Production of urothelial tumors in the heterotopic bladder of rat by benzidine derivatives

Male Fischer rats which had been implanted with a heterotopic bladder were randomly divided into five groups and their heterotopic bladders were instilled once a week for 20 weeks with 0.5 ml phosphate-buffered saline:dimethyl sulfoxide solution (4:1) or this solution containing 1 mumol benzidine (BZ), N'-hydroxy-N-acetylbenzidine, the N'-glucuronide of N'-hydroxy-N-acetylbenzidine, or the N-glucuronide of N-hydroxy-2-aminofluorene. These bladders were then instilled once a week for an additional 30 weeks with the phosphate-buffered saline without dimethyl sulfoxide. The experiment was terminated at the end of 50 weeks. Transitional cell carcinomas were observed in 1 of 39 (control), 1 of 29 (BZ), 18 of 30 (N'-hydroxy-N-acetylbenzidine), 28 of 28 (N'-hydroxy-N-acetylbenzidine N'-glucuronide), and 24 of 29 (N-hydroxy-2-aminofluorene N-glucuronide) rats. No histological alterations were observed in their natural bladders. These results demonstrate the urothelial carcinogenicity of the N-hydroxy metabolites of BZ and suggest that N'-hydroxy-N-acetylbenzidine N'-glucuronide may play a major role in the initiation of urothelial carcinogenesis by BZ in humans.     

Influence of dose of N-methyl-N-nitrosourea on the induction of urinary bladder cancer in rats

N-methyl-N-nitrosourea (MNU) was instilled by a urethral catheter into the urinary bladders of female Wistar rats in weekly doses of 0.5 mg for 1, 2, 3 and 4 weeks. At 75 weeks after the initial dose of MNU, the incidences of bladder cancer were 0, 7, 50 and 64% for the total doses of MNU of 0.5, 1.0, 1.5 and 2.0 mg, respectively. Control rats instilled with 0.9% NaCl only for 1--4 weeks did not develop bladder cancer by 75 weeks. Higher doses of MNU of 4.0 and 6.0 mg, given weekly in 0.5 mg amounts for 8 and 12 weeks, respectively, induced a higher incidence (nearly 90%) of urinary bladder cancer in rats at 22--28 weeks. However, it was shown that control rats given 12 weekly installations of solvent only developed a significant number (33%) of bladder cancers by 22--28 weeks.     

Marked enhancement of rat urinary bladder carcinogenesis by heat-killed Escherichia coli.

Chronic urinary tract infection is an important risk factor for the development of carcinoma in the human urinary bladder. To test the effect of chronic persistent inflammation on bladder carcinogenesis, we instilled heat-killed Escherichia coli (1 x 10(8) cells suspended in 0.5 ml of phosphate-buffered 2.1% NaCl solution) twice a week into the heterotopically transplanted rat urinary bladders in which carcinogenesis was initiated by a single dose (0.25 mg) of N-methyl-N-nitrosourea. When compared with the control animals, the rats treated with killed E. coli showed significantly enhanced bladder tumorigenesis, as reflected by an increase in the incidence of tumor (P = 0.05) and a 6- to 40-fold increase in the number of tumors per bladder (P less than 0.0001). The tumors were characterized by intraepithelial clusterings of neutrophils and by chronic inflammation and marked capillary proliferation in the tumor stroma. All of these features were rare in tumors in the control groups. The accelerated cell proliferation induced by killed E. coli treatment appears to play a significant role in the enhancement of tumorigenesis.     

Exogenous Epidermal Growth Factor Exerts Promoting Action during the Early Phase of Rat Urinary Bladder Carcinogenesis

Using the heterotopically transplanted rat urinary bladder (HTB) model that was developed in our laboratory, we examined the relationship between the duration of epidermal growth factor (EGF) treatment and acquisition of EGF-independence of urinary bladder tumors that were induced by EGF stimulation. After treatment with N -methyl- N -nitrosourea (MNU) (0.25 mg/0.5 ml of 0.9% NaCl once a week for 3 consecutive weeks), animals at week 3 received EGF [250 ng/ 0.5 ml phosphate-buffered saline (PBS)] into the HTBs once a week for 20, 28, or 36 weeks. For examination of the effect of EGF withdrawal, one half of the rats received the vehicle (PBS) only beginning at week 23 or week 31 for 8 weeks. When animals were examined at week 23, the incidence and the mean number of tumors per bladder were low, irrespective of EGF treatment. In the bladders that had been exposed to EGF during the first 20 weeks after MNU administration, however, both the incidence and the mean number of tumors per bladder had increased significantly at week 31, regardless of whether or not EGF treatment was continued beyond week 23. Between weeks 31 and 39, EGF treatment demonstrated no effect; both the incidence of tumors and the mean number of tumors were the same as those at week 31. These results suggest that EGF exerts its promoting effect only during the early phase of MNU-initiated bladder carcinogenesis, but that its effect becomes manifest during the subsequent 8 weeks. EGF independence may be due to establishment of an autocrine growth-stimulatory mechanism in bladder tumors.     

Effects of formalin-induced injuries on urinary bladder carcinogenesis

In multistage carcinogenesis, promotion is a long-term or repeated growth stimulation of initiated cells. Possible effects of regenerative hyperplasia induced by repeated intravesical instillation of 0.3% formalin solution on urinary bladder carcinogenesis were examined using heterotopically transplanted rat urinary bladders (HTBs) initiated by N-methyl-N-nitrosourea. The HTB system was chosen because in this system, transient generalized hyperplasia lasting less than a week can be induced readily and repeatedly by intravesical instillation of the formalin solution. No statistically significant tumor enhancement was observed after 15 formalin treatments administered in 30 weeks. It appears that regenerative cytotoxic stimuli, even multiple, may have no significant tumor-promoting activity. Discussed is the possibility that mild and, more importantly, persistent non-cytotoxic stimuli may be more effective as tumor promoters.     

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 ± 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.     

Comparative carcinogenicities of 1-, 2-, and 4-nitropyrene and structurally related compounds in the female CD rat

The comparative carcinogenicities of N-hydroxy-N-acetyl-1-aminopyrene, N-acetyl-1-aminopyrene, and 1-, 2-, and 4-nitropyrene were determined following i.p. injection into weaning female CD rats (67 mumol/kg body weight in dimethyl sulfoxide; 3 times/week for 4 weeks). At sacrifice 61 weeks after the first injection the incidences of malignant mammary tumors were increased significantly to 45 and 24% in the 4-nitropyrene- and N-hydroxy-N-acetyl-2-aminofluorene-treated groups, respectively. Cellular altered foci in the liver were increased significantly in the N-acetyl-1-aminopyrene-, N-hydroxy-N-acetyl-1-aminopyrene-, and N-hydroxy-N-acetyl-2-aminofluorene- treated groups; the latter two compounds also led to significantly increased formation of hyperplastic nodules in this organ. Significant increases in leukemia induction were observed in animals treated with 2-nitropyrene or N-hydroxy-N-acetyl-2-aminofluorene. In an experiment designed to compare the influence of the route of administration on the carcinogenic potential of this agent, 1-nitropyrene was injected i.p. or s.c. into weanling female CD rats (100 mumol/kg body weight; once a week for 4 weeks). The animals were sacrificed at 87 to 90 weeks after the first treatment. The incidences of mammary gland tumors in animals receiving injections of 1-nitropyrene by either route (59%) were significantly higher than in solvent-injected controls (37%). The incidences of adenocarcinoma in the i.p. 1-nitropyrene group (28%) and fibroadenoma in the s.c. 1-nitropyrene group (52%) were significantly higher than in the control animals (7 and 27%, respectively). These data suggest that the demonstration of the weak carcinogenicity of 1-nitropyrene is probably more a function of the length of the observation period than of the routes of administration used here. A further exploration of the effect of the route of administration involved treatment of weanling female CD rats by direct injection of 1-, 2-, or 4-nitropyrene into the mammary fat pads. A total of 2.04 mumol of the nitrocompound in dimethyl sulfoxide was injected into the mammary glands under each of the 6 left nipples. The right mammary glands were treated with the solvent only. Injections of the thoracic nipple areas were carried out on day 1; inguinal areas were treated on day 2. The animals were sacrificed after 77 weeks. The number of mammary tumor-bearing animals (23 of 28), the number with fibroadenoma (15 of 28), and the number with adenocarcinoma (19 of 28) were significantly increased in the 4-nitropyrene-treated group as compared with animals treated with only dimethyl sulfoxide.(ABSTRACT TRUNCATED AT 400 WORDS)     

膀胱内灌注吡柔比星预防膀胱癌术后复发的临床研究

目的研究吡柔比星膀胱灌注预防膀胱癌术后复发的疗效。方法自2002年1月至2004年12月对56例膀胱癌患者在经尿道膀胱癌电切术或膀胱部分切除术后,定期膀胱内灌注吡柔比星。每次用30mg,膀胱内保留半小时,术后第1周开始使用,每周1次,共6次;以后每2周1次,共6次;再以后每月1次,共2年。结果56例患者均随访观察2年以上,无肿瘤复发46例(82.2%),复发10例(17.9%),无全身毒副作用。结论吡柔比星膀胱内灌注预防膀胱癌术后复发疗效肯定,毒副作用小。     

Carcinogenesis in heterotopically-transplanted urinary bladder by N-methyl-N-nitrosourea: a model for initiation and promotion

The present investigation was conducted to establish a workingmodel to evaluate the role of potential modifiers (promotersand inhibitors) of urinary bladder carcinogenesis. A singledose (0.25 mg) of the carcinogen N-methyl-N-nitrosourea (MNU)when instilled directly into heterotopically transplanted urinarybladder (HTB) induced a 9% (2 of 22) incidence of urinary bladdertumors by 30 weeks in the urine free HTB environment, whereasrepeated instillation of urine into the HTB after MNU treatmentresulted in a 61% (14 of 23) incidence of these tumors. Threedoses of MNU (0.25 mg/dose once a week for 3 weeks) induced33% (6 of 18) and 89% (16 of 18) incidence of tumors, respectively,in the absence and presence of urine in the HTB. These dataconfirm our previous observation that normal urine enhancesurinary bladder carcinogenesis, and are consistent with thenotion that normal urine exerts a tumor promoting effect.     

Induction of high-grade, high-stage carcinomas in the rat urinary bladder

The hypothesis that biologic aggressiveness of bladder cancer is determined by carcinogen dose was tested using heterotopically transplanted rat urinary bladders (HTBs). Young male Fischer rats, which were recipients of normal bladders, were divided into three groups; the first group received 0.5 mg of N-methyl-N-nitrosourea (MNU) into HTBs for six doses, a second, 0.05 mg for six doses and the third, 1 mg for three doses. Separately, a group of animals received bladders from rats treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) in drinking water for 4 weeks; the transplanted bladders then were treated with 0.5 mg of MNU for six doses. Treatment with the larger dose of MNU resulted in a significant increase in tumor incidence and frequency of invasive carcinomas. The combination carcinogen treatment induced more invasive carcinomas than the single treatment. The data suggest that deeply invasive carcinomas may develop in two ways: the first is by emergence of a more anaplastic cell population within a pre-existing noninvasive carcinoma and the second is by the de novo development of an invasive carcinoma directly from a severely dysplastic urothelium, which is acceptable as carcinoma in situ. Squamous differentiation was characteristic of deeply invasive carcinomas. The dose of carcinogen(s) is a determinant of aggressiveness of bladder carcinomas.     

致敏树突细胞治疗膀胱肿瘤血液细胞毒性T淋巴细胞的变化

目的观察致敏的树突细胞（DC）治疗膀胱肿瘤（BT）后血液细胞毒性T淋巴细胞（CTL）的变化并探讨其机制。方法选取F344大鼠44只，称重后随机分为4组；均采用膀胱灌注N-甲基亚硝基脲（MNU）制成BT；第11周4组分别经皮下注射磷酸缓冲液（PBS）、未致敏DC、肿瘤抗原及致敏DC，每周1次，共4次；实验第15周，经显微镜和流式细胞仪（FCM）检测。结果致敏DC组膀胱重量比其他三组轻（P〈0．05）。致敏DC组BT病理分期低于PBS组和未致敏DC组（P〈0．05）；CD3^＋ T细胞在致敏DC组低于其余三组（P〈0．05）；CTL在致敏DC组高于其余三组（P〈0．001）。结论应用致敏DC经皮下注射治疗大鼠BT可降低肿瘤的病理分期，未致敏DC皮下注射对膀胱肿瘤治疗无效，肿瘤抗原皮下注射对膀胱肿瘤治疗效果欠佳。致敏DC可通过呈递抗原来激活CTL增生而发挥其免疫杀伤作用。     

Establishment of a Fluorescent Implantation Metastasis Model of Bladder Cancer and Real-time Microscopic Detection in Nude Mice

Objective: To establish a fluorescent implantation metastasis model of bladder carcinoma with high metastaticpotential in nude mice and observe development and metastasis. Methods: Human bladder cancer EJ cells withhigh invasive ability were screened and transfected with GFP plasmid to screen stable enhanced GFP-expressingclones instilled into the bladders of nude mice. Subsequent growth, invasion, and metastasis of the implantedtumors were observed and evaluated with a whole-body fluorescence optical imaging system. Results: Thetransfected bladder cancer EJ cells stably and efficiently expressed EGFP. The growth, invasion and metastasisof the implant bladder tumor were readily observed and accurately evaluated by fluorescent microscopy. Inthe bladders of nude mice, the rates of EGFP expression detected by flow cytometry at weeks 1-4 were 22.6%,46.7%, 62.3% and 72.7%, respectively, with clear increase over time. Conclusion: GFP-labeled bladder cancerEJ cells display green fluorescence under fluorescent microscopy and show stable GFP expression. The model willprovide a simple and reliable means for studying the mechanism of implantation metastasis of human bladdercancers in vivo.     

In vitro malignant conversion of low-grade rat urinary bladder carcinoma cells by exposure to N-methyl-N-nitrosourea

The cause of deeply invasive human bladder carcinoma is unknown. Animal studies suggest that a malignant (invasive) conversion is inducible in low-grade noninvasive tumors by further exposure to a chemical carcinogen. To elucidate what molecular mechanism(s) is involved in the conversion, an in vitro system has been established in which conversion from low- to high-grade carcinoma can be induced. A rat bladder carcinoma cell line D44, derived from an N-methyl-N-nitrosourea (MNU)-induced low-grade noninvasive rat bladder carcinoma was used in the present investigation. Cloned D44 cells (D44c) were exposed to MNU, 50 to 400 micrograms/ml, for 1 h at 37 degrees C once a week for up to 6 weeks. After exposure to MNU, cells with altered morphology were cloned. The yield of altered clones was highest after a total dose of 150 to 200 micrograms of MNU used in 1 to 3 doses. Of 21 clones with altered morphology, 4 clones were further treated with MNU at the initial dose once a week for up to 3 weeks and then subcloned. Thirty-three of these subclones were examined for tumorigenicity in athymic nude mice. Twenty-seven formed highly invasive carcinomas, mostly squamous type, whereas the parental D44c cells failed to develop tumors upon inoculation. Pulmonary metastases were observed in 17 of the 27 clones. Plasminogen activator activity was elevated 4- to 9-fold as compared to parent D44c cells. ras p21 mutations at codon 12 were detected in 5 of 30 clones. These results indicate that the in vitro system described here may provide a useful model to study the molecular mechanisms involved in the conversion of noninvasive bladder carcinomas to metastasizing ones.     

Conversion from low grade to high grade of rat urinary bladder carcinomas

The present study was conducted to test if low-grade carcinomas induced by a single dose of N-methyl-Nitrosourea (MNU) can be converted to high-grade carcinomas by a second identical dose of the carcinogen. The heterotopically transplanted rat urinary bladder system was used. Four wk after heterotopic bladder transplantation, the recipient male Fischer 344 rats were divided into 2 groups. The first group received 0.25 mg of MNU into heterotopically transplanted rat urinary bladder; the second group (controls) received 0.9% NaCl solution. At week 29 of the experiment, 1/3 of the animals from each group were killed for histological examination of the heterotopically transplanted rat urinary bladders. The remaining animals from each group were divided into 2 subgroups, the first receiving 0.25 mg MNU and the second, 0.9% NaCl solution. All animals were killed at 50 wk of the experiment. MNU-induced carcinomas at week 29 were all of low histological grade and were noninvasive. Longer follow-up without a second carcinogen administration resulted in both an increase in tumor incidence (P less than 0.005) and more tumors per bladder (P less than 0.001), but high-grade invasive carcinomas were rare. The second dose of MNU administered at the stage when low-grade carcinomas were prevalent (week 29) resulted in a significant increase in invasive high-grade carcinomas (P less than 0.01). Our data are consistent with the view that the second carcinogen administration induces a new mutation(s) within low-grade carcinomas which leads to invasive carcinomas.     

Differential induction of squamous cell carcinomas and adenocarcinomas in mouse lung by intratracheal instillation of benzo(a)pyrene and charcoal powder

Differential induction of squamous cell carcinomas, adenomas, and adenocarcinomas was observed in the lungs of male C57BL/6 and C3H/He mice after repeated intratracheal instillation of benzo(a)pyrene (BP) and charcoal powder suspended in 0.9% NaCl solution. when a high dose of BP (1.0 mg BP and 0.5 mg charcoal powder) was instilled intratracheally once a week for 8 weeks or when a low dose of BP (0.5 mg BP and 0.5 mg charcoal powder) was instilled once a week for 16 weeks, squamous cell carcinomas were induced in high incidence (77 to 87%) in the early period of observation, whereas pulmonary adenomas and adenocarcinomas were induced in low incidence (0. to 48%) in the late period of observation in both strains of mice. On the other hand, when a low dose of BP was instilled intratracheally once a week for 8 weeks, pulmonary adenomas and adenocarcinomas were induced in high incidence (76 to 91%), but squamous cell carcinomas were induced in low incidence (9 to 26%). These results show that a larger quantity of BP instilled intratracheally was needed for induction of squamous cell carcinomas than for induction of adenomas and adenocarcinomas in the lung of mice. Thus, when the carcinogen is administered to a single organ of a single mouse strain by the same route, different amounts of carcinogen have different effects on the incidences of various histological types of tumors.     

浅表性膀胱癌术后早期膀胱内灌注吡柔比星预防复发疗效观察

目的评价浅表性膀胱癌术后早期膀胱内灌注吡柔比星(THP)预防肿瘤复发的疗效和安全性。方法对18例浅表性膀胱癌患者行TUR-Bt或膀胱部分切除术,术后一周内开始30mgTHP/50ml蒸馏水膀胱内灌注,药物于膀胱内保留30分钟,每周一次,连续八周,以后每月一次,连续八个月。结果随访6～12个月,无一例复发,其中10例出现短时期的膀胱激惹症状,4例TUR-Bt术的患者创面修复延迟,其余病例均愈合良好。结论浅表性膀胱癌术后早期予THP膀胱内灌注预防肿瘤复发疗效肯定、安全可靠、对手术创面愈合影响不大。     

Monitoring therapeutic response to tamoxifen in NMU-induced rat mammary tumours by 31P MRS

Tamoxifen injections were given once a week for 4 weeks to 19 rats bearing N-methyl-N-nitrosourea (NMU)-induced mammary carcinomas. NMR spectra were collected on days 2, 7, 14, 21 and 28. Only 42% of the tumours responded to the tamoxifen in that they regressed significantly; another 21% did not change in size and 37% grew significantly. In the ones that did subsequently regress there were significant changes in the NTP/Pi ratio as early as 2 days after treatment, before any detectable change in volume was recorded, and continuing up to 21 days. The significance of these findings and the possible mechanisms underlying the changes are discussed.     

33例膀胱内灌注吡柔比星预防浅表性膀胱癌术后复发疗效观察

目的评价吡柔比星(THP)膀胱内灌注预防浅表性膀胱癌术后复发的近期疗效和不良反应。方法对33例浅表性膀胱癌患者行经尿道膀胱肿瘤电切术(TURBT),术后2周THP30mg膀胱灌注,每周1次,共8次;以后每月1次,共10个月,随访期间内行膀胱镜检查。结果30例可评价疗效,无肿瘤复发28例,复发2例,复发率为6.7%;发生不良反应者12例(40.0%),均为不同程度膀胱刺激症状。结论THP膀胱内灌注预防浅表性膀胱癌术后复发近期疗效满意,不良反应小,耐受性良好。     

Effect of leupeptin, a protease inhibitor, on induction of bladder tumors in rats by n-butyl-n-(4-hydroxybutyl)nitrosamine.

Leupeptin, isolated from Actinomycetes, is a potent and specific inhibitor of proteases. We found that the administration of leupeptin enhanced that size of urinary bladder tumors induced in rats by the oral administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). BBN was given as a 0.05% solution in the drinking water for 6 weeks, and then animals were fed a diet with or without 0.1% leupeptin for 30 weeks. The average weight of the bladders with tumors in rats fed a leupeptin diet was about eight times that of rats on a diet without leupeptin, though the incidences and average numbers of tumors in the bladders were similar in the two groups.     

Responses of rat urine and urothelium to bladder tumor promoters: possible roles of prostaglandin E2 and ascorbic acid synthesis in bladder carcinogenesis

An investigation of sequential changes in urine composition,levels of DNA synthesis and morphology of bladder epitheliumfollowing administration of the tumor promoters sodium ascorbate(AsA-Na) or butylated hydroxyanisole (BHA) and the non-promoterascorbic acid (AsA) for 36 weeks was performed. In addition,prostaglandin E₂ (PGE₂), cAMP and AsA content were assessedin bladder tissue after 16 weeks. While AsA-Na caused increasein pH, sodium content and volume, and a decrease in osmolalityof the urine throughout the study, these changes were not observedwith AsA administration which resulted in a decrease in urinarypH. BHA treatment was not associated with any urinary changes.AsA-Na brought about a significant elevation of DNA synthesisin the bladder epithelium from weeks 2 to 16 and was associatedwith simple hyperplasia at week 8, which, however, decreasedby week 16 and was no longer evident at weeks 24 and 36 whenDNA synthesis returned to normal. Under the scanning electronmicroscope (SEM), morphologic alterations of the urothelialsurface in rats given AsA-Na were observed at weeks 8 and 16,but the appearance at week 36 was almost normal. AsA did notcause any changes in these parameters at any time point. BHAinduced a significant elevation of DNA synthesis throughoutthe study, produced simple hyperplasia at week 36 and alterationsof the epithelial surface from weeks 4 to 36. Significant increasesof PGE₂ and AsA in bladder tissue were noted for the AsA-Naor BHA, but not AsA groups. Moreover, cAMP levels in bladdertissue of rats exposed to AsA-Na or BHA were slightly higherthan in the controls. The results suggest that changes in PGE₂,cAMP and AsA may be involved in promotion of rat bladder carcinogenesis.