[Changes in tissue and blood polyamines during N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinogenesis in rats].

BACKGROUND: Polyamine are recognized as cell growth factors. We studied in order to determine whether alterations in the levels of tissue and blood polyamines were useful biochemical markers for bladder tumor. METHODS: The concentrations of three polyamines, diamine, spermidine and spermine, in urinary bladder and blood were determined during N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder carcinogenesis in male F344 rats. At 5 weeks of age, rats were given 0.05% BBN in the drinking water for 20 weeks. RESULTS: BBN induced bladder hyperplasia in 4 of 5 rats at 8 weeks, papillomas in 2 of 5 rats at 12 weeks, and transitional cell carcinoma in all the rats by 20 weeks. The levels of total polyamine in both bladder and blood of the rats during 12-20 weeks were significantly higher than those of the control animals given water alone. The elevation of total polyamine was mainly due to the increase of spermidine of the three polyamines, which was coincident with the incidence of bladder tumors. CONCLUSION: The results indicated that the polyamines are excellent biochemical markers for bladder tumors.

Increased gap junctional intercellular communication capacity and connexin 43 and 26 expression in rat bladder carcinogenesis.

Many reports have suggested that gap junctional intercellular communication or gap junction proteins (connexins) could have tumor suppression characteristics. We investigated gap junctional intercellular communication capacity and connexin 26, 32 and 43 mRNA expression in four rat bladder cell lines and the results were compared to their tumorigenicity. We also examined connexin expression in rat bladder carcinomas induced by 3,2'-dimethyl-4-aminobiphenyl or N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and in normal bladders. There was clear tendency that cell lines with greater communication had stronger tumorigenicity and more expression of connexin 26 or 43. We could not detect connexin 32 in these cell lines. In normal bladder tissue, connexin 43 expression was barely detectable and there was no detectable connexin 26. However, in rat bladder carcinomas, especially the EHBN-induced carcinomas, abundant expression of both connexins was observed. These results indicate that increased gap junctional intercellular communication capacity or increased connexin(s) expression may give a growth advantage in rat bladder carcinogenesis.

[Study on the effects of the hypocholesteremic agents, cholestyramine and compactin on the induction of renal tumors in rats by N-ethyl-N-hydroxyethyl nitrosamine].

Male Wistar rats were administered with 0.1% N-ethyl-N-hydroxyethyl nitrosamine (EHEN)-containing diet for 2 weeks, and were then Rept for further 23 weeks on basal diet. At 25 weeks, the rats were divided into three groups. Group I was fed on basal diet to 40 weeks. Group II was fed on 2% cholestyramine-containing diet to 40 weeks. Group III was fed on 0.02% compactin-containing diet to 40 weeks. At 40 weeks after the start of experiment, the rats were sacrificed and examined histologically for the incidence of renal cell tumors (RCT) and dysplastic foci (DF) of the kidney. The serum levels of total cholesterol at 40 weeks were 108.9 +/- 23.4, 78.8 +/- 12.5, 95.6 +/- 43.4 mg/dl in Groups I, II and III, respectively. The levels were significantly lower in Groups II and III (p less than 0.01, 0.05, respectively, Wilcoxon test) than Group I. The average numbers of DF were 4.5 +/- 4.1, 1.3 +/- 0.3, 2.3 +/- 2.0 per cm2 of kidney slices in Groups I, II and III, respectively. DF were significantly less in Group II than Group I (p less than 0.025, Wilcoxon test), and less in Group III than Group I (not statistically significant). There was no statistical difference between Groups II and III. RCT were observed in 9 of 34 kidneys (26%), 2 of 18 (11%), 1 of 20 (5%) in Groups, I, II and III, respectively. The ratios of kidneys with RCT were lower in Group II than Group I (not statistically significant), and lower in Group III than Group I (p less than 0.05, qui-square test). There was no statistical difference between Groups II and III. RCT were classified into gross and microscopic lesions. Four gross RCT were found in 4 rats of Group I; no gross RCT was in Groups II and III. There were 5 microscopic RCT in 5 rats, 3 in 2, 1 in 1 in Groups I, II and III, respectively. In Group I, serum levels of total cholesterol were compared between the following subgroups; the rats with RCT and/or with more than 10 DF per kidney, and the rats without RCT and with less than 10 DF per kidney. The mean level was 120.8 +/- 21.8 mg/dl in the former subgroup, which was significantly higher than that (94.1 +/- 19.6 mg/dl) in the latter subgroup (p less than 0.025, Wilcoxon test). The serum cholesterol was suggested to act as a promoter in the development of DF and RCT.

Low susceptibility of nude mice to induction of invasive urinary bladder cancers by N-ethyl-N-(4-hydroxybutyl)nitrosamine.

A time- and dose-dependent study of N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) bladder carcinogenesis was performed in nude mice maintained on tap water containing 0.025% EHBN for 4, 12, and 20 weeks ad libitum. A total of 13 invasive tumors, comprising 11 transitional cell carcinomas (TCCs) (84.6%) and 2 squamous cell carcinomas (SCCs) (15.4%), were found. Compared with previous results for B6C3F1 mice exposed to the same EHBN insult, the numbers of invasive carcinomas induced in nude mice, and especially of SCCs, were low. In order to ascertain whether this difference in cancer incidence between nude and B6C3F1 mice was due to variation in urinary excretion, the metabolism of EHBN was also investigated and compared with that of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Respective total urinary excretions over 48 hr of N-ethyl-N-(3-carboxypropyl)nitrosamine (ECPN) or N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), the ultimate carcinogenic species of EHBN or BBN, were 822.4 +/- 41.4 micrograms and 530.4 +/- 81.0 micrograms, respectively, in nude mice, and 800.6 +/- 83.7 micrograms and 407.8 +/- 69.7 micrograms, respectively, in B6C3F1 mice. In conclusion, although it is apparent that nude mice have a low susceptibility to EHBN induction of urinary bladder cancer, this does not appear to be dependent on reduced metabolism to the active form.

Summation effects of uracil and other promoters on epithelial lesion development in the F344 rat urinary bladder initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Five non-genotoxic chemicals previously demonstrated to be bladder cancer promoters in 36-week in vivo assays for carcinogenesis were reevaluated in a 20-week experiment in order to assess the summation influence of dietary uracil, a component of RNA, on the development of (pre)neoplastic lesions. The test chemicals, sodium bicarbonate, sodium L-ascorbate, sodium citrate, butylated hydroxytoluene and ethoxyquin, were mixed into the diet at concentrations of 3%, 5%, 5%, 1% and 0.8%, respectively, and administered to male F344 rats after initiation with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks. The test chemicals were given from the 4th to the 8th and the 11th to 20th experimental weeks, uracil being administered at the level of 3% in the diet during the intervening period. Rats in the control group received only BBN and uracil. All animals were killed at week 20 and the bladders were evaluated for the occurrence of putative preneoplastic papillary or nodular (PN) hyperplasia and tumors. Significant increase in the occurrence of PN hyperplasia was observed in all groups initiated with BBN and fed uracil and test chemicals. Quantitative values for papillomas were also significantly increased except in the ethoxyquin-treated group. The results confirm that uracil given in the middle of the post-initiation stage enhances the promoting activity of chemicals and suggest that the use of this chemical might be useful to reduce the duration of current bioassays for bladder chemical carcinogens.

Timing effects of uracil-induced urolithiasis on amplification of second-stage promotion in rat bladder carcinogenesis.

The post-initiation enhancing activities of the non-genotoxic agent NaHCO3 and the genotoxic agent N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) in combination with uracil-induced urolithiasis were investigated in a rat bladder carcinogenesis model. Animals were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks, and then 3% uracil was given for 3 weeks in the early (weeks 4-7), middle (weeks 8-11) or late (weeks 12-15) post-initiation phase. In addition, administration of 3% NaHCO3, 20 ppm EHBN or no chemical supplement was performed for the 13 weeks when the rats were not receiving BBN or uracil. NaHCO3 in sequential combination with early and middle stages uracil treatment strongly enhanced tumorigenesis in the urinary bladder, while EHBN treatment amplified lesion development at the middle stage only of uracil treatment. DNA synthesis and associated epithelial surface alterations observed by scanning electron microscopy tended to be increased in the NaHCO3 and EHBN groups without BBN initiation, independently of uracil treatment timing. The present results demonstrated that uracil-induced urolithiasis during the middle post-initiation phase is highly active in enhancing bladder tumor development under the influence of a promoter or carcinogen.

Time- and dose-dependent induction of invasive urinary bladder cancers by N-ethyl-N-(4-hydroxybutyl)nitrosamine in B6C3F1 mice.

A sequential investigation of N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) bladder carcinogenesis was performed in male B6C3F1 mice maintained ad libitum on tap water containing 0.025% EHBN for 4, 12, 20, 28 and 36 weeks. A total of 81 invasive tumors, comprising 55 squamous cell carcinomas (SCCs) (68%), 25 transitional cell carcinomas (TCCs) (31%) and 1 adenocarcinoma (1%) were found. Of these, 23 (22 SCCs and 1 TCC) demonstrated invasion to the prostate, 3 metastasized to the lung, and 2 spread by peritoneal seeding. The anaplastic grade and extent of invasion of the SCCs significantly exceeded those of the TCCs. The results suggested a histogenetic pathway from simple dysplasia through papillary or nodular dysplasia and/or carcinoma in situ to eventual development of invasive carcinomas.

A molecular basis for target-cell toxicity and upper urothelial carcinoma in analgesic abusers and patients with Balkan endemic nephropathy.

Ochratoxin A is ubiquitous in regions where Balkan endemic nephropathy is common. It damages the kidney cortex in a range of experimental animals and induces renal parenchymal carcinoma in mice, but it is not a potent carcinogen, nor is there experimental evidence to link it to upper urothelial carcinoma (UUC). A model UUC can be induced experimentally in rodents by urothelial initiation, followed by an acutely induced papillary necrosis. This two-stage experimental model may help to clarify the role of ochratoxin A in initiating or promoting upper urothelial cells and increase our understanding of the development of UUC in patients with Balkan endemic nephropathy.

Correlation between medium-term liver bioassay system data and results of long-term testing in rats.

The effects of hepatocarcinogens (ethionine, thioacetamide, phenobarbital), non-hepatocarcinogens [N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and a hepatoinhibitor [(butylated hydroxyanisole (BHA)] were compared in medium- and long-term in vivo systems. In experiment I, 2 weeks after a single injection of diethylnitrosamine (DEN) groups of male F344 rats received chemical administration for 6 weeks, combined with partial hepatectomy at week 3 and were killed at the end of week 8. In experiment II, animals were treated in the same manner and then given basal diet and tap water (group 1) or chemical continuously (group 2) until the 2 year timepoint. Numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci developing in the liver under medium-term bioassay conditions (experiment I) were found to closely correlate with eventual hepatocellular carcinoma incidences after continuation of test chemical administration (experiment II). Thus all of the hepatocarcinogens enhanced both the induction of GST-P-positive focal lesions and liver tumors. While non-hepatocarcinogens exerted no such effects, their influence being limited to inducing lesions in their own respective target organs such as urinary bladder cancers in the EHBN case and glandular stomach adenocarcinomas with MNNG, BHA demonstrated inhibition potential in both experiments. The observed correlation between long- and medium-term results strongly indicates the applicability of our medium-term bioassay system for detection of liver carcinogens.

[Animal models of bladder cancer].

Treatment with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) or N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) in the drinking water induces bladder cancer of rats or mice with high incidence. Induced bladder cancers of rats are mostly papillary, non-invasive type, whereas those of mice are non-papillary, invasive type. Histologically, most of BBN-induced bladder cancers are transitional cell carcinoma in both rats and mice. On the other hand, the incidences of squamous cell carcinoma increased in cases of mice, especially in mice treated with EHBN. Metastases and invasions of bladder cancers are more common in mice than in rats. There are strain and species differences in the bladder response to BBN and EHBN. In addition, a putative preneoplastic lesion, papillary or nodular hyperplasia of the epithelium is a good marker for early detection of bladder cancer development.

Summation effect of uracil on the two-stage and multistage models of urinary bladder carcinogenesis in F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Uracil is known to cause reversible urolithiasis and to induce papillomatosis in the urinary bladder of F344 rats. We examined whether the marked urothelial cell proliferation caused by uracil, given in the middle of the post-initiation stages, enhances the promoting activity of a promoter in the two-stage model or the promoting and/or carcinogenic activity of a low-dose carcinogen in the multistage model of urinary bladder carcinogenesis. Rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks, and then 2% butylated hydroxyanisole (BHA) or 0.002% N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) were given during experimental weeks 4-9 and weeks 12-20. Uracil was given during weeks 9-12 at a level of 3% of the diet. Rats in the control group were treated with BBN and uracil. Rats were killed at weeks 16 and 20. At week 16, higher occurrences of papillary or nodular (PN) hyperplasia and papilloma were observed in uracil-BHA-treated rats than in the controls. At week 20, significantly higher incidences of PN hyperplasia and papilloma were observed in both uracil-EHBN- and uracil-BHA-treated groups, and a summation effect of uracil was observed. These results indicate that uracil given in the middle of the post-initiation stage enhanced the promoting activity of the compound through marked proliferation of the bladder epithelium.

Effect of acute and chronic butylated hydroxyanisole administration on in vivo glucuronidation of N-nitrosobutyl(4-hydroxybutyl)amine in rats.

The urinary metabolic pattern of N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) administered ip at a dose of 5 mg/kg body weight was studied in animals either pretreated with butylated hydroxyanisole (BHA) as a single oral dose of 50 or 250 mg/kg, or fed a diet containing 0.1 or 0.5% BHA. The 24-hr urinary excretion of NB4HBA, its glucuronic acid-conjugate (NB4HBA-G) and N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA) in control rats were 0.12, 0.75 and 30% of the administered dose, respectively, and were not changed after a single oral dose of 50 mg BHA/kg. NB4HBA-G was significantly reduced in the urine of rats given 250 mg BHA/kg. In vitro assays carried out using rat-hepatic microsomal preparations as the source of the enzyme UDP-glucuronyl transferases (GT) and NB4HBA as the substrate, suggest that a competition between NB4HBA and BHA for the same enzyme may be the cause of the decreased NB4HBA-G excretion observed in vivo. A fourfold increase in NB4HBA-G urinary excretion was observed after chronic 0.5% BHA feeding; moreover, the glucuronic acid-conjugate of NB3CPA (NB3CPA-G), which was not detected in the controls or after acute BHA treatment, appeared in the urine of rats given dietary BHA for 3 wk, accounting for about 10% of the administered NB4HBA. In vitro experiments indicate that the increased glucuronides excretion may be the result of an elevated hepatic GT activity.

Induction of repair synthesis of DNA in primary cultures of rat urothelial cells by derivatives of the bladder carcinogen, N-n-butyl-N-(4-hydroxybutyl)nitrosamine.

Urinary metabolites of N-n-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) which have been identified thus far did not induce repair synthesis of DNA in cultured rat urothelial cells. Urine of rats which had been administered with BHBN or BCPN did not induce repair synthesis of DNA either. However, all the synthetic nitrosamines that can produce 1-hydroxyalkylnitrosamine intermediates induced repair synthesis of DNA. The results suggest that rat urothelial cells, at best, may only have very limited capability of activating nitrosamines.

Promoting effects of unilateral ureteric ligation on two-stage urinary bladder carcinogenesis in rats.

The effect of ligation of the left ureter on the development of lesions in the urinary bladder were investigated in male F344 rats treated with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for 2 weeks followed by 0.002% N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) for 10 or 22 weeks. The lesions of the urinary bladder found in weeks 12 and 24 in rats treated with BBN and EHBN were preneoplastic papillary or nodular hyperplasia (PN hyperplasia), papilloma and cancer. The incidences and numbers of these lesions per 10 cm of basement membrane of urinary bladder were increased significantly by ureteric ligation. Treatment with BBN or EHBN only induced no remarkable change and thus the doses used were subcarcinogenic in the conditions used. The results show that unilateral ureteric ligation enhanced two-stage bladder carcinogenesis.

Species variations in the metabolism of N-butyl-N-(4-hydroxybutyl) nitrosamine and related compounds in relation to urinary bladder carcinogenesis.

Species variations in response to urinary bladder carcinogens, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN), and N,N-dibutylnitrosamine (DBN), were investigated in several animal species from the metabolic point of view. Since N-butyl-N-(3-carboxypropyl) nitrosamine (BCPN) and N-ethyl-N-(3-carboxypropyl) nitrosamine (ECPN) had been found to be the principal urinary metabolites which are responsible for the induction of bladder tumors by BBN or DBN and EHBN, respectively, in rats, acidic urinary metabolites with the N-nitroso moiety were isolated and determined by a colorimetric method after oral administration of these nitrosamines to rats, mice, hamsters, guinea pigs, and dogs. Qualitatively almost no species differences were observed among these animals in regard to the urinary metabolites except in the case of mice, in which the glycine conjugate of BCPN was isolated from the urine and identified as the principal metabolite of BBN and DBN. However, appreciable quantitative differences in the urinary excretion of BCPN or ECPN were found among these animal species, indicating that the differences in the susceptibilities of different animal species to urinary bladder carcinogenesis induced by BBN, DBN and EHBN may be closely related to the different extents of urinary excretion of the active metabolites of these nitrosamines.

Effect of partial cystectomy on the induction of pre-neoplastic lesions in rat bladder initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine followed by bladder carcinogens and promoters.

The effect of partial cystectomy on the occurrence of pre-neoplastic lesions, papillary or nodular hyperplasia (PN hyperplasia), of the bladder in male F344 rats was studied in an experiment in which bladder carcinogens and promoters were given to the rats after initiation with BBN. The bladder carcinogens tested were N-ethyl-N-(4-hydroxybutyl)nitrosamine (EHBN) and N-4[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and the bladder promoters were sodium saccharin, sodium cyclamate, and DL-tryptophan. Partial cystectomy significantly decreased the occurrence of PN hyperplasia in rats treated with EHBN and tended to inhibit that in rats given saccharin or tryptophan. Thus partial cystectomy inhibited rather than enhanced the induction of PN hyperplasia.

Bladder carcinogenesis in mice induced by N-butyl-N-(4-hydroxybutyl) nitrosamine and N-ethyl-N-(4-hydroxybutyl)-nitrosamine with reference to the effect of cyclophosphamide.

The carcinogenicities of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and N-ethyl-N-(4-hydroxybutyl) nitrosamine (EHBN) on the urinary bladder were compared in male BALB/c mice. The effect of cyclophosphamide (CPA) on the carcinogenicities was also investigated. The carcinogenic activity on the bladder mucosa of EHBN was similar to that of BBN, and there was no difference in the growth patterns, histological types or invasive characters of carcinomas induced by BBN and EHBN. Benign or malignant tumors of vascular origin developed in the urinary bladders of 15 mice treated with these carcinogens. The incidence of vascular tumors induced by EHBN was significantly higher than that of tumors induced by BBN. Intraperitoneal injections of CPA seemed to have no effect in the incidence of bladder carcinomas induced by these carcinogens, or on the development of vascular tumors in the bladder wall induced by EHBN.

Metabolic fate and carcinogenicity of N-(omega-hydroxyalkyl) N-(4-hydroxybutyl) nitrosamines analogs of N-butyl-N-(4-hydroxy-butyl) nitrosamine, in the rat.

The metabolic fate and carcinogenicity fo three omega-hydroxy derivatives of N-alkyl-N-(4-hydroxybutyl) nitrosamines, potent bladder carcinogens, were investigated in the rat. They were N-(2-hydroxyethyl)-N-(4-hydroxybutyl)-nitrosamine (HEHBN), N-(3-hydroxypropyl)-N-(4-hydroxybutyl) nitrosamine (HPHBN), and N-N-bis(4-hydroxybutyl) nitrosamine (BHBN). The principal urinary metabolite of HEHBN as well as HPHBN was identified as the corresponding 3-carboxypropyl compound, while the main metabolite of BHBN was N,N-bis (3-carboxypropyl) nitrosamine, indicating the preferential metabolic oxidation of the 4-hydroxybutyl chain to the 3-carboxypropyl group in the N-(omega-hydroxyalkyl)-N-(4-hydroxybutyl) nitrosamines. All three N-nitrosamines having the 4-hydroxybutyl chain induced neither bladder tumor nor any tumor in other organs under conditions similar to those used for N-alkyl-N-(4-hydroxy-butyl) nitrosamines. The essential structural and metabolic requirements in N-nitrosamines for the induction of bladder cancer in the rat are discussed.

Metabolic fate of N-alkyl-N-(3-hydroxypropyl and 2-hydroxy-ethyl)nitrosamines in the rat in relation to the induction of bladder cancer by N-butyl-N-(4-hydroxybutyl)nitrosamine and its homologs.

The metabolic fate of N-alkyl-N-(3-hydroxypropyl)nitrosamines and N-alkyl-N-(2-hydroxyethyl)nitrosamines (alkyl=butyl, ethyl) [analogs of N-alkyl-N-(4-hydroxybutyl)nitrosamines, which are potent bladder carcinogens] was investigated in the rat in order to elucidate a possible relationship between chemical structure, in vivo metabolism, and organotropic carcinogenicity to the urinary bladder of N-alkyl-N-(4-hydroxybutyl)nitrosamines. The principal urinary metabolites of N-alkyl-N-(3-hydroxypropyl)nitrosamines and N-alkyl-N-(2-hydroxyethyl)nitrosamines, which are not carcinogenic to the urinary bladder but are hepato-carcinogenic in rats, were the corresponding 2-carboxyethyl and carboxymethyl compounds. Urinary metabolites with a 2-carboxyethyl or carboxymethyl group are not important, as far as the induction of bladder cancer is concerned, and the urinary excretion of metabolites having a 3-carboxypropyl chain is essential for the induction of bladder cancer.