Carcinogenesis vol.22 no.3 pp.441–446, 2001
Inhibition of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus by dietary freeze-dried strawberries
Peter S.Carlton, Laura A.Kresty, Joseph C.Siglin1, Mark A.Morse, Jerry Lu, Charlotte Morgan and Gary D.Stoner2 Division of Environmental Health Sciences, The Ohio State University School of Public Health and The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Room 1148, 300 West 10th Avenue, Columbus, OH 43210, USA 1Present
address: Springborn Laboratories, Inc., Spencerville, OH 45887, USA 2To
whom correspondence should be addressed Email:
[email protected]
In the present study, we examined the ability of dietary freeze-dried strawberries to inhibit N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. Initially, we conducted a bioassay to determine the effects of dietary freeze-dried strawberries on esophageal tumor development. Two weeks prior to NMBA treatment, animals were placed on a control diet or diets containing 5 and 10% freeze-dried strawberries. NMBA treatment was once per week for 15 weeks. At 30 weeks, 5 and 10% freeze-dried strawberries in the diet caused significant reductions in esophageal tumor multiplicity of 24 and 56%, respectively. Based on these results, we conducted studies to determine potential mechanisms by which freeze-dried strawberries inhibit tumorigenesis. In a short-term bioassay, we evaluated the effects of dietary freeze-dried strawberries on the formation of O6-methylguanine in the rat esophagus. Animals were placed on control diet or diets containing 5 and 10% freeze-dried strawberries for two weeks. At the end of this period, animals received a single subcutaneous dose of NMBA and were killed 24 h later. A significant decrease in O6-methylguanine levels was observed in the esophageal DNA of animals fed strawberries, suggesting that one or more components in strawberries influence the metabolism of NMBA to DNAdamaging species. Finally, in order to evaluate post-initiation effects, we conducted a study where freeze-dried strawberries were administered in the diet only following NMBA treatment. Animals were placed on control diet and dosed with NMBA three times per week for 5 weeks. Immediately following NMBA treatment, animals were placed on control diet or diets containing 5 and 10% freezedried strawberries. At 25 weeks, 5 and 10% freeze-dried strawberries in the diet significantly reduced tumor multiplicity by 38 and 31%, respectively. Our data suggest that dietary freeze-dried strawberries effectively inhibit NMBAinduced tumorigenesis in the rat esophagus.
Introduction Epidemiologic evidence suggests an inverse relationship between consumption of fruit and vegetables and the occurrence of several types of cancer (1). In particular, the incidence of squamous cell carcinoma (SCC) of the esophagus, a neoplasm of variable geographic distribution, has been linked to diets deficient in fresh fruit and vegetables (2). This association is thought to be related to the abundance of naturally occurring preventive agents found in fruit and vegetables, including vitamins, minerals and numerous nonnutrient compounds. Recently, a study by Rijken et al. (3) demonstrated the efficacy of a food-based approach to cancer prevention in a rodent model of colon carcinogenesis. Dietary supplementation with various freeze-dried vegetables was shown to significantly reduce aberrant crypt multiplicity (ACM), a surrogate endpoint marker for azoxymethane (AOM)-induced adenomas and carcinomas in the rat colon. Studies have also shown that tomato juice, paprika juice, dry beans, garlic and soybeans have the ability to inhibit carcinogenesis in various animal model systems when added to the water supply or mixed in the diet (4–9). Our laboratory has applied a similar food-based approach to the prevention of cancer in a rat model of esophageal SCC (10). The present study stems from work by Daniel et al. (11) in which various fruits and nuts were characterized for their content of the phenolic compound, ellagic acid (EA). In particular, the strawberry (Fragaria ananassa) was shown to contain high levels of EA, with most of the compound in the pulp. This characteristic of the strawberry is thought to confer enhanced bioavailability of EA compared with fruit in which the compound is found predominantly within the seeds (11). Plant-derived EA is known to have chemopreventive effects in various in vitro and in vivo model systems. In studies by Mandel et al., EA was shown to inhibit N-nitrosomethylbenzylamine (NMBA) metabolism in cultured explants of the rat esophagus and to inhibit NMBA-induced tumorigenesis in the rat esophagus (12,13). In view of the substantial epidemiologic and experimental support for food-based cancer prevention with fruit and vegetables, and the data indicating that strawberries are a natural source of EA, a known inhibitor of tumor development in animal model systems, we evaluated the ability of a lyophilized preparation of strawberries to prevent NMBAinduced esophageal tumorigenesis in the F344 rat. Our results suggest that freeze-dried strawberries in the diet effectively inhibit tumor development in the rat esophagus and that this inhibition is probably due to the combined effects of multiple strawberry components. Materials and methods
Abbreviations: ACM, aberrant crypt multiplicity; AOM, azoxymethane; DMSO, dimethyl sulfoxide; EA, ellagic acid; NBF, 10% neutral buffered formalin; NMBA, N-nitrosomethylbenzylamine; O6-meGua, O6-methylguanine; SCC, squamous cell carcinoma. © Oxford University Press
Animals Male F344 rats, 5–6 weeks-of-age, were purchased from Harlan SpragueDawley (Indianapolis, IN). The animals were housed three per cage under
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Table I. Experimental design of the freeze-dried strawberry complete carcinogenesis study Group
Treatment
No. rats
Amount administered (ml)
Dose administered (mg/kg body weight)
Diet
1 2 3 4 5
DMSO ⫹ H2Oa None NMBA NMBA NMBA
15 15 15 15 15
0.2 0 0.2 0.2 0.2
0 0 0.25 0.25 0.25
Control AIN-76A AIN-76A ⫹ 10% Strw.b Control AIN-76A AIN-76A ⫹ 5% Strw. AIN-76A ⫹ 10% Strw.
⫹ H2O, vehicle for NMBA. freeze-dried strawberries.
aDMSO bStrw.,
standard conditions (20 ⫾ 2°C; 50 ⫾ 10% relative humidity; 12 h light/dark cycles). Rats were maintained on a modified AIN-76A purified diet containing 20% casein, 0.3% D,L-methionine, 52% cornstarch, 13% dextrose, 5% cellulose, 5% corn oil, 3.5% AIN salt mixture, 1% AIN vitamin mixture and 0.2% choline bitartrate (Dyets, Bethlehem, PA). Food and water were provided ad libitum. Hygienic conditions were maintained by twice weekly cage changes. Body weight and food consumption measurements were recorded weekly for the duration of the studies. Chemicals NMBA, obtained from Ash Stevens, Inc. (Detroit, MI), was determined to be ⬎98% pure by HPLC analysis. Preparation of strawberry diets Three harvests of strawberries (Commander variety) were provided by Driscoll Strawberry Associates (Watsonville, CA). Seed removal and freeze-drying (lyophilization) was performed by the Department of Food Science and Technology, University of California, Davis (Davis, CA) as detailed by Stoner et al. (10). Briefly, strawberries were washed and excess water drained. Capstems and seeds were removed and the strawberry pulp crushed by passing the whole fruit through a Brown pulper-finisher fitted with a 0.02 inch screen. The pulp was then freeze-dried in a Virtis Freeze Dryer model 50-SRC-5. Freeze-dried strawberries were stored at 4°C after lyophilization. The EA content of freeze-dried strawberry batches used in the adduct and postinitiation studies was determined in our laboratory by HPLC analysis as described previously (11). In addition, aliquots of freeze-dried strawberries were analyzed for other compounds with known chemopreventive activity by Covance Laboratories (Madison, WI). On a weekly basis, a Hobart mixer was used to combine portions of the freeze-dried strawberries with a modified AIN-76A purified diet in place of 5 and 10% cornstarch. Based on known calorie values for strawberries and cornstarch (14), we estimate that our freeze-dried strawberry diet preparations provide a caloric content comparable with the control diet. Portions of these diets were placed in glass feeding jars and fed to the rats. Previous studies in our laboratory indicate that EA in the freeze-dried strawberry diets remains stable at room temperature for at least 2 weeks (data not shown). Complete carcinogenesis study The effects of freeze-dried strawberries on initiation and post-initiation events of NMBA-induced rat esophageal tumorigenesis were evaluated in an initial 75 animal bioassay. Five groups of 15 animals were randomized and placed on AIN-76A diet or AIN-76A diets containing 5 and 10% freeze-dried strawberries (Table I). Rats were maintained on these diets for the duration of the 30 week bioassay. Two weeks following diet initiation, rats were injected subcutaneously with NMBA at 0.25 mg/kg body weight once per week for 15 weeks (Figure 1A). Vehicle controls received subcutaneous injections of 20% dimethyl sulfoxide (DMSO) in water, the solvent for NMBA. Thirty weeks following initiation of NMBA treatment, rats were killed by carbon dioxide asphyxiation. The esophagus of each animal was excised, opened longitudinally, fixed in 10% neutral buffered formalin (NBF) and processed for histopathological evaluation. Multiple H&E stained slides were prepared from the esophagi of each rat and used for the confirmation of neoplastic lesions (i.e. papillomas and carcinomas) and for the quantification of preneoplasia (i.e. hyperplasia, simple leukoplakia and dysplastic leukoplakia). In addition, sections of the liver, bladder, spleen, heart, lung, colon, stomach and small intestine were harvested and fixed in NBF in order to evaluate possible toxicity of dietary freeze-dried strawberries. DNA adduct study To evaluate the effects of freeze-dried strawberries on NMBA-induced formation of O6-methylguanine (O6-meGua) adducts in the rat esophagus, seven groups of animals were randomized and placed on either AIN-76A diet or AIN-76A diets containing 5 and 10% freeze-dried strawberries. A single
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Fig. 1. Experimental protocols for the complete carcinogenesis (A) and the post-initiation (B) studies. Rats were treated with NMBA at 0.25 (A) or 0.5 mg/kg body weight (B) once per week for 15 weeks (A) or three times per week for five weeks (B). Freeze-dried strawberry diets were administered 2 weeks prior to initiation of NMBA treatment and for the duration of the study (A) or only following NMBA treatment (B). subcutaneous dose of NMBA at 0.5 or 0.25 mg/kg body weight was administered 2 weeks after the initiation of control or freeze-dried strawberry diets. The vehicle controls received a single subcutaneous injection of 20% DMSO. Twenty-four hours after NMBA treatment, rats were killed by carbon dioxide asphyxiation, and the esophagus of each animal excised, opened longitudinally and the epithelium separated from the underlying sub-mucosa. The epithelial layer was immediately frozen in liquid nitrogen. DNA was isolated from four to five pooled mucosal samples, subjected to acid hydrolysis, and the levels of O6-meGua quantified using strong cation exchange HPLC as described previously (15). Post-initiation study To evaluate the effects of freeze-dried strawberries on post-initiation events of NMBA-induced rat esophageal tumorigenesis, 65 animals were randomized into five groups and placed on AIN-76A diet (Table II). According to a shortened dosing protocol developed in our laboratory by Siglin et al. (16), animals were injected subcutaneously with NMBA (0.5 mg/kg body weight) three times per week for 5 weeks (Figure 1B). Vehicle controls received subcutaneous injections of 20% DMSO. Immediately following NMBA treatment, animals were placed on AIN-76A diet or AIN-76A diets containing 5 and 10% freeze-dried strawberries. Rats were maintained on these diets for the duration of the bioassay. Twenty-five weeks after initiation of NMBA treatment, rats were killed by carbon dioxide asphyxiation. The esophagus of each animal was excised, opened longitudinally and fixed in NBF. Statistical analysis Body weight, food consumption, tumor multiplicity, DNA adduct and preneoplastic lesion data were analyzed for statistical significance (P ⬍ 0.05) using analysis of variance (ANOVA) followed by Neuman–Keuls’ multiple comparisons test. Differences in tumor incidence were determined by χ2 test.
Results Nutrient content of freeze-dried strawberries In the present study, freeze-dried strawberries were assayed for their content of several known chemopreventive agents in order to identify strawberry components that may be active in
Strawberries and NMBA-induced tumorigenesis in the rat esophagus
Table II. Experimental design of the freeze-dried strawberry post-initiation study Group
Treatment
No. rats
Amount administered (ml)
Dose administered (mg/kg body weight)
Diet
1 2 3 4 5
DMSO ⫹ H2Oa None NMBA NMBA NMBA
10 10 15 15 15
0.2 0 0.2 0.2 0.2
0 0 0.5 0.5 0.5
Control AIN-76A AIN-76A ⫹ 10% Strw.b Control AIN-76A AIN-76A ⫹ 5% Strw. AIN-76A ⫹ 10% Strw.
⫹ H2O, vehicle for NMBA. freeze-dried strawberries.
aDMSO bStrw.,
Table III. Contents of freeze-dried strawberries used in the DNA adduct study and the post-initiation study Assay
α-carotene β-carotene Lutein Zeaxanthin EA Ferulic acid Vitamin C Folic acid Calcium Selenium
Analysis (mg/g) Batch I
Batch II
0.0003 ⬍0.0002 ⬍0.0002 ⬍0.0002 1.41 ⬍0.025 1.41 0.0037 1.60 ⬍0.0002
⬍0.0002 0.0016 ⬍0.0002 ⬍0.0002 2.22 0.025 2.98 0.0020 1.74 ⬍0.0002
the inhibition of NMBA-induced tumorigenesis in the rat esophagus (Table III). Assays for various carotenoids, polyphenolics, vitamins and minerals were performed in our laboratory and by Covance Laboratories. The EA content of freeze-dried strawberry batches used in the adduct study and the post-initiation study was determined to be 1.41 and 2.22 mg/g dry weight, respectively. Other compounds present at relatively high concentrations included vitamin C (1.41 and 2.98 mg/g dry weight) and calcium (1.60 and 1.74 mg/g dry weight). Complete carcinogenesis study We conducted an initial bioassay to determine whether the addition of 5 and 10% freeze-dried strawberries in the diet would inhibit rat esophageal tumorigenesis. There were no remarkable differences in mean body weight (Figure 2A) or food consumption (data not shown) for the duration of the bioassay. Histopathological evaluation indicated no strawberry related toxicity in any of the tissues harvested. In order to determine the effects of dietary freeze-dried strawberries on microscopic NMBA-induced preneoplastic lesions in the rat esophagus, we have used a classification system based on work by Pozharisski (17) wherein progression of chemicallyinduced tumorigenesis in the rat esophagus is described in detail. At 10% in the diet (group 5), freeze-dried strawberries significantly reduced lesions classified as epithelial hyperplasia by 23% when compared with the NMBA controls (P ⬍ 0.05) (Table IV). In addition, freeze-dried strawberries at 5 and 10% in the diet (groups 4 and 5) significantly reduced lesions classified as dysplastic leukoplakia by 52 and 65%, respectively, when compared with NMBA controls (group 3) (P ⬍ 0.01). However, with respect to lesions classified as simple leukoplakia, 5 and 10% freeze-dried strawberries in the diet (groups 4 and 5) produced significant increases of 53
Fig. 2. Effects of freeze-dried strawberries on body weight gain in the complete carcinogenesis study (A) and the post-initiation study (B). Group symbols are as follows: vehicle control (r); 10% freeze-dried strawberry diet control (u); NMBA control (m); NMBA ⫹ 5% freeze-dried strawberry diet (⫻); and NMBA ⫹ 10% freeze-dried strawberry diet (e). Dashed lines indicate NMBA treated groups.
and 50%, respectively, when compared with the NMBA controls (group 3) (P ⬍ 0.05). The effects of dietary freezedried strawberries on NMBA-induced tumorigenesis in the rat esophagus are summarized in Table V. Tumor incidence among groups dosed with NMBA (groups 3–5) did not vary significantly. At 5% in the diet (group 4), freeze-dried strawberries significantly reduced tumor multiplicity by 24% compared with the NMBA controls (group 3) (P ⬍ 0.05). At 443
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Table IV. Effects of freeze-dried strawberries on NMBA-induced preneoplastic lesions in the rat esophagus Group
Treatment
1 2 3 4 5
Vehicle 10% Strw. NMBA NMBA ⫹ 5% Strw. NMBA ⫹ 10% Strw.
aSignificantly bSignificantly
No. lesions/rat (mean ⫾ SE)
No. rats
7 15 12 14 15
Epithelial hyperplasia
Simple leukoplakia
Dysplastic leukoplakia
0 0 43.3 ⫾ 2.4 40.6 ⫾ 2.6 33.5 ⫾ 2.1a
0 0 9.6 ⫾ 2.4 20.6 ⫾ 3.5a 19.3 ⫾ 2.8a
0 0 11.6 ⫾ 1.8 5.6 ⫾ 0.9b 4.1 ⫾ 0.5b
different from NMBA controls (P ⬍ 0.05). different from NMBA controls (P ⬍ 0.01).
Table V. Effects of freeze-dried strawberries on NMBA-induced tumorigenesis in the rat esophagus—complete carcinogenesis treatment protocol Group Treatment
1 2 3 4 5
Vehicle 10% Strw. NMBA NMBA ⫹ 5% Strw. NMBA ⫹ 10% Strw.
No. rats
15 14 14 15 15
Tumor incidence %
0 0 100 100 80
Tumor multiplicity Tumors/rat % (mean ⫾ SE) inhibition 0 0 4.1 ⫾ 0.3 3.1 ⫾ 0.2a 1.8 ⫾ 0.4b
24.4 56.1
Data taken from reference 10. aSignificantly different from NMBA controls (P ⬍ 0.05). bSignificantly different from NMBA controls (P ⬍ 0.01), NMBA ⫹ 5% Strw. (P ⬍ 0.05).
10% in the diet (group 5), freeze-dried strawberries significantly reduced tumor multiplicity compared with the NMBA controls (group 3) by 56% (P ⬍ 0.01), and compared with the NMBA ⫹ 5% freeze-dried strawberry group (group 4) by 42% (P ⬍ 0.05). Thus, the addition of freeze-dried strawberries in the diet before, during and after NMBA treatment modulated the development of microscopic preneoplastic lesions and, in a dose-dependent manner, reduced tumor multiplicity in the rat esophagus. DNA adduct study Based on the results of the complete carcinogenesis bioassay, we conducted studies to determine potential mechanisms by which dietary freeze-dried strawberries inhibit NMBA-induced tumorigenesis in the rat esophagus. Metabolic activation of NMBA in the rat esophagus results in DNA methylation at the N7 and O6 positions of guanine (18). While the N7 methyl adduct is observed more frequently, O6-meGua formation leads to single nucleotide mutations and, thus, is associated with carcinogenic effects (19). In the present study, we have measured O6-meGua levels in the rat esophagus in order to determine the effects of dietary freeze-dried strawberries on the metabolism of NMBA (Figure 3). There were no detectable levels of O6-meGua in the esophagi of vehicle controls. Rats dosed with 0.5 mg/kg body weight NMBA and fed freezedried strawberries had significantly reduced esophageal O6meGua levels compared with the NMBA controls (P ⬍ 0.05). At 5 and 10% in the diet, freeze-dried strawberries caused a 59 and 64% inhibition of esophageal O6-meGua, respectively. In rats dosed with 0.25 mg/kg body weight NMBA, freezedried strawberries at 5 and 10% in the diet significantly reduced 444
Fig. 3. Effects of freeze-dried strawberries on NMBA-induced O6-meGua levels in the rat esophagus. Rats were treated with NMBA at 0.5 (u) or mg/kg body weight (j). Means and standard errors are shown. *Significantly lower than controls dosed with the same concentration NMBA (P ⬍ 0.05). Data taken from reference 10.
Table VI. Effects of freeze-dried strawberries on NMBA-induced tumorigenesis in the rat esophagus—post-initiation treatment protocol Group Treatment
1 2 3 4 5
Vehicle 10% Strw. NMBA NMBA ⫹ 5% Strw. NMBA ⫹ 10% Strw.
aSignificantly
No. rats
10 10 14 15 15
Tumor incidence %
0 0 100 100 100
Tumor multiplicity Tumors/rat % (mean ⫾ SE) inhibition 0 0 4.8 ⫾ 0.6 3.0 ⫾ 0.3a 3.3 ⫾ 0.4a
37.5 31.3
different from NMBA controls (P ⬍ 0.05).
O6-meGua levels by 68 and 57%, respectively, compared with the NMBA controls (P ⬍ 0.05). Post-initiation study In order to better determine the effects of dietary freezedried strawberries on post-initiation events of NMBA-induced tumorigenesis in the rat esophagus, we conducted a bioassay where strawberry diets were administered only following NMBA treatment. There were no remarkable differences in mean body weight (Figure 2B) or food consumption (data not shown) for the duration of the study. The effects of freezedried strawberries on NMBA-induced tumor incidence and tumor multiplicity are summarized in Table VI. Tumor incidence was 100% for groups dosed with NMBA (groups 3–5).
Strawberries and NMBA-induced tumorigenesis in the rat esophagus
At 5 and 10% in the diet (groups 4 and 5), freeze-dried strawberries significantly reduced tumor multiplicity by 38 and 31%, respectively, when compared with the NMBA controls (group 3) (P ⬍ 0.05). There was no significant difference in tumor multiplicity between the NMBA ⫹ 5% freeze-dried strawberry group and the NMBA ⫹ 10% freezedried strawberry group (groups 4 and 5). Thus, when administered in the diet following NMBA treatment, freezedried strawberries effectively reduced tumor multiplicity in the rat esophagus, but this effect was not dose related. Discussion In the present study, we have shown that dietary administration of freeze-dried strawberries effectively inhibits NMBAinduced tumorigenesis in the rat esophagus. This inhibitory ability may be related to the EA content of strawberries, but is more likely the result of the combined anti-initiation and anti-promotion/progression effects of other known and yet to be identified strawberry components. Our data show that 5 and 10% freeze-dried strawberry diets inhibit initiation events associated with NMBA-induced tumorigenesis in the rat esophagus in a dose-dependent manner. We suggest that this occurs due to the ability of strawberry components to inhibit the metabolic activation of NMBA to DNA damaging species, and thus, prevent the formation of O6-meGua DNA adducts. Mechanistic studies are currently ongoing to determine whether these components influence the activation/detoxification of NMBA or modify DNA repair. The present study suggests that freeze-dried strawberries effectively modulate the formation of preneoplastic lesions in the rat esophagus, and thus, may be involved in the inhibition of post-initiation stages of tumorigenesis. In particular, the occurrence of simple leukoplakic lesions was significantly increased by freeze-dried strawberries in the diet, while lesions classified as dysplastic leukoplakia were reduced. A study by Pozharisski (17) suggests that the development of chemicallyinduced tumors in the rat esophagus begins with diffuse hyperplasia and proceeds to focal lesions of leukoplakia. These highly keratinized lesions are likely to progress to dysplastic lesions, followed by papillomas, and ultimately SCCs. We suggest that components of the strawberry act to arrest the progression of less advanced lesions, i.e. simple leukoplakia, to dysplastic lesions. Moreover, we believe that strawberry components may induce keratinocyte differentiation in dysplastic epithelium, thereby reversing the progression of these advanced lesions. A study by Babcock et al. (20) suggests that calcium, a compound we have shown to be present at relatively high amounts in strawberries, can induce terminal differentiation in cultured rat esophageal epithelial cells. In addition, another major component of strawberries, the antioxidant vitamin C, is thought to influence cancer cell proliferation and differentiation in vitro (21). Our laboratory has developed a modified NMBA dosing protocol that allows for the evaluation of putative chemopreventive agents in the post-initiation stages of esophageal tumorigenesis (16). In this protocol, freeze-dried strawberries were added to the diet only following NMBA treatment, and for the duration of the study. Our results suggest that freezedried strawberries in the diet effectively inhibit post-initiation events of esophageal tumorigenesis, but these effects are not dose dependent.
Previous studies in our laboratory have demonstrated that EA is an effective inhibitor of NMBA-induced tumorigenesis in the rat esophagus (13,22). When EA was added to the diet at 0.4 and 4.0 mg/g diet 2 weeks prior to NMBA treatment, and for the duration of the study, a respective 27 and 47% reduction in esophageal tumor multiplicity was observed (13). A subsequent study in our laboratory demonstrated that the administration of EA only following NMBA treatment produced modest reductions in tumor incidence and had no effect on tumor multiplicity (22). In the present study, freezedried strawberries in the diet, which impart actual dietary EA concentrations of ⬍0.4 mg/g diet, were shown to produce inhibitory effects equal to or greater than those achieved by EA alone. Moreover, freeze-dried strawberries in the diet significantly reduced tumor multiplicity when administered only following NMBA treatment. Similar to our findings with EA and strawberries, Rijken et al. (3) demonstrated that dietary freeze-dried vegetables more effectively inhibited AOMinduced ACM in the rat colon than vegetable-derived α- and β-carotene in the diet. Narisawa et al. (5,6) have also observed such an effect when evaluating the ability of tomato and paprika juice and their respective carotenoid constituents, lycopene and capsanthin, to inhibit N-methylnitrosoureainduced carcinogenesis in the rat colon. The present study provides further evidence in support of food-based cancer prevention with fruit and vegetables. In order to better understand, mechanistically, why certain foods inhibit various stages of tumorigenesis, known chemopreventive agents should be identified and measured in the foods under study. Moreover, efforts should be made to isolate, quantitate and evaluate novel food-based compounds that may contribute to cancer prevention. In the present study, we have measured the levels of a number of potential inhibitory agents in freeze-dried strawberries and have shown EA, vitamin C and calcium to be present in appreciable levels. We suggest that these components may, in part, be responsible for the anti-carcinogenic effects of strawberries. Ongoing studies in our laboratory are being conducted to isolate other major components of strawberries and to evaluate their antitumorigenic activity. Acknowledgements The authors wish to thank Dr Tom Sjulin at Driscoll Strawberry Associates for the generous contribution of strawberries for these studies and Jim Buhlert at the University of California, Davis Department of Food Science and Technology, for his involvement in the preparation of freeze-dried strawberries. By permission of Oxford University Press, data in Table V and Figure 3 have been adapted from a previous publication: Stoner,G.D., Kresty,L.A., Carlton,P.S., Siglin,J.C. and Morse,M.A. (1999) Isothiocyanates and freezedried strawberries as inhibitors of esophageal cancer. Toxicol. Sci., 52 (Suppl.), 95–100. This work was supported by a grant from the Ohio Department of Agriculture.
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