Carcinogenic and Mutagenic Activities of Safrole, Metabolites1

[CANCER RESEARCH 37, 1883-1891, June 1977] Carcinogenic and Mutagenic Activities of Safrole,

1 â€˜-Hydroxysafrole, and Some Known or Possible Metabolites1

Peter G. Wlslocki,2 Elizabeth C. Miller,3 James A. Miller, Elena C. McCoy, and Herbert S. Rosenkranz McArdleLaboratoryforCancerResearch,UniversityofWisconsinMedicalSchool,Madison,Wisconsin53706(P.G. W.,E.C.M.,J. A. M.J,andDepartmentof Microbiology,New YorkMedicalCollege,Valhalla,NewYork10595fE.C. McC.,H. S. R.J

SUMMARY INTRODUCTION

The carcinogenic and mutagenic activities of metabolites Safrobe is a hepatocarcinogen that occurs in oil of sassa and possible metabolites of safrole and 1â€˜-hydnoxysafrole fnas and certain spices and essential oils (9, 11-13), and were investigated as guides to their importance as possible recent studies have implicated 1â€˜-hydroxysafroleasa proxi proximate or ultimate carcinogens. 1â€˜-Acetoxysafroleand mate carcinogenic metabolite in the matand mouse (3, 4). the 2' ,3'-oxides of safrole, 1â€˜-hydroxysafrole,1â€˜-acetoxysaf The finding that the synthetic ester 1â€˜-acetoxysafrole,a robe, and 1â€˜-oxosafrole,weredirectly mutagenic for Sa!mo strong ebectrophilic reactant, induced sarcomas at the site nel!a typhimurium strains TA1535 and TA100. No significant of repeated s.c. injections in matsand papilbomas in the mutagenicity was detected with safrole, isosafrole, 2',3'- fonestomach on p.o. administration (3, 4) suggested that dihydrosafrole, 1â€˜-hydroxysafnole,3'-hydnoxyisosafnole, 3'- any metabolically formed ester of 1â€˜-hydroxysafrolewould acetoxyisosafrole, or 1â€˜-oxosafrobeeither with or without be a candidate ultimate carcinogenic metabolite. Evidence the addition of reduced nicotinamide adenine dinucleotide that an ester was formed in vivo was obtained by the release phosphate-fortified hepatic microsomes plus cytosol. of small amounts of 3'-methylmemcaptoisosafrobe from the Multiple topical applications to mice of 1â€˜-hydnoxysafrole liver protein of rats and mice given 1â€˜-hydroxysafrole(32). 2',3'-oxide, followed by repetitive doses of croton oil, The conversion of 1â€˜-hydroxysafroleto an ebectrophilic caused the formation of skin papilbomas. Under the same reactant in a reaction dependent on both liven cytosob and conditions significant numbers of papilbomas were not initi 3'-phosphoadenosine 5'-phosphosulfate implicated the sub ated by safrole, 1â€˜-acetoxysafrobe,1â€˜-hydmoxysafrole,1â€˜-ox fumicacid ester of 1â€˜-hydroxysafroleasa candidate ultimate osafrole, safmole-2',3'-oxide, or 1â€˜-acetoxysafrobe-2',3'-ox carcinogenic metabolite in vivo (32). ide. 1â€˜-Oxosafrolehad little or no carcinogenic activity on Three other electrophilic metabolites of safrole may also P.O.administrationtoratsonon s.c.administrationtopre be formed in vivo. The formation of 1'-oxosafmobewas mdi weanling mice or adult rats. The incidence of hepatocelbular cated by the excretion of small amounts of addition prod carcinomas in matsfed safrole was markedly increased by ucts of 1â€˜-oxosafroleand certain secondary amines in the simultaneous administration of phenobanbital. urine of rats and guinea pigs given safrole (2, 20, 22, 23). 1â€˜-Hydnoxysafrole p.o. was more hepatotoxic and hepato Epoxidation of 1â€˜-hydnoxysafroleto its 2' ,3'-oxide occurs carcinogenic for adult female mice than for adult male with both rat and mouse liver microsomes (32). The excre mice. Injection of [2',3'-'H]-1 â€˜-hydroxysafrolei.p. yielded at tion of 2',3'-dihydro-2',3'-dihydroxysafrole in the urine of least 10-fold greaten levels of hepatic DNA-, nibosomal rats and guinea pigs given injections of safrole and its RNA-, and protein-bound derivatives in preweanling male formation by liver cells in culture provide strong evidence or female mice and in adult female mice than in adult male for the epoxidation of safrobe (6, 29). mice. The levels of hepatic macromolecube-bound tnitiated The mutagenic and carcinogenic activities of the above derivatives in adult mice were produced by prefeeding of metabolites and of certain postulated metabolites of satrole nonradioactive 1â€˜-hydroxysafrobepriorto the administration and 1â€˜-hydmoxysafrolehave been studied as further ap of [3H1-1â€˜-hydnoxysafmole. proaches to assessment of their possible roles in the carci Multiple electrophiles, especially safrobe-1â€˜-sulfate,1â€˜-hy nogenic activities of safrobe and 1â€˜-hydroxysafrole.Thepre dmoxysafmole-2',3'-oxide , 1â€˜-oxosafrole,and safrole-2' ,3'- viously reported carcinogenic and mutagenic activities of oxide, must be considered as possible ultimate carcino 1â€˜-acetoxysafrole(3, 8, 19) were confirmed. Furthermore, genic metabolites of safrole in mouse and matliver. the 2' ,3'-oxides of safrole, 1â€˜-hydroxysafrole,1â€˜-oxosafrole, and 1â€˜-acetoxysafrole,all showed mutagenic activity; and

I This work was supported at the University of Wisconsin by Grants CA 1'-hydroxysafrole-2',3'-oxide initiated the formation of pap 07175, CA-15785, and CRTY-5002 of the National Cancer Institute, Depart ibbomasin mouse skin. ment of Health, Education and Welfare, and at New York Medical College by Grants Al-11470 and N01-CP-65855. MATERIALS AND METHODS 2 Present address: Eppley Institute for Research in Cancer, 42nd Ave. and Dewey Ave., Omaha, Neb. 68105. Safrole Derivatives 3 To whom requests for reprints should be addressed. Received January 17, 1977; accepted March 16, 1977. Safnole (4-allyl-1 ,2-methylenedioxybenzene), isosaf role

JUNE 1977 1883

(4-propenyb-1,2-methylenedioxybenzene), and 2' ,3'-dihy (approximately 50/group) were weaned; the surviving mice drosafnole (4-pnopyl-1 ,2-methylenedioxybenzene) were pun were killed for autopsy at 16 months. chased from Aldrich Chemical Co. (Milwaukee, Wis.) and Topical ApplicatIons to the Skin of Mice. The safrole estimated to be at least 99% pure by thin-layer chroma derivatives (1.5 or 3.0 @molespen0.1 ml acetone per dose) tography (3). The other safrole derivatives were synthesized were applied 5 times/week for 6 weeks to the shaved backs as described in previous papers from this laboratory (3, 4, of 2- to 3-month-old female mice. Another group of mice 32). received 1 application of 7,12-dimethylbenz(a)anth nacene (Eastman Organic Chemicals, Rochester, N. V.) in acetone. Carcinogenicity and ToxicIty Studies Starting 1 week after the last application of the safrole derivatives, all of the mice received twice-weekly applica Rats (CD, random-bred) and mice (CD-i) were obtained tions of 0.5% croton oil (Amend Drug and Chemical Co., from the Charles Riven Breeding Laboratory, Wilmington, New York, N. Y.) in redistilled acetone until the experiment Mass. Pregnant females and females with litters were kept was terminated at 36 weeks. The backs of the mice were on bedding in plastic cages. All other animals were housed shaved with an electric clipper before the 1st application of in screen cages with raised bottoms. The mice were kept in safrobe derivative and as needed throughout the expeni groups of 4 to 5, and the rats were housed individually. The ment. From the 13th week of the experiment (6th week of animal rooms were maintained at 22-24Â°with a 12-hr light croton oil treatment), the mice were examined biweekly for and 12-hr dark cycle. Except for the experiments in which papilbomas. the compounds were fed as a part of the diet, the rats and Autopsies. All of the matsandmice from the carcinogenic mice were fed Wayne Breeder Bbox (Allied Mill, Inc. , Chi ity experiments were subjected to routine gross autopsies cago, Ill.); food and water were available ad !ibitum. All of which included examination of the skin, s.c. injection site, the animals were weighed at least monthly; the mice used in mammary and ear duct gland tissues, and the organs of the the toxicity experiment were weighed weekly. Heat-stemi abdominal and thoracic cavities. Of the papibbomasof the lized tnioctanoin (Pfaltz and Bauer, Flushing, N. V.) was skin in mice treated topically, 10 to 20% were examined used for all injections. histologically on a random basis. In all other cases pieces of Dietary Administration to Rats and Mice. Groups of 18 each gross tumor or suspected tumor and of other abnor male rats (average initial weight of 250 g) and groups of 40 mabtissues were fixed in neutral 10% formalin, sectioned at to 50 male or female mice (average initial weights of 25 and 5 to 6 @m,and stained with hematoxylin and eosin. We are 21 g, respectively) were fed the test diets for the times indebted to Dr. Henry C. Pitot and his colleagues of the indicated in the tables; they were then fed the same diet McArdle Laboratory for the histological diagnoses. without the safrole derivative for the remainder of the exper iment. The safrole derivatives were dissolved without heat Nucleic Acid- and Protein-bound Derivatives of I â€˜-Hydrox in corn oil, which was then added to a grain diet (28) at a ysafrole In Mouse Liver bevelof 5%; 5% corn oil was added to the diet of the control Male and female mice that were 1 to 3 or 9 to 12 weeks old animals. The diets were prepared weekly and were stored in were given i.p. injections of 2 mg of [2',3'-3H]-i â€˜-hydmoxysaf closed cans at 5Â°.Theanimals were fed ad libitum daily with mole(32) (specific activity, 40 to 400 Ci/mmole) per 0.1 ml of only minimum carryover in the feeders from day to day. In 1 tnioctanoin per 100 g body weight. The mice were killed 16 experiment some of the rats received 0.1% of sodium phe hr after injection; and DNA, nRNA,and protein were isolated nobarbital (J. T. Baker Chemical Co., Phillipsbung, N. J.) in from the livers by a slight modification (32) of the procedure the drinking water throughout the experiment. The surviv of Irving and Veazey (14). The specific madioactivities of the ing rats in both experiments were killed at 22 months. The macromolecules were determined as previously described mouse experiment was terminated at 17 months. (32). The adult mice were fed a grain diet, and some of the Injection s.c. Into Rats. Groups of 18 matswith average mice were fed diets that contained nonmadioactive safrole on initial weights of 280 g (Experiment 1) and 220 g (Expeni 1â€˜-hydroxysafrole (see â€œResultsâ€•)priorto administration of ment 2) were given twice-weekly injections for 12 weeks s.c. the tnitiated compound. in the night meanthigh with 0.2 ml of tnioctanoin that con tamed 18.5 j.@molesof the test compound (equivalent to 3 Mutagenlclty Assays mg of safrobe). For each injection fresh solutions were pre pared without heat with the aid of a magnetic stirrer. After Cultures of Salmonella typhimurium TA1535, TA98, and 10 months, the injection sites were palpated at 2- to 4-week TA100 were kindly provided by Dr. Bruce Ames of the Uni intervals. The experiments were terminated at 21 and 19 vemsity of California at Berkeley. The procedures for the months, respectively. maintenance of the stock cultures and for the mutagenicity Injections Into Suckling Mice. The mice were bred in our assays were those described by Ames et a!. (1). Compounds laboratory, and the young were given injections s.c. at the that were not mutagenic directly were also assayed in the nape of the neck with a Hamilton syringe fitted with a 27- presence of the hepatic 5-9 fraction (mitochondnial super gauge needle on the following schedule: 0.21 j.@moIesof natant) from Arocbon-tneated rats and a NADPH-generating test compound in 0.025 ml of tnioctanoin within the 1st 24 hr system (1). after birth; 0.42 pmole in 0.05 ml on the 8th day; 0.85 j.@mole RESULTS in 0.05 ml on the 15th day; and 1.7 @molesin0.1 ml on the Carcinogenicity of Safrole Derivatives for Rats and Mice 22nd day. Control mice received injections of tnioctanoin on the same schedule. After the last injection the male mice p.o. Administration to Rats. When fed as 0.25% of the

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Carcinogenic and Mutagenic Safrole Derivatives diet for 17 months, 1'-oxosafrobe failed to induce hepatic groups showed areas of fatty degeneration, generalized tumors on gross liven damage by 22 months (Table 1). An hepatic cell necrosis, atypical hyperplasia, and microscopic equimobar level of safrole also failed to induce hepatic tu foci of hepatocelbular carcinomas. Only 1 carcinoma (a scm mors, while 1'-hydroxysafrole induced liver carcinomas in rhous adenocarcinoma) was found in the liver of a rat given 40 and 88% of the rats fed 0.25 or 0.55% of the compound, phenobanbital in the absence of safrole. Since the rats given respectively. The liver carcinomas developed much more phenobambitab and safrole weighed about 15% more than rapidly in the rats fed the higher dose (60% incidence by 12 those given only safrobe throughout most of the experiment, months), although the administration of 1'-hydmoxysafnobe it is possible that some of the increase in hepatic tumor to this group was stopped at 10 months as compared to the incidence in the former group could be related to an in 17-month feeding period for the bower level of 1â€˜-hydnoxy creased intake of safrole; food consumption data for this safrole and the other compounds. 1'-Oxosafnobe was not experiment were not taken. However, an increased conver assayed at a higher level in view of the weight loss and poor sion of safrole to its proximate carcinogen 1â€˜-hydroxysaf health of rats fed 0.55% of this compound in a preliminary role induced in these rats by phenobarbital (4) probably was study. At 4 months the weight gain for rats fed 0.25% of 1â€˜- a more important factor leading to this increased tumor oxosafrole was only 65% as great as for the matsfed an incidence. The livers of the rats given phenobambitab,with or equimobar level of safrole or 1â€˜-hydnoxysafnoleandonly 45% without safrobe, were generally larger than from the rats not that of the control rats. The poor health of the rats fed 1â€˜- given the barbiturate. oxosafrole was not associated with liven damage; detailed s.c. Injection Into Rats. Three of 36 rats that received pathological study of these animals was not carried out. repeated s.c. injections of 1â€˜-oxosafnobe(totald9se, 70 mg) Safrole induced gross hepatocellulan carcinomas in 3 of developed sarcomas at the injection site by the termination 18 rats fed the compound as 0.5% of the diet for 22 months, of the experiments at 19 or 20 months (Table 2). At the same while 12 of 18 rats fed this level of safrole and simultane dose level, 1â€˜-acetoxysafrobeinducedsarcomas in 11 of 36 ously given 0.1% of sodium phenobarbital in the drinking rats, and only 1 sarcoma developed in a control rat that water developed hepatocellulan carcinomas. These hepatic received only the solvent. While the development of 3 sarco carcinomas were found in rats killed for autopsy at 22 mas at the injection site in rats that received injections of 1â€˜- months. Multiple gross carcinomas were found in 1 nat fed oxosafrole may be indicative of low carcinogenic activity, safrole alone and in 6 rats given both safrole and phenoban the tumor incidence was not significantly greater than that bitab. In addition, a number of the livers from the rats of both for the control animals. In the 1st experiment the 4-month

Table 1 dietCharles Incidences of tumors in adult male rats fed safrole derivatives in the timesindicated,RivenCD random-bred male rats (averageinitial weight, 252 g) were fed the safrobederivativesin a grain diet for the after which they were fed the unsupplementedrats/group.Av. diet untilthe experiment was terminated at 22 months. There were 18 alivegainwt No. of rats with hepatic No. of rats in carcinomas by without tumors at 1st 4 No. of rats with fore Expeni-Otherment Compoundadded to Mos. mos. 15 stomach tumors by mos.1 diet fed (g) 12 mos. mos. 22 mos. 22 mos. tumors 18 mos. 22 0.25%130.22%safrobe 1â€˜-oxosafrobe 17 120 0 0 0 0 14 120.25% 17 183 0 0 0 0 a 13 4role0.55%1â€˜-hydnoxysaf- 17 176 0 1 7 2 (papillomas) 1? 12

@ 0role 1'-hydroxysaf- 10 124 11 12 16 2 (carcinomas) C (papillomas)(None) 2 142 267 0 0 0 0 d 17 0.5%safrobe50.5% 22 172 0 0 3@ 0 f 12 3phenobanbitalsafrole + sodium 22 235 0 0 12Â° 0 15 (0.1%in water)Sodiumdrinking 11(0.1%phenobanbital 22 323 0 0 1 0 h 13 in drinking wa ten)(None)' @ 288 0 0 0 0 13 12 a There was 1 rat with each of the following tumors: insuloma (pancreas), spindle cell sarcoma with metastases (arising in Zymbal's gland), and malignant lymphoma. b One rat had an insuloma (pancreas) and 1 had a leiomyosarcoma (forestomach). C There was 1 rat with each of the following: sarcoma (mesentery), reticuboendothelial sarcoma (spleen), and s.c. fibroma. d There was 1 rat with each of the following: sarcoma (lung), insuloma (pancreas), and adenocarcinoma (pancreas). e One rat had multiple hepatic carcinomas. I There was 1 nat with each of the following: squamous cell carcinoma of the ear duct gland, s.c. fibroma, and carcinoma of the adrenal cortex with metastasesto the liver.

p Six rats had multiple hepatic carcinomas. I' One rat had a s.c. fibroma. I There were only 15 rats in this group. aOne rat had an epidenmoidcarcinoma of the jaw and another had generalizedlymphoma.

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weights of the 1â€˜-oxosafrole-treatedrats were 20% less than these conditions to liver tumor induction by safrole, 1'- those of the control or 1â€˜-acetoxysafrole-treatedmats.There hydroxysafrobe, or 1â€˜-acetoxysafrobe(3). were no marked weight differences between the matsof In view of the toxicity of 1â€˜-oxosafrobe,thetotal dose of these 2 groups in the 2nd experiment. the safrole derivatives that was administered in this experi s.c. Injection Into Pieweanling Mice. Male mice that ne ment was one-third of that used in a previous experiment ceived s.c. 1â€˜-oxosafroleinjections at 1 to 22 days of age (3). The overall incidences of hepatic tumors were similar in (total dose, 0.52 mg) and that were killed at 16 months had the 2 experiments, but the assay time was 16 months in this approximately the same incidence of liver carcinomas (16%) experiment as compared to 12 to 14 months in the earlier as did the control mice that received only the solvent (13%) one. Of the newborn mice given 1â€˜-oxosafrobeonly83, 37, (Table 3). Incidences of 51, 82, and 74% were observed in and 26% survived to the 8th, 15th, and 22nd days, respec male mice that were given injections of the same dose of tiveby. At least 90% of the mice that received 1â€˜-hydroxysaf safrole, 1â€˜-hydmoxysafrole,or 1â€˜-acetoxysafrole, respec role, 1â€˜-acetoxysafrole,or only the solvent survived the Se tively. The frequency of multiple hepatic carcinomas was nies of injections. greaten in the mice that received 1â€˜-hydnoxysafroleon1â€˜- Topical Application to the Skin of Mice. Topical applica acetoxysafrole than in those given safrole. The female litter tion of 1'-hydroxysafrole-2',3'-oxide (total dose, 17.5 mg) mates were not studied in view of their refractoriness under and subsequent repeated applications of croton oil caused

Table 2 injectionsMale Incidences of tumors in adult male rats given safrole derivatives by s.c. s.c.injectionsrats (Charles Riven CD random-bred) with average initial weights of 285 g (Experiment 1) on 225 g (Experiment 2) were given twice weekly, 24 times, of 0.2 ml of tnioctanoinrats/group.No. containing 18.6 @moIesofcompound. There were 18 andAv. of rats with sarcomas at No. of rats alive wt gain injection site by tumor free at Expeni- in 1st 4 mos. mentmos.1 Compound (g) 12 mos. 16 mos. 21 mos. Other tumors 16 mos. 21

1'-Oxosafnole 292 0 0 1 141 bymphoma 12 s.c.frominjection fibnoma (distant site)1 â€˜-Acetoxysafnole 386 1 1 5 1 sebaceous1(injection cell carcinoma 10 site)(Solvent 72 only) 364 0 0 1 0 14 1â€˜-Oxosafnole 327 0 0 2â€• 1 s.c.14injection fibroma (distant from site)1-liposarcoma (abdominalcavity)1

9@(Solventâ€˜-Acetoxysafnole 31 9 2 4 6Â° 0 14 @ only) 320 0 0 @a 18 18@ a Experiment 2 was terminated at 19 mos.

Table 3 lncidences of liver carcinomas in male mice given injections of safrole or its derivatives at 1 to 22 days of age s.c.injectionsMice of the CharlesRivenCD-i stock were bred in the laboratory, and the young were given in0.05 on the following schedule:0.21 @moIein0.025ml tnioctanoinon the 1stday;0.42 @.tmole 22ndday;ml on the 8th day; 0.85 @mobein0.05 ml on the 15thday; and 1.7 @.tmoIesin0.1 ml of the forthetotal dose, 3.18 @mobes(0.52 mg safrole). The mice were weaned at 22 days. The data are mice autopsied at 16 months.No.ofmicewith @ 0 liver carcinomas /0 of mice with No. of mice examinedMultiple16 at mos./no. 1 carci- >1 carci- Liver car- liver car- Other tu morsSafnoleCompound weaned@ noma noma cinomas cinomas 41 35/44 13 5 51 1 C1â€˜-Hydnoxysafnole 40/50 11 22 82 55 dIâ€˜-Acetoxysafnole 34/48 8 17 74 50 0(Solventâ€˜-Oxosafnole 31 /53 5 0 16 only) 45/51 4 2 13 5 a The data are tabulated only for those mice which were killed at 16 months. b Two mice had generalized lymphoma. C One mouse had generalized lymphoma. d One mouse had a lung adenocarcinoma and 1 had a lung adenoma. e Three mice had lung adenomas. I One mouse had generalized lymphoma and 2 had lung adenomas.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Carcinogenic and Mutagenic Safrole Derivatives papillomas to develop in the skin of 10 of 27 mice with an diet containing 0.08% 1'-hydroxysafrobe, 1 died within 2 average of 1.6 papibbomas/tumom-beaning mouse (Table 4, weeks and none of the remaining 19 mice.@diedor lost Experiment 1) and in 10 of 30 mice with an average of 1.9 significant amounts of weight during a subsequent 2-week papilbomas/tumor-beaning mouse (Experiment 2). Six and 5 period in which they were fed 0.55% 1â€˜-hydroxysafrolein of the mice that received equimolar levels of safnole-2',3'- the diet. oxide or 1â€˜-acetoxysafrobe-2',3'-oxide, respectively, each Histopathobogicab studies showed that the toxicity of 1'- developed a single papilboma, and 0 to 3 mice treated only hydroxysafrole was associated with marked liver damage. with the solvent on with one of the other safrole derivatives Female mice fed 0.08% of the compound for 12 days showed developed papilbomas of the skin. Both 1'-oxosafrole and acute liver necrosis; after a subsequent 2-week period dun 1â€˜-acetoxysafrole,especially at the higher level (3 @mobes/ ing which they were fed a diet containing 0.55% of the dose), caused ulceration of the skin at the site of applica compound, the livers were yellow-tan and on histological tion; noneof the other safrole derivatives caused gross skin examination showed peniportal necrosis and small hyper lesions other than the papibbomasnoted above. plastic foci. Female mice fed a diet containing 0.55% 1'- P.O. Administration to Adult Mice. Preliminary expeni hydroxysafrole without prior administration of a lower level ments showed that adult female mice were much more of the compound died with extensive liver damage that was susceptible to the toxic effects of 1â€˜-hydmoxysafrolethan marked by hemorrhage, centrobobulan necrosis, and en were adult male mice. Thus, in 1 experiment all of 20 adult banged and degenerating hepatocytes. The livers of male female mice fed a diet that contained 0.55% 1â€˜-hydroxysaf mice fed 0.55% 1â€˜-hydroxysafroleshowed similar, but much role died within 72 hr, while none of 20 male mice fed the less severe, hepatic damage. The heart, small and large same diet showed appreciable weight loss or died within 2 intestine, stomach, lungs, spleen, and kidneys showed no weeks. In a 2nd experiment in which groups of 10 female significant histological abnormalities. mice were fed diets containing 0.55, 0.33, 0.17, and 0.08% For determination of carcinogenicity adult male and fe of 1â€˜-hydmoxysafrobe8,6, 2, and none, respectively, of the male mice were fed for successive 10-day periods with diets mice died within 8 days. Female mice were much more that contained 0.08% and 0.17% 1'-hydroxysafrole or the tolerant of high doses of 1â€˜-hydnoxysafrobeifthey were first equivalent levels of safrobe before the bong-term feeding of fed the compound at bower levels. Thus, of 20 mice fed a 0.55 and 0.50% of these 2 compounds was started (Table 5).

Table 4 Incidences of papillomas in adult mice treated topically with safrole derivatives The safrole derivatives,dissolved in 0.1 ml of acetone per dose, were applied 5 times per week for 6 weeksto the shavedbacks of Charles River CD-I female mice (30 mice/group). Starting 1 week after the last treatment with the safrolederivatives,all mice receivedtwice-weeklyapplications of 0.5 mg cnotonoil pen0.1 ml acetonepenmouseuntil the expenimentwas terminated at36 weeks.Incidences papilbomas24 of skin wkâ€•No.TotalNo.TotalEx wkâ€•36

ofpen @tmoIesNo. ofwithno. ofwithno. /appbi appli No.papillo papilbo No.papilbo papillo mentCompoundcationcationsalivemasmasalivemasmas11 â€˜-Hydroxysafrole3.03027252710162' ,3'-oxide1 â€˜-Hydroxysafnole3.030290029221 â€˜-Acetoxysafrole3.030300030331 .530301129121'-Oxosafnoleâ€•3.030300029221â€˜-Acetoxysafrole1 .530301128117,12-Dimethylâ€˜-Oxosafroleâ€•1 0.213012712920C110benz(a)anthracene(Solvent only)303000300021 3.03030563010192',3'-oxideSafnole-2',3'-oxide3.030301130661â€˜-Hydnoxysafrole

â€˜-Acetoxysafrole3.030300027552' ,3'-oxide1 â€˜-Hydroxysafrole3.030300030001 â€˜-Acetoxysafrobeâ€•3.030300028221 â€˜-Oxosafrole3.03029002900Safnole'@3.03030113023(Solvent only)3030002922

a Weeks from beginning of experiment. b A lymphoma was found in 1 mouse. C Three mice in this group had skin carcinomas at 36 weeks. d One mouse had a reticulum cell sarcoma in the abdominal cavity.

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Table 5 â€˜-hydroxysafro!eSafroleIncidences of tumors in adult male and femalemice fed safrole and 1 to10; and 1â€˜-hydroxysafrobewerefed to adult CharlesRivenCD-i mice at 0.075and 0.083%,respectively,of a grain diet for Days1 thesameat 0.15 and 0.17%for Days11to 20; and thereafter up to 12 monthsat 0.50 and 0.55%.From 12to 17months,the mice were fed diet without additives.The averageinitial weights wererespectively.. 21 and 25 g for the female and male mice, Av.wtgain No.ofmlceallveat. No. of mice withby liver tumors in 1st 4 No. of mice with other mos.1'-Hydroxy-Compound Sex mos. (g) 0 mos. 12 mos. 14 mos. 17 mos. 12 mos. 14 mos. 17 mos. tumors by 17 angiosarcomassafroleF 9 50 33 22 10 2 12 30 5 s.c. carcinomaSafrole 1 mammary 25(None) F 9 50 36 19 12 1 ii carcinomas1'-Hydroxy-F 17 55 53 47 42 0 0 0 2 mammary kidneysafrole M 13 45 25 13 3 0 0 0 1 mesenchymal (malignant)Safrole tumor 11(None) M 11 46 26 12 7 3 7 M 17 55 44 36 18 0 0 0

Only 50% of the male and 70% of the female mice fed satrobe mice for 18 or 50 days prior to the i.p. injection of a single or its 1â€˜-hydroxyderivativewere alive at 12 months, the time dose of [3H]-1â€˜-hydroxysafrobereducedthe amount of mac at which the 1st hepatic tumors were noted and the admin momolecube-bound 3Hderivatives to 25 to 50% of the control istration of the test compounds was stopped. The survivals levels. Prefeeding of the above safrole or 1â€˜-hydroxysafrole of the control male and female mice at 12 months were 80 diets to adult female mice reduced the levels of hepatic and 96%, respectively. Of the female mice that survived at macromolecule-bound derivatives from a dose of [3H]-1â€˜- least 12 months, 90 and 70%, respectively, of those fed 1â€˜- hydroxysafrole to 10% or less of the levels observed in hydmoxysafmoleor safrole developed hepatocelbular carcino female mice not pretneated with these compounds. Pread mas by 17 months. None and 40% of the male mice fed 1'- ministration of 0.08% 1â€˜-hydroxysafroleorsafrole in the diet hydroxysafrole or safrole, respectively, and that survived for of adult female mice for only 8 days also decreased the at least 12 months developed liver carcinomas, but few of hepatic macromolecule tnitiated derivatives to no more than the male mice survived to the termination of the experiment 10% of the control levels, but this treatment was without at 17 months. These data for the male mice fed safnole and effect on the levels of macromolecule-bound tnitiated me 1â€˜-hydnoxysafrolearesimilar to those reported earlier, ex tabobites in the livers of male mice (data not presented). cept that in the previous experiment a few liver carcinomas and a moderate incidence of s.c. angiosarcomas were Mutagenicity of Known and Possible Metabolites of Saf found in the male mice fed 1'-hydroxysafrole (3). In the role experiment reported herein, the only s.c. angiosarcomas were obtained in female mice fed 1â€˜-hydmoxysafrole.The 1â€˜-Acetoxysafrobeandthe 2',3'-oxides of safrobe, 1â€˜-hy reason for the lack of angiosancomas in the male mice in droxysafrole, 1â€˜-acetoxysafrole,and 1â€˜-oxosafrole,were this experiment is not apparent. This experiment differed mutagenic for S. typhimurium TA100 without fortification from the previous one only in that the mice were prefed with tissue preparations; except for 1â€˜-acetoxysafrolethese diets containing lower doses of safrole and 1â€˜-hydmoxysaf compounds were also mutagenic for TA1535 (Table 7). The role for 20 days prior to the start of long-term feeding of mutagenicity of safrole-2' ,3'-oxide for S. typhimurium diets containing 0.50 and 0.55%, respectively, of these sub TA1535 has also been demonstrated by Domangeet a!. (7). stances. With the epoxides the mutagenicity was approximately a linear function of concentration up to about 250 @g/pbate; Covalent Binding of [2',3'-3H]-1 â€˜-HydroxysafroletoMouse with 1â€˜-acetoxysafrolea linear dose response with TA100 Hepatlc Macromolecules was observed only from 5 to 50 pg/plate. Under these conditions the numbers of mevertantsof TA100 per nmole of The amounts of covalent binding of 3H from a single compound added to the plate were in the range of 1 to 6 for injection of [2',3'-3H]-1'-hydnoxysafrole to DNA, rRNA, and the above 5 compounds. When the epoxides were assayed protein of mouse liver were consistent with the hepatotoxic in the presence of the NADPH-fortified S-9 fraction, theme ity and hepatocancinogenicity of 1â€˜-hydmoxysafnoleforadult were fewer revertant colonies per plate than in its absence. mice. Thus, the levels of the DNA- and rRNA-bound deniva No appreciable mutagenicity was observed for any of these tives were 40 to 50 times greaten and those of the pro compounds in assays with strain TA98. tein-bound derivatives were about 12 times greater for No mutagenic activity was observed for the other safrole the livers of adult female mice than for the livers of adult derivatives studied with S. typhimurium TA100 on TA98 male mice (Table 6). The levels of the hepatic macnomole whether the compounds were assayed directly or with the cube-bound derivatives were similar for 1- to 3-week-old addition of the S-9 fraction of liver from Aracbom-treatedrats male and female mice, and these values were similar to and an NADPH-generating system (1). These inactive com those obtained for adult female mice. Administration of pounds were safrobe, isosafrole, 2',3'-dihydrosafrobe, 1'- either safrobe or 1â€˜-hydroxysafrobeinthe diet of adult male hydroxysafrole, 3'-hydroxyisosafnole , 3'-acetoxyisosafnole,

1888 CANCERRESEARCHVOL. 37

Table 6 macromoleculesEachIn vivo binding of f2',3'-3H)-1 â€˜-hydroxysafroleresiduesto mouse hepatic bodyweightmouse received an i.p. injection of 2 mg of [2',3'-3H]-1'-hydroxysafrole per 100 g DNA,rRNA,pen0.1 ml tnioctanoin.The micewerekilled 16hr after injection for isolation of hepatic and protein by a slight modification (32) of the(14).Macromobecube-bound procedure of Irving and Veazey 3H(pmobes1â€˜-hydroxy macromolecube)aAge safrobe residues/mg Protein1@3b(wk) Sex Pretreatment DNA nRNA

140Â±32FM None 69Â±6 130Â±22 120Â±509-12 None 63Â±9 130Â±28

M None6Pnefed 2 Â±0.8 3 Â± 0.6 10 Â± 5PnefedsafnoIe'@ 0.6, 1 0.9, 1 5, 5F i'-hydnoxysafnoIe'@ 0.6, 0.5 0.7, 0.6 6,

None120Â±28Prefed 100Â±21 120Â±17 16Pnefedsafnoleâ€• 12, 6 12, 8 16, 1l@hydnoxysafnoleC 9, 4 14, 4 15, 14 a Averages Â± S.D. for analyses on 3 pools of liven. Where no standard deviation is given, each value is for an analysis of a pool of livers. Analyses were carried out on pools of 5 to 20 livers from 1- to 3-week-old mice and of 3 livers from 9- to 12-week-old mice. b Analyses were made on 1 pool of livers each from male and female mice 1 , 2, and 3 weeks old. Since there was no trend in the values as a function of age, the data for each sex were averaged. C The mice were fed 1 â€˜-hydnoxysafrole diets containing either 0.08% for Days 1 to 10, 0.17% for Days11 to 20, and 0.55%for Days21 to 50 onequimolar levelsof safrole. In each casethe 1st and 2nd values are for mice that received i.p. injections of [2',3'-3H]-1'-hydnoxysafrole on Days 18 and 50, respectively.

Table 7 0-CH2 Themutagenicityof safrolederivativesfortyphimuriumTA1535 Salmonella c@rb TA100The and Thenumberassayswerecarried out asdescribedby Ameset al. (1). H@C-CH=CH2 H-@CHaCH2 of nevertantspennmobewas calculated from linear dose SAFROLE I@-HYDROXYSAFROLE doselevelsresponse curves based on the data obtained ton at least 3 between5compounds.Reventants/nmoleSafrole and 250 .@g/plateforthe 1st 5 I /1 0â€”@H2 0â€”@H2 0-CH2 O@2 001 derivative TA1 535 TA1 1@Jo@1O@1O c@rO 2Safrole-2',3'-oxideâ€˜-Acetoxysafnole NSa 61 6 H@C-C@-$H2 @-CH=CH2H-g-CH:CH@H-Ã§-@-$H2 31â€˜-Hydroxysafrole-2' ,3'-oxide 2 0 -SO3H OH 0 11â€˜-Acetoxysafrole-2',3'-oxide 0.8 SAFROLE- i@-OXO- SAFROLE I@HYDROXY 2Safrobe,â€˜-Oxosafnole-2',3'-oxide 0.2 2:3@oxIDE SAFROLE ISULFATE SAFROLE- NSrole,isosafrole, 2',3'-dihydnosaf- NS 2:3@oxIDE 1'-hydroxysafnole,3'-hydnox Chart 1. Structures of the proximate and possible electrophilic ultimate yisosafnole,nob,1 3'-acetoxyisosaf carcinogenic metabolites of safrobe in the rat and mouse. â€˜-oxosafrol&'

a NS, no significant increase oven the spontaneous number of s.c. injection into preweanling mice indicate that 1â€˜-hydmox nevertants. ysafrole (Chart 1) is a proximate carcinogenic metabolite of b These compounds were assayed in log steps from 0.1 to 250 @g safrole (3, 4). The presence of sulfotransferase activity for on 1 mg of compound pen plate with and without addition of liven S 9 preparationsand a NADPH-generatingsystem(1). 1'-hydnoxysafmole in nat and mouse liver (32), the apparent formation of an ester of 1â€˜-hydnoxysafroleinrat and mouse and 1â€˜-oxosafrole.Theassays were run in bogsteps from 0.1 livers in vivo (32), and the strong electrophilicity of the to 250 /.Lgor 1 mg of compound pen plate. Except for 1â€˜- acetic acid and sulfuric acid esters of 1â€˜-hydroxysafrole(4, oxosafrobe, which caused a reduction in the number of 32) are all consistent with the designation of the sulfuric revertant colonies and a bossof the bacterial lawn as com acid ester of 1â€˜-hydmoxysafrole(Chart 1) as an ultimate pared to the control plates at levels in excess of 2.5 @g, hepatocarcinogenic metabolite. The carcinogenicity of the toxicity of the compounds was not evident. model ester 1â€˜-acetoxysafrolein preweanling mice and at the site of s.c. injection into adult mats(Ref. 3; this paper) further supports this assignment and suggests that any DISCUSSION ester formed in vivo might be an ultimate carcinogen. Like wise, the formation of 1'-hydroxysafrobe-2',3'-oxide (Chart The metabolism of safrole to 1â€˜-hydroxysafroleby rats 1) by liver microsomes in vitro (32), its apparent formation in and mice and the greaten hepatocancinogenic activity of the vivo (6, 29), its electrophilic reactivity (32), and its ability to battercompound on p.o. administration to adult rats or a initiate papilbomas in mouse skin make this compound a

JUNE 1977 1889

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. P. G. Wis!ocki et a!. candidate ultimate carcinogenic metabolite of safrole and similar levels of [3H]-1â€˜-hydmoxysafrole,butthe female mice 1 â€˜-hydroxysafnole . Although safnole-2' ,3'-oxide (Chart 1) did developed very few tumors (3). Likewise, while male rats not induce a significant incidence of papilbomas of the skin were more susceptible to the carcinogenic activity of dietary in our test, its apparent formation in vivo (29), its ebectro 1â€˜-hydroxysafrobethan were female mice, the bevels of mac philicity (32), and its mutagenic activity (Ref. 7; this paper) nomolecule-bound derivatives were very similar (Ref. 32; are consistent with a moleas an ultimate carcinogen. The this paper). It seems likely that a major share of the bound apparent formation in vivo of 1â€˜-oxosafrobe(20,22, 23) and derivatives may have little role in the initiation of hepatic its electrophilic reactivity (32) would be consistent with the tumors onthat the promotional phase of hepatic tumonigen assignment of this compound as a possible ultimate canci esis is deficient under the test conditions. Characterization nogenic metabobite. The lack of carcinogenic activity of 1â€˜- of these bound derivatives should help to elucidate the oxosafrobe neither supports nor mulesout this possibility. reasons for these differences. The mutagenicities of the safrole derivatives for S. typhi The protective effect of low doses of safrole or 1â€˜-hydrox murium TA1535 and TA100 are generally consistent with the ysafrole against the acute toxicity of subsequent higher available data on the reactivities of these compounds and doses of 1â€˜-hydnoxysafroleandthe enhancing effect of phe their carcinogenic activities. Thus, 1â€˜-acetoxysafnoleand nobambital administration on the carcinogenicity of safnole the 2',3'-oxides of the various safrole derivatives were all provide further examples of the intemplays that can occur mutagenic without enzymatic activation. As noted above, 1'- between various nonnutritive dietary ingredients. The acetoxysafrobe and 1â€˜-hydroxysafmole-2',3'-oxideare canci mechanisms involved in these interactions are not clear, nogenic in appropriate assays. The strong electrophile 1'- although both safrole and phenobarbital are inducers of oxosafrole failed to induce mutations in the S. typhimurium mixed-function oxidases in the endoplasmic reticulum (5, strains. The lack of mutagenicity and of carcinogenicity of 15, 17, 24). Administration of phenobanbital on other in 1 â€˜-oxosafrole may result from an inability of the exoge ducens of these enzymes prior to on concurrently with var nously administered compound to penetrate cellular mem ious carcinogens has previously led to either no effect or branes and to reach critical intracellular targets. The 2',3'- reduced incidences of tumors both in the liven and in extra double bond in 1'-oxosafnole readily reacts with amines hepatic tissues of rats or mice (31). Despite the fact that such as dimethylamine, pipenidine, and pynrolidine at room activation reactions for a number of chemical carcinogens temperature to form the corresponding 3'-addition prod are induced by these chemicals, the findings have led to the ucts (2, 20). The major product of the reaction of 1â€˜-acetoxy expectation that enhanced mixed-function oxidase activity safrobe with guanylic acid is substitution on the oxygen at would inhibit chemical carcinogenesis if it had any effect on position 6 (4, 32), a reaction site that has been correlated tumor induction (31). The enhancement of hepatic safrole with mutagenic and carcinogenic activity with simple alkyl carcinogenesis in the matwith concurrent administration of ating agents (10, 16, 18, 21). The backof mutagenic activity phenobanbital is the 1st clear exception; others probably of 1â€˜-hydnoxysafrolein systems fortified with the 5-9 frac exist. Furthermore, the promoting activity of phenobambital tion of liven and a NADPH-generating system or with cytosol for hepatic carcinogenesis in the rat when treatment with and 3'-phosphoadenosine 5'-phosphosulfate (Refs. 3, 7, phenobambitabfollows carcinogen treatment (25, 27) and the 8, and 19; this paper) provides an exception to the general greatly increased incidence of liver tumors in mice treated correlation between carcinogenic activity and mutagenic with phenobanbital in the absence of any other known activity of chemicals for S. typhimurium in the presence of carcinogen (26, 30) emphasize the complexity of the possi activating systems (19). The known activation reactions for ble sequelae to the administration of enzyme inducens. 1â€˜-hydroxysafrobe(sulfuric acid estenification of the 1â€˜-hy The carcinogenicity of safrole and its derivatives and of droxy group and epoxidation at position 2',3') proceed at estragole and 1'-hydmoxyestnagole (Refs. 3, 8, and 13; this slow rates in vitro (about 0.1 to 0.3 @molepeng liver per hn) paper) point to the need for further studies on the cancino (32). These rates of formation of mutagen would be man genic activities of the various ablylic and propenylic anenes ginal for the detection of a significant number of revertant that may occur in natural foodstuffs on be used as food colonies for chemicals with the mutagenic activity of 1'- additives (9, 11, 12). Studies of the major food spice compo hydroxysafrobe-2',3'-oxide, even if the activation system ne nents anethobe (trans-i -pmopenyl-4-methoxybenzene) and tamed the above reaction rate for 1 hr after the plates were eugenol (1-albyI-3-hydnoxy-4-methoxybenzene) are in prog poured. Further approaches to metabolic activation in the mess. detection of these relatively weak premutagens are needed. The bevels of hepatic DNA-, nRNA-, and protein-bound ACKNOWLEDGMENTS derivatives from [2' ,3'-'H]-l â€˜-hydroxysafrolearesurprisingly high (1 mole/104 to 10@moles of monomer in these poly We are grateful to Mary Kolstad, Lona Barsness, and Diane Chambliss for mers) (Ref. 32; this paper) for a carcinogen that is weakly expert technical assistance and to Dr. Henry C. Pitot and his associates for active in adult matsand mice. Furthermore, while the higher the histopathological studies. levels of macnomobecube-bound denivativesin female mouse liver as compared to male mouse liven paralleled the greater REFERENCES susceptibility of the adult female mouse to the development of liver tumors, a similar correlation between hepatocellular 1 . Ames, B. N., McCann, J., and Yamasaki, E. Methods for Detecting carcinomas and macromobeculan binding was not observed Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31: 347-364, 1975. on comparison of male and female mice given the com 2. Borchert, P. 1â€˜-Hydroxysafrole:AProximate Carcinogenic Metabolite of pound pniorto weaning. Infant male and female mice bound Safrole in the Rat and Mouse. Ph.D. Dissertation, University of Wiscon

1890 CANCERRESEARCHVOL. 37

sin, Madison, Wis., 1972. 18. Margison, G. P., and Kleihues, P. Chemical Carcinogenesis in the Nerv 3. Borchent, P., Miller, J. A., Miller, E. C., and Shires, T. K. 1'-Hydnoxysaf ous System. Preferential Accumulation of O'-Methybguanine in Rat Brain role, a Proximate Carcinogenic Metabolite of Safrole in the Rat and Deoxyribonucleic Acid during Repetitive Administration of N-Methyl-N- Mouse. Cancer Res., 33: 590-600, 1973. nitrosourea. Biochem. J., 148: 521-525, 1975. 4. Borchert, P., Wisbocki, P. G., Miller, J. A., and Miller, E. C. The Metabo 19. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. The Detection of lism of the Naturally Occurring Hepatocarcinogen Safrole to 1â€˜-Hydroxy Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of safrole and the Electrophilic Reactivity of 1â€˜-Acetoxysafrole.Cancer 300 Chemicals. Proc. NatI. Acad. Sci. U. S., 72: 5135-5139, 1975. Res., 33: 575-589, 1973. 20. McKinney, J. D., Oswald, E., Fishbein, L., and Walker, M. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1977 American Association for Cancer Research. Carcinogenic and Mutagenic Activities of Safrole, 1′ -Hydroxysafrole, and Some Known or Possible Metabolites

Peter G. Wislocki, Elizabeth C. Miller, James A. Miller, et al.

Cancer Res 1977;37:1883-1891.

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