Effects of phenylethyl isothiocyanate on early molecular events in N-nitrosomethylbenzylamine-induced cytotoxicity in rat esophagus

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone.     

Chemopreventive effects of a selective nitric oxide synthase inhibitor on carcinogen-induced rat esophageal tumorigenesis

The inducible nitric oxide synthase (iNOS) generates a high concentration of nitric oxide (NO) in tissues. Increased NO production is associated with many disorders including esophageal cancer. Previous studies in our laboratory demonstrated an association between increased iNOS expression and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. On the basis of these observations, we initiated a bioassay to evaluate the ability of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, to prevent the progression of esophageal tumors in rats preinitiated with NMBA. Rats were given s.c. injections of NMBA (0.25 mg/kg body weight) three times per week for 5 weeks. One week later, they were fed a synthetic diet containing either 50 or 100 ppm PBIT until the end of the bioassay (25 weeks). PBIT reduced the incidence of esophageal cancer from 96% in NMBA-treated rats to 83% and 77% (P < 0.05) in rats treated with 50 and 100 ppm PBIT, respectively. Tumor multiplicity was reduced from 3.64 +/- 0.42 tumors per esophagus in NMBA-treated rats to 1.79 +/- 0.25 (P < 0.001) and 1.50 +/- 0.24 (P < 0.0001) in rats treated with 50 and 100 ppm PBIT, respectively. PBIT reduced the production of NO in NMBA-induced preneoplastic and papillomatous esophageal lesions when compared with comparable lesions in rats treated with NMBA only. iNOS mRNA expression was not modulated by PBIT. These observations suggest that iNOS plays a role in tumor development and that its selective inhibitor, PBIT, significantly inhibits esophageal tumor progression presumably through reducing the production of NO.     

The effects of L-748706, a selective cyclooxygenase-2 inhibitor, on N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis

Epidemiological studies suggest that the frequent intake of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a decreased risk of developing esophageal squamous cell carcinoma (SCC). This decrease is thought to correlate with the inhibition of cyclooxygenase (COX) activity. The production of prostaglandin E2 (PGE2), a major metabolite of COX, is increased in numerous human cancers including esophageal SCC, therefore, inhibition of COX activity and subsequent suppression of the formation of PGE2 may be chemopreventive in the esophagus. The objective of the present study was to determine whether L-748706 (L-706), a novel selective COX-2 inhibitor, would prevent N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor progression in the Fischer 344 (F344) rat. In rats pretreated with a low-dose of NMBA (0.25 mg/kg body weight), L-706 at 100 p.p.m. in the diet significantly reduced tumor multiplicity but not tumor incidence. At 150 p.p.m. in the diet, L-706 alone and in combination with 200 p.p.m. piroxicam produced significant reductions in both tumor incidence and multiplicity. Inhibition of tumor development in low-dose NMBA-treated rats was associated with reductions in esophageal cell proliferation rates and PGE2 levels in preneoplastic tissues. In contrast, in rats treated with a higher dose of NMBA (0.5 mg/kg body weight), neither L-706 alone nor in combination with piroxicam reduced esophageal tumor incidence or multiplicity in spite of the fact that they reduced esophageal PGE2 levels in preneoplastic tissues and in papillomas. Cell proliferation rates were reduced only in animals treated with L-706 + piroxicam. Our data suggest that the chemopreventive treatments were effective in inhibiting tumor development in NMBA-treated animals only when they reduced PGE2 levels in preneoplastic esophageal tissues approximately to those levels found in normal esophagus.     

Alpha-difluoromethylornithine induction of apoptosis: a mechanism which reverses pre-established cell proliferation and cancer initiation in esophageal carcinogenesis in zinc-deficient rats.

Alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis. Previous work showed simultaneous administration of DFMO and a zinc-deficient (ZD) diet to weanling rats from the beginning inhibited the onset of zinc-deficiency-induced esophageal cell proliferation by activating apoptosis and reduced the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer. Because esophageal cancer initiation by NMBA is very rapid in ZD rats, this study determined whether DFMO is effective in preventing esophageal carcinogenesis when administered after the establishment of a carcinogenic environment. Weanling rats were given a ZD diet for 5 weeks to establish sustained increased esophageal cell proliferation and then an intragastric dose of NMBA. Thereafter, 20 rats were switched to DFMO-containing water while nine control ZD animals remained on deionized water; all of the animals continued on the ZD diet. Esophagi were collected 15 weeks later. The upper portion was processed for immunohistochemical analysis of cell proliferation, apoptosis, and expression of related genes, and the lower was processed for polyamine content. DFMO substantially reduces the levels of esophageal putrescine and spermidine and esophageal tumor incidence from 89 to 10% in ZD rats. Importantly, DFMO-treated ZD esophagi display increased rate of apoptosis accompanied by intense bax expression and greatly reduced cell proliferation by proliferating cell nuclear antigen expression. In addition, the p16(ink4a)/retinoblastoma control at G1 to S, deregulated in ZD esophagi, is restored after DFMO treatment. These results demonstrate that DFMO, a highly effective chemopreventive agent in esophageal carcinogenesis, reverses and counteracts esophageal cell proliferation/cancer initiation in ZD animals by way of stimulating apoptosis.     

Piroxicam is an ineffective inhibitor of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus

Epidemiological studies indicate an association between the frequent use of nonsteroidal anti-inflammatory drugs and decreased risk for esophageal cancer. These studies suggest that limiting excess prostaglandin production, via inhibition of cyclooxygenase (COX)-mediated arachidonic acid metabolism, may be an important strategy for the prevention of this type of malignancy. N-Nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus is a model of human esophageal squamous cell carcinoma used for investigations of chemical carcinogenesis and for the evaluation of putative chemopreventive agents. In this study, we characterized COX-mediated arachidonic acid metabolism in NMBA-induced rat esophageal tumorigenesis by measuring COX-1 and COX-2 expression and prostaglandin E(2) production. In addition, we evaluated the ability of piroxicam, a potent COX inhibitor, to prevent postinitiation events of NMBA-induced tumorigenesis in the rat esophagus. After a 2-week acclimatization period, groups of 30 male F344 rats received s.c. injections of NMBA (0.5 mg/kg b.w.) three times/week for 5 weeks. Seventy-two h after the final NMBA treatment and for the remainder of the study, piroxicam was administered in the diet at 200 and 400 ppm. Twenty-five weeks after the initiation of NMBA treatment, we observed an elevation in COX mRNA and protein expression and prostaglandin E(2) production in NMBA-treated esophageal tissues compared with normal epithelium. However, these changes were associated with data indicating that a COX inhibitor is not preventive in NMBA-induced rat esophageal tumorigenesis. Administration of piroxicam in the diet produced no significant reductions in esophageal tumor incidence, multiplicity, or size. The reasons for the lack of effect are largely unknown but may be related to the inability of piroxicam to modulate other biochemical pathways involved in NMBA-induced tumorigenesis.     

Induction of esophageal tumors in zinc-deficient rats by single low doses of N-nitrosomethylbenzylamine (NMBA): analysis of cell proliferation, and mutations in H-ras and p53 genes

Dietary zinc deficiency in rats induces hyperplasia in the esophagus and increases N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence. Previous work showed a direct relationship between epithelial cell proliferation and esophageal tumor incidence in rats given multiple doses of NMBA. We investigated the effects of single low doses of NMBA in zinc-deficient rats since a single dose of 5.0 mg/kg was reported to be non-carcinogenic in rats. Zinc-sufficient and deficient rats received a single i.g. dose of NMBA at 0.5 or 2.0 mg/kg. At week 14, tumor incidence was 50% with 0.8 +/- 1.0 tumors/rat, and 80% with 2.2 +/- 1.9 tumors/rat, in deficient groups, D(0.5) and D(2.0), that received the lower and higher dose, respectively. In addition, two small papillomas were found in one out of eight untreated zinc-deficient rats. None of the NMBA-treated or untreated zinc- sufficient rats had any tumors. Esophageal cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry, showed that, irrespective of NMBA treatment, deficient esophagi had significant increases in the number of labeled cells, the total number of cells, and the labeling index, as compared with zinc-sufficient ones. Mutations in Ha-ras and p53 genes in esophageal tumors were detected by single strand conformation polymorphism (SSCP) analysis. DNA sequencing of variant conformers revealed a point mutation (GGA-->GAA, codon 12) in Ha-ras in 4/5 (80%) and 5/8 (63%) tumors, from D(0.5) and D(2.0) rats, respectively. Three out of eight tumors from D(2.0) rats exhibited SSCP mobility shifts within p53 exons 5 and 7: two tumors (2/8, 25%) had missense mutations and the third, a silent mutation. Of the two tumors with p53 mutations, one had a double mutation (transition at codon 164, TCA-->TTA; transversion at codon 241, AGT-->TGT), and the other tumor, a transition at codon 172 (AGA-->GGA), with amino acid changes in all cases. In parallel with PCNA expression, elevated p53 expression was associated with hyperplastic and dysplastic regions, as well as with tumors, in deficient esophagi. In short, these data indicate that dietary zinc deficiency, with its associated sustained increased cell proliferation in the esophagus, can drive an otherwise non-tumorigenic dose of NMBA into a highly tumorigenic one.      

Overexpression of Cyclin D1 in Rat Esophageal Carcinogenesis Model

Overexpression of cyclin D1 in human esophageal carcinomas has been well documented. The aim of the present study was to assess the expression of cyclin D1 in different types of esophageal epithelial lesions induced by N -nitrosomethylbenzylamine (NMBA) in rats. A total of 30 rats received s.c.-injections, five times/week, of 1.0 mg/kg NMBA for a period of 5 weeks followed by the same dose once per week for another 10 weeks. An additional 15 rats were given saline and used as controls to provide normal epithelium. The tumor incidence was 100% at the termination point of 21 weeks. Seventeen rats (57%) showed nuclear staining for cyclin D1, with a great variation in the intensity, as demonstrated by using an immunohistochemical technique. The cyclin D1 positive indices were in the range of 0% to 60% of the individual cells. Negligible staining was observed for normal esophageal epithelium, with a minimal increase in hyperplastic and dysplastic lesions. A significant elevation of cyclin D1 levels was observed in tumors. However, no significant differences were found between papillomas and carcinomas. The immunohistochemical results were confirmed by western blotting analysis. Tumors, papillomas and carcinomas overexpressing cyclin D1 had elevated proliferating cell nuclear antigen (PCNA) indices (P<0.05). The correlation coefficient of overexpressions of PCNA and cyclin D1 was r =0.7 for papillomas, but only r =0.3 for carcinomas. The study thus provides strong evidence of relatively early Overexpression of cyclin D1 during tumorigenesis in the present rat esophageal model. Cyclin D1 expression is not simply a direct consequence of increase cell proliferation.     

Enhancement of esophageal carcinogenesis in male F344 rats by dietary phenylhexyl isothiocyanate

The purpose of this study was to evaluate the potential effectsof dietary 6-phenylhexyl isothiocyanate (PHITC) on N-nitrosomethylbenzylamine(NMBA)-induced esophageal carcinogenesis in rats. Groups of15 male F344 rats received weekly s.c. injections of NMBA in20% dimethylsulfoxide or the vehicle alone for 15 consecutiveweeks. Two weeks prior to initiation of carcinogen or vehicleinjections rats were provided with modified AIN-76A diet ormodified AIN-76A diet containing PHITC at levels of 0.4, 1.0or 2.5 µmol/g diet. Experimental controls consisted ofgroups that received only the vehicle (vehicle controls), NMBA(carcinogen controls) or PHITC at the high dose level of 2.5µmol/g diet. No esophageal tumors or preneoplastic lesionswere detected in rats that received the vehicle or PHITC alone.In contrast, all rats treated with NMBA alone or PHITC + NMBAexhibited esophageal tumors and preneoplastic esophageal lesions.In groups that received PHITC + NMBA tumor multiplicity wasincreased by 21–69% when compared with rats treated withNMBA alone, indicating that PHITC enhanced esophageal tumorigenesisin this model system. These results, in conjunction with ourprevious work, demonstrate that arylalkyl isothiocyanates mayinhibit or enhance esophageal tumorigenesis in the NMBA-treatedrat. The ability of isothiocyanates to inhibit or enhance experimentaltumorigenesis may depend on alkyl chain length of the isothiocyanate,the animal species examined and the specific carcinogen employed.     

G→A mutations in p53 and Ha-ras genes in esophageal papillomas induced by N-nitrosomethylbenzylamine in two strains of rats

In human esophageal cancers, no ras gene mutations but a relatively high prevalence of p53 gene mutations have been reported. We found a high prevalence of point mutations in Ha-ras and p53 genes in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in two strains of rats (BD VI and F344). Our analysis showed the point mutation GGA←GAA (expected from the known mechanisms of action of NMBA) at Ha-ras codon 12 in 22 of 46 (48%) and 22 of 38 (58%) papillomas from BD VI and F344 rats, respectively. There was no significant difference in the prevalence of ras mutations in tumors induced by high doses (5.0 mg/kg) and low doses (2.5 mg/kg) of NMBA. Eleven papillomas from each strain were analyzed for p53 mutations. The prevalent mutations found were G←A and C←T transitions. The frequency of p53 mutation was 36% (four of 11) for each strain. No apparent hot-spot codon or exon was found in the p53 gene, and two papillomas contained double mutations in this gene. The high prevalence of G←A mutations in the rat Ha-ras gene contrasts with that in the human gene, in which no ras mutations have been found in primary tumors, and suggests either that the biology of esophageal carcinogenesis differs in humans and rats or that nitrosamines are not the major etiological risk factor for human esophageal cancers. © 1994 Wiley-Liss, Inc.     

Suppression of N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis by dietary feeding of auraptene

The modifying effects of auraptene on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis were investigated in male F344 rats. At 5 weeks of age, all animals, except those with the test chemical alone and control rats, received s.c. injections of NMBA (0.5 mg/kg body weight/injection, three times per week) for 5 weeks. At the end of the study (20 weeks), 75% of the rats treated with NMBA alone had esophageal neoplasms (papillomas). However, the groups who received a dose of 500 ppm auraptene during the initiation phase developed significantly reduced incidence of tumors (39%; P<0.05). Exposure to auraptene (500 ppm) during the post-initiation phase also decreased the frequency of the tumors (29%; P<0.01). The reduction of the incidence of severe dysplasia was obtained when auraptene was administered in the post-initiation phase (P<0.05). Cell proliferation in the esophageal epithelium determined by proliferating cell nuclear antigen (PCNA) was lowered by auraptene (P<0.01). Blood polyamine contents in rats who received NMBA and the test compound were also smaller than those of rats that received the carcinogen (P<0.05). These findings suggest that dietary auraptene is effective in inhibiting the development of esophageal tumors by NMBA when given during the initiation as well as post-initiation phases, and such inhibition is related to suppression of cell proliferation in the esophageal epithelium.     

Suppression of N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis by dietary feeding of 1'-acetoxychavicol acetate.

The modifying effects of 1'-acetoxychavicol acetate (ACA) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis were investigated in male F344 rats. At 5 weeks of age, all test animals, except those given the test chemical alone, and the control rats received s.c. injections of NMBA (0.5 mg/kg body weight/injection, three times per week) for 5 weeks. At the termination of the study (20 weeks), 75% of rats treated with NMBA alone had esophageal neoplasms (papillomas). However, the groups given a dose of 500 ppm ACA during the initiation phase developed a significantly reduced incidence of tumors (29%; P<0.01). Exposure to ACA (500 ppm) during the post-initiation phase also decreased the frequency of the tumors (38%; P<0.05). A reduction of the incidence of preneoplastic lesions (hyperplasia or dysplasia) was obtained when ACA was administered in the initiation phase (P<0.01). Cell proliferation in the esophageal epithelium, determined by assay of proliferating cell nuclear antigen (PCNA), was lowered by ACA (P<0.05). Blood polyamine contents in rats given NMBA and the test compound were also smaller than those of rats given the carcinogen (P<0.05). These findings suggest that dietary ACA is effective in inhibiting the development of esophageal tumors by NMBA when given during the initiation or post-initiation phase, and such inhibition is related to suppression of cell proliferation in the esophageal epithelium.     

Effects of dietary N-(4-hydroxyphenyl)retinamide on N-nitrosomethylbenzylamine metabolism and esophageal tumorigenesis in the Fischer 344 rat.

BACKGROUND: 9-cis-Retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive agents against epithelial tumors in the oral cavity, breast, and prostate. We tested the inhibitory activity of these retinoids against N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. METHODS: Male Fischer 344 rats were randomly assigned to receive diets either lacking or containing 9-cis-RA or 4-HPR for 1 week before tumor initiation with NMBA and then for the duration of the study. NMBA metabolism, O(6)-methylguanine adduct formation, and cytochrome P450 messenger RNA (mRNA) expression in the esophagi of the rats were studied to investigate the mechanisms by which dietary 4-HPR affects tumorigenesis. All statistical tests were two-sided. RESULTS: Dietary 4-HPR resulted in a dose-dependent and statistically significant enhancement (P<.05) of tumorigenesis in response to NMBA. In two different tumor bioassays, the mean tumor multiplicity for rats fed the highest concentration of dietary 4-HPR (0.8 g/kg diet) was increased by 5.9 tumors (95% confidence interval [CI] = 1.7 to 10.1 tumors) and 6.7 tumors (95% CI = 5.6 to 7.8 tumors) compared with the mean tumor multiplicity for rats that received the control diet lacking 4-HPR. Animals fed diets containing 9-cis-RA displayed no statistically significant increase in tumorigenesis. Compared with animals fed a diet lacking 4-HPR, animals fed 4-HPR had increased NMBA metabolism in esophageal explant cultures and had higher levels of O(6)-methylguanine DNA adducts and CYP2A3 mRNA in their esophagi. CONCLUSIONS: Dietary 4-HPR enhances tumorigenesis in response to NMBA in the rat esophagus by increasing tumor initiation events. Dietary 4-HPR may exert paradoxical effects at some sites, such as the aerodigestive tract, by modulating the bioactivation of carcinogens in target tissues.     

Dietary zinc deficiency fuels esophageal cancer development by inducing a distinct inflammatory signature

Chronic inflammation is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC). The causes of inflammation in ESCC, however, are undefined. Dietary zinc (Zn)-deficiency (ZD) increases the risk of ESCC. We have previously shown that short-term ZD (6 weeks) in rats induces overexpression of the proinflammatory mediators S100a8 and S100a9 in the esophageal mucosa with accompanying esophageal epithelial hyperplasia. Here we report that prolonged ZD (21 weeks) in rats amplified this inflammation that when combined with non-carcinogenic low doses of the environmental carcinogen, N-nitrosomethylbenzylamine (NMBA) elicited a 66.7% (16/24) incidence of ESCC. With Zn-sufficiency, NMBA produced no cancers (0/21) (P<0.001). At tumor endpoint, the neoplastic ZD esophagus, as compared with Zn-sufficient esophagus, had an inflammatory gene signature with upregulation of numerous cancer-related inflammation genes (CXC and CC chemokines, chemokine receptors, cytokines and Cox-2) in addition to S100a8 and S100a9. This signature was already activated in the earlier dysplastic stage. Additionally, time-course bioinformatics analysis of expression profiles at tumor endpoint and before NMBA exposure revealed that this sustained inflammation was due to ZD rather than carcinogen exposure. Importantly, Zn replenishment reversed this inflammatory signature at both the dysplastic and neoplastic stages of ESCC development, and prevented cancer formation. Thus, the molecular definition of ZD-induced inflammation as a critical factor in ESCC development has important clinical implications with regard to development and prevention of this deadly disease.     

Esophageal cancer prevention in zinc-deficient rats: rapid induction of apoptosis by replenishing zinc

BACKGROUND: Nutritional zinc deficiency in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors. Replenishing zinc with a zinc-sufficient diet reduces these effects in zinc-deficient (ZD) rats. We investigated whether apoptosis was involved in the reduction of NMBA-induced esophageal tumors when ZD rats consumed a zinc-sufficient diet. METHODS: Weanling rats were fed a ZD diet (zinc at 3-4 ppm) for 5 weeks to establish esophageal cell proliferation, then treated once with NMBA (2 mg/kg body weight), and divided into the following five groups (47-100 per group). One ZD group was fed the ZD diet, and four zinc-replenished (ZR) groups, ZR(1), ZR(24), ZR(72), and ZR(432), were fed a zinc-sufficient diet (zinc at 74-75 ppm) beginning 1, 24, 72, and 432 hours, respectively, after NMBA treatment. From 24 hours to 2 weeks after beginning a zinc-sufficient diet, esophagi from all ZR groups were analyzed for apoptosis and cell proliferation; ZD esophagi were the controls. Tumor incidence was determined 15 weeks after zinc replenishment. All statistical tests were two-sided. RESULTS: Zinc replenishment initiated shortly after NMBA treatment effectively reduced esophageal tumorigenesis; 8% (three of 37) of ZR(1), 14% (five of 37) of ZR(24), 19% (five of 26) of ZR(72), and 48% (19 of 40) of ZR(432) rats developed esophageal tumors compared with 93% (14 of 15) of ZD animals (all P<.001). Importantly, 24 and 30 hours after zinc replenishment, esophagi had numerous apoptotic cells (% apoptotic cells: 0 hour = 2.9%, 95% confidence interval [CI] = 2.5% to 3.3%; 24 hours = 9.4%, 95% CI = 8.2% to 10.6%), and the expression of the proapoptotic Bax protein doubled. Within 48 hours, the ZR(1) epithelium was three to five cell layers thick compared with 10-20 layers before zinc replenishment. CONCLUSIONS: Zinc replenishment of NMBA-treated ZD rats rapidly induces apoptosis in esophageal epithelial cells and thereby substantially reduces the development of esophageal cancer.     

Suppression of N-Nitrosomethylbenzylamine-induced Rat Esophageal Tumorigenesis by Dietary Feeding of 1'-Acetoxychavicol Acetate

The modifying effects of 1'-acetoxychavicol acetate (ACA) on N -nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis were investigated in male F344 rats. At 5 weeks of age, all test animals, except those given the test chemical alone, and the control rats received s.c. injections of NMBA (0.5 mg/kg body weight/injection, three times per week) for 5 weeks. At the termination of the study (20 weeks), 75% of rats treated with NMBA alone had esophageal neoplasms (papillomas). However, the groups given a dose of 500 ppm ACA during the initiation phase developed a significantly reduced incidence of tumors (29%; P < 0.01). Exposure to ACA (500 ppm) during the post-initiation phase also decreased the frequency of the tumors (38%; P < 0.05). A reduction of the incidence of preneoplastic lesions (hyperplasia or dysplasia) was obtained when ACA was administered in the initiation phase ( P < 0.01). Cell proliferation in the esophageal epithelium, determined by assay of proliferating cell nuclear antigen (PCNA), was lowered by ACA ( P < 0.05). Blood polyamine contents in rats given NMBA and the test compound were also smaller than those of rats given the carcinogen ( P < 0.05). These findings suggest that dietary ACA is effective in inhibiting the development of esophageal tumors by NMBA when given during the initiation or post-initiation phase, and such inhibition is related to suppression of cell proliferation in the esophageal epithelium.     

Cell proliferation and esophageal carcinogenesis in the zinc-deficient rat

Target cell proliferation was investigated throughout the developmentof esophageal cancer induced by N-nitroso-methylbenzylamine(NMBA) in weanhing rats maintained on zinc-deficient or sufficientdiets. Deficient rats were fed ad libitum, while zinc-sufficientrats were either pair-fed to the deficient animals or fed adlibitum. After 5 weeks, half of the animals in each dietarygroup were given six intragastric doses of NMBA (2 mg/kg; twiceweekly). The remaining rats were untreated by carcinogen. Atweeks 1, 2, 3, 4, 5, 7, 9 and 11 post first dose, esophagealcell proliferation was assessed in rats from each group by invivo bromodeoxyuridine (BrDU) labeling followed by immunohistochemicaldetection of cells in S-phase. At 11 weeks, the tumor incidencewas 100, 23 and 6%, respectively, in the zinc-deficient, zinc-sufficient,ad libitum and pair-fed groups. In vivo BrDU labeling revealedthat in the NMBA-untreated groups, the labeling index (LI),the number of labeled cells, and the total number of cells percross section of entire esophagi were significantly increasedby zinc deficiency at all time points; LI was lowest in zinc-sufficient,pair-fed rats. During NMBA treatment (weeks 6, 7 and 8), increasedcell proliferation occurred in both groups of zinc-sufficientesophagi but only during week 6 in the deficient ones. In theweeks following the cessation of NMBA treatment, zinc-deficientesophagi showed significantly increased LI and greater numberof labeled cells than the carcinogen treated, zinc-sufficientpair-fed or ad libitum fed groups. On the other hand, NMBA-treatedzinc-sufficient pair-fed rats showed lower LI and smaller numberof labeled cells than their zinc-sufficient ad libitum counterparts.Most importantly, esophageal papillomas were found in two zinc-deficientanimals that had received no NMBA treatment, after 10–11weeks of experimental diet These data support a direct relationshipbetween cell proliferation and tumor incidence, and also provideevidence that zinc deficiency and its associated cell proliferationcould be carcinogenic.     

Inhibition of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus by dietary freeze-dried strawberries

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 DNA-damaging 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% freeze-dried 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 NMBA-induced tumorigenesis in the rat esophagus.