Inhibitory Effects of Phenethyl Isothiocyanate on Jy-Nitrosobenzylmethylamine Carcinogenesis in the Rat Esophagus1

ICANCER RESEARCH 51. 2063-2068. April 15. 1991| Inhibitory Effects of Phenethyl Isothiocyanate on jY-Nitrosobenzylmethylamine Carcinogenesis in the Rat Esophagus1

Gary D. Stoner,2 Diana T. Morrissey, Young-Hun Heur, Elaine M. Daniel, Anthony J. Calati, and Steven A. Wagner

Department of Pathology, Medical College of Ohio, Toledo, Ohio 43614 [G. D. S., D. T. M., Y-H. H., E. M. D., A. J. G.J, and Division of Epidemiology and Toxicology, Ohio Department of Health, Columbus. Ohio 43266 [S. A. W.]

ABSTRACT found in the diet in the People's Republic of China (8, 12). F-344 rats fed diets containing phenethyl isothiocyanate (PEITC; 3 Investigations in experimental animals have demonstrated that foods contain a number of substances with the ability to and 6 Mmol/g diet), a naturally occurring constituent of cruciferous vegetables, before and during treatment with the carcinogen .V-niiniso- inhibit chemically induced cancer (13). Among these are the benzylmethylamine (NBMA), developed 99-100% fewer esophageal tu isothiocyanates, including PEITC (14-17). PEITC is a primary mors than NBMA-treated control rats. PEITC exhibited inhibitory ef product of thioglucosidase-catalyzed hydrolysis of gluconastur- fects against both preneoplastic lesions (acanthosis and hyperkeratosis, tiin, a naturally occurring compound found in certain crucifer leukoplakia, leukokeratosis) and neoplastic lesions (papilloma, carci ous vegetables (18). Pretreatment of rats with PEITC inhibited noma). Tumors were not observed in rats treated with PEITC alone. The the induction of mammary tumors by DMBA (14). Addition of effects of PEITC (10, 25, 50, 100 JZM)on the metabolism and DNA PEITC to the diet inhibited DMBA-induced lung and forestom- binding of NBMA in cultured expiants of rat esophagus were also ach tumors in mice (14) and lung tumors in rats induced by the investigated. PEITC produced a marked (53-97%) dose-dependent inhi tobacco-specific nitrosamine NNK (15). In addition, PEITC bition in the binding of NBMA metabolites to DNA and in the levels of DNA methylation at the /V7 (20-89%) and O" (55-93%) positions of inhibited NNK-induced lung tumors in mice, and the extent of inhibition correlated with a reduction in the formation of the guanine. This isothiocyanate also reduced the metabolism of NBMA by O6-methylated guanine adduci in lung DNA (16). The closely esophageal tissues as indicated by increased amounts of unmetabolized NBMA in the medium of cultures containing PEITC. Collectively, these related isothiocyanates benzyl isothiocyanate and phenyl iso data indicate that PEITC is a potent inhibitor of NBMA-induced esoph thiocyanate were both found to inhibit DMBA-induced mam ageal carcinogenesis in rats. mary tumors in rats (14) but did not inhibit NNK-induced lung tumors in mice (16). INTRODUCTION In this paper we report the results of testing PEITC in a 25- week bioassay for its ability to inhibit NBMA tumorigenesis in Esophageal cancer occurs worldwide and ranks seventh the esophagus of F-344 rats. Results from the in vivo bioassay among cancers in order of frequency of occurrence in both sexes are compared with the inhibitory effects of PEITC on the combined (1). The geographical distribution of the disease metabolism and DNA-damaging effects of NBMA in cultured varies considerably and is more diverse than for any other rat esophageal tissues. cancer of the digestive tract (2). The disease occurs most fre quently in areas of the world where the diet is often restricted and nutritional imbalance is common (3). MATERIALS AND METHODS EpidemiolÃ³gica! studies indicate that nutritional and environ Chemicals mental factors play a major role in the etiology of esophageal cancer. An increased risk for developing esophageal cancer has [methyI-Ã¬H\NBM\ (5 Ci/mmol; purity, >97%) was purchased from been associated with the smoking and chewing of tobacco (4, Moravek Biochemicals, Inc. (Brea, CA). Unlabeled NBMA (purity, 5) and the consumption of alcoholic beverages (6, 7) and salt- >98%) was obtained from the National Cancer Institute Chemical cured, salt-pickled, and moldy foods (8). The molds which can Carcinogen Repository at Midwest Research Institute (Kansas City, MO). Standards for /V7-MeGua and O6-MeGua (purity, 98%) were contaminate foods include members of the Fusarium species purchased from Chemsyn Science Laboratories (Lenexa, KS). PEITC (9), which produce several toxins, and Geotrichum candidum (purity, >99%) was obtained from Aldrich Chemical Company (Mil (10), which promotes the formation of Â¿V-nitrosaminecom waukee, WI). The purity of all chemicals was checked by HPLC. pounds. Research in the Transkei region of South Africa and in the People's Republic of China suggests that A'-nitroso In Vitro Studies compounds and their precursors are probable etiological factors Expiant Culture. Male F-344 rats, 9-10 weeks old, were purchased in esophageal cancer in these high-incidence areas (5, 8). Ani from HarÃanSprague-Dawley (Indianapolis. IN). The rats were killed mal model studies of esophageal cancer have shown that several by sodium pentobarbital injection, and the esophagi were removed and A'-nitroso compounds are esophageal carcinogens in rodents, transported to the tissue culture laboratory in L-15 medium (19). especially in the rat (11). Among the most potent inducers of Esophageal expiants were prepared and cultured in chemically defined esophageal tumorigenesis in the rat is NBMA,1 a compound CMRL-1066 medium as described previously (19). To determine non- toxic concentrations of PEITC for subsequent inhibition experiments, Received 10/19/90; accepted 2/6/91. The costs of publication of this article were defrayed in part by the payment the expiants were treated with PEITC at concentrations of 10, 25, 50, of page charges. This article must therefore be hereby marked advertisement in 100, and 200 ^M for 48 h. After treatment, the tissues were examined accordance with 18 U.S.C. Section 1734 solely to indicate this fact. by light microscopy for PEITC-induced histopathological effects. 1This research was supported by National Cancer Institute Grants CA28950 Incubation of Esophageal Expiants with PEITC and Radiolabeled and CA46535. 2To whom requests for reprints should be addressed, at Department of NBMA. Rat esophageal expiants were cultured for 6 h and then exposed Pathology. Medical College of Ohio. Health Education Building. Room 202. for 12 h to 1 /iM [inrtA>-/--'H]NBMA concurrently with PEITC at P.O. Box 10008. Toledo. OH 43699. concentrations of 10, 25, 50. and 100 p\\. Control expiant cultures 'The abbreviations used are: NBMA. iV-nitrosoben/.ylmethylamine; PEITC, were incubated for 12 h in medium containing l //M [m?/A)7-'H]NBMA phenethyl Â¡sothiocyanate:A"-MeGua, A/7-methylguanine: O'-MeGua. O6-methylguanine; NNK, 4-(mcthylnilrosamino)-l-(3-pyridyl)-l-butanone; DMBA, 7,12- and 1% dimethyl sulfoxide, the solvent for NBMA. Medium controls dimethylbenz[a]anthracene: HPLC. high-performance liquid chromatography. consisted of culture medium containing 1 ^M [mertj'/-3H]NBMA and 2063

l % dimethyl sulfoxide (i.e., no explants). After incubation for 12 h, the Table 1 Incidence and multiplicity of esophageal tumors after treatment with medium and tissues from each series were pooled and stored at -85Â°C vehicle, PEITC, NBMA, and NBMA plus PEITC for subsequent analysis. TreatmentGroup1 incidence% multiplicityTumors/rat Isolation of Radiolabeled Carcinogen Bound to DNA. Explant DNA of rats was isolated by a modification of the method of Gupta (20). The with tumors expiants were thawed in 1% sodium dodecyl sulfate-1 m\i EDTA and bodyweight)0 diet)0 (total no. ofrats)0(15) (mean Â±SD)0 homogenized, and the homogenate was then treated for 30 min with proteinase K (500 Â¿ig/ml).The homogenate was then extracted succes 2 0 3 0(15) 0 sively with 1 volume each of phenol, a 1:1 mixture of phenol:sevag 3 0 6 0(15) 0 4 0.5 0 100(15) 11.5 Â±4.5 (chloroform:isoamyl alcohol, 24:1), and sevag. The aqueous phase was 5 0.5 3 13(15) 0.1 Â±0.3f(99)'/ separated from the organic phase by centrifugation and transferred to 6NBMAÂ°(mg/kg0.5PEITC*(Â»imol/g6Tumor 0(15)Tumor 0.0(100) a fresh tube to which 0.1 volume of 5 M NaCl was added. The DNA " Administered s.c. once per week for 15 weeks. was precipitated with I volume of ice-cold ethanol, and the aliquot was * PEITC was fed in the diet at concentrations of 3 and 6 ^mol/g 2 weeks kept at -20Â°C for 30 min. After centrifugation, the ethanol was re before and during and for 8 weeks after administration of NBMA. c Significantly different from group 4 (P < 0.001). moved, and the pellet was rinsed with 0.5 ml of cold 70% ethanol to d Numbers in parentheses, percentage of inhibition. remove the salt. The residue was dissolved in 1 ml of 0.01 x standard saline-citrate (0.15 M NaCl, 0.015 M Na citrate)-! HIM EDTA and digested with RNase T, (50 units/ml) and RNase A (100 Mg/ml) for 30 pletion of the treatment with either NBMA or vehicle, all groups were min at 37Â°Cwith shaking. One volume of sevag was added to the maintained on their respective diets (with or without PEITC) for 8 reaction mixture, and the mixture was vortexed and centrifuged. The additional weeks. Food consumption was measured weekly, and body aqueous layer was transferred to a fresh tube to which was added 0.1 weights were recorded every 2 weeks during the 25-week bioassay. volume of 5 M NaCl and 1 volume of ice-cold ethanol. After centrifu At the termination of the bioassay, a complete gross autopsy was gation, the ethanol was removed, and the DNA pellets were rinsed with performed on each animal. The esophagus was excised in toto, opened 0.5 ml of cold 70% ethanol. The DNA was dissolved in 200 Mlof 0.01 x longitudinally, placed flat on white index cards with the epithelium standard saline-citrate and quantitated spectrophotometrically, and the uppermost, and fixed in 10% buffered formalin. Quantification of radioactivity associated with the DNA was determined in a liquid esophageal tumors (>0.5 mm in diameter) was performed with a scintillation counter. dissecting microscope and a millimeter ruler (22). Each esophagus was Analysis of DNA Adducts. /V7-MeGua and O6-MeGua adducts in rat then divided into three parts (upper, middle, and lower), embedded in esophageal DNA were analyzed as described previously (21). Briefly, paraffin blocks, serially sectioned, and stained for histopathological DNA isolated as described above was hydrolyzed with 0.1 M HC1 at 70Â°Cfor 30 min. The hydrolysates were analyzed by HPLC (Waters evaluation. Sections of esophagi from each group were scored for the presence of preneoplastic lesions (i.e., acanthosis and hyperkeratosis, Associates, Milford, MA) using a Partisil cation exchange column leukoplakia, and leukokeratosis) and neoplastic lesions (i.e., papilloma (SCX-10; 4.5 x 250 mm) and a radiomatic detector (Radiomatic Instru and carcinoma) (23). ments and Chemical Co., Inc., Tampa, FL). Samples were eluted isocratically with a buffer solution containing 75 mM ammonium Statistical Analysis phosphate:methanol (85:15) buffer, pH 2.5, at a flow rate of 1.0 ml/ min, and the column effluent was monitored at 254 nm. Standards of All data were analyzed using SAS on a personal computer (24). The nonradioactive /V7-MeGua and O6-MeGua were added to all the sam data on the metabolism and DNA binding of NBMA were analyzed ples to ascertain retention times of the major NBMA-DNA adducts. utilizing a test of linear contrast (25). These data, as well as the N1 and Analysis of NBMA in Culture Medium. The concentration of [methyl- O6 adduct formation data, were expressed in terms of percentage of 'HjNBMA in the medium at time zero and after incubation with inhibition. Ninety-five % confidence intervals were calculated. Food expiants (with or without PEITC) for 12 h was measured by HPLC consumption and body weight data were analyzed using analysis of using a Beckman reverse-phase C18column (4.5 x 250 mm). Samples variance. The food intake over the study period was evaluated on a per were eluted isocratically with acetonitrile:water (1:1) at a flow rate of 1 Ã§agebasis. Weight gain was evaluated over the study period as the ml/min and monitored spectrophotometrically at 254 nm, and the difference in weight between week 25 and week 1. Four predefined 1 radioactivity was quantitated with a radiomatic detector. Nonradioac d.f., contrasts of mean weight gain were calculated: NBMA-treated tive NBMA was used as a standard. groups versus groups not treated with NBMA; PEITC-treated groups versus groups not treated with PEITC; the interaction between treat In Vivo Bioassay ment with PEITC and treatment with NBMA; and low-dose PEITC groups versus high-dose PEITC groups. Tumor incidence data were Animals. Male F-344 rats at 5 weeks of age were housed 2/cage and analyzed using x2 tests. The data on frequency of tumors per rat were maintained under standard conditions [20 Â±2Â°C;50 Â±10% (SD) relative humidity; 12-h light/dark cycle]. AIN-76A diet (Teklad Labo evaluated using a Wilcoxon rank sum test. ratory Diets, Madison, WI), provided in glass feeders to allow meas urement of food consumption, and water were available ad libitum. RESULTS Bioassay. After 2 weeks of acclimatization to the animal facility, the rats were randomized into 6 groups, each consisting of 15 animals. The In Vivo Studies. At the beginning of the bioassay, the weights results from these six groups are summarized in Table 1. Groups 1 and of animals in all six groups were the same (Fig. 1). However, 4 were given AIN-76A diet, while groups 2, 3, 5, and 6 received AIN- weight gain throughout the study was not equivalent for all 76A diet containing PEITC at concentrations of either 3 or 6 ^mol/g groups. There was a significant reduction \P < 0.05] in weight diet. PEITC was mixed in the diet weekly, using a Hobart mixer, and stored at 4Â°Cbefore use. Previous studies have shown that PEITC is gain in all groups receiving PEITC [groups 2, 3, 5, and 6 (Table 1)] compared to those not receiving PEITC in the diet [groups stable in the diet for at least 10 days (15). The concentration of PEITC 1 and 4 (Table 1)]. Furthermore, a significant [P < 0.05] in the diet was monitored by random sampling of portions of the mixed diet and was found to be homogeneous throughout. After 2 weeks of difference in weight gain was observed between groups fed the feeding the respective diets, NBMA (0.5 mg/kg body weight) was high dose of PEITC [groups 3 and 6 (Table 1)] and those given administered by s.c. injection once per week for 15 weeks. The total the low dose of PEITC [groups 2 and 5 (Table 1)]. No other dose of NBMA administered per rat was 7.42 mg (0.050 mmol)/kg differences between the groups were observed. body weight. Vehicle-control rats were given s.c. injections of dimethyl The dietary intake was the same for all groups (Fig. 2). A sulfoxide in distilled water (1:4, v/v) for the same period. After corn- reduction in the dietary intake of all groups was noted at week 2064

In Vitro Studies. Rat esophageal expiants treated for 48 h

400- with 10, 25, 50, and 100 UM PEITC showed no evidence of histopathological effects (data not shown). However, expiants (fi treated with 200 UM PEITC exhibited a loss of the surface epithelium and retention of only a single basal layer of epithelial cells (data not shown). Many of these cells contained pyknotic nuclei and showed intense eosinophilic staining, indicative of o o CONTROL coagulative necrosis. In view of these results, all subsequent â€¢â€”â€¢PEITC(H.D.) experiments utilized PEITC at concentrations ranging from 10 *â€”* PEFTC(LD.) tO 100 ÃŸM. HPLC analysis of culture medium collected from dishes without expiants (i.e., medium controls) showed that [methyl- 100 'HJNBMA was stable in the medium for a period of at least 12 10 15 20 25 h. PEITC elicited a dose-dependent inhibition (P < 0.01) in the metabolism of NBMA by rat esophageal expiants (Fig. 3). The 400 â€¢â€¢ mean percentage of inhibition (95% confidence interval) cal culated from four experiments was 42 (18-59), 60 (43-72), 71 to (59-80), and 82% (74-87) for 10, 25, 50, and 100 Â¿i 300 â€¢â€¢

e> * * NBMA 125-- 200- oâ€”Ã¼ NBMA + PETO (H.D.) â€¢â€”â€¢NBMA+ PEfTC (L.D.) 100-

100 10 15 20 25 75-- WEEKS CO o o CONTROL Fig. 1. Average body weights of the animals in the various control and treated â€¢â€”â€¢PEFTC(H.D.) groups for the duration of the dose-response bioassay. HD, high-dose or 6 ^mol/ 50-â€¢ g diet; LD, low-dose or 3 /imol/g diet. Bars, SE. * Â»PEFTC(LD.)

24. This reduction was due to problems with humidity control 25 10 15 20 25 in the animal facility. Food placed in the animal cages absorbed moisture, which gave the false impression that food consump tion was reduced. 125- At necropsy, the esophageal mucosa of control animals ap peared normal. No tumors were seen in vehicle-treated rats or i in rats that received PEITC (Table 1). Tumors were observed 100- â€¢ only in the esophagi of NBMA-treated animals; no mÃ©tastases or invasion into adjacent tissue was found. Most tumors were exophytic and were distributed along the entire length of the 75-â€¢ esophagus, without predilection for the upper, middle, or lower CO NBMA region. 50-- NBMA + PEfTC (H.D.) The tumor incidence in NBMA-treated rats (group 4) was NBMA + PEFTC(LD.) 100% (Table 1). PEITC, at concentrations of 3 and 6 /Â¿mol/g diet, decreased the tumor incidence to 13 and 0%, respectively. 25 The tumor incidence in group 5 (3 Â¿Â¿mol/gPEITC)was not 10 15 20 25 significantly different from that in group 6 (6 nmo\/g PEITC). WEEKS The observed average number of 11.5 esophageal tumors per Fig. 2. Average dietary intake of animals in the various control and treated rat in NBMA-treated animals (Table 1) was similar to that groups for the duration of the dose-response bioassay. HD, high-dose or 6 Â¿

100 DISCUSSION

In the present study, PEITC was found to be a potent 80-- inhibitor of NBMA-induced esophageal carcinogenesis in male F-344 rats. When fed in the diet at concentrations of 3 and 6 Â¡60 + /Â¿mol/g,PEITC caused a 99 and 100% inhibition in the multi m plicity of NBMA-induced esophageal tumors, respectively (Ta i Z 40-- ble 1). Histopathological analysis indicated that the isothiocyanate inhibited the development of both preneoplastic and neoplastic lesions in the esophagus (Table 2). In comparison, 20-- ellagic acid, added to the diet at approximately 13.2 ^mol/g, inhibited the induction of esophageal tumors in F-344 rats given a similar amount of NBMA by the same route (s.c.) by 25 50 75 100 only 47% (22). Ellagic acid also inhibited both preneoplastic PEITC (Â¿Â¿M) and neoplastic lesions, but appeared to be more effective against Fig. 3. Effect of PEITC on the metabolism of NBMA by expiants of rat preneoplastic lesions. The differences in inhibitory activity be esophagus. See "Materials and Methods" for experimental details. Points, means tween PEITC and ellagic acid could be due to differences in from four experiments. Bars, SE. absorption rate and/or in mechanism(s) of action. Diallyl sulfide, a thioether found in garlic, was also found to be potent inhibitor of NBMA-induced esophageal tumorigenesis in Spra- 60 gue-Dawley rats (26). Unfortunately, due to the fact that the diallyl sulfide was administered intragastrically shortly before s.c. injection of the rats with NBMA and, in the present study, PEITC was administered continuously in the diet, a comparison of the relative chemopreventative efficacy of diallyl sulfide and PEITC cannot be made. In vitro studies using explant cultures of rat esophagus indi cated that PEITC inhibits the metabolism of NBMA and the binding of NBMA metabolites to esophageal DNA. When added to the medium at a concentration of 100 pM, PEITC inhibited the metabolism of NBMA by approximately 80% and the formation of DNA adducts by NBMA metabolites by ap 10 20 30 40 50 60 70 80 90 100 proximately 90%. Since NBMA was added to the medium at a PEITC f>M) concentration of only l /uM,the data suggest that PEITC must be present in more than 100-fold excess to elicit complete Fig. 4. Effect of PEITC on the binding of NBMA metabolites to the DNA of rat esophageal expiants. See "Materials and Methods" for experimental details. inhibition of NBMA metabolism and DNA binding. However, Points, means from four experiments. Bars, SE. preliminary studies showed that approximately 90% of the PEITC added to the medium becomes rapidly bound (within Table 3 Effect of PEITC on DNA audaci formation in cultured rat esophagus" 30 min) to the surface of the culture dish, and only 10% (i.e., Adduci formation (pmol/mg 10 AIMwhen added at 100 /UM)remains in the medium. Pretreat Concentration (>iM) DNA) ment of the dishes with either fetal bovine serum or bovine |3H]NBMA1 albumin did not reduce the amount of bound PEITC. Since [methyPH]NBMAi does not bind to culture dishes (21) it would 1 10100A"-MeGua8.817.06(19.9)* 0.18(55.0)* appear that PEITC is capable of eliciting a strong inhibition of 1PEITC0 0.94 (89.3)O'-MeGua0.400.03 (92.5) NBMA metabolism and DNA adduci formation when present " Data were derived from four experiments. in 10-fold excess. In contrast, ellagic acid at 100 /UM(ellagic * Numbers in parentheses, percentage of inhibition. acid does not bind to culture dishes) (21) produced only a 45% inhibition in the formation of yV7-MeGua and O6-MeGua ad DNA binding studies showed that PEITC elicited a dose- ducts in the DNA of rat esophageal expiants treated with 1 Â¿iM dependent inhibition (P < 0.01) of NBMA metabolite binding [methyl-*H]NBMA (21). These data are in agreement with the to the DNA of rat esophageal expiants (Fig. 4). The mean observation that PEITC is a more potent inhibitor of NBMA- percentage of inhibition (95% confidence interval) calculated induced tumors in the rat esophagus than ellagic acid. They from four experiments was 54 (23-72), 77 (62-86), 91 (84-94), also suggest that DNA methylation could be used as a means and 97% (95-98) at doses of 10, 25, 50, and 100 MMPEITC, of screening potential inhibitors of NBMA carcinogenicity. respectively. It is likely that PEITC inhibits NBMA tumorigenesis in the The effect of PEITC (10 and 100 /UM)on the formation of rat esophagus through its ability to inhibit the metabolism of /V7-MeGua and O6-MeGua adducts by NBMA in esophageal NBMA. However, the mechanism by which PEITC inhibits explant DNA is shown in Table 3. At 10 and 100 /IM, PEITC NBMA metabolism in the esophagus is unknown. PEITC could inhibited the formation of W-MeGua by 19.9 and 89.3%, and influence the detoxification of NBMA and/or elicit specific of O6-MeGua by 55 and 92.5%, respectively, in esophageal effects on the cytochrome P-450 enzymes involved in NBMA DNA. The inhibition in the formation of both adducts was activation. Labuc et al. (27) have shown that microsomes pre significant (P < 0.05) only in expiants treated with 100 Â¿Â¿Mpared from rat esophageal mucosa metabolize NBMA at a high PEITC. rate to yield, almost exclusively, benzaldehyde and a methylat- 2066

ing agent in a cytochrome P-450-dependent reaction (NBMA inhibit NBMA tumorigenesis in the esophagus, as was done by debenzylase). NBMA is also metabolized at a high rate in rat Morse et al. (17) in ihe mouse lung. In addition, they provide liver, but it is not toxic or carcinogenic for the liver (28). Based a basis for the conduci of biochemical and enzymalic sludies lo upon its structure, PEITC and its metabolites are unlikely to delermine the mechanisms(s) of PEITC inhibition of NBMA act as scavengers of the reactive intermediates produced in the melabolism and lumorigenesis in Ihe rat esophagus. metabolic activation of NBMA. It is more likely that PEITC inhibits the oxidative metabolism of NBMA. The binding of ACKNOWLEDGMENTS isothiocyanates to protein through sulfhydryl or amino groups has been demonstrated (29). As suggested by Morse et al. (15), We thank Dr. Herman Schut for comments on the preparation of it is conceivable that this type of conjugation could occur the manuscript and Anna Conley for secretarial assistance. between PEITC and specific esophageal cytochrome P-450 isozymes responsible for NBMA activation. Further studies are REFERENCES required to elucidate the specific biochemical mechanisms for the inhibition of NBMA-induced esophageal tumorigenesis by 1. Parkin, D. M, Stjernsward, J., and Muir, C. S. Estimates of the worldwide frequency of twelve major cancers. Bull. WHO, 62: 163-182, 1984. PEITC. 2. Silber, W. Carcinoma of the oesophagus: aspects of epidemiology and aetiol One of the most important considerations in the development ogy. Proc. Nutr. Soc., 44: 101-110, 1985. and use of inhibitors of carcinogenesis is their potential toxic 3. Rothman, K. J. Alcohol. In: ). F. Fraumeni Jr. (ed.). Persons at High Risk effect. In a previous 13-week toxicity study, we found that the of Cancer: An Approach to Cancer Etiology and Control, pp. 139-150. New York: Academic Press, 1975. only significant toxic effects of PEITC (at 3 and 6 nmol/g diet) 4. Wynder, E. L., and Bross, I. J. A study of etiological factors in cancer of the were fatty changes in the liver (15). In general, fatty metamor esophagus. Cancer (Phila.), 14: 389-413, 1961. 5. Warwick, G. P., and Harington, J. S. Some aspects of the epidemiology and phosis in the liver is considered to be a reversible lesion; etiology of esophageal cancer with particular emphasis on the Transkei, however, as a severe form of injury, this lesion may be an South Africa. Adv. Cancer Res., 17: 81-229, 1973. indicator of cell death (30). The small but significant (P < 0.05) 6. Tuyns, A. J., Pequignot, G., and Abbatucci, J. S. Oesophageal cancer and alcohol consumption: importance of type of beverage. Int. J. 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Effects of aromatic 3-carbinol induced them. Another factor to be considered in isothiocyanates on tumorigenicity. O'-methylguanine formation, and metab relating these results to humans is that the doses of both olism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone in A/J mouse lung. Cancer Res., 49: 2894-2897, 1989. inhibitor and carcinogen used in the bioassay are likely to be 17. Morse, M. A., Elkind, K. I., Amin, S. G.. Hecht, S. S., and Chung, F-L. considerably higher than the doses to which humans are ex Effects of alkyl chain length on the inhibition of NNK-induced lung neoplasia posed. The effect of lower concentrations of both inhibitor and in A/J mice by arylalkyl isothiocyanates. Carcinogenesis (Lond.), 10: 1757- 1759, 1989. carcinogen cannot be assessed with certainty from these results. 18. Van Etten, C. H., Daxenbichler, M. E., Williams, P. H., and Kwolek, W. F. This study demonstrates that feeding PEITC in the diet Glucosinolates and derived products in cruciferous vegetables. Analysis of results in a 99-100% inhibition in the incidence of NBMA- the edible part from twenty-two varieties of cabbage. J. Agrie. Food Chem., 24:452-455, 1976. induced esophageal tumors in F-344 rats. The inhibition of 19. Stoner. G. D., Pettis, W., Haugen, A., Jackson, F., and Harris, C. C. Expiant esophageal tumor incidence by dietary PEITC correlated with culture of rat esophagus in a chemically defined medium. In Vitro (Rockville), / 7:681-688, 1981. the reduction in DNA adduci formation by NBMA in cultured 20. Gupta, R. C. Nonrandom binding of the carcinogen A'-hydroxy-2-acetylami- expiants of rat esophagus. These data are consistent with the nofluorene to repetitive sequence of rat liver DNA in vivo. Proc. Nati. Acad. results of Morse et al. (15, 16), who found a close correlation Sci. USA, 81: 6943-6947, 1984. 21. Mandai, S., Shivapurkar, N. M., Galati. A. J., and Stoner. G. D. Inhibition between the ability of PEITC to inhibit adduci formation by of yV-nitrosobenzylmethylamine metabolism and DNA binding in cultured NNK in rat and mouse lung DNA and its ability to inhibit rat esophagus by ellagic acid. Carcinogenesis (Lond.). 9: 1313-1316, 1988. NNK lung tumorigenesis. The demonstrated inhibition of 22. Mandai, S., and Stoner, G. D. Inhibition of /V-nitrosobenzylmethylamine- induced esophageal tumorigenesis in rats by ellagic acid. Carcinogenesis NBMA-induced esophageal tumorigenesis is important since, (Lond.), //: 55-61, 1990. in certain areas of the world, humans are exposed to NBMA in 23. Pozharisski, K. M. Tumors of the oesophagus. In: V. S. Turusov (ed.). Pathology of Tumors in Laboratory Animals. Vol. I, Part 1. IARC Scientific the diet (8, 12). Our studies provide a basis for comparing the Publication No. 5, pp. 87-100. Lyons, France: International Agency for relative abilily of PEITC and longer-chain isolhiocyanales lo Research on Cancer, 1973. 2067

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. INHIBITION OF ESOPHAGEAL CARCINOGENESIS BY PEITC 24. SAS Institute, Inc. SAS Procedures for Personal Computers. Ed. Version 6. 29. Drobnica, L., and Gemeiner, P. Use of isothiocyanates as "reporter" groups Cary. NC: SAS Institute, Inc., 1985. in modification of enzymes. In: 3. L. Fox, Z. Devi et al. (eds.). Protein 25. Snedecor. G. W., and CochraÃ±.W. G. Statistical Methods, Ed. 7. Ames, IA: Structure and Evolution, pp. 105-115. New York: Marcel Dekker, Inc., Iowa State University Press, 1980. 1976- 26. Wargovich, M. J., Woods. C, Eng, V. W. S., Stephens. L. C, and Gray. K. 30- ?obb"!s- ,S-hfiS&S; ' a"d1K"mar-, V:nCeJ'u\" inJ"r> and adaptation. Chemoprevemion of A'-nitrosobenzylmethylamine-induccd esophageal can- /Â«:Patologie Bas.s of D.sease, Ed. 3, pp. 1-39. Ph.ladelph.a: W. B. Saunders Ã•n bh'nrally Â°CCUrringthioether- dia"yl Sulfide- CanCer ReS" 31. Tookey.' H. L.. Van Etten. C. H., and Daxenbichler. M. E. Glucosinolates. /â€ž., E Liener (ed ( Toxic Constitutents of Plant Foodstuffs. Ed. 2, pp. 27. Labile. G. E., and Archer. M. C. Esophageal and hepatic microsomal metab- 103-142 New York' Academic Press 1980 olism of A'-nitrosomethylbenzylamine and /v'-nitrosodimethylamine in the 32. Morse, M. A., Wang, C-X., Amin, S.. Hecht, S. S., and Chung, F-L. Effects rat. Cancer Res., 42: 3181-3186. 1982. of dietary sinigrin or indole-3-carbinol on O'-methylguanine-DNA-trans- 28. Mehta, M., Labuc.G. E., Urbanski, S.J., and Archer, M.C.Organ specificity methylase activity and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone-in- in the microsomal activation and toxicity of A/-nitrosomethylbenzylamine in duced DNA methylation and tumorigenicity in F344 rats. Carcinogenesis various species. Cancer Res.. â€¢Â«.â€¢4017-4022. 1984. (Lond.), 9: 1891-1895, 1988.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1991 American Association for Cancer Research. Inhibitory Effects of Phenetyl Isothiocyanate on N -Nitrosobenzylmethylamine Carcinogenesis in the Rat Esophagus

Gary D. Stoner, Diana T. Morrissey, Young-Hun Heur, et al.

Cancer Res 1991;51:2063-2068.

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