Inhibitory effects of epigallocatechin-3-gallate on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats

The present study was conducted to assess the inhibitory effects of EGCG (epigallocatechin-3-gallate) on NMBA-induced rat esophageal tumorigenesis and to seek the potential mechanisms. In experiment I, 81 F344 rats were randomly divided into seven experimental groups according to the different regiments of NMBA 1 mg/kg subcutaneously (s.c.) and EGCG 4 mg/kg or 10 mg/kg orally or intraperitoneally (i.p.). The experiment was terminated at 24 weeks. In experiment II, 48 rats were allocated into two groups, each group contained 24 rats, in which the rats were injected with NMBA 1 mg/kg only or a combination of NMBA 1 mg/kg and EGCG 4 mg/kg i.p. Six rats from each group were sacrificed at the 12th, 16th, 20th and 24th week, respectively. The expression of cyclin D1 and cyclooxygenases (COX-2 and COX-1) was detected using semi-quantitative RT-PCR, and the production of prostaglandin E2 (PGE2) was measured by ELISA. In the groups which were treated with EGCG at a dose of 4 mg/kg i.p., or 10 mg/kg both orally and i.p., the mean number of tumors per rat was significantly reduced to 48, 56 and 61%, respectively (p<0.05). The incidence rate of esophageal carcinomas in the rats that were treated with EGCG 4 mg/kg i.p., was significantly lower than that in the rats which only received NMBA 1 mg/kg (p<0.05). The expression of cyclin D1 and COX-2, and the levels of PGE2 were also decreased by EGCG treatment. These results indicated that EGCG significantly inhibits the NMBA-induced rat esophageal carcinogenesis and it inhibitory effects may partly target cyclin D1 and COX-2 expression, and PGE2 production.     

Suppression of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in F344 rats by JTE-522, a selective COX-2 inhibitor

Recent studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) and elevation of COX-2-mediated synthesis of prostaglandin E(2) (PGE(2)) were observed in various cancers including esophageal cancer, but their roles in carcinogenesis of the esophagi still remain unclear. To address the issue, we observed the reduction of N:-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in rat esophagi via JTE-522 (4-[4-cyclohexyl-2-methyloxazol-5-yl]-2-fluorobenzenesulfonamide), a selective COX-2 inhibitor. In this study, 54 F344 male rats were divided into nine groups; JTE-522 (3, 9 and 30 mg/kg) was administered orally. We also examined the effects of JTE-522 on COX-2 mRNA and synthesis of PGE(2). In the group in which JTE-522 was administered intermittently at a daily dose of 30 mg/kg, the number of NMBA-induced esophageal tumors per rat significantly reduced, to 62% (P< 0.05), but the size of the tumors was not significantly inhibited. In the group in which JTE-522 was administered continuously five times weekly for 24 weeks at a daily dose of 9 mg/kg, both the number and size of tumors significantly reduced, to 29 and 44%, respectively (P<0.05). Furthermore, JTE-522 suppressed not only tumor formation but also developing carcinomas (P<0.0021) [corrected]. In this study, treatment with NMBA alone resulted in an approximately 5-fold rise in expression of COX-2 mRNA detected by semi-quantitative RT-PCR analysis and an approximately 7-fold increase in the production of PGE(2) measured by ELISA compared with the normal esophageal mucosa. The up-regulated COX-2 expression did not decrease with the treatment of JTE-522 at the 3, 9 and 30 mg/kg doses; however, the increased levels of PGE(2) synthesis were significantly decreased by administering JTE-522 (P<0.01). Our study suggests that COX-2-mediated PGE(2) is important in NMBA-induced esophageal tumorigenesis in rats, and therefore may be a promising chemotherapeutic target for the prevention and treatment of esophageal cancer, especially with selective COX-2 inhibitors.     

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.     

香加皮三萜类化合物对实验性大鼠食管癌的阻断作用及机制

目的探讨中药香加皮提取物三萜类化合物（TCCP）对甲基苄基亚硝胺（NMBA）诱导F344大鼠食管癌前病变形成的阻断作用及其机制。方法健康雄性F344大鼠90只,随机分为模型组、TCCP干预组、大豆油对照组和正常对照组。模型组大鼠皮下注射0.5 mg/kg NMBA,TCCP干预组大鼠同时给予0.5 mg/kg NMBA皮下注射及香加皮三萜类化合物20 mg/kg肌肉注每周给药3次,连续5周;大豆油对照组大鼠肌注大豆油1 ml/kg,正常对照组大鼠常规饲养。分别在给药后第9、15和25周,麻醉后解剖大鼠,HE染色后光镜下观察食管上皮组织病理学变化;用Western blot法检测GSK-3β和β-catenin蛋白表达水平;用RT-PCR法检测c-myc mRNA表达水平。结果(1)正常对照组及大豆油对照组在整个实验过程中未发现食管异常变化,模型组大鼠随诱癌时间延长食管病变逐渐加重。TCCP干预可缓解食管上皮的病变。(2)正常大鼠食管上皮中有少量β-catenin蛋白表达,模型组大鼠食管上皮β-catenin蛋白表达显著增强（P<0.05）;正常大鼠食管上皮GSK-3β的蛋白表达丰富,随着诱癌时间延长表达水平逐渐降低;与模型组相比,在诱癌9、15和25周时TCCP干预组大鼠食管上皮组织中β-catenin蛋白表达水平均显著下降（P<0.05）;而GSK3β蛋白表达显著升高（P<0.05）。(3)与正常对照组相比,各时间点模型组大鼠食管上皮c-myc mRNA表达水平均显著升高。与模型组相比,在诱癌9、15周时TCCP干预组大鼠食管上皮组织中c-myc mRNA表达水平均显著下降,而诱癌25周时差异无统计学意义（P>0.05）。结论TCCP可能通过促进Wnt信号分子GSK-3β的表达,抑制β-catenin蛋白的表达,进而下调下游靶基因c-myc的转录,干扰细胞周期转化,逆转细胞的分化,可能是其抑制食管癌变细胞生长机制之一。     

Weak enhancing effects of simultaneous ethanol administration on chemically induced rat esophageal tumorigenesis

Excessive alcohol consumption is associated epidemiologically with an elevated risk of esophageal cancer. In this study, we examined the effects of simultaneous administration of ethanol on N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. Groups 1-3 were treated with NMBA at a dose of 0.5 mg/kg body weight (high dose), and groups 4-6 received a dose of 0.1 mg/kg body weight (low dose), by s.c.-injection, 3 times per week for the first 5 weeks. Groups 1 and 4 were given ethanol free water as controls. Groups 2 and 5 were treated with 10% ethanol in their drinking water only at the time of NMBA treatment, while groups 3 and 6 were administrated the supplement continuously up to the end of the experiment. Macroscopically, with high dose NMBA-initiation, simultaneous 5-week and continuous 24-week ethanol administration demonstrated a tendency to increase the incidence and multiplicity of tumors, and also microscopically the multiplicity of papillary hyperplasias. In low dose groups, the incidence of esophageal papillary hyperplasias was significantly increased by continuous 24-week ethanol administration. Immunohistochemistry, proliferating cell nuclear antigen (PCNA) positive indices tended to be increased in tumors by simultaneous 5-week and continuous 24-week ethanol administration, but cyclin D1 expression was not affected. These data suggest that simultaneous ethanol administration have weak enhancing effects, and also promoting effects in post-initiation phase is present on NMBA-induced rat tumorigenesis.     

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.     

Promotion effects of hot water on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in F344 rats

This study is to determine the effects of hot water on N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis model. F344 rats received one treatment of hot water 1 ml/kg and NMBA 1 mg/kg, or a combination treatment of NMBA 1 mg/kg pus hot water 1 ml/kg, or/and EGCG (epigallocatechin-3-gallate) 10 mg/kg. The experiment was concluded at the 20th week. Our results showed that the number of tumors and incidence of carcinomas were significantly increased by hot water (65 degrees C) (p<0.05, p<0.03, respectively), as compared with the group which received NMBA injections only. EGCG treatment did not significantly reduce the number or the size of tumours as the temperature of added hot water increased. In addition, PGE2 production was induced by NMBA, and further significantly increased by added hot water (p<0.05). On the other hand, EGCG slightly decreased the elevated PGE2 production, however, this effect of EGCG was offset by hot water. Our study further confirmed that the drinking of hot beverages increased the risk of esophageal carcinogenesis, and the drinking hot tea will abolish the inhibitory effects of EGCG on this disease.     

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.     

Preventive effect of fermented brown rice and rice bran on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats

Modifying effect of fermented brown rice by Aspergillus Oryzae (FBRA) during the initiation or post-initiation phase of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis was investigated in rats. Five-week-old male F344 rats were divided into 7 groups, and groups 1-5 were given subcutaneous injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks starting at 7 weeks of age. Groups 2 and 4 were fed the diet containing 5 and 10% FBRA during the initiation phase, respectively, whereas groups 3 and 5 were fed these diets during the post-initiation phase. Group 6 was given the diet containing 10% FBRA throughout the experiment, and group 7 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 89% and 1.63+/-1.01/rat, respectively. Those of groups 3 (65%, 1.04+/-1.04) and 5 (58%, 0.77+/-0.86) were significantly less than those of group 1. Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (dysplasia) of group 5 were less than those of group 1. Post-initiation feeding of 10% FBRA significantly decreased BrdU incorporation in the non-lesional esophageal tissues when compared to that of the control. In addition, the analysis of expression levels of phase I enzymes of livers at the termination of experiment showed no clear differences among the groups. These observations indicate for the first time that FBRA inhibits NMBA-induced esophageal tumor development in rats possibly through inhibition of cell proliferation in the post-initiation phase, and suggest that FBRA is a promising dietary agent for prevention of human esophageal cancer.     

Effects of dietary phenethyl isothiocyanate, ellagic acid, sulindac and calcium on the induction and progression of N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats

The potential inhibitory effects of phenethyl isothiocyanate(PEITC), ellagic acid (EA), sulindac and supplemental dietarycalcium (SDC) on N-nitrosomethylbenzylamine (NMBA)-induced esophagealcarcinogenesis were evaluated in rats utilizing an abbreviated(5 week) NMBA treatment protocol which allowed administrationof the putative inhibitors throughout the experiment (i.e. beginning2 weeks prior to NMBA treatment) or following completion ofNMBA dosing only. PEITC at 500 p.p.m. significantly inhibitedtumor incidence and multiplicity when given before and during,but not following, NMBA treatment. Neither sulindac at 125 p.p.m.nor SDC (2% versus 0.5% in control diet) inhibited tumor developmentwhen given during or following NMBA treatment. EA, which wasadministered only following NMBA treatment, significantly reducedthe incidence (66.7% versus 100% in NMBA controls), but notthe multiplicity, of esophageal tumors at the high-dose (4000p.p.m.) level. Together these findings indicate that: (i) PEITCselectively inhibits the induction but not the subsequent progressionof NMBA-induced esophageal tumors; (ii) EA may repress esophagealtumor development when administered following NMBA treatment;(iii) at the doses administered, neither sulindac nor SDC possesssignificant inhibitory activity against NMBA-induced esophagealcarcinogenesis in the rat.     

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.     

香加皮三萜类化合物对甲基苄基亚硝胺诱导的食管癌大鼠调节性T细胞功能的影响

目的：探讨香加皮三萜类化合物（triterpenes compound of cortex periplocae,TCCP）对甲基苄基亚硝胺（N_nitrosomethylbenzylamine,NMBA）诱导的食管癌大鼠CD4^＋CD25^＋调节性T细胞功能的影响。方法：健康雄性F344大鼠40只,随机分为模型组（0.5mg/kg NMBA皮下注射）、治疗组（0.5mg/kg NMBA皮下注射及10mg/kg TCCP肌肉注射）、大豆油对照组（肌肉注射大豆油1ml/kg）和正常对照组,每组10只。各组每周给受试物3次,连续5周。分别在给受试物后第9周和第15周抽取4组大鼠外周血各1ml。用流式细胞仪检测大鼠外周血中CD4^＋T细胞和CD4^＋CD25^＋调节性T细胞（Tr细胞）的水平。另留取血清用ELISA法检测大鼠外周血白细胞介素IL-2、IL-10和转化生长因子TGF_β1的含量。结果：与正常对照组相比,NMBA给药后第9周和第15周,模型组外周血中CD4^＋T细胞比例均显著降低（P均〈0.05）,而CD4^＋CD25^＋Tr细胞比例均显著升高（P均〈0.05）。与同时期模型组比较,TCCP治疗组在给药后第9周和第15周外周血中CD4^＋T细胞比例升高,CD4^＋CD25^＋Tr细胞比例降低,差异均具有统计学意义（P均〈0.05）。NMBA给药后第9周和第15周,模型组大鼠外周血中IL-10、TGF-β1水平显著高于正常对照组（P均〈0.05）,经TCCP治疗后显著下降（P均〈0.05）;大鼠外周血中IL-2水平则呈现与之相反的趋势。结论：NMBA诱导大鼠食管癌后外周血中CD4^＋CD25^＋Tr细胞水平增加,TCCP可以通过抑制CD4^＋CD25^＋Tr细胞活化来纠正食管癌发生、发展过程中所致免疫细胞抑制状态,改善NMBA诱导的食管癌大鼠细胞免疫功能紊乱。     

香加皮三萜类化合物对大鼠食管癌增殖细胞核抗原表达的影响

目的:探讨中药香加皮提取物三萜类化合物(triterpenes compound of cortex periploeae,TCCP)对甲基苄基亚硝胺(N-nitrosomethylbenzylamine,NMBA)诱导的大鼠食管癌组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达的影响.方法:雄性F344大鼠120只,随机分为NMBA模型组、TCCP干预组、大豆油对照组和正常对照组.模型组大鼠皮下注射NMBA,TCCP干预组大鼠同时给予皮下注射NMBA及肌注TCCP,大豆油对照组大鼠肌注大豆油,正常对照组大鼠常规饲养.分别在给药后第9、15和25周,H-E染色检测大鼠食管上皮组织病理变化,免疫组织化学SP法检测大鼠食管组织中PCNA的表达.结果:正常对照组及大豆油对照组大鼠食管未发现异常变化,NMBA模型组大鼠9周时,食管癌前病变发生率为20.0％,15周时为46.7％,25周时达93.3％.与NMBA模型组相比,第9、15周时TCCP干预组癌前病变大鼠的比例明显降低(0、0 vs 20.0％、46.7％,P＜0.05).NMBA模型组第9、15和25周时,大鼠食管上皮组织PCNA表达水平显著高于正常对照组(213.17 ±29.74 vs 167.96 ±20.16,268.35±39.56 vs 170.76±14.79,327.24±28.19 vs 172.49 ±17.49;P＜0.05);与NMBA模型组相比,TCCP干预组大鼠食管上皮组织中PCNA表达水平显著降低(P＜0.05).结论:TCCP可抑制NMBA诱导的大鼠食管癌前病变,该作用可能与其抑制PCNA的表达有关.     

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.     

Evaluation of dose and treatment duration on the esophageal tumorigenicity of N-nitrosomethylbenzylamine in rats

N-Nitrosomethylbenzylamine (NMBA) is a potent esophagus-specificcarcinogen that has been utilized extensively in the study ofesophageal carcinogenesis in rats. While many studies have focusedon the pathogenesis of NMBA-induced esophageal tumors, the tumorigenicityof NMBA itself has not been thoroughly investigated in any single,systematic dose-response study. Therefore, in this study weevaluated NMBA tumorigenicity in rats following various short-terms.c. treatment regimens with the aim of developing an abbreviatedtreatment protocol which could be used in future studies. Toassess the possible correlation of basal cell proliferationwith NMBA tumorigenicity, we evaluated the expression of proliferatingcell nuclear-antigen (PCNA) in both control and NMBA-treatedrats. In rats which received a cumulative NMBA dosage of 7.5mg/kg over the course of 5 weeks, tumor incidence and multiplicitywere as follows: 40% with 0.4 ± 0.3 tumors/rat at week10; 100% with 2.2 ± 1.0 tumors/rat at week 20; and 100%with 2.3 ± 1.0 tumordrat at week 30. These rats exhibitedmarked increases in basal cell labeling, with indices that were1.5- to 1.8-fold higher than controls. NMBA treatment regimensof shorter duration with equivalent or higher cumulative dosageswere generally ineffective in producing esophageal tumors, eventhough significantly elevated levels of basal cell proliferationoccurred. Together, these findings indicate that the durationof NMBA treatment is of critical importance in the tumorigenicpotential of the carcinogen.     

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.     

Chemopreventive Effect of Curcumin on N-Nitrosomethylbenzylamine-induced Esophageal Carcinogenesis in Rats

Modifying effects of curcumin (derived from the rhizome of Curcuma longa L.) during the initiation or post-initiation phase of N -nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis were investigated in male F344 rats. Five-week-old rats were divided into 5 groups, and groups 1, 2 and 3 were given intraperitoneal injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks from 7 weeks old to induce esophageal neoplasms. Groups 2 and 3 were fed the diet containing 500 ppm curcumin during the initiation and post-initiation phases, respectively. Group 4 was given the diet containing curcumin throughout the experiment, and group 5 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 66.7% and 0.83 ±0.70, respectively. Those of groups 2 and 3 were significantly less than those of group 1 (39.3%, 0.46±0.64, P < 0.05; 33.3%, 0.36±0.56, P < 0.05, respectively). Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (moderate or severe epithelial dysplasia) of group 2 (57.1%, 0.61±0.57; 40%, 0.29±0.46) or 3 (56.7%, 0.67±0.66; 23.3%, 0.23±0.43) were less than those of group 1 (100%, 1.67±0.70; 70.8%, 0.92±0.72) ( P < 0.05). In this experiment, feeding of curcumin significantly decreased the expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling index) in the non-lesional esophageal epithelium ( P < 0.01). These findings indicate that curcumin inhibits NMBA-induced esophageal carcinogenesis when given during the post initiation as well as initiation phase. This inhibition may be related to suppression of the increased cell proliferation induced by NMBA in the esophageal epithelium.