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.     

A dynamic switch in Rb+/- mediated neuroendocrine tumorigenesis

Rb+/- mice develop a complex spectrum of neuroendocrine tumors on a mixed genetic (129Sv x C57BL/6) background. To understand how the 129Sv and C57BL/6 contributions affect Rb+/- tumorigenesis, we serially backcrossed Rb+/- animals to the 129Sv or C57BL/6 strain, and analysed their pathological profiles. Strikingly, the length of survival and the penetrance, severity and multiplicity of neuroendocrine tumors switch dramatically between Rb+/- animals from the two genetic backgrounds. In fact, the 129Sv background significantly enhances both the initiation and progression of tumorigenesis in the intermediate lobe of the pituitary (ILP) in Rb+/- animals. This is due to the surprising fact that ILPs from wild-type 129Sv animals are inherently abnormal, and thus greatly predisposed to neoplasia. This is likely to explain the high incidence of ILP tumors, an otherwise rare tumor type in wild-type mice, in numerous knockout studies performed on the 129Sv strain, and raises the intriguing possibility that the classic Rb+/- neuroendocrine tumors may fade away in another as of yet unidentified inbred strain. Finally, we have increased the utility of the Rb+/- tumor model, since Rb+/- animals on the C57BL/6 background develop high-penetrance tumors of the anterior lobe of the pituitary, a class of tumors estimated to occur in 20-25% of humans.     

High susceptibility of an analbuminemic congenic strain of rats with an F344 genetic background to induced bladder cancer and its possible mechanism

The susceptibility of an analbuminemic congenic strain of rats (F344-alb) originating from the F344 strain to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. F344-alb rats were found to be highly susceptible to induction of urinary bladder cancers. The incidences of bladder cancers in F344-alb and F344 rats were 94% (15/16) and 31% (5/16) in males and 100% (16/16) and 19% (3/16) in females. The bladder weights of these rats, including tumors, were 307 +/- 294 mg, 123 +/- 26 mg, 183 +/- 80 mg and 93 +/- 11 mg, respectively. Administration of 0.05%, 0.1% and 0.3% BBN in the drinking water for 2 weeks resulted in greater increases in the bladder content of N-butyl-N-(3-carboxypropyl)nitrosamine in F344-alb rats than in F344 rats. This increase was prevented by the presence of rat albumin.     

Significant overexpression of metallothionein and cyclin D1 and apoptosis in the early process of rat urinary bladder carcinogenesis induced by treatment with N-butyl-N-(4-hydroxybutyl)nitrosamine or sodium L-ascorbate

Effects of a genotoxic bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and a non-genotoxic bladder promoter, sodium L-ascorbate (Na-AsA), on protein expression, cell proliferation and apoptosis of the bladder epithelium with or without the influence of testicular castration were investigated. Male F344 rats were divided into six groups (groups 1-6). BBN was given with 0.05% drinking water to groups 1 and 4 for 8 weeks, groups 2 and 5 received diet with 5% Na-AsA. Then the animals were treated without any chemicals. Groups 3 and 6 were non-treated controls. Testicular castration was carried out 2 weeks before commencement of chemical treatment on groups 4-6. The total observation period was 18 weeks. Overexpression of cyclin D1 was induced by BBN but not Na-AsA and the degree of overexpression was higher in the order simple hyperplasia, papillary or nodular hyperplasia, papilloma and carcinoma. Metallothionein (MT) was also overexpressed in bladder epithelium treated with BBN but not Na-AsA, but was decreased in papillomas and never found in a carcinoma. Cyclin D1-positive cells were essentially MT-negative. Therefore, it is speculated that MT protects genes from insult by genotoxic carcinogens and its lack is associated with tumor development. Apoptotic cell death occurred during treatment with BBN and Na-AsA and after their withdrawal. Chromatin condensation of many G0/G(1) cells was particularly marked on flow cytometry analysis 1 week after cessation of treatment, this being considered as an early apoptotic change. Although testicular castration had no influence on the above events, it resulted in decreased tumor formation as compared with the case of similarly treated intact animals. Our data demonstrate that overexpression of MT and cyclin D1 is specific for treatment with a genotoxic carcinogen, and suggest that MT overexpression may play an important suppressive role in the early stages of rat urinary bladder carcinogenesis.     

Uracil-induced calculi and carcinogenesis in the urinary bladder of rats treated simultaneously with N-butyl-N-(4-hydroxybutyl)nitrosamine

Urinary bladder carcinogenicity of uracil and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in combination was investigated in male F344 rats. Two experiments were performed: in the first animals were treated with 3.0, 1.0 and 0.3% uracil in the diet and/or 0.005% BBN in the drinking water for 36 weeks followed by a 4 week period without chemicals, the total observation time being 40 weeks; in the second rats received 3.0% uracil in the diet or 0.05% BBN in the drinking water for 4 weeks. In the first experiment, a markedly high incidence of carcinomas was observed in the urinary bladder with both 3.0% uracil alone (11/15, 73%) and 3.0% uracil plus 0.005% BBN (18/20, 90%) treatments. The multiplicity, i.e. numbers of carcinomas induced per 10 cm of basement membrane, was significantly higher with the latter treatment (P less than 0.05), as were proliferative lesions in the renal pelvis. Stone formation was observed in all 3.0% uracil treatment animals. In the second experiment, prostaglandin E2 levels in bladder tissue of uracil-treated animals proved to be significantly higher than in BBN-treated or untreated animals. Thus, the results clearly indicated that calculi associated with 3.0% uracil ingestion can themselves cause carcinoma development in the urinary bladder, while also acting as a potent co-carcinogen agent for BBN carcinogenesis.     

Renal Pelvic Carcinoma which Shows Metastatic Potential to Distant Organs, Induced by N-Butyl-N-(4-hydroxybutyl)nitrosamine in NON/Shi Mice

Renal pelvic carcinoma was induced in mice by giving N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Initially, differences in renal pelvic carcinogenesis by BBN were examined in three male mouse strains: NON/Shi, which demonstrate spontaneous hydronephrosis with incidences of 10-30%, and DS/Shi and B6C3F1, which do not exhibit hydronephrosis. When mice of these strains were given 0.05% BBN in the drinking water for 12 weeks followed by water without BBN for 8 weeks, renal pelvic carcinoma morphologically similar to human carcinomas developed in 8 of 23 NON/Shi mice (35%). Metastasis to the lung was found in one of them (13%). B6C3F1 and DS/Shi mice had no pelvic tumors, but the response to urinary bladder carcinogenesis in NON/Shi mice was nearly equal to that in DS/Shi and B6C3F1 mice. These results suggest that renal pelvic carcinogenesis is related to the presence of stagnant urine containing carcinogen in the renal pelvis. In a second experiment, we examined renal pelvic carcinogenesis in NON/Shi mice given BBN for 4 weeks followed by water without BBN for 32 weeks. The incidence of renal pelvic carcinoma (28%) was similar to that in the first experiment, but the incidence of metastasis was markedly elevated to 60%. These results indicate that BBN treatment can induce renal pelvic carcinoma which often metastasizes to the lung in NON/Shi mice.     

Renal pelvic carcinoma which shows metastatic potential to distant organs, induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in NON/Shi mice.

Renal pelvic carcinoma was induced in mice by giving N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Initially, differences in renal pelvic carcinogenesis by BBN were examined in three male mouse strains: NON/Shi, which demonstrate spontaneous hydronephrosis with incidences of 10-30%, and DS/Shi and B6C3F1, which do not exhibit hydronephrosis. When mice of these strains were given 0.05% BBN in the drinking water for 12 weeks followed by water without BBN for 8 weeks, renal pelvic carcinoma morphologically similar to human carcinomas developed in 8 of 23 NON/Shi mice (35%). Metastasis to the lung was found in one of them (13%). B6C3F1 and DS/Shi mice had no pelvic tumors, but the response to urinary bladder carcinogenesis in NON/Shi mice was nearly equal to that in DS/Shi and B6C3F1 mice. These results suggest that renal pelvic carcinogenesis is related to the presence of stagnant urine containing carcinogen in the renal pelvis. In a second experiment, we examined renal pelvic carcinogenesis in NON/Shi mice given BBN for 4 weeks followed by water without BBN for 32 weeks. The incidence of renal pelvic carcinoma (28%) was similar to that in the first experiment, but the incidence of metastasis was markedly elevated to 60%. These results indicate that BBN treatment can induce renal pelvic carcinoma which often metastasizes to the lung in NON/Shi mice.     

Effect of FT-207 on bladder cancer in rats induced by BBN. II: The effect of oral administration of FT-207 during oncogenesis of bladder cancer

The effect of Tegafur (FT-207) by oral administration on the development of urinary bladder tumors in Wistar strain male rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was studied. Urinary bladder tumors were induced in 18 of 20 rats (90.0%) when rats were given 0.05% BBN in the drinking water for 8 weeks and then given water without BBN for 12 weeks. When FT-207 100mg/kg B. W./day was given in their diet after treatment with 0.05% BBN for 8 weeks, tumors developed in the urinary bladder with low incidence (9 of 16 rats: 56.3%). Hematotoxicity was not observed in all animals treated with FT-207. These results shows that FT-207 also inhibited the development of urinary bladder tumors treated with BBN in rats by oral administration, which were similar those our previous results showed by intraperitoneal administration of FT-207.     

Loss of Heterozygosity in (Lewis×F344)F1 Rat Urinary Bladder Tumors Induced with N-Butyl-N-(4-hydroxybutyl)nitrosamine Followed by Dimethylarsinic Acid or Sodium L-Ascorbate

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor-promoting activity on rat urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Sodium L-ascorbate (Na-AsA) is also a strong tumor promoter in this animal model. In this study, we used (Lewis×F344)F₁ rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na-AsA. Male, 6-week-old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na-AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na-AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na-AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na-AsA, may differ in rat urinary bladder carcinogenesis.     

Trisodium nitriloacetate monohydrate: promoting effect in urinary bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine

The effect of trisodium nitrilotriacetate monohydrate [(Na3NTA X H2O) CAS: 18662-53-8] on development of urinary bladder tumors in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine [(BBN) CAS: 3817-11-6] was studied. Twenty-one male inbred W rats 6 weeks of age were given drinking water containing 500 ppm of BBN for 4 weeks and then put on diet containing 10,000 ppm of Na3NTA X H2O for 28 weeks. Na3NTA X H2O promoted the development of urinary bladder tumors in rats treated with BBN. The incidences of papilloma and transitional cell carcinomas in the urinary bladder were 90% (18/20) and 25% (4/20), respectively, in rats treated with BBN and then Na3NTA X H2O and 0 in those treated with BBN or Na3NTA X H2O alone. The incidence of papillary or nodular hyperplasia in week 32 was 100% (20/20) in rats treated with BBN and then Na3NTA X H2O and 61% (13/21) in rats treated with BBN only.     

Lack of synergism in rat urinary tract carcinogenesis between prior uracil treatment and N-butyl-N-(4-hydroxybutyl)-nitrosamine administration

Combination effects of sequential treatment with uracil prior to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) with regard to carcinogenesis in the urinary bladder and renal pelvis were investigated in male F344 rats. Uracil was administered for 15, 10 or 5 weeks followed by BBN for 23 weeks, the total observation time being 40 weeks. Carcinoma(s) and papilloma(s) were induced in the urinary tract by the 15-week uracil treatment independent of subsequent BBN administration. It was concluded that uracil administration prior to BBN treatment is not associated with any synergistic effects, although both uracil and BBN alone exerted carcinogenicity.     

Absence of ras oncogene activation in rat urinary bladder carcinomas induced by N-methyl-N-nitrosourea or N-butyl-N-(4-hydroxybutyl)nitrosamine.

Previously, we demonstrated point mutations of the H-ras gene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat urinary bladder carcinomas. In this study, ras oncogene activation was examined in urinary bladder carcinomas induced by N-(4-hydroxybutyl)nitrosamine (BBN) or N-methyl-N-nitrosourea (MNU) administration followed by uracil treatment. In the first experiment, MNU (20 mg/kg body wt) was i.p. injected into 11 male F344 rats twice a week for 4 weeks, followed by feeding 3% uracil for 20 weeks (MNU/uracil group). Ten rats were given only 3% uracil without MNU pretreatment. In the second experiment, 20 male F344 rats were given 0.05% BBN in the drinking water for 4 weeks, then fed 3% uracil for 20 weeks (BBN/uracil group). Another 20 rats were fed 3% uracil without the BBN pretreatment. Transitional cell carcinomas were induced in the urinary bladder of all rats in the MNU/uracil and BBN/uracil groups. Papillomas and hyperplasias were present in the rats given uracil without prior BBN or MNU. DNA and protein were extracted from the tumors (MNU/uracil or BBN/uracil groups) or from the scraped bladder epithelium (uracil alone groups). Sequences around codons 12, 13 and 61 of H-, K- and N-ras genes were examined by direct sequencing after polymerase chain reaction, and p21 was examined by Western blotting. No mutation was found within the examined sequences and p21 showed no changes in mobility. There was no difference in the level of p21 expression between rats treated with MNU/uracil or BBN/uracil compared to corresponding uracil alone groups. These results indicate that the ras oncogene was not activated in urinary bladder carcinomas induced by BBN or MNU in combination with uracil treatment, in contrast to previous findings with FANFT.     

Loss of heterozygosity in (LewisxF344)F1 rat urinary bladder tumors induced with N-butyl-N-(4-hydroxybutyl)nitrosamine followed by dimethylarsinic acid or sodium L-ascorbate.

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor-promoting activity on rat urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Sodium L-ascorbate (Na-AsA) is also a strong tumor promoter in this animal model. In this study, we used (LewisxF344)F, rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na-AsA. Male, 6-week-old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na-AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na-AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na-AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na-AsA, may differ in rat urinary bladder carcinogenesis.     

Effect of UFT on urinary bladder tumors in rats induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The effect of combined tegafur and uracil (UFT) on the development of rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was studied. Two hundred F344 male rats were divided into 10 groups. Groups 1 to 5 were given 0.05% BBN in drinking water for the initial 8 weeks of the experiment, and Groups 6 to 10 were the controls of the prior 5 groups treated with BBN. UFT in commercial diet was administered at daily doses of 100mg (30.9 mg as FT-207) per kg of body weight in Groups 2 and 4 and 200 mg (61.7 mg as FT-207) per kg of body weight in Groups 3 and 5. Groups 2 and 3 received UFT throughout the period of the experiment, and Groups 4 and 5 for 12 weeks after 8 week treatment with BBN. All animals were sacrificed at 20 weeks, and studied histopathologically. In Groups 1 to 5, urinary bladder tumors developed in 20 of 20, 13 of 20, 6 of 20, 14 of 20 and 6 of 20, respectively. Incidences of tumors in the 4 groups treated with UFT were significantly lower than that in Group 1 treated with BBN alone. This result shows that UFT inhibits the development of urinary bladder tumors in rats induced by BBN.     

Dose response of N-butyl-N-(4-hydroxybutyl)nitrosamine on urinary bladder carcinogenesis in mutant ODS rats lacking L-ascorbic acid synthesizing ability

With a daily intake of 250 ppm total ascorbic acid, ODS and F344 male rats were given 0.0125%, 0.025% or 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in the drinking water for 12 weeks, and additional groups received 0.05% BBN for 2, 4 or 8 weeks. The experiment was terminated after a total of 36 weeks. A greater response to urinary bladder carcinogenesis was observed in both strains with increasing dose of BBN or longer treatment period. However, the magnitude of urinary bladder carcinogenesis in ODS rats given the higher BBN concentrations and/or long periods of BBN treatment was less than in comparably treated F344 rats, but not with lower concentrations of BBN and/or shorter periods of BBN treatment.     

Stimulatory effects of growth hormone on rat bladder carcinogenesis

The authors investigated the influences of recombinant human growth hormone (rh-GH) on rat urinary bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Rats belonging to the control group (Group I, n = 19) were given 0.05% BBN in drinking water for 9 weeks, and the bladder was excised on the 22nd week after the initiation of BBN administration and inspected. All animals developed visible tumors in the bladder. The mean number of tumors per bladder was 11.26 +/- 5.21, and the mean total volume of tumors per bladder was 126.1 +/- 212.7 microliters. In all but one of the experimental groups (Group V) and in the control group, all animals developed visible tumors in the bladder. When 0.5 units of rh-GH was injected subcutaneously once a week from the week 1 through the week 6 (Group II; n = 20), the mean number of tumors and mean total volume of the tumors were 12.15 +/- 6.59 and 206.6 +/- 318.0 microliters, respectively. When the administration period of rh-GH was changed to between week 7 through the week 12 (Group III; n = 19), the mean number of tumors and mean total volume of the tumors were 16.95 +/- 7.07 and 204.5 +/- 317.7 microliters, respectively. When rh-GH was administered from the week 13 through the week 18 (Group IV; n = 19), the respective values were 16.79 +/- 10.75 and 213.4 +/- 274.6 microliters. In Group V (n = 19), which received only rh-GH from week 1 through the week 6, no tumors were detected. There were statistically significant differences in the mean tumor numbers between Groups I and III, Groups I and IV, and Groups II and III. The mean volume of individual tumor was the greatest in Group II, although the differences were not statistically significant in comparison with the other groups. Histologically, all tumors were transitional cell carcinoma in every group. There were no statistically significant differences in distributions of tumor stage and tumor grade between any groups. These findings suggest that rh-GH enhances the promotion of carcinogenesis of chemically induced rat urinary bladder cancer. It will be necessary to elucidate whether this effect of rh-GH is expressed by the somatostatin hypothesis of GH action, its direct action, or some other mechanisms.     

LOH and mutational analysis of p53 alleles in mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine

In human urinary bladder carcinogenesis, alterations in the p53 tumor suppressor gene are common events. We have previously reported that they are also frequent in invasive urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in NON/Shi mice. To further investigate the significance of the p53 gene status for mouse urinary bladder carcinogenesis, we examined both allele loss and mutational alterations in urinary bladder cancers of (NON/Shi x C3H/He/Shi) F1 hybrid mice exposed to the carcinogen for 12 weeks and then maintained for a further 9 weeks without treatment. An intragenic silent polymorphism within exon 7 of the p53 gene between NON/Shi and C3H/He/Shi mice allows assessment of allele loss of the p53 gene and determination of the parental origin of mutated and/or lost alleles. A tissue microdissection method was employed to obtain carcinoma samples without excessive contamination with normal tissue. Allele losses were detected in one of 14 tumors (7.1%) and nine mutations in eight of 14 (57%) tumors were found in exons 5-8 by polymerase chain reaction-single strand conformation polymorphism followed by DNA direct sequencing analysis. All mutations involved one base substitution with an amino acid change, although the types of base substitution were random. In conclusion, the high incidence of p53 alterations suggests a significant role in the genesis of invasive urinary bladder tumors in BBN-treated mice.     

Parp-1 deficiency does not increase the frequency of tumors in the oral cavity and esophagus of ICR/129Sv mice by 4-nitroquinoline 1-oxide, a carcinogen producing bulky adducts

The impact of poly(ADP-ribose) polymerase-1 (Parp-1)-deficiency on 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis was studied in mice with an ICR/129Sv mixed genetic background. Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) animals given 4NQO for thirty-two weeks at 0.001% in their drinking water developed papillomas and squamous cell carcinomas of the tongue, palate and esophagus, but with no statistically significant variation with the Parp-1 genotype. Thus Parp-1 deficiency does not elevate susceptibility to carcinogenesis induced by a carcinogen which gives rise to bulky DNA lesions. This study also indicated that the ICR/129Sv mixed genetic background is associated with high yield induction of esophageal tumors by 4NQO.