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Dose-related inhibition by dietary phenethyl isothiocyanate of esophageal tumorigenesis and DNA methylation induced by N-nitrosomethylbenzylamine in rats
Cancer Lerters, 72 (1993) 103-l 10 Elsevier Scientific Publishers Ireland
103 Ltd.
Dose-related inhibition by dietary phenethyl isothiocyanate of esophageal tumorigenesis and DNA methylation induced by IV-nitrosomethylbenzylamine in rats Mark A. Morse”., Hongxiang Zua, Anthony J. Galatib, Carl J. Schmidtb and Gary D. Stonera aLaboratory of Cancer Chemoprevention and Etiology. Department of Preventive Medicine. Ohio State University. CHRI Suite 1148. 300 West Tenth Avenue, Columbus, Ohio 43210 and bDepartment of Pathology, Medical College of Ohio, Toledo, Ohio 43614 (USA) (Received 5 March 1993) (Revision received 14 May 1993) (Accepted
15 May 1993)
Summary
The purpose of th:s investigation was to establish a dose response for the effects of dietary phenethyl isothiocyanate (PEITC) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis and D’NA methylation. Groups of 13-27 rats were randomly assigned to AIN-76A diets containing 0, 0.325, 0.75, 1.5 or 3.0 pmol PEITC/g. Two weeks later, rats were administered NMBA subcutaneously at a dose of 0.5 mg/kg once a week for 15 weeks. Animals were maintained on control or experimental diets for an additional 8 weeks and were terminated at week 25 of the experiment. No significant effects on weight gain or food intake were noted for any of the experimental diets when compared with control values. Animals receiving only NMBA developed 9.3 f 0.9 tumors/rat, with an incidence of 100%. Dietary PEITC at concentrations of 0.75, 1.5 and 3.0 pmol/g inhibited NMBA-induced esophageal tumor multiplicity by 39%, 90% and loo%, respectively. Esophageal tumor incidence in these groups was reduced by O%, 40% and lOO%, respectively. The 0.325 pmol/g PEITC diet did not significantly Correspondence to: Gary D. Stoner, Laboratory of Cancer Chemoprevention and Etiology, Department of Preventive Medicine, Ohio State University. CHRI Suite 1148. 300 West Tenth Avenue, Columbus, OH 43210, USA. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
affect NMBA-induced esophageal tumorigenesis. These results indicate that the minimum inhibitory dietary concentration of PEITC is between 0.325 and 0.75 pmollg. Groups of 20 rats were assigned to diets containing O-3.0 pmol PEITCig for two weeks as described above, and then sacrificed 24 hours after administration of [ 3H-methyl]NMBA. The esophageal DNA was isolated, purified, hydrolyzed, and analyzed by HPLC. PEITC inhibited DNA methylation in a dose-dependent manner, as was found in the tumor bioassay. The inhibition of tumor incidence was highly correlated with the percentage inhibition of either 7methylguanine or 06-methylguanine. These latter results suggest that the inhibitory activity of PEITC in this model is manifested, at least in part, during the functional equivalent of tumor initiation
Keywords: phenethyl isothiocyanate
nitrosomethylbenzylamine tumor; DNA adducts
(PEITC); N(NMBA); esophageal
Introduction
Esophageal cancer ranks seventh in frequency throughout the world for both sexes [20]. Human risk of esophageal cancer is positively associated Ltd.
104
with smoking [34], alcohol consumption [26,28] and consumption of salt-pickled and mouldy foods [35]. Investigations conducted in the Transkei region of South Africa and northern China, areas of high incidence of esophageal cancer, indicate that N-nitroso compounds and their precursors are possible etiological factors [8,30,35]. In rats, several asymmetric nitrosamines are potent inducers of esophageal tumors [6]. Among the most potent of these nitrosamines is Nnitrosomethylbenzylamine (NMBA) [ 111. The induction of esophageal tumors by NMBA has proved to be a valuable model for screening the chemopreventive efficacy of several naturally occurring compounds, including diallyl sulfide [29], ellagic acid [4,12] and phenethyl isothiocyanate (PEITC) [25]. Isothiocyanates are naturally occurring constituents of cruciferous vegetables that are released from their glucosinolate precursors by hydrolysis catalyzed by the enzyme myrosinase (P-thioglucosidase) [ 1,9,27]. Isothiocyanates possess significant antitumorigenic activities in a number of animal models. PEITC, the hydrolysis product of gluconasturtiin, has proved to be particularly effective as a chemopreventive agent in various animal models. Wattenberg demonstrated that phenyl isothiocyanate, benzyl isothiocyanate and PEITC inhibited 7,12_dimethylbenz(a)anthracene (DMBA)-induced mammary tumor formation in female Sprague-Dawley rats when administered orally shortly before carcinogen administration [31]. In the same report, dietary benzyl isothiocyanate or PEITC inhibited DMBA-induced forestomach and pulmonary tumors in ICR mice. Benzyl isothiocyanate also inhibited forestomach tumors induced by N-nitrosodiethylamine and forestomach and pulmonary tumors induced by benzo(a)pyrene [33]. Dietary PEITC inhibited metabolic activation of 4-(methylnitrosamino)- l(3-pyridyl)-1-butanone (NNK), NNK-induced DNA adduct formation and NNK-induced lung tumors in F344 rats [2,13]. PEITC also inhibited metabolic activation, DNA adduct formation and lung tumorigenesis of NNK in A/J mice [14,15,17,18,22]. Dietary PEITC has recently been shown to be a potent inhibitor of NMBA-induced esophageal
tumorigenesis in rats. At 3 pmol/g diet, PEITC inhibited the incidence of esophageal tumorigenesis induced by NMBA by 87% in F344 rats and reduced tumor multiplicity by 99%. When fed at 6 pmol/g diet, no esophageal tumors were observed [25]. As a prelude to comparative studies with PEITC and other isothiocyanates on NMBAinduced esophageal tumorigenesis, dietary concentrations of PEITC that are significantly, albeit not completely, inhibitory should be established. Thus, the effect of dose on inhibition by dietary PEITC of NMBA-induced esophageal tumors in F344 rats was investigated. Since it was unclear which stage or stages of the tumorigenic process are affected by PEITC in the rat esophageal tumor model, and since PEITC can inhibit NMBAinduced DNA methylation in vitro in esophageal explant cultures [25], we have also investigated whether PEITC could inhibit NMBA-induced esophageal DNA methylation in vivo. Materials and Methods Chemicals Unlabeled NMBA was synthesized essentially as described previously [7]. [3H-methyl]NMBA was obtained from Moravek Biochemicals, Inc. (Brea, California). Phenethyl isothiocyanate and dimethyl sulfoxide (DMSO) were obtained from Aldrich Chemical Company (Milwaukee, Wisconsin). Animals All experimental protocols were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the Medical College of Ohio, where all animal experiments were performed. Male F344 rats were obtained from HarlanSprague Dawley (Indianapolis, Indiana). On arrival, animals were acclimatized to the animal facility for 7-10 days. Rats were housed two per cage and maintained on a 12-h light-dark cycle. Hygienic conditions were maintained by changing cages twice weekly and daily changes of water bottles. AIN-76A diet and water were provided ad libitum.
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Experimental diets
Experimental diets were prepared every two weeks with the aid of a Hobart D-300 compact mixer. AIN-76A diet was mixed with the appropriate amounts of PEITC and blended at a setting of ‘2’ for 15 minutes. Diets were stored at 4°C before feeding. NMBA Esophageal tumorigenesis bioassa_v
In separate experiments, groups of 13-27 rats (6-7 weeks of age) were placed on control (AIN76A) or experimental diets containing 0.325, 0.75, 1.5 or 3.0 pmol PEITC/g AIN-76A diet for 2 weeks before NMBA dosing. NMBA was administered subcutaneously once each week at a dose of 0.5 mg/kg (in 0.1 ml of 10% DMSO in water) for 15 weeks. Three additional groups of 15 rats were fed PEITC at concentrations of 0.0, 0.75 and 3.0 pmolig and administered 10% DMSO subcutaneously for 15 weeks. All animals were maintained on control or experimental diets during NMBA dosing and for an additional 6 weeks. Body weights were measured weekly until week 19 and every two weeks thereafter. Food consumption was monitored weekly throughout the course of the experiment. At the end of this 25-week protocol rats were killed. The esophagi were excised, opened longitudinally, placed flat on white index cards and fixed in 10% buffered formalin. Lesions at the surface of each esophagus that were ~0.5 mm in diameter were counted by means of a dissecting microscope. Representative tumors were examined histologically, and most were found to be papillomas. Previous studies have shown that more than 95% of these lesions are squamous papillomas [4,12,25]. Isolation and quantitation of DNA adducts
Groups of twenty rats were acclimatized to diets containing 0.0, 0.325, 0.75, 1.5 and 3.0 pmol PEITC/g of AIN-76A diet. Two weeks after being maintained on the diets the rats were administered NMBA subcutaneously in 10% DMSO at a dose of 0.5 mg/kg (specific activity = 0.375 mCi/ymol). Twenty-four hours after NMBA dosing the rats were killed and the esophagi harvested. The esophagi of five rats were homogenized in 3 ml of 0.01 M Tris and 0.001 M EDTA, pH 7.0.
DNA was isolated and purified as described previously. Aliquots (20 ~1) of each sample were analyzed spectrophotometrically to determine DNA content and purity. Aliquots of 40 ~1 were taken to determine total radioactivity. The remainder of each sample was pooled with a randomly chosen sample to yield two samples per group. DNA samples were hydrolyzed as described previously [ 171. After hydrolysis, 10 ~1 of 100 &ml of authentic standards of 7-methylguanine (7-meG) and 06-methylguanine (06-meG) were added. Samples were chromatographed on a Waters HPLC equipped with a Whatman 5 p strong cation exchange column. Adducts were eluted isocratically with 0.05 M ammonium phosphate (pH 2.3) - 10% methanol. Both the injection port and the column were maintained at a temperature of 30°C. Radioactive peaks coeluting with the methylated guanine standards were quantitated using an IN/US flow-through radioactivity detector. Results were expressed as pmol adduct/mg DNA. Statistical analysis
In the tumor bioassay, statistical comparisons of tumor multiplicities were made by analysis of variance followed by Newman-Keuls’ ranges test. Comparisons of tumor incidence were performed by the use of Fisher’s exact probability test. DNA adduct levels were compared by analysis of variance and Duncan’s multiple ranges test. Correlations between inhibition of tumor incidence and inhibition of formation of 7-meG or 06-meG were made by linear regression.
Effect of PEITC tumorigenesis
on NMBA-induced
esophageal
There were no statistically significant effects of the various diets on body weight gain (Fig. 1). The greatest weight deviations occurred towards the end of the bioassay; the magnitude of these deviations was 5% or less compared with animals fed AIN-76A. Similarly, there were no significant differences among groups in food consumption throughout the course of the bioassay (data not shown).
106
450
400 A E 2 0, V
350
z *gJ
300
AIN-76A 0.75 pmol/g PEITC 3.00 pmol/g PEITC AIN-76A + NMBA 3.00 pmol/g PEITC + NMBA 1.50 pmol/g PEITC + NMBA 0.75 pmol/g PEITC + NMBA 0.325 pmol/g PEITC + NMBA
; g cz & F 0, k
250
200
150
100 0
I
I
I
10
20
30
Weeks Fig. 1. Effect of dietary PEITC on body weight gain. Groups of 13-27 rats were administered control or experimental diets for 25 weeks. After two weeks, NMBA was administered once weekly for 15 weeks at a dose of 0.5 mg/kg by subcutaneous injection. Eight weeks later the experiment was terminated. Body weights were measured weekly until week 18. and every two weeks thereafter. Values are given as mean f standard error.
Table I depicts the tumor incidence and multiplicity data for the bioassay. Rats fed PEITC at concentrations of 0.0, 0.75 and 3.0 pmollg and administered vehicle developed no esophageal tumors, while rats fed the control diet and administered NMBA had a tumor multiplicity of 9.3 f 0.9 (mean f SE.) tumors/rat. Dietary PEITC inhibited NMBA-induced esophageal tumors in a dose-related manner. Statistically significant reductions in both tumor incidence and multiplicity were evident at 0.75, 1.5 and 3.0 Fmollg diet. The 0.325 pmollg diet had no signiticant inhibitory effects on tumor multiplicity or incidence.
Effect of PEITC on NMBA-induced DNA methylation Control animals had an average total binding of 133.0 pmol/mg (data not shown), less than 50% of which could be accounted for by 7-meG and 06meG. However, much of the total could reflect tritium that was incorporated into normal (i.e. unmethylated) bases and additional adducts that are not released by acidic hydrolysis (e.g. 04methylthymidine), as well as the presence of partially undigested DNA or ring-opened ‘I-meG. As can be seen in Fig. 2, PEITC decreased both 7-meG levels and 06-meG levels, exhibiting a dose-response relationship. Significant reductions
107
Table 1.
Effect of dietary
Dietary PEITC concentration (pmollg diet) 0.00 0.75 3.00 0.00 0.325 0.75
PEITC
NMBA
+ + + + +
1.50 3.00
on NMBA-induced Number of rats
IS 15 15 27 I3 I5 I5 I5
esophageal
tumorigenesis.
Percentage of rats with tumors*
Tumors
0
0
0
0
0
0
100” looa 1ooa 60b OC
9.3 10.7 5.7 0.9 04
f f f *
per rat7
0.9’ 1.1’ I.22 0.23
*Values that bear different superscripts are statistically different from one another as determined by Fisher’s exact probability test (P < 0.01). tMean f standard error. Values that bear different superscripts are statistically different from one another as determined by analysis of variance and Newman-Keuls ranges test.
r
PEITC
Concentration
(pmol/g
Fig. 2. Effect of dietary PEIITC on NMBA-induced DNA methylation. diets for I4 days before administration of [3H-methyl]NMBA. Animals isolated and analyzed as described in Materials and Methods.
diet) Groups of 20 rats were administered control or experimental were killed 24 h later. Esophageal ‘I-meG and 06-meG were
108
in 7-meG and 06-meG levels were achieved at the 3.0 pmolig concentration. Interestingly, maximal inhibition of DNA methylation was only approximately 60% at 3.0 pmollg, a PEITC concentration that totally inhibits NMBA-induced esophageal tumorigenesis. However, inhibition of tumor incidence by PEITC and inhibition of DNA methylation by PEITC were well correlated. When the percentage inhibition of tumor incidence was plotted against either the percentage inhibition of 7meG or 06-meG (data not shown), the corresponding correlation coefficients were 0.98 (P = 0.014) and 0.99 (P = 0.009). respectively. Discussion
The esophageal tumor response (9.3 f 0.9 tumors/rat) of the rats treated only with NMBA in this study was in agreement with earlier studies conducted using this model system [4,25]. As was found previously (251, the 3.0 pmol/g dietary concentration of PEITC completely inhibited NMBAinduced tumors. The 1.5 pmol/g concentration yielded a 90% reduction in tumor multiplicity and a 40% reduction in incidence, while the 0.75 pmolig diet reduced tumor multiplicity by only 39% and had no effect on esophageal tumor incidence. No significant effects were observed at the 0.325 Gmol/g concentration of PEITC. Thus, the minimum inhibitory concentration of PEITC (i.e. the dose of PEITC that will yield a statistically significant reduction in tumor multiplicity) apparently lies between 0.325 and 0.75 pmol/g. In the current model, NMBA is administered repetitively over 15 weeks, with termination of the experiment at week 25 (4,251. Thus it is virtually impossible to discern at what time the functional equivalent of initiation ends and at what time promotion or progression begins. As a result, since PEITC was fed during the entire course of the experiment in this study and previously [25], the DNA methylation studies were conducted to examine the possible effects of PEITC on the initiation stage (or its functional equivalent). A doserelated inhibition of NMBA-induced DNA methylation was observed, in agreement with the tumor bioassay results obtained for the 0.75-3.0 pmol/g diets. This data suggests that PEITC affects initiation in this model.
Of greater interest is the finding that 06-meG levels at 24 hours at PEITC dietary concentrations of 1.5 and 3.0 lmol/g, concentrations that yield reductions of NMBA-induced tumor multiplicity of 90% and loo%, are reduced by only 34% and 64%, respectively. A similar phenomenon has been observed with isothiocyanates and methylation of murine pulmonary DNA. A dose of phenylhexyl isothiocyanate that resulted in 100% inhibition of NNK-induced pulmonary adenomas in A/J mice only inhibited pulmonary 06-meG levels by approximately 50% at 6 h [18]. However, NNK doses that produced 06-meG levels that were onehalf or less of that of a lo-pmol dose (i.e. the bioassay dose) produced much lower tumor multiplicities than the 10 pmol dose, because the methylation produced at the lower doses was repaired much more rapidly [21]. If a similar chain of events occurs in the rat esophagus, the present results are quite credible. Detailed time course studies of rat esophageal DNA methylation after feeding of control or PEITC-containing diets would be necessary to confirm such a possibility. Isothiocyanates are known to be potent inhibitors of enzymes, including cytochrome P-450. PEITC and other isothiocyanates inhibit microsoma1 metabolism of many carcinogens, whether the isothiocyanates are administered in vivo before microsomal isolation or added to the microsomal reaction mixtures in vitro [2,3,10,14,22]. Similar inhibitory effects of PEITC on nitrosamine metabolism have been reported for cultured rat tissues as well (5,19,25]. Isothiocyanates also induce phase II enzymes such as glutathione S-transferases [24]. However, phase II enzymes are not known to play a significant role in the metabolism of most nitrosamines. Thus we believe that the inhibition of DNA methylation observed here is most probably due to a direct inhibition of the cytochrome P45Os responsible for NMBA activation, an assertion that remains to be proved. Of course, with the difficulties in delineating the various stages of NMBA tumorigenesis in this model, one cannot rule out possible antipromotional activity by PEITC. Although benzyl isothiocyanate has been shown to possess antipromotional activity [32], no such effects for PEITC have been demonstrated in any animal model [ 181. Future experiments with this model system will
109
involve the comparison of PEITC with other isothiocyanates, and th.e development of NMBA dosing regimens that will allow testing of isothiocyanates and other chemopreventive agents at various stages of NMBA tumorigenesis, including promotion or progression.
11
Acknowledgements
13
This work was supported by NIH grants CA28950 and CA-46535. The authors wish to thank Young-Sun Heur for Irechnical assistance in the DNA methylation experiment.
12
14
References 1
2
3
4
5
6
7
8
9
10
Carlson, D.G., Daxenbichler, M.E., VanEtten, C.H.. Tookey, M.L. and Williams, P.H. (1981) Glucosinolates in crucifer vegetables: turnips and rutabagas. J. Agric. Food Chem.. 29, 1235- 1239. Chung, F-L., Juchatz, A., Vitarius, J. and Hecht. S.S. (1984) Effects of dietary compounds on o-hydroxylation of N-nitrosopyrrolidine ;and N’nitrosonornicotine in rat target tissues. Cancer Res., 44, 2924-2928. Chung, F.-L., Wang, M. and Hecht, S.S. (1985) Effects of dietary indoles and iz.othiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-I-(3-pyridyl)-lbutanone o-hydroxylation and DNA methylation in rat liver. Carcinogenesis (London), 6. 539-543. Daniel, E.M. and Stoner, G.D. (1991) The effects of ellagic acid and I3-cis-mtinoic acid on N-nitrosobenzylmethylamine-induced esophageal tumorigenesis in rats. Cancer Lett.. 56. 117-124. Doerr-O’Rourke, K., Tr.tshin, N., Hecht, S.S. and Stoner. G.D. (1991) Effect of phenethyl isothiocyanate on the menitrosamine 4of the tobacco-specific tabolism (methylnitrosamino)-1-(:l-pyridyl)-1-butanone by cultured rat lung tissue. Carcinogenesis. 12, 1029-1034. Druckrey, H., Preussme.nn. R., Blum. G., Ivankovic. S. and Afkham, J. (1963) Erzeugung von karzinoman der speiserohre durch unsymmetrische nitrosamine. Naturwissenschaften, 50. loo-- 101. Druckrey. H., Preussman, R., Ivankovic, S. and Schmahl, D. (1967) Organotrope carcinogene wirkungen bei 65 verschiedenen iV-nitroso-verbindungen an BD-ratten. Z. Krebsforsch.. 69, 103-201. DuPlessis, L.S.. Nunn, J.R. and Roach, W.A. (1969) Carcinogen in a Transkeian Bantu food additive. Nature, 222, 1198. Hanley, A.B., Heaney, R.K. and Fenwick. G.R. (1983) Improved isolation of glucobrassicin and other glucosinolates. J. Sci. Food Agric., 34, 869-873. Ishizaki, H., Brady. J.F., Ning, S.M. and Yang, C.S. (1990) Effect of phenethyl isothiocyanate on microsomal N-nitrosodimethylamine metabolism and other monooxygenase activities, Xenobiotica, 20, 255-264.
15
16
17
18
19
20
21
22
Lijinsky, W., Saavedra, J.E., Rueber, M.D. and Singer. S.S. (1982) Esophageal carcinogenesis in F344 rats by nitrosomethylethylamines substituted in the ethyl group. J. Nat. Cancer Inst., 68, 681-684. Mandal, S. and Stoner, G.D. (1990) Inhibition of Nnitrosobenzylmethylamine-induced esophageal tumorigenesis in rats by ellagic acid. Carcinogenesis (London), 11. 55-61. Morse, M.A., Wang, C-X., Stoner, G.D., Mandal, S., Conran, P.B., Amin, S.G., Hecht, S.S. and Chung, F.-L. (1989) Inhibition of 4-(methylnitrosamino)-I-(3-pyridyl)I-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate. Cancer Res., 49, 549-553. Morse, M.A., Amin, S.G., Hecht, S.S. and Chung. F.-L. ‘(1989) Effect of aromatic isothiocyanates on tumorigenicity, 06-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone in A/J mouse lung. Cancer Res., 49, 2894-2897. Morse, M.A., Eklind, H.I., Amin, S.G., Hecht, S.S. and Chung, F.-L. (1989) Effects of alkyl chain length on the inhibition of NNK-induced lung neoplasia in A/J mice by arylalkyl isothiocyanates. Carcinogenesis (London), 10. 1757-1759. Morse, M.A., Reinhardt, J.C.. Amin, S.G., Hecht, S.S., Stoner, G.D. and Chung, F.-L. (1990) Effects of dietary aromatic isothiocyanates fed subsequent to the administration of 4-(methylnitrosamino)-I-(3-pyridyl)-l-butanone on lung tumorigenicity in mice. Cancer Lett., 49, 225-230. Morse, M.A.. Eklind, K.I., Hecht. S.S., Jordan. K.G., Chdi, C.-I., Desai, D.H., Amin, S.G. and Chung. F.-L. (1991) Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-I-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice. Cancer Res., 51, 1846-1850. Morse. M.A., Eklind, K.I., Amin, S.G. and Chung, F.-L. (1992) Effect of frequency of isothiocyanate administration on inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)I-butanone-induced pulmonary adenoma formation in A/J mice. Cancer Lett., 62, 77-81. Murphy, SE., Heiblum. R., King, P.C., Bowman, D.. Davis, W.J. and Stoner, G.D. (1991) Effect of phenethyl isothiocyanate on the metabolism of tobacco-specific nitrosamines by cultured rat oral tissue. Carcinogenesis, 12. 957-961. Parkin. D.M., Stjernsward, J. and Muir, C.S. (1984) Estimates of the worldwide frequency of twelve major cancers. Bull. WHO, 62, 163-182. Peterson. L.A. and Hecht. S.S. (1991) 06-methylguanine is a critical determinant of 4-(methylnitrosamino)-i-(3pyridyl) I-butanone tumorigenesis in A/J mouse lung. Cancer Res., 51, 5557-5564. Smith, T.J., Guo. Z., Thomas, F.E., Chung, F.-L., Morse, M.A., Eklind, K. and Yang, C.S. (1990) Metabolism of 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes and its inhibition by isothiocyanates. Cancer Res.. 50, 6817-6822.
110
24
25
26
27
28
29
Spamins, V.L., Venegas, F.L. and Wattenberg. L.W. (1982) Glutathione S-transferase activity: enhancement by compounds inhibiting chemical carcinogenesis and by dietary constituents. J. Nat. Cancer Inst.. 68. 493-496. Stoner, G.D., Morrissey, D.T.. Heur. Y.-H., Daniel, E.M., Galati, A.J. and Wagner, S.A. (1991) Inhibitory effects of phenethyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus. Cancer Res., 51, 2063-2068. Tuyns, A.J., Pequignot, G. and Abbatuci, J.S. (1979) Oesophageal cancer and alcohol consumption: importance of type of beverage. Int. J. Cancer, 23, 443-447. VanEtten, C.H., Daxenbichler, M.E., Williams, P.H. and Kwolek, W.F. (1976) Glucosinolates and derived products in cruciferous vegetables: analysis of the edible part from twenty-two varieties of cabbage. J. Agric. Food Chem., 24, 452-455. Walker, E.A., Castegnario, M.. Garren, L., Toussaint. G. and Kowalski, B. (1979) Intake of volatile nitrosamines from consumption of alcohols. J. Nat. Cancer Inst., 63, 947-951. Wargovich, M.J.. Woods, C., Eng, V.W.S.. Stephens, L.C. and Gray, K. (1988) Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by
30
31
32
33
34
35
the naturally occurring thioether. diallyl sulfide. Cancer Res., 48, 6872-6875. Warwick, G.P. and Harington. J.S. (1973) Some aspects of the epidemiology and etiology of esophageal cancer with particular emphasis on the Transkei. South Africa. Adv. Cancer Res.. 17. 18-229. Wattenberg, L.W. (1977) Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J. Nat. Cancer Inst.. 58, 395-398. Wattenberg. L. W. ( 198 1) Inhibition of carcinogeninduced neoplasia by sodium cyanate. rert-butyl isocyanate. and benzyl isothiocyanate adminstered subseCancer Res.. 41. quent to carcinogen exposure. 299 l-2994. Wattenberg, L.W. (1987) Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo(a)pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis (London). 8. 1971-1973. Wynder, E.L. and Bross, I.J. (1961) A study of etiological factors in cancer of the esophagus. Cancer (Phila.). 14. 389-413. Yang, C.S. (1980) Research on esophageal cancer in China: a review. Cancer Res., 40, 2633-2644.
103 Ltd.
Dose-related inhibition by dietary phenethyl isothiocyanate of esophageal tumorigenesis and DNA methylation induced by IV-nitrosomethylbenzylamine in rats Mark A. Morse”., Hongxiang Zua, Anthony J. Galatib, Carl J. Schmidtb and Gary D. Stonera aLaboratory of Cancer Chemoprevention and Etiology. Department of Preventive Medicine. Ohio State University. CHRI Suite 1148. 300 West Tenth Avenue, Columbus, Ohio 43210 and bDepartment of Pathology, Medical College of Ohio, Toledo, Ohio 43614 (USA) (Received 5 March 1993) (Revision received 14 May 1993) (Accepted
15 May 1993)
Summary
The purpose of th:s investigation was to establish a dose response for the effects of dietary phenethyl isothiocyanate (PEITC) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis and D’NA methylation. Groups of 13-27 rats were randomly assigned to AIN-76A diets containing 0, 0.325, 0.75, 1.5 or 3.0 pmol PEITC/g. Two weeks later, rats were administered NMBA subcutaneously at a dose of 0.5 mg/kg once a week for 15 weeks. Animals were maintained on control or experimental diets for an additional 8 weeks and were terminated at week 25 of the experiment. No significant effects on weight gain or food intake were noted for any of the experimental diets when compared with control values. Animals receiving only NMBA developed 9.3 f 0.9 tumors/rat, with an incidence of 100%. Dietary PEITC at concentrations of 0.75, 1.5 and 3.0 pmol/g inhibited NMBA-induced esophageal tumor multiplicity by 39%, 90% and loo%, respectively. Esophageal tumor incidence in these groups was reduced by O%, 40% and lOO%, respectively. The 0.325 pmol/g PEITC diet did not significantly Correspondence to: Gary D. Stoner, Laboratory of Cancer Chemoprevention and Etiology, Department of Preventive Medicine, Ohio State University. CHRI Suite 1148. 300 West Tenth Avenue, Columbus, OH 43210, USA. 0304-3835/93/$06.00 0 1993 Elsevier Scientific Printed and Published in Ireland
Publishers
Ireland
affect NMBA-induced esophageal tumorigenesis. These results indicate that the minimum inhibitory dietary concentration of PEITC is between 0.325 and 0.75 pmollg. Groups of 20 rats were assigned to diets containing O-3.0 pmol PEITCig for two weeks as described above, and then sacrificed 24 hours after administration of [ 3H-methyl]NMBA. The esophageal DNA was isolated, purified, hydrolyzed, and analyzed by HPLC. PEITC inhibited DNA methylation in a dose-dependent manner, as was found in the tumor bioassay. The inhibition of tumor incidence was highly correlated with the percentage inhibition of either 7methylguanine or 06-methylguanine. These latter results suggest that the inhibitory activity of PEITC in this model is manifested, at least in part, during the functional equivalent of tumor initiation
Keywords: phenethyl isothiocyanate
nitrosomethylbenzylamine tumor; DNA adducts
(PEITC); N(NMBA); esophageal
Introduction
Esophageal cancer ranks seventh in frequency throughout the world for both sexes [20]. Human risk of esophageal cancer is positively associated Ltd.
104
with smoking [34], alcohol consumption [26,28] and consumption of salt-pickled and mouldy foods [35]. Investigations conducted in the Transkei region of South Africa and northern China, areas of high incidence of esophageal cancer, indicate that N-nitroso compounds and their precursors are possible etiological factors [8,30,35]. In rats, several asymmetric nitrosamines are potent inducers of esophageal tumors [6]. Among the most potent of these nitrosamines is Nnitrosomethylbenzylamine (NMBA) [ 111. The induction of esophageal tumors by NMBA has proved to be a valuable model for screening the chemopreventive efficacy of several naturally occurring compounds, including diallyl sulfide [29], ellagic acid [4,12] and phenethyl isothiocyanate (PEITC) [25]. Isothiocyanates are naturally occurring constituents of cruciferous vegetables that are released from their glucosinolate precursors by hydrolysis catalyzed by the enzyme myrosinase (P-thioglucosidase) [ 1,9,27]. Isothiocyanates possess significant antitumorigenic activities in a number of animal models. PEITC, the hydrolysis product of gluconasturtiin, has proved to be particularly effective as a chemopreventive agent in various animal models. Wattenberg demonstrated that phenyl isothiocyanate, benzyl isothiocyanate and PEITC inhibited 7,12_dimethylbenz(a)anthracene (DMBA)-induced mammary tumor formation in female Sprague-Dawley rats when administered orally shortly before carcinogen administration [31]. In the same report, dietary benzyl isothiocyanate or PEITC inhibited DMBA-induced forestomach and pulmonary tumors in ICR mice. Benzyl isothiocyanate also inhibited forestomach tumors induced by N-nitrosodiethylamine and forestomach and pulmonary tumors induced by benzo(a)pyrene [33]. Dietary PEITC inhibited metabolic activation of 4-(methylnitrosamino)- l(3-pyridyl)-1-butanone (NNK), NNK-induced DNA adduct formation and NNK-induced lung tumors in F344 rats [2,13]. PEITC also inhibited metabolic activation, DNA adduct formation and lung tumorigenesis of NNK in A/J mice [14,15,17,18,22]. Dietary PEITC has recently been shown to be a potent inhibitor of NMBA-induced esophageal
tumorigenesis in rats. At 3 pmol/g diet, PEITC inhibited the incidence of esophageal tumorigenesis induced by NMBA by 87% in F344 rats and reduced tumor multiplicity by 99%. When fed at 6 pmol/g diet, no esophageal tumors were observed [25]. As a prelude to comparative studies with PEITC and other isothiocyanates on NMBAinduced esophageal tumorigenesis, dietary concentrations of PEITC that are significantly, albeit not completely, inhibitory should be established. Thus, the effect of dose on inhibition by dietary PEITC of NMBA-induced esophageal tumors in F344 rats was investigated. Since it was unclear which stage or stages of the tumorigenic process are affected by PEITC in the rat esophageal tumor model, and since PEITC can inhibit NMBAinduced DNA methylation in vitro in esophageal explant cultures [25], we have also investigated whether PEITC could inhibit NMBA-induced esophageal DNA methylation in vivo. Materials and Methods Chemicals Unlabeled NMBA was synthesized essentially as described previously [7]. [3H-methyl]NMBA was obtained from Moravek Biochemicals, Inc. (Brea, California). Phenethyl isothiocyanate and dimethyl sulfoxide (DMSO) were obtained from Aldrich Chemical Company (Milwaukee, Wisconsin). Animals All experimental protocols were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the Medical College of Ohio, where all animal experiments were performed. Male F344 rats were obtained from HarlanSprague Dawley (Indianapolis, Indiana). On arrival, animals were acclimatized to the animal facility for 7-10 days. Rats were housed two per cage and maintained on a 12-h light-dark cycle. Hygienic conditions were maintained by changing cages twice weekly and daily changes of water bottles. AIN-76A diet and water were provided ad libitum.
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Experimental diets
Experimental diets were prepared every two weeks with the aid of a Hobart D-300 compact mixer. AIN-76A diet was mixed with the appropriate amounts of PEITC and blended at a setting of ‘2’ for 15 minutes. Diets were stored at 4°C before feeding. NMBA Esophageal tumorigenesis bioassa_v
In separate experiments, groups of 13-27 rats (6-7 weeks of age) were placed on control (AIN76A) or experimental diets containing 0.325, 0.75, 1.5 or 3.0 pmol PEITC/g AIN-76A diet for 2 weeks before NMBA dosing. NMBA was administered subcutaneously once each week at a dose of 0.5 mg/kg (in 0.1 ml of 10% DMSO in water) for 15 weeks. Three additional groups of 15 rats were fed PEITC at concentrations of 0.0, 0.75 and 3.0 pmolig and administered 10% DMSO subcutaneously for 15 weeks. All animals were maintained on control or experimental diets during NMBA dosing and for an additional 6 weeks. Body weights were measured weekly until week 19 and every two weeks thereafter. Food consumption was monitored weekly throughout the course of the experiment. At the end of this 25-week protocol rats were killed. The esophagi were excised, opened longitudinally, placed flat on white index cards and fixed in 10% buffered formalin. Lesions at the surface of each esophagus that were ~0.5 mm in diameter were counted by means of a dissecting microscope. Representative tumors were examined histologically, and most were found to be papillomas. Previous studies have shown that more than 95% of these lesions are squamous papillomas [4,12,25]. Isolation and quantitation of DNA adducts
Groups of twenty rats were acclimatized to diets containing 0.0, 0.325, 0.75, 1.5 and 3.0 pmol PEITC/g of AIN-76A diet. Two weeks after being maintained on the diets the rats were administered NMBA subcutaneously in 10% DMSO at a dose of 0.5 mg/kg (specific activity = 0.375 mCi/ymol). Twenty-four hours after NMBA dosing the rats were killed and the esophagi harvested. The esophagi of five rats were homogenized in 3 ml of 0.01 M Tris and 0.001 M EDTA, pH 7.0.
DNA was isolated and purified as described previously. Aliquots (20 ~1) of each sample were analyzed spectrophotometrically to determine DNA content and purity. Aliquots of 40 ~1 were taken to determine total radioactivity. The remainder of each sample was pooled with a randomly chosen sample to yield two samples per group. DNA samples were hydrolyzed as described previously [ 171. After hydrolysis, 10 ~1 of 100 &ml of authentic standards of 7-methylguanine (7-meG) and 06-methylguanine (06-meG) were added. Samples were chromatographed on a Waters HPLC equipped with a Whatman 5 p strong cation exchange column. Adducts were eluted isocratically with 0.05 M ammonium phosphate (pH 2.3) - 10% methanol. Both the injection port and the column were maintained at a temperature of 30°C. Radioactive peaks coeluting with the methylated guanine standards were quantitated using an IN/US flow-through radioactivity detector. Results were expressed as pmol adduct/mg DNA. Statistical analysis
In the tumor bioassay, statistical comparisons of tumor multiplicities were made by analysis of variance followed by Newman-Keuls’ ranges test. Comparisons of tumor incidence were performed by the use of Fisher’s exact probability test. DNA adduct levels were compared by analysis of variance and Duncan’s multiple ranges test. Correlations between inhibition of tumor incidence and inhibition of formation of 7-meG or 06-meG were made by linear regression.
Effect of PEITC tumorigenesis
on NMBA-induced
esophageal
There were no statistically significant effects of the various diets on body weight gain (Fig. 1). The greatest weight deviations occurred towards the end of the bioassay; the magnitude of these deviations was 5% or less compared with animals fed AIN-76A. Similarly, there were no significant differences among groups in food consumption throughout the course of the bioassay (data not shown).
106
450
400 A E 2 0, V
350
z *gJ
300
AIN-76A 0.75 pmol/g PEITC 3.00 pmol/g PEITC AIN-76A + NMBA 3.00 pmol/g PEITC + NMBA 1.50 pmol/g PEITC + NMBA 0.75 pmol/g PEITC + NMBA 0.325 pmol/g PEITC + NMBA
; g cz & F 0, k
250
200
150
100 0
I
I
I
10
20
30
Weeks Fig. 1. Effect of dietary PEITC on body weight gain. Groups of 13-27 rats were administered control or experimental diets for 25 weeks. After two weeks, NMBA was administered once weekly for 15 weeks at a dose of 0.5 mg/kg by subcutaneous injection. Eight weeks later the experiment was terminated. Body weights were measured weekly until week 18. and every two weeks thereafter. Values are given as mean f standard error.
Table I depicts the tumor incidence and multiplicity data for the bioassay. Rats fed PEITC at concentrations of 0.0, 0.75 and 3.0 pmollg and administered vehicle developed no esophageal tumors, while rats fed the control diet and administered NMBA had a tumor multiplicity of 9.3 f 0.9 (mean f SE.) tumors/rat. Dietary PEITC inhibited NMBA-induced esophageal tumors in a dose-related manner. Statistically significant reductions in both tumor incidence and multiplicity were evident at 0.75, 1.5 and 3.0 Fmollg diet. The 0.325 pmollg diet had no signiticant inhibitory effects on tumor multiplicity or incidence.
Effect of PEITC on NMBA-induced DNA methylation Control animals had an average total binding of 133.0 pmol/mg (data not shown), less than 50% of which could be accounted for by 7-meG and 06meG. However, much of the total could reflect tritium that was incorporated into normal (i.e. unmethylated) bases and additional adducts that are not released by acidic hydrolysis (e.g. 04methylthymidine), as well as the presence of partially undigested DNA or ring-opened ‘I-meG. As can be seen in Fig. 2, PEITC decreased both 7-meG levels and 06-meG levels, exhibiting a dose-response relationship. Significant reductions
107
Table 1.
Effect of dietary
Dietary PEITC concentration (pmollg diet) 0.00 0.75 3.00 0.00 0.325 0.75
PEITC
NMBA
+ + + + +
1.50 3.00
on NMBA-induced Number of rats
IS 15 15 27 I3 I5 I5 I5
esophageal
tumorigenesis.
Percentage of rats with tumors*
Tumors
0
0
0
0
0
0
100” looa 1ooa 60b OC
9.3 10.7 5.7 0.9 04
f f f *
per rat7
0.9’ 1.1’ I.22 0.23
*Values that bear different superscripts are statistically different from one another as determined by Fisher’s exact probability test (P < 0.01). tMean f standard error. Values that bear different superscripts are statistically different from one another as determined by analysis of variance and Newman-Keuls ranges test.
r
PEITC
Concentration
(pmol/g
Fig. 2. Effect of dietary PEIITC on NMBA-induced DNA methylation. diets for I4 days before administration of [3H-methyl]NMBA. Animals isolated and analyzed as described in Materials and Methods.
diet) Groups of 20 rats were administered control or experimental were killed 24 h later. Esophageal ‘I-meG and 06-meG were
108
in 7-meG and 06-meG levels were achieved at the 3.0 pmolig concentration. Interestingly, maximal inhibition of DNA methylation was only approximately 60% at 3.0 pmollg, a PEITC concentration that totally inhibits NMBA-induced esophageal tumorigenesis. However, inhibition of tumor incidence by PEITC and inhibition of DNA methylation by PEITC were well correlated. When the percentage inhibition of tumor incidence was plotted against either the percentage inhibition of 7meG or 06-meG (data not shown), the corresponding correlation coefficients were 0.98 (P = 0.014) and 0.99 (P = 0.009). respectively. Discussion
The esophageal tumor response (9.3 f 0.9 tumors/rat) of the rats treated only with NMBA in this study was in agreement with earlier studies conducted using this model system [4,25]. As was found previously (251, the 3.0 pmol/g dietary concentration of PEITC completely inhibited NMBAinduced tumors. The 1.5 pmol/g concentration yielded a 90% reduction in tumor multiplicity and a 40% reduction in incidence, while the 0.75 pmolig diet reduced tumor multiplicity by only 39% and had no effect on esophageal tumor incidence. No significant effects were observed at the 0.325 Gmol/g concentration of PEITC. Thus, the minimum inhibitory concentration of PEITC (i.e. the dose of PEITC that will yield a statistically significant reduction in tumor multiplicity) apparently lies between 0.325 and 0.75 pmol/g. In the current model, NMBA is administered repetitively over 15 weeks, with termination of the experiment at week 25 (4,251. Thus it is virtually impossible to discern at what time the functional equivalent of initiation ends and at what time promotion or progression begins. As a result, since PEITC was fed during the entire course of the experiment in this study and previously [25], the DNA methylation studies were conducted to examine the possible effects of PEITC on the initiation stage (or its functional equivalent). A doserelated inhibition of NMBA-induced DNA methylation was observed, in agreement with the tumor bioassay results obtained for the 0.75-3.0 pmol/g diets. This data suggests that PEITC affects initiation in this model.
Of greater interest is the finding that 06-meG levels at 24 hours at PEITC dietary concentrations of 1.5 and 3.0 lmol/g, concentrations that yield reductions of NMBA-induced tumor multiplicity of 90% and loo%, are reduced by only 34% and 64%, respectively. A similar phenomenon has been observed with isothiocyanates and methylation of murine pulmonary DNA. A dose of phenylhexyl isothiocyanate that resulted in 100% inhibition of NNK-induced pulmonary adenomas in A/J mice only inhibited pulmonary 06-meG levels by approximately 50% at 6 h [18]. However, NNK doses that produced 06-meG levels that were onehalf or less of that of a lo-pmol dose (i.e. the bioassay dose) produced much lower tumor multiplicities than the 10 pmol dose, because the methylation produced at the lower doses was repaired much more rapidly [21]. If a similar chain of events occurs in the rat esophagus, the present results are quite credible. Detailed time course studies of rat esophageal DNA methylation after feeding of control or PEITC-containing diets would be necessary to confirm such a possibility. Isothiocyanates are known to be potent inhibitors of enzymes, including cytochrome P-450. PEITC and other isothiocyanates inhibit microsoma1 metabolism of many carcinogens, whether the isothiocyanates are administered in vivo before microsomal isolation or added to the microsomal reaction mixtures in vitro [2,3,10,14,22]. Similar inhibitory effects of PEITC on nitrosamine metabolism have been reported for cultured rat tissues as well (5,19,25]. Isothiocyanates also induce phase II enzymes such as glutathione S-transferases [24]. However, phase II enzymes are not known to play a significant role in the metabolism of most nitrosamines. Thus we believe that the inhibition of DNA methylation observed here is most probably due to a direct inhibition of the cytochrome P45Os responsible for NMBA activation, an assertion that remains to be proved. Of course, with the difficulties in delineating the various stages of NMBA tumorigenesis in this model, one cannot rule out possible antipromotional activity by PEITC. Although benzyl isothiocyanate has been shown to possess antipromotional activity [32], no such effects for PEITC have been demonstrated in any animal model [ 181. Future experiments with this model system will
109
involve the comparison of PEITC with other isothiocyanates, and th.e development of NMBA dosing regimens that will allow testing of isothiocyanates and other chemopreventive agents at various stages of NMBA tumorigenesis, including promotion or progression.
11
Acknowledgements
13
This work was supported by NIH grants CA28950 and CA-46535. The authors wish to thank Young-Sun Heur for Irechnical assistance in the DNA methylation experiment.
12
14
References 1
2
3
4
5
6
7
8
9
10
Carlson, D.G., Daxenbichler, M.E., VanEtten, C.H.. Tookey, M.L. and Williams, P.H. (1981) Glucosinolates in crucifer vegetables: turnips and rutabagas. J. Agric. Food Chem.. 29, 1235- 1239. Chung, F-L., Juchatz, A., Vitarius, J. and Hecht. S.S. (1984) Effects of dietary compounds on o-hydroxylation of N-nitrosopyrrolidine ;and N’nitrosonornicotine in rat target tissues. Cancer Res., 44, 2924-2928. Chung, F.-L., Wang, M. and Hecht, S.S. (1985) Effects of dietary indoles and iz.othiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-I-(3-pyridyl)-lbutanone o-hydroxylation and DNA methylation in rat liver. Carcinogenesis (London), 6. 539-543. Daniel, E.M. and Stoner, G.D. (1991) The effects of ellagic acid and I3-cis-mtinoic acid on N-nitrosobenzylmethylamine-induced esophageal tumorigenesis in rats. Cancer Lett.. 56. 117-124. Doerr-O’Rourke, K., Tr.tshin, N., Hecht, S.S. and Stoner. G.D. (1991) Effect of phenethyl isothiocyanate on the menitrosamine 4of the tobacco-specific tabolism (methylnitrosamino)-1-(:l-pyridyl)-1-butanone by cultured rat lung tissue. Carcinogenesis. 12, 1029-1034. Druckrey, H., Preussme.nn. R., Blum. G., Ivankovic. S. and Afkham, J. (1963) Erzeugung von karzinoman der speiserohre durch unsymmetrische nitrosamine. Naturwissenschaften, 50. loo-- 101. Druckrey. H., Preussman, R., Ivankovic, S. and Schmahl, D. (1967) Organotrope carcinogene wirkungen bei 65 verschiedenen iV-nitroso-verbindungen an BD-ratten. Z. Krebsforsch.. 69, 103-201. DuPlessis, L.S.. Nunn, J.R. and Roach, W.A. (1969) Carcinogen in a Transkeian Bantu food additive. Nature, 222, 1198. Hanley, A.B., Heaney, R.K. and Fenwick. G.R. (1983) Improved isolation of glucobrassicin and other glucosinolates. J. Sci. Food Agric., 34, 869-873. Ishizaki, H., Brady. J.F., Ning, S.M. and Yang, C.S. (1990) Effect of phenethyl isothiocyanate on microsomal N-nitrosodimethylamine metabolism and other monooxygenase activities, Xenobiotica, 20, 255-264.
15
16
17
18
19
20
21
22
Lijinsky, W., Saavedra, J.E., Rueber, M.D. and Singer. S.S. (1982) Esophageal carcinogenesis in F344 rats by nitrosomethylethylamines substituted in the ethyl group. J. Nat. Cancer Inst., 68, 681-684. Mandal, S. and Stoner, G.D. (1990) Inhibition of Nnitrosobenzylmethylamine-induced esophageal tumorigenesis in rats by ellagic acid. Carcinogenesis (London), 11. 55-61. Morse, M.A., Wang, C-X., Stoner, G.D., Mandal, S., Conran, P.B., Amin, S.G., Hecht, S.S. and Chung, F.-L. (1989) Inhibition of 4-(methylnitrosamino)-I-(3-pyridyl)I-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate. Cancer Res., 49, 549-553. Morse, M.A., Amin, S.G., Hecht, S.S. and Chung. F.-L. ‘(1989) Effect of aromatic isothiocyanates on tumorigenicity, 06-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone in A/J mouse lung. Cancer Res., 49, 2894-2897. Morse, M.A., Eklind, H.I., Amin, S.G., Hecht, S.S. and Chung, F.-L. (1989) Effects of alkyl chain length on the inhibition of NNK-induced lung neoplasia in A/J mice by arylalkyl isothiocyanates. Carcinogenesis (London), 10. 1757-1759. Morse, M.A., Reinhardt, J.C.. Amin, S.G., Hecht, S.S., Stoner, G.D. and Chung, F.-L. (1990) Effects of dietary aromatic isothiocyanates fed subsequent to the administration of 4-(methylnitrosamino)-I-(3-pyridyl)-l-butanone on lung tumorigenicity in mice. Cancer Lett., 49, 225-230. Morse, M.A.. Eklind, K.I., Hecht. S.S., Jordan. K.G., Chdi, C.-I., Desai, D.H., Amin, S.G. and Chung. F.-L. (1991) Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-I-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice. Cancer Res., 51, 1846-1850. Morse. M.A., Eklind, K.I., Amin, S.G. and Chung, F.-L. (1992) Effect of frequency of isothiocyanate administration on inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)I-butanone-induced pulmonary adenoma formation in A/J mice. Cancer Lett., 62, 77-81. Murphy, SE., Heiblum. R., King, P.C., Bowman, D.. Davis, W.J. and Stoner, G.D. (1991) Effect of phenethyl isothiocyanate on the metabolism of tobacco-specific nitrosamines by cultured rat oral tissue. Carcinogenesis, 12. 957-961. Parkin. D.M., Stjernsward, J. and Muir, C.S. (1984) Estimates of the worldwide frequency of twelve major cancers. Bull. WHO, 62, 163-182. Peterson. L.A. and Hecht. S.S. (1991) 06-methylguanine is a critical determinant of 4-(methylnitrosamino)-i-(3pyridyl) I-butanone tumorigenesis in A/J mouse lung. Cancer Res., 51, 5557-5564. Smith, T.J., Guo. Z., Thomas, F.E., Chung, F.-L., Morse, M.A., Eklind, K. and Yang, C.S. (1990) Metabolism of 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes and its inhibition by isothiocyanates. Cancer Res.. 50, 6817-6822.
110
24
25
26
27
28
29
Spamins, V.L., Venegas, F.L. and Wattenberg. L.W. (1982) Glutathione S-transferase activity: enhancement by compounds inhibiting chemical carcinogenesis and by dietary constituents. J. Nat. Cancer Inst.. 68. 493-496. Stoner, G.D., Morrissey, D.T.. Heur. Y.-H., Daniel, E.M., Galati, A.J. and Wagner, S.A. (1991) Inhibitory effects of phenethyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus. Cancer Res., 51, 2063-2068. Tuyns, A.J., Pequignot, G. and Abbatuci, J.S. (1979) Oesophageal cancer and alcohol consumption: importance of type of beverage. Int. J. Cancer, 23, 443-447. VanEtten, C.H., Daxenbichler, M.E., Williams, P.H. and Kwolek, W.F. (1976) Glucosinolates and derived products in cruciferous vegetables: analysis of the edible part from twenty-two varieties of cabbage. J. Agric. Food Chem., 24, 452-455. Walker, E.A., Castegnario, M.. Garren, L., Toussaint. G. and Kowalski, B. (1979) Intake of volatile nitrosamines from consumption of alcohols. J. Nat. Cancer Inst., 63, 947-951. Wargovich, M.J.. Woods, C., Eng, V.W.S.. Stephens, L.C. and Gray, K. (1988) Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by
30
31
32
33
34
35
the naturally occurring thioether. diallyl sulfide. Cancer Res., 48, 6872-6875. Warwick, G.P. and Harington. J.S. (1973) Some aspects of the epidemiology and etiology of esophageal cancer with particular emphasis on the Transkei. South Africa. Adv. Cancer Res.. 17. 18-229. Wattenberg, L.W. (1977) Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J. Nat. Cancer Inst.. 58, 395-398. Wattenberg. L. W. ( 198 1) Inhibition of carcinogeninduced neoplasia by sodium cyanate. rert-butyl isocyanate. and benzyl isothiocyanate adminstered subseCancer Res.. 41. quent to carcinogen exposure. 299 l-2994. Wattenberg, L.W. (1987) Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine or benzo(a)pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis (London). 8. 1971-1973. Wynder, E.L. and Bross, I.J. (1961) A study of etiological factors in cancer of the esophagus. Cancer (Phila.). 14. 389-413. Yang, C.S. (1980) Research on esophageal cancer in China: a review. Cancer Res., 40, 2633-2644.