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
Katsumi Takaba1,2,3,
Koji Saeki1,
Kazuo Suzuki1,
Hideki Wanibuchi2 and
Shoji Fukushima2
1 Toxicological Research Laboratories, Kyowa Hakko Kogyo Co. Ltd, 2548 Fujimagari, Ube, Yamaguchi 755-8501 and
2 Department of Pathology, Osaka City University Medical School, Osaka, Japan
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Abstract
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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 16). 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 46. 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/G1 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.
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; BrdU, 5-bromo-2'-deoxyuridine; FCM, flow cytometry; LI, labeling index; MT, metallothionein; Na-AsA, sodium L-ascorbate; PN, papillary or nodular; SEM, scanning electron microscope; TdT, terminal deoxynucleotidyl transferase; TEM, transmission electron microscope; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
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Introduction
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Since the work of Berenblum (1), the two-stage mechanism of carcinogenesis, which begins with initiation events followed by a period of promotion, has been studied in great detail (27). It is possible that preneoplastic lesions grow in the promotion stage due not only to increased cell proliferation, but also to a decrease in cell death or apoptosis. In urinary bladder carcinogenesis, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) acts as a genotoxic initiator and sodium L-ascorbate (Na-AsA) as a non-genotoxic promoter (79). In rats, the two chemical treatments for over 4 weeks both induce the same morphological changes, i.e. epithelial hyperplasia of urinary bladder (10,11). However, there is a qualitative difference in that only the lesions induced by BBN treatment subsequently develop into neoplasms after cessation of the chemical insult.
The purpose of the present experiment was to clarify the effects of BBN and Na-AsA on cell proliferation and cell death (apoptosis) using immunohistochemical and flow cytometric (FCM) techniques. Rapid cell proliferation is usually achieved by an accelerated cell transit through G1 phase and results in a relative increase in the cell fraction in S phase, which can be easily calculated from the DNA content distribution measured by FCM (12,13). Another means of assessing cell movement through the G1/S boundary is to count the number of cells which express cyclin D1, which is expressed around this period (14,15). Actually, overexpression of cyclin D1 has been observed in various human malignant tumors (1621) and is considered as a putative preneoplastic alteration in murine chemical carcinogenesis (2228).
Recently, apoptotic cell loss in carcinogenesis has been examined not only by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and FCM detection of sub-G1 populations but also by immunohistochemical demonstration of metallothionein (MT), a cysteine-rich protein of low molecular weight capable of binding several heavy metals such as copper, zinc and cadmium (29,30). MT expression has been found in various types of human tumors and hyperplastic lesions and its function has mainly been investigated in terms of inhibition of apoptotic transduction signals (3134). However, it was also shown to act as an endogenous defensive factor in a mouse skin tumor model (35). Although its biological significance is still unclear, we evaluated its expression in this study.
In addition, the susceptibility to induction of transitional epithelium proliferation by sodium salt-type compounds (e.g. Na-AsA and sodium saccharin) is greater in male than in female rats and mice, while hamsters and monkeys are resistant (3639). This has recently been explained on the basis of
2u-globin (4043), which is synthesized in large amounts in the livers of rats, secreted into the blood and excreted into the urine (4244), and amorphous precipitates, including calcium phosphate, in the urine (47,48). Female and castrated male rats generally have only a weak capacity for
2u-globin synthesis because this is under androgen control (41,44). Therefore, we also investigated whether testicular castration might influence the response to Na-AsA exposure.
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Materials and methods
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Animals
A total of 320 6-week-old male F344 rats were obtained from Charles River Japan Inc. (Shiga, Japan). They were housed at a rate of 4 animals/cage in stainless steel wire mesh cages and placed in a controlled environment, maintained under a 12 h lightdark cycle (lights on 8:0020:00), air conditioned at 24 ± 2°C and 55 ± 20% humidity. Food (FR-2; Funahashi Farm Inc., Chiba, Japan) and water were available ad libitum throughout the period of the experiment.
Chemicals
BBN was obtained from Tokyo Kasei Co. Ltd (Tokyo, Japan) and Na-AsA from Wako Pure Chemical Industries Ltd (Osaka, Japan).
Experimental design
The experimental protocol is shown in Figure 1
. After 2 weeks quarantine, rats were divided into six groups, 16, containing 68, 63, 34, 63, 63 and 29 animals, respectively. Rats in groups 46 underwent testicular castration 2 weeks before commencement of chemical treatment. Dose levels and duration of BBN and Na-AsA treatments, selected with reference to the results of earlier studies (10,49,50), were as follows. Drinking water containing 0.05% BBN was given to rats of groups 1 and 4 from weeks 2 to 10 (for 8 weeks). Powdered diet supplemented with 5% Na-AsA was given to groups 2 and 5 for 8 weeks. Groups 3 and 6 were maintained without chemical treatment as controls. The total observation period was 18 weeks.

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Fig. 1. Experimental design. Groups 1 and 4, 0.05% BBN in drinking water; groups 2 and 5, 5% Na-AsA in powdered basal diet; groups 3 and 6, non-treatment control. Arrowheads, testicular castration; arrows, death time points.
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During the experiment, a daily health check was performed and body weights were measured at weeks 0, 2, 6, 10, 11, 12, 14 and 18. Consumption of food and water was recorded over 2 days in weeks 6, 10, 14 and 18.
Urinalysis
Fresh urine samples were taken by forced urination from 10 rats of each group in the morning (8:309:00) in weeks 10 and 18 and urinary pH was measured with a pH meter (Twin pH; Horiba Ltd, Kyoto, Japan). In parallel, 10 rats of each group were kept separately in metabolic cages without feeding and urine was collected over a period of 18 h (15:0009:00) for volume measurement and electrolyte analysis. Sodium and potassium were analyzed with an atomic absorption spectrophotometer (Z8000; Hitachi Ltd, Tokyo, Japan) and chloride with a chloride meter (CL-7; Hiranuma Sangyo Co. Ltd, Ibaragi, Japan).
Histopathology
DNA synthesis in urinary bladder epithelium was examined with five rats of each group at the ends of weeks 6, 10, 11, 12, 14 and 18 as follows. 5-Bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co., St Louis, MO) solution was i.p. injected at a dose of 100 mg/kg body wt, followed by killing 1 h later under ether anesthesia. The urinary bladder, liver and kidney from each rat were removed and fixed in 10% phosphate-buffered formalin solution. Particular care was taken with the urinary bladders, which were inflated by intraluminal injection of 10% phosphate-buffered formalin solution and then cut into 10 strips. Tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Five control animals were examined at the ends of weeks 10 and 18, respectively. In addition, small samples of colon from each animal were removed as positive control tissue for BrdU immunohistochemistry.
Electron microscopy
Two rats each in groups 1, 2, 4 and 5 were killed under anesthesia at the ends of weeks 10, 11, 12 and 18, along with group 3 and 6 controls at the ends of weeks 10 and 18. The urinary bladders were removed from each rat after inflation by intraluminal injection of 2.5% glutaraldehyde solution and immersed in the same solution for 2 h. Each bladder was cut longitudinally into halves and post-fixed in 1% osmium tetroxide. One half was embedded in Epok 812 (Oken, Tokyo, Japan). Ultrathin sections were obtained with an ultramicrotome (Ultracut E; Reichert-Jung, Nussloch, Germany), double stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (TEM) (JEM-1200EX; JEOL, Tokyo, Japan). The other half was critical point dried (HCP-1; Hitachi Ltd, Tokyo, Japan) with liquid CO2 after dehydration through an ascending alcohol series. The specimens were cemented to aluminum specimen stubs, spatter-coated with gold in a vacuum evaporator and examined at 10 keV in a scanning electron microscope (SEM) (S-450; Hitachi Ltd, Tokyo, Japan).
Immunohistochemistry and the TUNEL method
Paraffin sections of the urinary bladder were spread on silanized slides. They were immunohistochemically stained for incorporated BrdU, cyclin D1 and MT using a peroxidase-labeled polymer reagent (EnvisionTM; Dako Japan Co. Ltd, Kyoto, Japan) together with mouse monoclonal antibodies against BrdU (clone Bu20a; Dako A/S, Glostrup, Denmark), cyclin D1 (clone 5D4; IBL, Gunma, Japan) and MT (clone E9, reacts with both MT-1 and MT-2; Zymed Laboratories Inc., CA), respectively. Before immunostaining, slides for cyclin D1 immunohistochemistry were incubated in 0.01 mol/l citric buffer (pH 6.0) for 5 min at 121°C in an autoclave to effect antigen retrieval. Nick ends of DNA in the nuclei were detected by the TUNEL method using a TACSTM in situ apoptosis detection kit (Trevigen Inc., MD). Sections were developed with a diaminobenzidine hydrogen peroxidase substrate and counterstained with either hematoxylin or methyl green.
To score each parameter, the positive cell ratio (labeling index, LI) was determined by randomly observing at least 2500 epithelial cells (>5000 cells in many sections) using a x200 magnification (over 50 fields). LI were calculated as numbers per 100 cells.
In addition, to achieve immunohistochemical staining of
2u-globulin, liver and kidney sections from each animal at week 10 were visualized using the EnvisionTM reagent together with rabbit anti-
2u-globulin serum (51).
Flow cytometry
Cell suspensions of urinary bladder epithelium were prepared by killing five animals each in the treated groups (groups 1, 2, 4 and 5) at the ends of weeks 6, 10, 11, 12 and 18 and five rats of the control groups (groups 3 and 6) at the ends of weeks 10 and 18 according to the method reported previously (52), as follows. The urinary bladders were removed, everted and immersed in an enzyme cocktail solution of collagenase/dispase (Boehringer Mannheim Biochemica GmbH, Mannheim, Germany) at a final concentration of 2 mg/ml in 0.25% trypsin Hank's () solution and incubated at 37°C for 90 min. After centrifugation of the cell suspension (2000 r.p.m., 4°C, 5 min), pellets were resuspened in 2 mM EDTA (Wako Pure Chemical Industries Ltd) in Hank's () solution and incubated at 37°C for 20 min. After recentrifugation (2000 r.p.m., 4°C, 5 min), cells were fixed in 5 ml of ice-cold 70% ethanol solution and stored at 20°C before staining.
Cells were stained with a propidium iodide solution (20 µg/ml) in phosphate-buffered saline containing RNase (100 µg/ml) for 15 min. DNA content distributions were determined using a flow cytometer (Coulter EPICS Elite; Coulter Co., FL), with the excitation wavelength set at 488 nm. Data for >3000 cells were collected from each sample and analyzed with software for automatic analysis (MultiCycle; Phoenix Flow System, CA). Sub-G1 cells were defined as those in the region below 0.8 times the G0/G1 peak DNA channel.
In addition, cells were taken and dispersed enzymatically in the same way from five animals each of the treated and control groups (groups 1 and 3) at week 11 for terminal deoxynucleotidyl transferase (TdT) assay. After fixation in ice-cold 1% paraformaldehyde in phosphate-buffered saline for 15 min and post-fixation in 5 ml of 70% ethanol solution at 20°C overnight, TdT assays were performed using a commercially available kit which results in staining of DNA with propidium iodide and apoptotic cells with fluorescein (Apo-DirectTM; Phoenix Flow System). Fluorescence intensities of at least 10 000 cells were measured with a Coulter EPICS Elite flow cytometer and analysed with Multigraph software.
Statistical analysis
The statistical significance of the differences in incident data between the control and experimental groups was examined using the
2 test with the Yates correction. For other measurements Student's t-test and/or Welch's t-test were applied in combination with the F-test. For treated group data at weeks 6 and 10 comparison was made with control values at week 10 and for weeks 11, 12, 14 and 18, control data at week 18 were employed.
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Results
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General condition
During the experiment, no deaths occurred and no abnormalities were noted on clinical observation. Representative data for body weight and food and water consumption are shown in Table I
. In castrated animals (groups 46), the values were decreased in comparison with those of non-castrated animals (groups 13) from week 6 to the end of the experiment. There were no differences ascribable to the chemical treatments in either castrated or non-castrated animals.
Urinalysis
The results of urinalysis at week 10 are also shown in Table I
. Castrated and non-castrated animals treated with Na-AsA (groups 2 and 5) showed significantly elevated pH values and Na ion concentrations and decreased Cl ion concentrations when examined at the end of the treatment period. However, the data returned to normal levels by week 18. There were no differences ascribable to castration.
Histopathology
Histopathological changes of urinary bladder epithelium were classified into four categories, i.e. simple hyperplasia, papillary or nodular (PN) hyperplasia, which is a putative neoplastic lesion, papilloma and carcinoma, as described by Fukushima et al. (11). The data are summarized in Table II
. Most of the treated animals (groups 1, 2, 4 and 5), whether castrated or non-castrated, showed simple hyperplasia. The degree of change and/or distribution density decreased after cessation of treatment. Particularly, simple hyperplasia in Na-AsA-treated animals (groups 2 and 5) disappeared completely by the end of the experiment. In contrast, BBN-treated animals (groups 1 and 4) began to develop PN hyperplasia and neoplasms (papilloma and carcinoma) from week 10. The distribution densities and incidences of PN hyperplasia were not different between castrated and non-castrated animals and were also decreased after cessation of BBN treatment. However, in castrated animals (group 4) total numbers of neoplasms (five papillomas and one carcinoma in 24 animals) were less (14 papillomas in 25 animals) than in non-castrated animals (group 1).
Electron microscopy
Upon SEM examination, the normal luminal surface epithelium of the urinary bladder was shown to be lined by polygonal cells with a complex network of microridges (Figure 2A
). SEM examination performed at week 10 revealed loss of the normal microridge network in Na-AsA-treated animals (groups 2 and 5) and formation of ropey or leafy microridges and/or short, uniform microvilli in BBN-treated animals (groups 1 and 4; Figure 2B
). After cessation of treatment, the lesions disappeared rapidly (Figure 2C
).
TEM examination demonstrated the presence of apoptotic bodies within the cytoplasm of transitional cells in BBN-treated animals (groups 1 and 4) at week 11 (Figure 2D
).
Immunohistochemistry and the TUNEL method
The LIs for BrdU and cyclin D1 increased significantly in hyperplastic lesions in BBN-treated animals (groups 1 and 4) when examined at weeks 6 and 10 (Figures 3A and B, 4A and B and 5B and C

). They decreased after cessation of BBN treatment but remained significantly higher than in untreated controls. The LIs in Na-AsA-treated animals (groups 2 and 5) changed in a similar way except that cyclin D1 was not overexpressed and they rapidly recovered after cessation of Na-AsA treatment. Sequential changes in each LI indicated no differences ascribable to castration. The LIs for BrdU and cyclin D1 in BBN-induced papillomas ranged from 1.8 to 22.9% (mean ± SD 7.10 ± 5.64%; incidence 15/15 papillomas) and from 0 to 12.4% (mean ± SD 4.10 ± 3.69%; incidence 16/17 papillomas), and in one case of carcinoma were 26.3 and 38.0%, respectively.

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Fig. 3. Sequential changes in non-castrated animals (groups 13). (A) BrdU LI. (B) Cyclin D1 LI. (C) TUNEL LI. (D) MT LI. Data presented are means ± SD values.
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Fig. 4. Sequential changes in castrated animals (groups 46). (A) BrdU LI. (B) Cyclin D1 LI. (C) TUNEL LI. (D) MT LI. Data presented are means ± SD values. No differences ascribable to non-castration were found for any parameter in comparison with Figure 3 .
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The TUNEL LI increased in hyperplastic lesions in BBN-treated animals (groups 1 and 4; Figures 3C, 4C and 5D

). After cessation of BBN treatment, the LI began to decrease slightly but 1 or 2 weeks afterwards it increased transiently. Thus, in week 18, hyperplastic lesions still demonstrated high levels of apoptosis. In Na-AsA-treated animals (groups 2 and 5), hyperplastic lesions also showed an increase in TUNEL-positive cells. No differences ascribable to castration were noted. In neoplastic lesions induced by BBN treatment values ranged from 0 to 2.2% (mean ± SD 1.040.74%; incidence 15/17 papillomas), and in one case of carcinoma it was 0.9%.
Regions of MT overexpression began to appear in hyperplastic areas in BBN-treated animals (groups 1 and 4) from the end of the treatment period and remained thereafter (Figures 3D and 4D
). The distribution of MT overexpression showed a characteristic patchy staining (Figure 5E
). In Na-AsA-treated animals (groups 2 and 5), MT was not overexpressed and no influence of castration was observed. In tumors, levels were low (incidence 6/16 papillomas, positive range 0.415.82%; carcinomas negative). There was no obvious overlap between cyclin D1-positive and MT-positive regions in hyperplastic and neoplastic epithelium (Figure 5F and G
).
A stronger reaction for
2u-globulin in hyaline droplets in the renal proximal tubular epithelium and in hepatocytes was demonstrated in non-castrated animals (groups 13) than in castrated animals (groups 46; Figure 5H and I
).
Flow cytometry
The results for cell cycle distribution are shown in Table III
and DNA histograms and cytograms at week 11 are demonstrated in Figure 6
. During the period of treatment with BBN and Na-AsA, the percentage of S phase cells measured by FCM was higher than the control level, but it returned to normal after cessation of treatment with a sharp peak appearing after 1 week in close proximity to the G0/G1 peak (not in the sub-G1 area), due probably to chromatin condensation in the G0/G1 cells (Figure 6A, B, D and E
). It then moved further to the left to form a wide sub-G1 region in the DNA histogram, representing apoptotic cells (5355). The cells in the sub-G1 area increased in number during treatment but decreased gradually to the control level by week 12, although a transient rise was noted in the first week after cessation of treatment. The above phenomena were observed with all treated groups (groups 1, 2, 4 and 5) and there were no differences ascribable to castration.

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Fig. 6. FCM findings for urinary bladder epithelial cells. (A and D) DNA histogram (A) and cytogram (D) for cells from a non-castrated rat 1 week after withdrawal of BBN. (B and E) DNA histogram (B) and cytogram (E) for cells from a non-castrated rat 1 week after withdrawal of Na-AsA. (C and F) The normal features of the DNA histogram (C) and cytogram (F) for cells from a non-castrated rat not receiving chemicals at the end of the experiment. The sub-G1 area (arrowheads) is increased with both BBN and Na-AsA and an apparently sharp peak (large arrows) is shown in close proximity to the normal G0/G1 peak (small arrows). More than 50% of cells in the G0/G1 population show clearly reduced volumes (FS values) but little decrease in their DNA content, indicating nuclear and/or cytoplasmic condensation.
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TdT assays for detection of DNA fragmentation revealed a significant increase in TdT-positive cells to 1.78 ± 0.67% 1 week after cessation of BBN treatment (group 1) from a control value of 1.01 ± 0.58% (group 3).
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Discussion
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In accord with earlier findings (7,9,10,49,50), the present investigation showed an increase in the number of S phase cells incorporating BrdU due to treatment with BBN or Na-AsA. Worthy of note in this connection is the fact that the changes were reversible or irreversible depending on whether induced by the non-genotoxic Na-AsA or the genotoxic BBN (11,42,49,50). Overexpression of cyclin D1 was also induced with BBN but not with Na-AsA. The distribution and sequential changes in cells expressing cyclin D1 were analogous to those of BrdU-positive cells. The degree of cyclin D1 overexpression increased in the order simple hyperplasia, PN hyperplasia, papilloma and carcinoma. It has been reported that cyclin D1 overexpression might play a critical role in urinary tumor progression in both humans and rats (21,28). Therefore, its overexpression could be a useful marker for histological diagnosis of malignancy in the early stage of urinary carcinogenesis.
MT, found to be overexpressed in bladder epithelium of rats treated with BBN but not with Na-AsA, has been considered to protect against oxidative stress (3133,56). However, the distribution and sequential changes in cells expressing MT were different from those of both BrdU- and cyclin D1-positive cells. The fact that cyclin D1-positive cells were generally lacking MT and that MT overexpression was little observed in papillomas and never found in carcinomas is suggestive in this respect. The observed strong contrast between positive and negative regions in the bladder epithelium uniformly exposed to BBN suggests that there may be differential sensitivity, with heterogeneous mutational and/or loss of heterozygosity patterns within single BBN-induced lesions. Hence, it should be very interesting to determine whether regions susceptible to carcinogens exist in two-dimensional units, analogous to the epidermal proliferation units described by Potten (57). In addition, the present experiment showed no relationship between MT and TUNEL. We have no explanation for the discrepancy with earlier reports that MT synthesis is characteristic of malignant bladder tumor cells in rats (34). This contrasts with a possible inhibition of hyperplastic laryngeal cells in man (35), and also requires further investigation.
In the present study, apoptotic cell death occurred during and after treatment with BBN or Na-AsA, as demonstrated using a variety of methods. TUNEL-positive cells made their appearance in simple hyperplasia induced by BBN. During this stage in the process of carcinogenesis, apoptotic cell loss may play an important role in the balance with cell production. Although the proportions of cells exhibiting chromatin condensation did not directly match those of apoptotic cells measured by the TUNEL and TdT methods, this feature is generally considered as an early maker of this type of cell death (58). This suggests that cessation of BBN and Na-AsA treatments may rapidly derange the cell cycle and finally induce apoptotic cell death. However, most cells with nuclear condensation may not pass through the point of no return leading to cell death.
From the standpoint of variation in cell proliferation and apoptosis between hyperplastic lesions of urinary bladder induced by genotoxic and non-genotoxic agents, the findings that overexpression of cyclin D1 and MT were only induced by BBN in the present study is of interest.
A number of experiments have shown that sodium salt-type compounds stimulate cell proliferation in male rats but are not effective in the NCI-Black-Reiter (NBR) strain (41,42), which lacks
2u-globulin synthesizing ability (59,60). Actually, Na-AsA has no promoter activity during two-stage urinary carcinogenesis in male NBR rats after initiation with BBN (43), while BBN induces cell proliferation in male rats of the same strain (61). Since urinary excretion of
2u-globulin is reduced by castration to ~10% of the control level (41,46), Na-AsA might be expected to be less effective in castrated rats. However, this was not the case with the F344 rats used in the present experiment. This discrepancy in results is probably due to genetic factors or calcium phosphate-containing precipitates in the urine. Male ODS/Shi-od/od and WS/Shi rats, which have levels of
2u-globulin synthesis equal to that of F344 rats, also demonstrated resistance to the promoting effects of Na-AsA in two-stage urinary bladder carcinogenesis (62,63). Precipitates formed at high urinary pH might contribute to urothelial proliferation by direct mechanical stimulation and variation of this parameter could therefore be important (44,45). Our data are consistent and suggest a hypothesis that the susceptibility to Na-AsA depends on factors other than urinary
2u-globulin.
In BBN-treated animals, the finding that total numbers of neoplasms in the castrated groups were less than in the non-castrated group is also in line with the reports of Okajima et al. (64) and Murai et al. (65), that a sex factor influences the progression of BBN-induced urinary bladder tumor in rodents.
In conclusion, our data demonstrate the following: overexpression of both MT and cyclin D1 in hyperplastic bladder epithelium is associated with BBN but not Na-AsA treatment, MT being suggested as playing an important suppressive role in the early stages of rat urinary carcinogenesis. Induction of cell proliferation and apoptotic changes (especially in G0/G1 cells) in hyperplastic epithelium is common to treatment with and after withdrawal of both chemicals. However, cell proliferation in hyperplastic lesions induced by BBN is irreversible, while being reversible in those caused by Na-AsA. Testicular castration had no influence on these events, although it decreased tumor formation due to BBN treatment.
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Acknowledgments
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We acknowledge the valuable advice of Dr Manabu Takahashi, Professor Emeritus, Yamaguchi University, and are grateful to Dr Koichi Saito of Sumitomo Chemical Co. Ltd, Osaka, Japan, for providing rabbit anti-
2u-globulin serum. In addition, we thank Messrs Takahisa Asamatsu, Hirofumi Misaka and Teruyoshi Imada and Miss Megumi Kawano for their valuable technical assistance.
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Notes
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3 To whom correspondence should be addressed at: Toxicological Research Laboratories, Kyowa Hakko Kogyo Co. Ltd, 2548 Fujimagari, Ube, Yamaguchi 755-8501, Japan Email: katsumi.takaba{at}kyowa.co.jp 
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Received June 1, 1999;
revised November 30, 1999;
accepted December 1, 1999.