gas Chromatography of Natural Toxicity of Some Allyl, Propenyl, and Propyl Compounds in the Volatile Oils. IV. Gas Chromatography of the Volatile Oils of Rat. Toxicol. Appi. Pharmacol., 6: 378-387, 1964. Plants Belonging to the Heterotropa Genus. 2. Yakugaku Zasshi, 64. Tomasz, M. Extreme Liability of the C-8 Proton: A Consequence 87: 1539-1543, 1967. Abstracted in Chem. Abstr., 68: of 7-Methylation of Guanine Residues in Model Compounds and in 11301-11302, 1968. DNA and Its Analytical Application. Biochim. Biophys. Acta, 199: 54. Saiki, Y., Akahori, Y., Morinaga, K., Taira, T., Noro, T., 18-28, 1970. Fukushima, S., and Harada, T. Gas Chromatography of Natural 65. Tsuboi, M., Kyogoku, Y., and Shimanouchi, T. Infrared Absorp Volatile Oils. V. Gas Chromatography of the Volatile Oils of Plants tion Spectra of Protonated and Deprotonated Nucleosides. Belonging to the Heterotropa Genus. 3. Yakugaku Zasshi, 87: Biochim. Biophys. Acta, 55: 1-12, 1962. 1544-1547, 1967. Abstracted in Chem. Abstr., 68: 11302, 1968. 66. Wagstaff, D. J., and Short, C. R. Induction of Hepatic Microsomal 55. Saiki, Y., Akahori, Y., Noro, T., Morinaga, K., Taira, T., Hydroxylating Enzymes by Technical Piperonyl Butoxide and Fukushima, S., and Harada, T. Gas Chromatography of Natural Some of Its Analogs. Toxicol. Appi. Pharmacol., 19: 54-61, 1971. Volatile Oils. II. Gas Chromatography of the Volatile Oils of Plants 67. Weisburger, J. H., Yamamoto, R. S., Williams, G. M., Grantham, P. Belonging to Asiasrum and Asarum genera. Yakugaku Zasshi, 87: H., Matsushima, T., and Weisburger, E. K. On the Sulfate Ester of 1529-1534, 1967. Abstracted in Chem. Abstr., 68: 11301, 1968. JV-Hydroxy-jV-2-fluorenylacetamide as a Key Ultimate Hepato- 56. Scribner, J. D., Miller, J. A., and Miller, E. C. Nucleophilic carcinogen in the Rat. Cancer Res., 32: 491-500, 1972. Substitution on Carcinogenic JV-Acetoxy-jV-arylacetamides. Cancer 68. Wislocki, P. G., Borchert, P., Miller, E. C., and Miller, J. A. Res., 30: 1570-1579, 1970. l'-Hydroxysafrole: A Proximate Carcinogenic Metabolite of 57. Seabra, A. P., Guimaraes, E. C., and Mors, W. B. Gas-liquid Safrole. Proc. Am. Assoc. Cancer Res., 13: 12, 1972.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1973 American Association for Cancer Research. The Metabolism of the Naturally Occurring Hepatocarcinogen Safrole to 1 ′-Hydroxysafrole and the Electrophilic Reactivity of 1 ′-Acetoxysafrole

Peter Borchert, Peter G. Wislocki, James A. Miller, et al.

Cancer Res 1973;33:575-589.

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