Effects of promoters on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in the rat.

It has been shown that the occurrence of the preneoplastic lesion, papillary or nodular hyperplasia (PN hyperplasia) in rat urinary bladder induced by carcinogens is correlated with that of cancer. Therefore, the promoting effects of chemicals in two-stage bladder carcinogenesis were judged by measuring their ability to induce PN hyperplasia in rats. Male rats were given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks and then one of 16 test chemicals for 32 to 34 weeks. Saccharin, ascorbate, DL-tryptophan, allopurinol, and diphenyl promoted development of PN hyperplasia. The dose-response of the promoters were examined in both sexes of rats by administration of saccharin at doses of 0.04, 0.2, 1.0 and 5.0% for 32 weeks after BBN treatment. The occurrence of PN hyperplasia was significantly increased in the group given 5% saccharin. Dose-response curves showed enhanced hyperplastic responses in both sexes given 0.2 to 5% saccharin. The organ specificities of promoters were studied in rats initiated with BBN or 2-acetylamino-fluorene (2-AAF) followed by phenobarbital or saccharin for 32 weeks. Phenobarbital greatly enhanced hepatocarcinogenesis. Saccharin significantly enhanced the occurrence of both BBN-induced and 2-AAF-induced PN hyperplasia. However, there was no effect of phenobarbital on the urinary bladder or of saccharin on the liver. The rats showed a strain difference in susceptibility of the urinary bladder to saccharin; ACI rats were most susceptible and Sprague Dawley rats were most resistant to saccharin. The membrane potential of superficial epithelial cells in the urinary bladder of rats treated with saccharin was measured with an intracellular microelectrode and found to be higher than that of controls.


Introduction
The two-stage process of chemical carcinogenesis, initiation and promotion, was initially proposed from studies on mouse skin and has subsequently been demonstrated in other organs, such as the liver, colon and urinary bladder (1)(2)(3). The two-stage process of urinary bladder carcinogenesis has been demonstrated in rats using single intravesical instillation of methylnitrosourea (4) or initiation by N-[4-(5-nitro-2-furyl)2-thiazolyl]formamide (FANFT) (5), followed by treatment with saccharin. In the present studies we examined the promoting effects of various chemicals on urinary bladder carcinogenesis in rats initiated by N-butyl-N-4-hydroxybutyl)nitrosamine (BBN) and evaluated the in vivo biological characteristics of tumor promoters in urinary bladder carcinogenesis using saccharin as a typical promoter. Effects were mainly judged by measuring the formation of the preneoplastic lesion and papillary and nodular hyperplasia of the urinary bladder.

The Preneoplastic Lesion and Histological Observation
BBN is a potent carcinogen in the urinary bladder of animals. There have been many reports on the carcinogenicity of BBN in rat urinary bladder (6)(7)(8). Histological lesions in the urinary bladder of rats induced by BBN were classified into four types, simple hyperplasia, PN hyperplasia, papilloma and cancer (9). As shown in Figure 1, PN hyperplasia of the urinary bladder in rats treated with 0.05% BBN developed before the induction of papilloma or cancer (6). The incidence of PN hyperplasia in the uri-nary bladder of rats treated with various doses of BBN for different periods is shown graphically in Figure 2 (7). A high dose of BBN induced a high incidence of PN hyperplasia in a short period. Dose-response relationships were observed for the induction of PN hyperplasia, the dose of BBN, and the period of BBN treatment. Moreover, a good correlation was found between the inductions of PN hyperplasia and cancer with different doses of BBN. Thus, it is clear that PN hyperplasia is a preneoplastic lesion of rat urinary bladder, and can be used as a marker in studies on the 2-stage process of urinary bladder carcinogenesis.
For histological examination of the urinary bladder, rats were killed with ether, 10% phosphatebuffered formalin (pH 7.4) was injected into the uri-100 * -eI U 50 -U C FIGURE 1. Incid 0.05% N-buty nary bladder through the urethra, and the urinary bladder was then divided in half sagitally. Each half was cut into four to six strips, which were embeddee in paraffin. Tissue sections were stained with hematoxylin and eosin. In quantitative analyses, the numbers of lesions were counted under a light microscope, the total length of the basement membrane was measured with a color video image processor (VIP-21 or 21C; Olympus-Ikegami Tsushin Co., Tokyo, Japan), and the results were expressed as the number of lesions per 10 cm of basement membrane.  16 20 was initiated in male 6-week-old F344 or Wistar rats Weeks by adding 0.01°/ BBN to their drinking water for 4 lence of urinary bladder lesions in rats given weeks, and then the animals were given a test ,l-N-(4-hydroxybutyl)nitrosamine. chemical to examine its promoting activity. The Wks. FIGURE 2. Incidence of PN hyperplasia in the urinary bladder of rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). 62 ITO ET AL. r chemicals tested, their dose levels and the observation periods used are shown in Figure 3. The incidence and number of PN hyperplasia per 10 cm of basement membrane are shown in Table 1. Administration of 5% saccharin, but not 0.5% saccharin, significantly increased the incidence and number of PN hyperplasia. This finding could be related to the induction of cancers in rat urinary bladder by high levels of saccharin. Sodium ascorbate, DL-tryptophan, allopurinol and diphenyl also increased the numbers of PN hyperplasia signifi-  cantly, but other test chemicals, such as acetazolamide, quercetin, cyclamate, o-phenylphenol, and caffeine did not. Results on saccharin and tryptophan are consistent with previous findings (5) and suggest that sodium ascorbate, allopurinol and diphenyl have promoting activities in urinary bladder carcinogenesis in rats. No correlation was found between the number of crystals in the urine sediment and promotion of preneoplastic lesion, but further studies are required on this problem. In general, chemical carcinogens show doseresponse relation for both tumor induction and the length of the induction period (8,11). Dose-response studies have been undertaken with the use of saccharin as a promoter for preneoplastic lesions in the urinary bladder (12). F344 rats of both sexes were initiated by treatment with 0.01% BBN in their drinking water for 4 weeks and then given diets containing saccharin at concentrations of 5, 1, 0.2, 0.04 and 0% for 32 weeks. The animals were killed at the end of week 36. The incidences and average numbers of PN hyperplasia per 10 cm of basement membrane in males and females treated with saccharin after BBN are shown in Tables 2 and 3, respectively. The incidences in males with a dose of 5% saccharin and in females with doses of 5% and 1% were significantly higher than those with BBN alone. Plots of the incidence of PN hyperplasia against the dose of saccharin gave parabolic curves in both sexes (Fig. 4); those dose-response curves showed enhanced hyperplastic responses in both sexes given 0.2 to 5% saccharin. Administration of various doses of saccharin without BBN did not cause any changes in the urinary bladder of rats of either sex. Although no increase in the induction of papilloma or cancer by saccharin was noted in either sex after BBN administration, the results suggest that if the experimental period had been longer, a dose-response relationship for the induction of papilloma and cancer might have been ob-served. A similar dose-response relation for the effect of a promoter has been reported (13) for liver carcinogenesis initiated by 2-acetylaminofluorene (2-AAF).
Promoting Effects of Saccharin with Two Different Initiating Doses of BBN It is generally accepted that initiation of chemical carcinogenesis is brought about by chemicals that are mutagenic in a short-term in vitro assay. Doserelated mutagenic effects have been reported for many chemical carcinogens, and in vivo doseresponse relations of initiation activities have been studied in two-stage carcinogenesis in the skin and liver (14,15).  bValues are means ± SD. ***p < 0.05 compared with BBN alone. *p < 0.001 compared with BBN alone.  We examined the promoting effect of saccharin after initiation with two different doses of BBN. Male 6-week-old F344 rats were divided into five groups. In the initiation stage, the rats were given drinking water containing solutions of 0.05%, 0.01% or 0% BBN for 4 weeks. Then in the promotion stage, they were given diet containing 5% saccharin or basal diet for 32 weeks.
The incidences and numbers of PN hyperplasia per 10 cm of basement membrane in the urinary bladder are shown in Table 4. The incidence and number of PN hyperplasia and cancer in the groups that received 0.01 or 0.05% BBN followed by saccharin was significantly higher than in the respective BBN groups without saccharin. In the groups treated with 0.01% BBN, administration of saccharin also increased the occurrence of PN hyperplasia significantly. The inductions of PN hyperplasia and cancer in the group treated with the high dose of BBN followed by saccharin were greater than those in the group treated with the low dose of BBN followed by saccharin. Thus, in the present study, the two different doses of BBN showed a dose-response relationship for initiation of urinary bladder carcinogenesis that was promoted by saccharin. These results are consistent with those on the promoting effect of phenacetin in two-stage carcinogenesis or rat urinary bladder initiated by two different doses of BBN (9).

Organ-Specific Effects of Promoters
For study of the organ specific effects of two different tumor promoters, rats were divided into eight groups. In the initiation stage, the rats were given 0.02% 2-AAF in the diet for 4 weeks, or 0.01% BBN in the drinking water for 4 weeks, or neither 2-AAF nor BBN for 4 weeks. Then, in the promotion stage, they were given 0.05% phenobarbital in the diet, or 5.0% saccharin in the diet, or basal diet for 32 weeks. The experimental protocol is shown in Figure 5. Phenobarbital greatly en-   hanced hepatocarcinogenesis and induced hepatocellular carcinoma after 2-AAF treatment, and it significantly increased formation of hyperplastic (neoplastic) liver nodules after BBN treatment (Table 5). Saccharin significantly enhanced the induction of PN hyperplasias of the urinary bladder after BBN or 2-AAF treatment (Table 6). However, after 2-AAF or BBN treatment, there was no effect of phenobarbital on the urinary bladder or of saccharin on liver neoplasia induction. These data indicate that although 2-AAF and BBN have tumor-initiating effects in both liver and urinary bladder, the tumor-promoting effects of phenobarbital and saccharin are organ specific. The results are consistent with the findings that phenobarbital did not enhance 7,12-dimethylbenz(a)anthracene-induced skin tumors (16) or dimethylnitrosamine-induced lung tumors and that phenobarbital increased the incidence of spontaneous liver tumors, but had no effect on the incidence of other spontaneous tumors (17).
Saccharin also did not enhance induction of forestomach tumors by benzo(a)pyrene (18). Therefore, the tumor-promoting effects of phenobarbital and saccharin may be restricted to the liver and urinary bladder, respectively.
Strain Differences in Susceptibility of the Urinary Bladder to Saccharin There are many reported differences in chemical carcinogenesis between strains of animals (7,19). Strain difference is one of the factors that modify chemical carcinogenesis. Strain differences appear to modify promotion as well as initiation in twostage carcinogenesis. Therefore, we tested whether there was any strain difference in the effect of saccharin as a promoter.
aThe rats were initiated with either 0.02% 2-AAF in their diet or 0.01% BBN in their drinking water for 4 weeks followed by either 0.05% phenobarbital or 5.0% saccharin in their diet for 32 weeks.
trol, and then their urinary bladder was examined by light and scanning electron microscopy.
Histological examination showed that saccharin induced urinary bladder lesions in ACI rats, but no changes in Wistar, F344 or SD rats (Table 7). PN hyperplasia and papilloma were increased significantly in the urinary bladder of ACI rats given saccharin. Scanning electron microscopy showed mucosal foci with slightly elevated cells, giving a cobblestone appearance, on the luminal surface of the urinary bladder. The superficial epithelial cells were covered with short, uniform microvilli and with ropy or leafy microridges. In addition, several superficial epithelial cells with pleomorphic microvilli on their luminal surface were found in these foci, as described previously (20). These scanning electron microscopic findings were greatest in ACI rats among the groups given saccharin. Slight formation of pleomorphic microvilli and short, uniform microvilli were seen in Wistar and F344 rats treated with saccharin, but were not seen in SD rats. Thus, there was a clear strain difference in rats in susceptibility of the urinary bladder to saccharin. It is explained that the promoting effect of saccharin in the urinary bladder was greatest in ACI rats after initiation with naturally occurring compounds. Strain differences may be one of the important factors in explaining differences of exertion of promoting effects.

Membrane Potentials of Urinary Bladder Epithelium in Rats Treated with Saccharin
Scanning electron microscopic studies have shown morphological alterations, such as pleomorphic microvilli and short, uniform microvilli on the bladder surface of rats treated with BBN (21), FANFT (22), or saccharin in the early stage of the urinary bladder carcinogenesis (20) Kakizoe et al. (3) reported that Concanavalin A agglutinated isolated bladder cells of rats treated with BBN or saccharin. These findings suggest that urinary bladder carcinogens or promoters might induce changes in physiological function of the surface membrane of the epithelium.
We investigated the electrophysiological changes in the membrane potential of superficial epithelial cells of the urinary bladder in rats exposed to BBN or saccharin. Male 6-week-old F344 rats were given 0.05% BBN in their drinking water or 5% sodium saccharin in their diet for 8 weeks. Untreated rats were used as controls. After 1, 2, 4 or 8 weeks on test, groups of five rats were killed, their urinary bladders removed, everted, and placed on a flat plastic sheet, they were then placed in a chamber filled with modified Krebs solution (350C) into which 95%0 2 and 5% CO2 was bubbled. Membrane potentials of superficial epithelial cells of the bladder were measured with a microelectrode filled with 3M KCI, and with an impedance of approximately 20 megohms.
The results are summarized in Table 8. Increases in the membrane potential of superficial epithelial cells of the urinary bladder in rats treated with BBN was observed from as early as week 1 to the end of the 8 weeks experiment (p<0.001). Saccharin administration also increased membrane potential, the increase being significant in weeks 2 and 4 (p< 0.05). However, the membrane potentials induced by saccharin were consistently lower than those induced by BBN. In general, change in membrane potential occur due to a change in K+ permeability and/or change of Naelectrogenic pump activity so that the increase of membrane potential in groups treated with BBN and saccharin might reflect these changes. Differences between the membrane potentials of groups treated with BBN and saccharin, therefore, may be related to differences in membrane components.

Conclusions
The two-stage process, initiation and promotion, applies to urinary bladder carcinogenesis. The promoting effects of 16 test chemicals on urinary bladder carcinogenesis in rats initiated by BBN were examined, and the in vivo biological characteristics of the promoter saccharin were examined.
The results showed that various environmental chemicals may have promoting effects on urinary bladder carcinogenesis in rats. The promoting effect of saccharin was dose-dependent and the effects of tumor promoters were organ specific. Moreover, there were strain differences in susceptibility to saccharin.
These findings show that urinary bladder carcinogenesis proceeds by a two-stage process. The concept of promotion is important in assessing the carcinogenic risk to humans of environmental factors. To counteract this risk we must detect not only environmental carcinogens but also environmental promoters. Some short-term in vitro tests are recommended for definite evidence of genotoxicity. However, promoters which are epigenetic cannot be detected by short-term in vitro test. Therefore, an in vivo bioassay using a marker of a preneoplastic lesion, as in the present studies, is useful for detection of promoters of urinary bladder carcinogenesis.