Urinary Bladder Carcinogenesis Research Articles

Chemoprevention of mouse urinary bladder carcinogenesis by fermented brown rice and rice bran

Fermented brown rice by Aspergillus oryzae (FBRA) has been shown to be a potent anti-carcinogenic compound. Here, we investigated the modifying effects of dietary feeding with a naturally occurring anti-oxidant FBRA on N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN)-induced urinary bladder carcinogenesis in male ICR mice. Five-week-old male ICR mice were divided into 7 groups, and groups 1-5 were given OH-BBN (500 ppm) in drinking water for 6 weeks starting at 7 weeks of age. Groups 2 and 3 were fed the diet containing 5% and 10% FBRA during the initiation phase, respectively, whereas groups 4 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. At the end of the study (week 32), the incidences of simple hyperplasia, dysplasia and carcinoma in the bladders of group 1 (OH-BBN alone) were 92%, 49% and 38%, respectively. Those of group 5 (64%, 23% and 10%) and the incidence of carcinoma of group 4 (17%) was significantly less than that of group 1. Furthermore, the multiplicity of simple hyperplasia and carcinoma of group 5 was significantly less than that of group 1. Post-initiation exposure of 10% FBRA significantly decreased the number/nucleus of silver-stained nucleolar organizer region proteins (AgNORs), an index of cell proliferation, in the non-lesional transitional epithelium when compared to that of the control. Our results indicate that FBRA exerts chemopreventive effects against chemically induced urinary bladder carcinogenesis through anti-proliferative mechanisms. FBRA could be a promising chemopreventive agent for human urinary bladder cancer.

Chemopreventive effects of cyclooxygenase‐2 inhibitor and epidermal growth factor‐receptor kinase inhibitor on rat urinary bladder carcinogenesis

To examine the chemopreventive effects of a selective cyclooxygenase (COX)-2 inhibitor, meloxicam, and a selective epidermal growth factor (EGF)-receptor tyrosine kinase inhibitor, gefitinib (as a single agent) on a carcinogen-induced rodent bladder carcinogenesis model. The study comprised 103 male Fisher-344 rats (8 weeks old); after initial carcinogen treatment for 8 weeks with 0.05%N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, the rats were divided into five groups, i.e. group 1, control (vehicle only); group 2, gefitinib high-dose (15 mg/kg by gavage once daily); group 3, gefitinib low-dose (5 mg/kg); group 4, meloxicam high-dose (1.8 mg/kg by gavage once daily); and group 5, meloxicam low-dose (0.6 mg/kg). Twelve weeks later the rats were killed; after fixing the bladder in 10% formalin, the number and size of hyperplasia and carcinoma foci were recorded microscopically in sections stained with haematoxylin and eosin, submitted entirely as multiple strips. The incidence of carcinoma, confirmed microscopically, was: control 14/20 (70%); high-dose gefitinib, 7/20 (35%); low-dose gefitinib, 7/20 (35%); high-dose meloxicam 7/21 (33%); and low-dose meloxicam, 12/20 (60%). The mean numbers of carcinomas per bladder in groups 1-5 were 1.2, 0.5, 0.4, 0.5 and 1.1, respectively. The incidence and the mean number of carcinomas per bladder were significantly lower in the treatment groups (P < 0.05) than in the control group, except in the low-dose meloxicam group. There were no significant adverse effects. Both meloxicam and gefitinib have inhibitory effects on rat bladder carcinogenesis with no significant adverse effects. A combination of these drugs would be worth studying for their synergistic effects.

Dietary administration with prenyloxycoumarins, auraptene and collinin, inhibits colitis‐related colon carcinogenesis in mice

We previously reported the chemopreventive ability of a prenyloxycoumarin auraptene in chemically induced carcinogenesis in digestive tract, liver and urinary bladder of rodents. The current study was designed to determine whether dietary feeding of auraptene and its related prenyloxycoumarin collinin can inhibit colitis-related mouse colon carcinogenesis. The experimental diets, containing the compounds at 2 dose levels (0.01 and 0.05%), were fed for 17 weeks to male CD-1 (ICR) mice that were initiated with a single intraperitoneal injection of azoxymethane (AOM, 10 mg/kg body weight) and promoted by 1% (w/v) DSS in drinking water for 7 days. Their tumor inhibitory effects were assessed at week 20 by counting the incidence and multiplicity of colonic neoplasms and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA)-labeling index, apoptotic index, cyclooxygenase (COX)-2, inducible nitric oxide (iNOS) and nitrotyrosine in colonic epithelial malignancy. Feeding with auraptene or collinin, at both doses, significantly inhibited the occurrence of colonic adenocarcinoma. In addition, feeding with auraptene or collinin significantly lowered the positive rates of PCNA, COX-2, iNOS and nitrotyrosine in adenocarcinomas, while the treatment increased the apoptotic index in colonic malignancies. Our findings may suggest that certain prenyloxycoumarins, such as auraptene and collinin, could serve as an effective agent against colitis-related colon cancer development in rodents.

Susceptibility of Four F1 Hybrids of Male Rats to the Promoting Effects of Sodium L-ascorbate in Two-Stage Urinary Bladder Carcinogenesis

In the two-stage rat urinary bladder carcinogenesis model with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as an initiator and sodium L-ascorbate (Na-AsA) as a promoter, we previously reported that F344/DuCrj (F344) and LEW/Crj (Lewis) rats were sensitive, whereas WS/Shi (WS) and ODS/Shi-od/od (ODS) rats were resistant. In the present study, for the development of a model useful to the QTL analysis of host genes, we examined the susceptibility to Na-AsA promotion in (F344 × WS)F1, (F344 × ODS)F1, (Lewis × WS)F1, and (Lewis × ODS)F1 hybrids of male rats. Rats were given 0.05% BBN in their drinking water for 4 weeks and then a basal diet with or without a 5% Na-AsA supplement for 32 weeks. In urine of all F1 hybrids, Na-AsA elevated pH and concentrations of sodium ion and total ascorbic acid. There were not significant differences for susceptibilities to BBN alone among the four F1 hybrids. In all F1 hybrids, administration of Na-AsA increased urinary bladder carcinogenesis when compared to the matched control rats given BBN alone. Susceptibilities to Na-AsA promotion were high in (F344 × WS)F1 and (F344 × ODS)F1 hybrids, whereas they were mild in (Lewis × WS)F1 and (Lewis × ODS)F1 hybrids. The present results therefore indicate that F1 hybrids among the F344, ODS, Lewis and WS strains may be a useful model for analyzing host genes susceptible to Na-AsA promotion in rat bladder carcinogenesis.

Transitional cell carcinomas and nonurothelial carcinomas of the urinary bladder differ in the promoter methylation status of the caveolin-1, hDAB2IP and p53 genes, but not in the global methylation of Alu elements

Tumor suppressor genes play a prominent role in the modification and progression of urinary bladder carcinogenesis as a result of classic genetic alterations. Little is known about the potential significance of epigenetic events, mediated by DNA hypermethylation. This prompted our investigation to explore the global Alu methylation and the promoter methylation of the novel putative tumor suppressor genes caveolin-1 and hDAB2IP, and of p53 in transitional cell carcinomas (TCC), squamous cell carcinomas and undifferentiated small cell carcinomas of the urinary bladder. Quantitative GeneScan analysis revealed that the various histopathological tumor entities showed considerable interindividual variations in the global methylation, but the overall rate did not significantly differ between the various cancer subtypes. With methylation-specific PCR, a high frequency of methylation of the promoter region of the caveolin-1 gene was detected in undifferentiated small cell carcinomas (50%) and in squamous cell carcinomas (25.9%), while TCC were found not to be methylated. By immunohistochemistry, all squamous cell carcinomas showed a strong diffuse overexpression of caveolin-1, whereas undifferentiated small cell cancers lacked any expression. High-grade, high-stage TCC disclosed a higher incidence (60%) and a substantially stronger expression than low-grade, low-stage TCC (42.9%). Our findings suggest that hypermethylation of the caveolin-1 gene and an abnormal protein expression play a crucial role in cell differentiation, and in the phenotypical conversion of TCC into nonurothelial carcinomas. Promoter methylation of the hDAB2IP gene occurred more frequently in advanced muscle invasive (72.7%) than in superficial noninvasive (50%) TCC. DNA hypermethylation of p53 was detected in a quarter of the low-grade, low-stage TCC and undifferentiated small cell carcinomas, but only sporadically in squamous cell carcinomas, and was absent in high-grade, high-stage TCC. In conclusion, aberrant methylation and abnormal protein expression of the caveolin-1-gene is involved in the formation of nonurothelial carcinomas of the urinary bladder and promoter methylation of the hDAB2IP gene in the progression of TCC from a low to a high malignant potential.

Aberrant expression of E‐cadherin and β‐catenin in association with transforming growth factor‐β1 in urinary bladder lesions in humans after the Chernobyl accident

This study examines the molecular pathways of cell-cell communication in chronic inflammatory processes associated with long-term low-dose urinary bladder exposure to ionizing radiation in people without major disease living more than 19 years in radio-contaminated areas of Ukraine after the Chernobyl accident. Patterns of components of the E-cadherin/beta-catenin complex, and transforming growth factor-beta1 (TGF-beta1) and inducible nitric oxide synthase (iNOS) expression were immunohistochemically evaluated in urinary bladder biopsies from 52 males with benign prostate hyperplasia and 8 females with chronic cystitis (group 1). For comparison, 25 males and 6 females living in non-contaminated areas of Ukraine were also investigated (group 2). Fourteen patients with primary urothelial carcinomas, which were operated on before the Chernobyl accident, were included as a carcinoma group. Chronic proliferative atypical cystitis ('Chernobyl cystitis') was observed in group 1 patients. Foci of dysplasia and carcinoma in situ were found in 51 (85%) and 34 (57%) of the 60 cases, respectively. Chronic cystitis with areas of dysplasia was detected in only 4 (13%) cases of 31 group 2 patients. Statistically significant differences in immunohistochemical scores for TGF-beta1 in the urothelium and lamina propria, iNOS in the urothelium and both beta-catenin and E-cadherin in the cytoplasm were observed between groups 1 and 2 with marked expression in group 1. Furthermore, TGF-beta1 overexpression and alteration in E-cadherin/beta-catenin complexes in bladder urothelium might play a crucial role in urinary bladder carcinogenesis in humans exposed to long-term low-dose ionizing radiation.

Differences in susceptibility to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine‐induced urinary bladder carcinogenesis between SD/gShi rats with spontaneous hypospermatogenesis and SD/cShi rats with spontaneous hydronephrosis

Differences in susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis between two substrains of male Sprague-Dawley rats were examined. One substrain was SD/gShi, which has spontaneous hypospermatogenesis, and the other was SD/cShi, which is a sister strain of SD/gShi, and has normal testis but spontaneous hydronephrosis. SD/gShi rats had a lower incidence of urinary bladder tumors and had lower 5-bromo-2'-deoxyuridine labeling indices in the urinary bladder epithelium than SD/cShi rats when BBN was given. SD/gShi rats had significantly lower urinary concentrations of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which is a metabolite and proximate carcinogen of BBN. In vitro analysis also showed significantly less BCPN formation, using an S9 mix derived from the liver and kidney, in SD/gShi rats than in SD/cShi rats. BCPN formation in vitro was markedly inhibited by non-selective cytochrome P450 (CYP) inhibitors, but not alcohol dehydrogenase inhibitor. However, analysis of CYP proteins including hepatic CYP1A1/2, 2B1/2, 2E1, and 3A2 and renal CYP2E1 and 3A2 revealed no significant variation in levels in either tissue in the groups. There were also no significant intergroup differences in the mutagenicity of carcinogens, including heterocyclic amines and N-nitrosamines, activated by CYP1A1/2 and CYP2E1 and/or CYP2B1/2, respectively. These results suggest that SD/gShi rats are less susceptible to BBN, possibly because less BCPN is produced by CYP isoforms other than those investigated. A contribution of CYP4B1 to the strain difference is also possible.

Modification of N-Methyl-N-Nitrosourea initiated bladder carcinogenesis in Wistar rats by terephthalic acid

The effect of terephthalic acid (TPA) on urinary bladder carcinogenesis was examined. Male Wistar rats were initiated by injection of N-Methyl-N-Nitrosourea (MNU) (20 mg/kg b.w. ip) twice a week for 4 weeks, then given basal diet containing 5% TPA, 5% TPA plus 4% Sodium bicarbonate (NaHCO3) or 1% TPA for the next 22 weeks, and then euthanized. 5% TPA treatment induced a high incidence of urinary bladder calculi and a large amount of precipitate. Though 5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment did not induce urinary bladder calculi formation, they resulted in a moderate increase in urinary precipitate. Histological examination of urinary bladder revealed that MNU-5% TPA treatment resulted in a higher incidence of simple hyperplasia, papillary or nodular hyperplasia (PN hyperplasia), papilloma and cancer than MNU control. MNU-5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment increased slightly the incidence of simple hyperplasia and PN hyperplasia (not statistically significant). The major elements of the precipitate are phosphorus, potassium, sulfur, chloride, calcium and TPA. The present study indicated that the calculi induced by TPA had a strong promoting activity on urinary bladder carcinogenesis and the precipitate containing calcium terephthalate (CaTPA) may also have weak promoting activity on urinary bladder carcinogenesis.

Erratum to Sugie et al., “Suppressive effect of 1,4‐phenylene diisothiocyanate on N‐butyl‐N‐(4‐hyroxybutyl)nitrosamine‐induced urinary bladder carcinogenesis in male 1CR mice.” Int J Cancer 2004;117:published online 31 May 2005; DOI:10.1002/ijc.21233

AbstractThe original article to which this Erratum refers was published in International Journal of Cancer (2005) 117(4) DOI:10.1002/ijc.21233 DOI: 10.1002/ijc.21233

767: NRF2 is Essential for the Chemopreventive Efficacy of Oltipraz Against Urinary Bladder Carcinogenesis

767: NRF2 is Essential for the Chemopreventive Efficacy of Oltipraz Against Urinary Bladder Carcinogenesis

Promoter Hypermethylation: A New Therapeutic Target Emerges in Urothelial Cancer

Significant changes in the global levels and regional patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancers. 1 Alterations in DNA methylation have a direct impact on both the mutational and epigenetic components of neoplastic transformation.ImportanteffectsofDNAmethylationonthe genome include mutational burden of 5-methylcytosine, epigenetic effects of promoter methylation on gene transcription and potential gene activation, and induction of chromosomal instability by DNA hypomethylation. 2,3 Hypermethylationofcytosine-guaninedinucleotide(CpG)islands is associated with transcriptional repression, whereas hypomethylation may lead to increased potential for gene activity or chromosomal instability. CpG islands located in tumor suppressor gene promoters are normally unmethylated. Abnormal methylation of these regions may lead to progressive reduction in gene expression, thus transcriptionally silencing suppressor genes by a variety of mechanisms, including remodeling of local chromatin structure or inhibition of transcription factor binding, ultimately altering normal cellular growth properties. Application of novel molecular biologic techniques has increased our appreciation of the widespread changes in methylation patterns that occur during urinary bladder carcinogenesis. Epigenetic events, such as methylation, occur throughout all stages of tumorigenesis, including the early phases, and are increasingly recognized as major mechanisms involved in silencing tumor suppressor genes. A large number of genes have been reported to be hypermethylated in bladder cancer. The frequency of such events suggests that widespread alterations in the patterns of DNA methylation are common in bladder carcinogenesis. Some examples include the p16 gene (at CDKN2A INK4a locus on chromosome 9p21), E-cadherin gene (CDH1, encoding a transmembrane glycoprotein that modulates calcium-dependent intercellular adhesion), and RASSF1A gene (ras association domain family gene 1, isoform A). 4-6 Diminished CDH1 expression may lead to increase in betacateninactivityandproliferationinurothelialcarcinomas. 7 Aside from deletion and mutation, the p16 gene has been shown to be inactivated by promoter methylation. 8-11 Mutationordeletionofonep16alleleandhypermethylationof the remaining allele may be sufficient for loss of functional activity. Progressive inhibition of p16 expression by DNA methylation may result in loss of adequate growth regulatory function. Methylation of p16 has been observed in 27% to 60% of primary urothelial carcinomas. 12,13 Both

Suppressive effect of 1,4-phenylene diisothiocyanate onN-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in male ICR mice

The modifying effects of dietary administration of 1,4-phenylene diisothiocyanate (DITC) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis during the initiation and post-initiation phases were examined in male ICR mice. Five-week-old animals were divided into 5 groups. Groups 1-3 were given BBN (500 ppm) in drinking water for 6 weeks starting at age 6 week. Mice in Group 2 were given the diet containing 100 ppm DITC for 8 weeks during the initiation phase, starting 1 week before BBN exposure. Animals in Group 3 were fed the experimental diet for 24 weeks during the post-initiation phase starting 1 week after the cessation of BBN exposure. Mice in Group 4 were given only the diet containing the test compound, and those in Group 5 were given the basal diet alone throughout the experiment (32 weeks). The frequency of bladder lesions, neoplasms, dysplasia and hyperplasia, was analyzed histopathologically. The cell-proliferation activity estimated by the 5-bromodeoxyuridine labeling index (BrdU-LI), and cell cycle progression by counting cyclin D1-positive cell ratios were compared among the groups using immunohistochemistry. Administration of DITC in the initiation phase reduced significantly the incidence of urinary bladder carcinoma and dysplasia. The frequencies of any lesions of urinary bladder were not reduced by DITC in post-initiation phase. Dietary exposure of this agent in initiation phase reduced significantly both BrdU-LI and cyclin D1-positive cell ratios in any bladder lesions. Administration of DITC in post-initiation phase also significantly reduced BrdU-LI in bladder neoplasms and hyperplasia and cyclin D1-positive cell ratios in urinary bladder carcinoma as well as dysplasia. These results suggest that dietary DITC could be a preventive agent against BBN-induced bladder carcinogenesis in mice when fed during the initiation phase.

Influence of Strain and Diet on Hepatocarcinogenicity of N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in Rats: Slight Inhibition of Preneoplastic Liver Lesion Development by Uracil

To assess sensitivity to hepatocarcinogenesis by BBN, formalin-fixed and paraffin-embedded rat liver tissues from two initiation/promotion (I/P) bioassays for urinary bladder carcinogenesis were sectioned, immunohistochemically stained for glutathione S-transferase placental form (GST-P), and quantitatively analyzed. In Experiment 1, male Sprague-Dawley (SD)/cShi strain rats (highly sensitive to urinary bladder carcinogenesis by BBN) and SD/gShi rats (insensitive), were treated with 0.05% BBN in the drinking water for 4 weeks and then housed for up to week 36, with or without exposure to bladder tumor promoters. Slightly higher quantitative values for small GST-P positive (GST-P+) foci were found in SD/gShi rats (8.57/cm2) compared to SD/cShi rats (2.48/cm2). In Experiment 2, F344 and Lewis rats, subjected to a similar I/P protocol, were maintained on MF or CA-1 diet for 36 weeks. Higher quantitative values of GST-P + hepatocytic foci (more than 0.1 mm in diameter) were found in Lewis rats (9.51/cm2 for MF diet; 7.95/cm 2 for CA-1 diet) compared to F344 rats (2.82/cm2 and 1.30/cm2, respectively). Slight inhibiting effects of GST-P+ foci were apparent in rats receiving uracil, but not sodium L-ascorbate, in the present experiment. Thus, it was confirmed that the bladder carcinogen BBN also targets the liver. The results, from quantitative analysis of small GST-P+ foci as end-point marker lesions, indicate considerable strain differences, but that liver tumor modifying potential of test chemicals can be evaluated in I/P protocols for urinary bladder carcinogenesis using any strain of rat.

High Susceptibility of Human c-Ha-ras Proto-oncogene Transgenic Rats to Carcinogenesis

Transgenic animals carrying human c-Ha-ras proto-oncogene, v-Ha-ras transgenic mice, pim-1 transgenic mice have been shown to exhibit increased carcinogen susceptibility. Based on this characteristics, studies aimed for application to medium-term screening with from 20 to 30 weeks duration for various environmental carcinogens are under way in many institutes. On the other hand, rat models are advantageous by larger organ size, abundant information regarding preneoplasias and virus-free constitution, we have generated transgenic rats carrying copies of the human c-Ha-ras proto-oncogene (Hras128) and shown to be extremely sensitive to mammary, and to a lesser extent, lesions in the urinary bladder, oesophagus and skin carcinogenesis. In most of mammary carcinomas, mutations of the transgene but not the endogenous H-ras gene are present, appearing to occur early in the process of carcinogenesis, which involves proliferation of cells in terminal end buds (TEBs) . Further findings suggest that this is independent of endogenous ovarian hormones, although inhibited by soy isoflavones. Although further studies of mechanistic aspect are clearly necessary, the model appears to have great potential for short-to long-term screening purposes, not only for modifiers active in the breast, but also other organs where tumors develop characterized by ras gene mutations.

Expression of Survivin in transitional cell carcinoma of urinary bladder and its clinical significance

Survivin is a member of the inhibitors of apoptosis protein (IAP) family. Recent researches had shown that survivin plays an important role in oncogenesis. This study was designed to investigate the expression of survivin in transitional cell carcinoma (TCC) of urinary bladder and its clinical significance. Immunohistochemical assay was used to detect the expression of survivin in 75 cases of tumor tissue and 7 cases of normal bladder tissue. The expression rates of survivin were 77.3% (58/75) in TCC of urinary bladder, whereas no expression of survivin was detected in the 7 cases of normal bladder tissue. Expression of survivin protein was observed in the tumor tissue derived from the patients with bladder TCC, indicating that this protein may play an important rule in carcinogenesis of human urinary bladder. The expression of survivin was statistically significant associated with tumor grade. Our results suggested that the expression of survivin may be considered as a prognostic factor for bladder TCC.

Alteration of the MDM2-p73-P14ARF pathway related to tumour progression during urinary bladder carcinogenesis

Transitional cell carcinomas (TCC) of the urinary bladder develop by a multistep process characterized by various stages of transformation differing in their grade of malignancy and biological behaviour. Since the prospective clinical outcome cannot be reliably predicted on histopathological grounds, we analysed the mRNA expression of the MDM2-p73-P14ARF tumour surveillance pathway in an attempt to detect alterations of gene activity, allowing a better understanding of the mechanisms responsible for conversion of low to high malignant TCC. Expression of the mRNA was determined in 71 TCC of various grades and stages using the real-time quantitative reverse transcription-polymerase chain reaction. The MDM2-p73-P14ARF pathway was dominated by the MDM2 gene, the mRNA expression of which proved to be significantly (5-fold) lower in advanced high-grade, high-stage than in superficial low-grade, low-stage TCC. Conversely, the expression of p73 mRNA increased with increasing tumour grades and stages, while the activity of the P14ARF gene was not substantially altered during early and late phases of urothelial carcinogenesis. Analysing the expression of spliced variants of MDM2 mRNA, we found a heterogeneous pattern including a novel splicing transcript coding for an abnormal protein. Promoter hypermethylation of P14ARF occurred in 10% of the TCC with an under-expression of mRNA. An analysis of the effects of lifestyle and occupational bladder cancer risk factors revealed that TCC of smokers showed a 2-fold elevated expression of MDM2 mRNA and an approximately 2-fold lower expression of P14ARF mRNA, whereas the activity of the p73 gene was unchanged. Heavy coffee consumption was associated with a 2-fold decreased expression level of P14ARF mRNA. Exposure to certain occupational hazards (plastic products, paints and lacquer, polycyclic hydrocarbons, chemical solvents) was observed to modulate the activity of the genes analysed. Our findings suggest that an alteration in the MDM2-p73-P14ARF pathway is involved in the progression of bladder cancer to a more malignant and aggressive form.

Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis.

The induction of phase 2 detoxifying enzymes, such as UDP-glucuronosyltransferases (UGTs), in response to an array of naturally occurring and synthetic agents, such as oltipraz (4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione), provides an effective means of protection against a variety of carcinogens. Transcription factor Nrf2 is an essential regulator of the inducible expression of detoxifying enzyme genes by chemopreventive agents. In this study, we investigated in Nrf2-deficient mice the susceptibility to the urinary bladder-specific carcinogen N-nitrosobutyl(4-hydroxybutyl)amine (BBN) and the chemopreventive efficacy of oltipraz. The incidence of urinary bladder carcinoma by BBN was significantly higher in Nrf2-/- mice than in wild-type mice; invasive carcinoma was found in 24.0 and 38.5% of wild-type and Nrf2-/- mice, respectively. Oltipraz induced the phase 2 enzymes responsible for BBN detoxification in the liver and urinary bladder in an Nrf2-dependent manner. As expected, therefore, oltipraz decreased the incidence of urinary bladder carcinoma by BBN in wild-type mice but had little effect in Nrf2-/- mice. In wild-type mouse liver, oltipraz significantly induced BBN glucuronidation and decreased the urinary concentration of N-nitrosobutyl(3-carboxypropyl)amine, a proximate carcinogen of BBN. Importantly, BBN was found to suppress the expression of UGT1A specifically in the urinary bladder. This suppression was counteracted by oltipraz in wild-type mice but not in Nrf2-/- mice. These results show that Nrf2 and its downstream target genes are responsible for BBN detoxification. Furthermore, oltipraz prevents carcinogenesis by BBN by enhancing detoxification of this carcinogen in the liver and urinary bladder.

Limited tumor-initiating activity of phenylethyl isothiocyanate by promotion with sodium l-ascorbate in a rat two-stage urinary bladder carcinogenesis model

Initiation activity of phenylethyl isothiocyanate (PEITC) was examined in a two-stage urinary bladder carcinogenesis model. Male 6-week-old Fischer 344 rats were fed diet containing 0.1% PEITC for 12 or 24 weeks, with or without subsequent administration of 5% sodium l-ascorbate (Na-AsA) in diet until week 48, or for the entire experimental period. After 12 weeks of PEITC-treatment, both simple hyperplasia and papillary or nodular (PN) hyperplasia had developed in all animals, but the majority of these lesions had disappeared at week 48, irrespective of the Na-AsA-treatment. The same lesions after 24 weeks of PEITC-treatment had progressed to dysplasia and carcinoma, in a small number of cases by week 48 (6% in incidence for each lesion), but enhancement by the Na-AsA-treatment was evident only with simple hyperplasias (from 56 to 100% in incidence) and PN hyperplasias (from 19 to 56%). The results suggest a limited initiation activity of PEITC with induction of irreversible lesions by 24 weeks of exposure.

Significance of overexpression of metallothionein in mouse urinary bladder focal lesions induced by treatment with N-butyl-N-(4-hydroxybutyl)-nitrosamine.

Metallothionein (MT) is expressed in various types of human tumors, including transitional cell carcinomas of the urinary bladder, but its biological significance remains unclear. In the present study, the role of MT in urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) treatment was investigated using C57BL/6 mice. One hundred 5-week-old male C57BL/6 mice were divided into two groups, which were given drinking water with or without 0.05% BBN throughout the experimental period. Subgroups of ten animals from each group were sacrificed at weeks 5, 10, 15, 20 and 25, and urinary bladder samples were examined immunohistochemically for MT, proliferating cell nuclear antigen (PCNA) and apoptosis. MT was found to be abundant in normal-looking mucosa, but decreased with progression from precancerous lesions to invasive carcinoma in the urinary bladder obtained from BBN-treated mice. Lesions could be divided into MT-positive and negative. There was a tendency for greater MT expression in PCNA-positive lesions, while apoptosis was rather associated with MT-negativity. These data suggest that the overexpression of MT may play a role in mouse urinary bladder carcinogenesis.

Enhancement of urinary bladder carcinogenesis by combined treatment with benzyl isothiocyanate and N-butyl-N-(4-hydroxybutyl)nitrosamine in rats after initiation.

Previously we reported that benzyl isothiocyanate (BITC) strongly enhanced rat urinary bladder carcinogenesis after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), while potently inhibiting BBN-induction of lesions when given simultaneously with the carcinogen. In the present experiment, the effects of simultaneous treatment with BITC and low-dose BBN on the post-initiation period of rat urinary bladder carcinogenesis were examined. After treatment with 500 ppm BBN for 4 weeks for initiation, groups of 20, 6-week-old, F344 male rats were given 25 ppm BBN alone, basal diet alone, or 100 or 1000 ppm BITC in the diet together with or without 25 ppm BBN in their drinking water for 36 weeks and then killed for autopsy. Further groups consisting of 10 rats each were similarly given BITC or the basal diet together with or without 25 ppm BBN, without initiation treatment. In the initiated groups receiving subsequent BBN exposure, papillary and nodular hyperplasia, dysplasia and carcinoma incidences were significantly increased, and they were further increased by the combined treatment with 100 and 1000 ppm BITC in a dose-dependent manner. In the non-initiation groups, carcinomas were only observed in a single rat in each of the BBN-treated control and BBN/BITC 100 ppm treatment groups. The results indicate that simultaneous treatment with BITC and a low dose of BBN does not inhibit, but rather enhances rat urinary bladder carcinogenesis after appropriate initiation, and further suggest that BITC may be a human risk factor, at least in high-risk populations.