Chapter 13
Inhibition of Esophageal Tumorigenesis by Phenethyl Isothiocyanate 1
G. D. Stoner , A. J. Galati, C. J. Schmidt, and M . A. Morse
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Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch013
Department of Pathology, Medical College of Ohio, Toledo, OH 43614
Phenethyl isothiocyanate, a naturally occurring constituent of cruciferous vegetables, is a potent inhibitor of nitrosamine-induced esophageal cancer. F-344 rats fed diets containing phenethyl isothio cyanate at 1.5, 3 and 6 mmol/kg diet, before and during treatment with the carcinogen N-nitrosobenzylmethylamine, developed 89100% fewer esophageal tumors than carcinogen-treated control rats. Phenethyl isothiocyanate exhibited inhibitory effects against both preneoplastic lesions and neoplastic lesions. The effects of phen ethyl isothiocyanate (10, 25, 50 and 100 μΜ) on D N A methylation by N-nitrosobenzylmethylamine in cultured expiants of rat esopha gus were also investigated. Phenethyl isothiocyanate produced a dose-dependent inhibition in the levels of D N A methylation at the N (20-89%) and O (55-93%) positions of guanine. Therefore, a strong correlation was observed between the inhibitory effects of phenethyl isothiocyanate in vivo and in vitro. 7
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Esophageal cancer in humans occurs worldwide and ranks seventh among cancers in order of frequency of occurrence in both sexes combined (7). Epidemiological studies indicate that nutritional and environmental factors play a major role in the etiology of esophageal cancer. A n increased risk for developing esophageal cancer is correlated with tobacco smoking (2,3), consumption of alcoholic beverages (4,5) and of salt-pickled and moldy foods (6). The molds which can contaminate foods include members of the Fusarium species (7), which produce several toxins, and Geotrichum candidum (8), which promotes the formation of nitrosamine carcin ogens. Research in the Transkei region of South Africa and in China suggests that 7V-nitroso compounds and their precursors are probable etiological factors in esopha geal cancer in these high-incidence areas (6,8). Most esophageal tumors in these high-incidence areas are classified histologically as squamous cell carcinomas. 1
Current address: Department of Preventive Medicine, The Ohio State University, 300 West 10th Avenue, Columbus, OH 43210
0097-6156/94/0546-0173$06.00/0 © 1994 American Chemical Society
Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES
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Animal model studies have shown that many nitrosamines are esophageal carcinogens in the rat (9,10). Early studies by Druckrey, et al. (9) indicated that asymmetrical nitrosamines could readily induce squamous cell carcinomas in the rat esophagus, the most potent being N-nitrosobenzylmethylamine (NBMA, Figure 1) (77). In metabolism studies of several nitrosamines in expiant cultures of rat esophagus, the highest level of metabolite binding to D N A was observed with N B M A (72); therefore, a strong correlation exists between the carcinogenic potency of N B M A in vivo and its binding to esophageal D N A in vitro.
A/-Nitrosobenzylmethylamine (NBMA)
Phenethyl isothiocyanate (PEITC)
Figure 1. Structures of N B M A and PEITC.
In vitro studies and investigations in experimental animals have shown that foods contain a number of compounds with the ability to inhibit chemicallyinduced cancer (13). Among these are the isothiocyanates, including phenethyl isothiocyanate (PEITC, Figure I) (14-18). PEITC is a primary product of thioglucosidase-catalyzed hydrolysis of gluconasturtiin, a glucosinolate compound found in certain cruciferous vegetables (79). Pretreatment of rats with PEITC inhibited the induction of mammary tumors by 7,12-dimethylbenz[a]anthracene (DMBA) (14). Addition of PEITC to the diet inhibited D M B A and benzo[a]pyrene (BP)-induced lung and forestomach tumors in mice (14). Recent studies have demonstrated the ability of PEITC to inhibit the metabolism and D N A methylation of a series of nitrosamines both in vivo and in vitro (20-23). Moreover, PEITC was shown to inhibit lung tumor induction in rats (75) and in mice (17) by the tobaccospecific nitrosamine, 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK). Finally, when provided in the diet at concentrations of 3 and 6 mmol/kg, PEITC inhibited the induction of esophageal tumors in rats by N B M A (18). In this paper, we report the results of testing PEITC at five concentrations (0.33, 0.75, 1.5, 3 and 6 mmol/kg) in the diet for its ability to inhibit N B M A tumorigenesis in the esophagus of F-344 rats. Results from the in vivo bioassay are compared with the inhibitory effects of PEITC on the metabolism and D N A damaging effects of N M B A in cultured rat esophageal tissues. Materials and Methods 3
Chemicals. [methyl- H]NBMA (5 Ci/mmol; purity, >97%) was purchased from Moravek Biochemicals, Inc., Brea, California. Unlabeled N B M A (purity, >98%) was obtained from the National Cancer Institute Chemical Carcinogen Repository at Midwest Research Institute, Kansas City, Missouri. Standards for N -methylguanine (N -MeGua) and 0 -methylguanine (0 -MeGua) (purity, 98%) were purchased from Chemsyn Science Laboratories, Lenexa, Kansas. PEITC (purity, >99%) was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin. 7
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Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
13. STONER ET AL.
Esophageal Tumorigenesis & Phenethyl Isothiocyanate 175
The purity of all chemicals was checked by high performance liquid chromato graphy (HPLC). DNA Binding and Adduct Studies. PEITC was evaluated for its ability to inhibit the interaction of N B M A metabolites to the D N A of rat esophagus. The protocol for these studies has been described in detail (18), and is summarized briefly as follows: Expiant Culture. Esophageal expiants were prepared and cultured in chemically defined CMRL-1066 medium as described (24). The expiants were cultured for 6 hours and then exposed for 12 hours to 1 μΜ [methyl- H]NBMA concurrently with PEITC at concentrations of 10, 25, 50 and 100 μΜ. Control expiant cultures were incubated for 12 hours in medium containing 1 μΜ [methylH ] N B M A and 1% dimethyl sulfoxide, the solvent for N B M A . After incubation, the tissues from each series were pooled and stored at -85°C for subsequent analyses.
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Isolation of Radiolabeled Carcinogen Bound to DNA. Explant D N A was isolated by a modification of the method of Gupta (25). The purified D N A was dissolved in 200 μΐ of 0.01 X standard saline-citrate and quantitated spectrophotometrically. The radioactivity associated with the D N A was determined in a liquid scintillation counter. 7
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Analysis of DNA Adducts. N -MeGua and 0 -MeGua adducts in rat esophageal D N A were analyzed as described previously (18). Briefly, D N A isolated as described above was hydrolyzed with 0.1 M HC1 at 70°C for 30 minutes. The hydrolysates were analyzed by H P L C using a Whatman Partisil strong cation exchange column (SCX-10; 4.5 χ 250 mm) and a Radiomatic FloOne/Beta detector. Samples were eluted isocratically with a buffer solution containing 75 m M ammonium phosphate:methanol (85:15), pH 2.5, at a flow rate of 1.0 ml/minute, and the column effluent was monitored at 254 nm. Standards of nonradioactive N -MeGua and 0 - M e G u a were added to all the samples to ascertain retention times of the major N B M A - D N A adducts. 7
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Bioassay. Male F-344 rats, 6-8 weeks old, were purchased from Harlan SpragueDawley, Indianapolis, Indiana. After two weeks of acclimatization to the animal facility, the rats were randomized into 10 groups, each consisting of 15 animals. The treatments administered to the groups are summarized in Table I. Groups 1 and 5 were given AIN-76A diet, while groups 2-4 and 6-10 received AIN-76A diet containing PEITC at concentrations ranging from 0.33 to 6 mmol/kg diet. PEITC was mixed in the diet weekly, using a Hobart mixer, and stored at 4°C before use. Previous studies have shown that PEITC is stable in the diet for at least 10 days (75). The concentration of PEITC 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 feeding the respective diets, N B M A (0.5 mg/kg body weight) was administered by s.c. injection once per week for 15 weeks. The total dose of N B M A administered per rat was 7.5 mg (0.050 mmol)/kg body weight. Vehiclecontrol rats were given s.c. injections of dimethyl sulfoxide in distilled water (1:4, v:v) for the same period. After completion of the treatment with either N B M A or vehicle, all groups were maintained on their respective diets (with or without
Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
Huang et al.; Food Phytochemicals for Cancer Prevention I ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
a
d
c
b
a
0 0 0 0 0.5 0.5 0.5 0.5 0.5 0.5
b
0 0.75 3.00 6.00 0 0.33 0.75 1.50 3.00 6.00
C
Treatment NBMA PEITC (mg/kg body wt.) (mmol/kg diet) 0 0 0 0 100 100 100 60 13 0 — — — — 0 0 40 87 100
Tumor Incidence Inhibition Percent of rats with tumors (%)
d
d
d
0 0 0 0 11.5 ± 4 . 5 10.7 + 1.1 5.7 ± 1.2 0.9±0.2 0.1 ± 0.3 0.0
— — — — — 7 50 93 99 100
Tumor Multiplicity Inhibition Tumors/rat (mean ± S.D.) (%)
Each group contained 15 animals. Administered s.c. once per week for 15 weeks. PEITC was fed in the diet 2 weeks before, during and for 8 weeks after administration of N B M A . Significantly different from Group 5 (p0.5 mm in diameter) was per formed as described previously (18,26). Sections of esophagi from each group were scored for the presence of preneoplastic lesions (i.e., acanthosis and hyperkeratosis, leukoplakia, and leukokeratosis) and neoplastic lesions (i.e., papilloma and carcinoma) (27).
Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch013
Statistical Analysis A l l data were analyzed using SAS on a personal computer (18). The data on D N A binding of N B M A were analyzed utilizing a test of linear contrast. These data, as well as the N and O adduct formation data, were expressed in terms of percentage of inhibition. Ninety-five percent confidence intervals were calculated. Food consumption and body weight data were analyzed using analysis of variance. The food intake over the study period was evaluated on a per-cage basis. Weight gain was evaluated over the study period as the difference in weight between week 25 and week 1. Four predefined 1 d.f., contrasts of mean weight gain were calculated: NBMA-treated groups versus groups not treated with N B M A ; PEITC-treated groups versus groups not treated with PEITC; the interaction between treatment with PEITC and treatment with N B M A ; and low-dose PEITC groups versus highdose PEITC groups. Tumor incidence data were analyzed using χ tests. The data on frequency of tumors per rat were evaluated using a Wilcoxon rank sum test. 7
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Results In Vitro Studies. D N A binding studies showed that PEITC elicited a dosedependent inhibition (p
