Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors

Min Wei1, Hideki Wanibuchi1, Keiichirou Morimura1, Shuji Iwai1, Kaoru Yoshida2, Ginji Endo2, Dai Nakae3 and Shoji Fukushima1,4

1 Department of Pathology, Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585,
2 Department of Preventive Medicine and Environment Health, Osaka City University Medical School, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585 and
3 Department of Oncological Pathology, Cancer Center, Nara Medical University, Nara 634, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arsenic is a well-documented human carcinogen, and contamination with this heavy metal is of global concern, presenting a major issue in environmental health. However, the mechanism by which arsenic induces cancer is unknown, in large part due to the lack of an appropriate animal model. In the present set of experiments, we focused on dimethylarsinic acid (DMA), a major metabolite of arsenic in most mammals including humans. We provide, for the first time, the full data, including detailed pathology, of the carcinogenicity of DMA in male F344 rats in a 2-year bioassay, along with the first assessment of the genetic alteration patterns in the induced rat urinary bladder tumors. Additionally, to test the hypothesis that reactive oxygen species (ROS) may play a role in DMA carcinogenesis, 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in urinary bladder was examined. In experiment 1, a total of 144 male F344 rats at 10 weeks of age were randomly divided into four groups that received DMA at concentrations of 0, 12.5, 50 and 200 p.p.m. in the drinking water, respectively, for 104 weeks. From weeks 97–104, urinary bladder tumors were observed in 8 of 31 and 12 of 31 rats in groups treated with 50 and 200 p.p.m. DMA, respectively, and the preneoplastic lesion, papillary or nodular hyperplasias (PN hyperplasia), was noted in 12 and 14 rats, respectively. DMA treatment did not cause tumors in other organs and no urinary bladder tumors or preneoplastic lesions were evident in the 0 and 12.5 p.p.m.-treated groups. Urinary levels of arsenicals increased significantly in a dose-responsive manner except for arsenobetaine (AsBe). DMA and trimethylarsine oxide (TMAO) were the major compounds detected in the urine, with small amounts of monomethylarsonic acid (MMA) and tetramethylarsonium (TeMa) also detected. Significantly increased 5-bromo-2'-deoxyuridine (BrdU) labeling indices were observed in the morphologically normal epithelium of the groups treated with 50 and 200 p.p.m. DMA. Mutation analysis showed that DMA-induced rat urinary bladder tumors had a low rate of H-ras mutations (2 of 20, 10%). No alterations of the p53, K-ras or ß-catenin genes were detected. Only one TCC (6%) demonstrated nuclear accumulation of p53 protein by immunohistochemistry. In 16 of 18 (89%) of the TTCs and 3 of 4 (75%) of the papillomas, decreased p27kip1 expression could be demonstrated. Cyclin D1 overexpression was observed in 26 of 47 (55%) PN hyperplasias, 3 of 4 (75%) papillomas, and 10 of 18 (56%) TCCs. As a molecular marker of oxidative stress, increased COX-2 expression was noted in 17 of 18 (94%) TCCs, 4 of 4 (100%) papillomas, and 39 of 47 (83%) PN hyperplasias. In experiment 2, 8-OHdG formation in urinary bladder was significantly increased after treatment with 200 p.p.m. DMA in the drinking water for 2 weeks compared with the controls. The studies demonstrated DMA to be a carcinogen for the rat urinary bladder and suggested that DMA exposure may be relevant to the carcinogenic risk of inorganic arsenic in humans. Diverse genetic alterations observed in DMA-induced urinary bladder tumors imply that multiple genes are involved in stages of DMA-induced tumor development. Furthermore, generation of ROS is likely to play an important role in the early stages of DMA carcinogenesis.

Abbreviations: AP-1, activating protein-1 transcription factor; AsBe, arsenobetaine; DMA, dimethylarsinic acid; BBN, N-butyl-N-(4-hydroxybutyl) nitrosamine; BrdU, 5-bromo-2'-deoxyuridine; CDK, cyclin dependent kinase; COX-2, cyclooxygenase-2; IC-ICP-MS, ion chromatography with inductively coupled plasma mass spectrometry; MMA, monomethylarsonic acid; MSI, microsatellite instability; NF-{kappa}B, nuclear factor-{kappa}B; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; PN hyperplasia, papillary or nodular hyperplasia; ROS, reactive oxygen species; TCC, transitional cell carcinoma; TeMA, tetramethylarsonium; TMAO, trimethylarsine oxide


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arsenic is a known human carcinogen with the development of neoplasms in humans exposed to arsenic in the general environment and in industry (1,2). Unfortunately, acute and chronic arsenic exposure still remains a major public health problem in many countries. In particular, drinking water contamination results in increased incidences of cancers at multiple organ sites, especially the skin and urinary bladder, but possibly also in the liver, kidney, lung, nasal cavity, and prostate, in exposed populations from Taiwan, China, Eastern Europe, Southwestern United States, and South America (3,4). However, unlike most substances classified as carcinogens, carcinogenesis in laboratory animals by the metalloid arsenic has proved elusive (5). In fact, attempts to induce tumors in experimental animals with inorganic arsenic compounds have mostly failed, except for a few studies in which animals were given arsenic trioxide by intratracheal instillation (6,7). Due to certain deficiencies, the findings were not considered sufficient evidence of carcinogenicity in animals (1,2). The lack of a properly designed bioassay was of integral importance in this respect (8).

We have focused on dimethylarsinic acid (DMA), a major metabolite of arsenic in most mammals, including humans (9–12). Humans have continuous exposure to DMA from arsenic in their drinking water, production or use of arsenic-containing herbicides and ingestion of foods contaminated with these herbicides. We previously have demonstrated that DMA promotes carcinogenesis in the urinary bladder, kidney, liver, and thyroid gland of F344 rats in an in vivo multi-organ carcinogenesis bioassay (13). Wanibuchi et al.(14,15) indicated that DMA exerts promoting potential in a dose-dependent manner with regard to urinary bladder and liver carcinogenesis in F344 rats, possibly via a mechanism involving stimulation of cell proliferation and DNA damage caused by oxygen radicals. Of particular importance, the findings from our animal studies revealed effects at sites where human arsenic-associated cancers develop, such as the urinary bladder, liver and kidney, suggesting that DMA exposure may be relevant to the carcinogenic risk of arsenic to humans. This led us to conduct a 2-year bioassay to determine the carcinogenicity of DMA in male F344 rats. Previously, we reported that administration of DMA induced urinary bladder tumors in male F344 rats at doses of 50 and 200 p.p.m. in drinking water for 2 years (16). However, so far no detailed information for DMA carcinogenicity in long-term studies is available, especially in organs other than urinary bladder. A brief report of a 2-year carcinogenicity study in F344 rats fed DMA in the diet showed that it produced bladder tumors at doses of 40 or 100 p.p.m., and the effect was greater in female rats compared with males (17). In contrast, no bladder tumors or treatment-related tumors in other tissues were observed in mice treated with DMA in the diet for a 2-year period (17). Administration of DMA in the drinking water to mice caused an increase in the total numbers of spontaneous tumors in wild type mice and significantly accelerated tumor induction in both p53 knockout and wild type mice when all neoplastic lesions were combined in a one and a half years study, while there was no evidence of DMA-related tumors (18). Considering the multi-site promotional effects of DMA, one might question logically whether DMA treatment induces tumors or not in other organs in rats (8).

A better understanding of the carcinogenic mechanisms of arsenic will facilitate not only cancer risk assessment but also cancer prevention strategies. The possibility exists that DMA plays a role in arsenic carcinogenicity in humans and therefore, elucidation of the kinds of genetic alterations involved in DMA-induced rat urinary bladder carcinogenesis is of interest. So far, a variety of mechanisms have been proposed by which arsenic may exert carcinogenic activity. Based on evidence largely from in vitro studies, Goering et al. (5) have suggested that induction of chromosome abnormalities, altered DNA repair, altered DNA methylation, oxidative stress, and increased cell proliferation could be involved. Although DMA, arsenite and other arsenic compounds are not believed to directly bind to DNA (19), DNA single-strand breaks and crosslink formation between DNA and protein have been observed (20,21). DMA is considered to be a clastogenic agent (22). Increasing in vivo evidence, furthermore, indicates that it stimulates cell proliferation (15,23,24) while generating ROS (14,25).

The first objective in the present study was to determine the carcinogenicity of DMA in F344 rats when orally administrated in the drinking water for a 2-year period. The pathology of tumor development at a number of sites is provided in detail for the first time.

The second objective was to elucidate possible mechanisms involved in DMA bladder carcinogenesis by analyzing DMA-induced urinary bladder tumors for mutations of p53, ras, and ß-catenin genes. As described above, the effect of DMA might be mediated as a result of interference with multiple pathways encompassing the cell cycle, DNA damage/repair, and generation of reactive oxygen species (ROS). Protein expression of cell cycle regulatory factors p53, p27kip1, and cyclin D1 in normal epithelium, preneoplastic and neoplastic lesions of rat urinary bladder was assessed by immunohistochemistry. To test the hypothesis that ROS may play a role in DMA bladder carcinogenesis, we evaluated formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urinary bladder after short-term DMA treatment, this lesion being most commonly used as a marker for evaluation of oxidative DNA damage (26,27). Moreover, cyclooxygenase-2 (COX-2) expression was examined in DMA-induced rat bladder tumors as it has been shown to play a role in development of preneoplastic and neoplastic lesions in the human and rat urinary bladder (28,29). ROS is known to play a crucial role in the expression of COX-2 through activating nuclear factor-{kappa}B (NF-{kappa}B), which acts as a positive regulatory element of expression (30,31). We also examined the possible role of defective DNA mismatch repair activity in DMA-induced urinary bladder tumors by analyzing microsatellite instability (MSI), reported in various human malignant tumors including human urinary bladder tumors (32,34). Finally, we discuss the possible implications of the results in DMA urinary bladder carcinogenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
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 References
 
Chemical
DMA was purchased from Wako Pure Chemical Industries, Japan (purity, 99%).

Experiment 1
The animal experimental protocol was previously described in detail (16). A total of 144 male, 6 week old, F344/DuCrj rats were obtained from Charles River Japan, Hino, Japan, and divided randomly into four groups, each consisting of 36 rats. At the age of 10 weeks, rats received DMA at concentrations of 0, 12.5, 50 and 200 p.p.m., respectively, in the drinking water for 104 weeks. Urine was collected by forced urination at the end of weeks 30, 60 and 100; pH was immediately measured with a pH meter (Horiba model F-15, Tokyo, Japan), and then samples were stored at –80°C until analyzed for urine chemistry and DMA metabolites. Rats that had died or were killed under ether anesthesia when becoming moribund during the study or killed at the end of the study at week 104 were autopsied for macroscopic and histopathological examination. Blood samples were taken from all surviving rats from each group at week 104 for biochemical and hematological analysis. Ten rats from each group received an i.p. injection of 100 mg/kg body weight of BrdU (Sigma Chemical Co., St Louis, MO) 1 h before autopsy. All major organs were excised and fixed in 10% buffered formalin. After adequate fixation, they were cut and processed for paraffin embedding and routinely stained with hematoxylin and eosin for histological examination.

Experiment 2
Forty 10-week-old male F344 rats were used for detection of 8-OHdG formation in urinary bladder DNA after DMA treatment. They were divided into two equal groups, and given DMA at concentrations of 0 and 200 p.p.m., respectively, in the drinking water for 2 weeks. The rats were killed under ether anesthesia and their urinary bladder removed, immediately frozen in liquid nitrogen and stored at –80°C until used for analysis. Tissues from pairs of urinary bladders were pooled as samples for detection of 8-OHdG formation in nuclear DNA.

Urinary analysis
Urinary samples from the rats at weeks 30, 60 and 100 were used for assessment of urine chemistry, including sodium, potassium, chloride, and calcium (Hitachi-710 Electrolyte Analyzer, Tokyo Japan). Combined ion chromatography (IC, model 7000, Yokogawa Analytical Systems, Tokyo, Japan) with inductively coupled plasma mass spectrometry (ICP-MS, model 4500; Hewlett-Packard, DE, USA) were used for the determination of arsenic species in the urine samples. Analysis conditions of the IC-ICP-MS system were described previously (35).

DNA extraction
In the present study, 20 tumors (18 carcinomas and two papillomas) from different rats were available for mutation analyses of p53, H- and K-ras, and the ß-catenin genes. Sixteen carcinomas were used for analysis of MSI. DNA was extracted from formalin-fixed, paraffin embedded urinary bladder tumors with microdissection. DNA concentrations were determined with a spectrophotometer (Ultraspec 3000, UV/Visible Spectrophotometer; Pharmacia Biotech, Tokyo, Japan) and adjusted to a final concentration of 50 ng/µl for PCR-SSCP and MSI analyses.

PCR-SSCP analysis of p53, H-ras, K-ras, and ß-catenin
Exons 5–8 of p53, exons 1 and 2 of the H-ras and K-ras, and the ß-catenin genes were analyzed by the PCR-SSCP method. For the ß-catenin gene, the primers were designed to cover the 5' terminal region of the gene corresponding to functionally important phosphorylation sites. Sequences of primers and conditions used for PCR were described previously (36,37). PCR-SSCP was repeated at least twice to confirm the results. If shifted bands were observed on PCR-SSCP analysis, they were cut out from the acrylamide gel to be sequenced. All mutations were confirmed by repeating the PCR from fresh microdissected tumor tissues to avoid PCR-related artifacts.

DNA sequencing
DNA sequences were determined with an ABI PRISM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase (Perkin Elmer, Applied Biosystems, Foster City, CA) on the ABI PRISM 310 genetic analyzer (Perkin Elmer, Applied Biosystems).

Microsatellite instability analysis
Analysis of MSI was performed by PCR using 18 microsatellite loci interspersed throughout the rat genome including D2MIT2 and D2MIT12 on Chr.2; D3MGH9 on Chr.3; D6MGH3, D6MGH7 and IGHE on Chr.6; D8MGH3 on Chr.8; D9MIT1 and D9MGH1 on Chr.9; D13UWM1 on Chr.13; D14MGH2 on Chr.14; D15MGH14 on Chr.15; D16MGH3 on Chr.16; D18MGH3 on Chr.18; TAT on Chr.19; D20MIT1 and D20UW1 on Chr.20; and DXMGH1 on Chr.X. Primer sets were chosen based on previous publications (38,39) and obtained from Research Genetics (Huntsville, AL). Hot start PCR was performed in a 5 µl reaction volume containing 50 ng DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.2 mM MgCl2, 50 µM each of dATP, dGTP, dTTP, and dCTP, 0.2 µCi of [32P]dCTP, 4 pM of each primer, and 0.5 U of AmpliTaq GoldTM polymerase. Reactions were carried out in a thermal cycler (MJ Research, Inc.) at 95°C for 10 min, followed by 38 cycles (95°C for 30 s, 52–56°C for 30 s, 72°C for 30 s), and final incubation for elongation at 72°C for 12 min. The PCR products were diluted with 20 µl stop solution, denatured, separated in two kinds of 6% polyacrylamide gel containing either 5 M urea or 30% formamide. Comparing tumors and normal liver tissues, a tumor was diagnosed as positive for MSI if one or more abnormal band-shifts were observed.

Immunohistochemical analysis
Urinary bladders from a total of 40 rats from the groups in which bladder tumors were observed (50, 200 p.p.m. DMA-treated groups), as well as 10 rats from the 12.5 p.p.m. DMA and control groups, respectively, were examined for p53, p27kip1, cyclinD1 and COX-2 by immunohistochemistry. BrdU incorporation in morphologically normal bladder epithelium was examined in 10 rats of each group, respectively. Serial sections (4 µm) were cut from paraffin-embedded urinary bladder tissues and mounted on poly-L-lysine-coated slides. Established procedures for the immunohistochemical staining with the avidin–biotin–peroxidase complex (ABC) method were used with minor modification (36,40,41). Paraffin sections were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% H2O2 in distilled water for 5 min, and then antigen retrieval was performed by microwaving at 98°C for 20 min in 0.01 M citrate buffer (pH 6.0). For Cox-2 immunohistochemistry, sections were trypsinated at 37°C in 0.4% trypsin, 0.01% CaCl2 in Tris-buffered saline for 30 min before microwave. After blocking non-specific binding with serum at 37°C for 30 min, sections were incubated with mouse monoclonal anti-BrdU antibody (M0744, Dako. A/S, Denmark) at 1:500 dilution, rabbit polyclonal p53 antibody (FL-393, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution, rabbit polyclonal anti- p27kip1 antibody (C-19, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2000 dilution, mouse monoclonal cyclin D1 antibody (A-12, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 dilution, or mouse monoclonal COX-2 antibody (Transduction Laboratories, Lexington, KY) at 1:500 dilution, overnight at 4°C. Immunoreactivity was detected using a Vectastain Elite ABC Kit (PK-6102; Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine hydrochloride (Sigma Chemical Co., St Louis, MO) followed by counterstaining with Mayer’s hematoxylin. A negative control was included with each staining procedure by omitting the primary antibody. All specimens were evaluated independently by two of the authors (M.Wei and H.Wanibuchi).

Assessment of staining patterns
For BrdU labeling index, 3000–5000 urothelial cells in morphologically normal urothelia for each rat were counted. The BrdU labeling indices were scored as the number of positive cells per 100 urothelial cells. Overexpression of p53 positive or cyclin D1 in lesions was defined as positive when nuclear staining was evident in >5% of the cells (36,40). The level of p27kip1 immunoreactivity was high in nuclei of the transitional epithelial cells in normal specimens and preneoplatic lesions, providing a positive internal control for each specimen. The specimens with p27kip1 expression <50% of the neoplastic cells were considered to have decreased p27kip1 expression (42,43). For COX-2 staining, normal urothelium consistently demonstrated no COX-2 immunoreactivity, providing a negative internal control for each specimen. Lesions with increased expression of COX-2 included tumors with weak immunoreactivity (cytoplasmic staining for >5% cells).

Detection of 8-OHdG formation in nuclear DNA of the urinary bladder
DNA of the urinary bladders from experiment 2 was extracted by chaotropic NaCl isolation method using a DNA Extractor WB kit (Wako Pure Chemical Industries, Kyoto, Japan). DNA hydrolysis and microfiltration of the resultant samples were subsequently conducted by the method of Helbock et al (44,45). The levels of 8-OHdG were then determined by the HPLC-ECD method as described elsewhere (27). Peaks detected with electrochemical (for 8-OHdG) and UV (for dG) detectors were integrated with a background noise correction loaded on an integrator. Values for 8-OHdG per 105 cells were obtained by calibration against curves from runs of standard samples containing known amounts of authentic 8-OHdG (Wako Pure Chemical Industries, Kyoto, Japan) and dG (Sigma Chemical Co., St Louis, MO). During the assays, light and air contamination were avoided as strictly as possible.

Statistical analysis
Statistical comparisons between the different groups were completed with StatView J-5.0 software (Abacus Concepts, Berkeley, CA) for the Macintosh computer. Inter-group relationships for lesion incidences and immunohistochemical variables were determined by {chi}2 probability analysis or Fisher’s exact probability test. For assessment of the mean values of the BrdU labeling index and 8-OHdG formation, Dunnett analysis and Welch’s t-test were used, respectively. A result was only considered as statistically significant if P < 0.05.


    Results
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 Materials and methods
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General conditions
There was no evidence of increased morbidity or mortality after DMA treatment. There were no statistically significant differences for the overall survival rates between groups during the experiment. The most common cause of death in all groups was leukemia, with grossly enlarged spleen and usually diffuse infiltrates of leukemic cells in the liver and spleen. DMA did not have adverse effects on food consumption, but caused an increase in water consumption at 50 and 200 p.p.m.. The final body weights were not significantly different between the groups. No treatment-related adverse effects were apparent in the hematological and serum biochemical data among groups (data not shown).

Tumorigenicity of DMA
Data for the numbers and incidences of rats with urinary bladder lesions are summarized in Table IGo. The reported rates are for animals surviving until at least week 97, when the first urinary bladder tumor was found. A total of 22 urinary bladder tumors including 18 TCCs and four papillomas were observed in 8 of 31 and 12 of 31 animals in 50 and 200 p.p.m. DMA groups, respectively. Two rats in 200 p.p.m. DMA group with bladder papillomas also had carcinomas. PN hyperplasias, preneoplastic lesions in the urinary bladder, were observed in 12 and 14 rats in 50 and 200 p.p.m. DMA groups, respectively. No such lesions were observed in groups treated with 0 or 12.5 p.p.m. DMA.


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Table I. Incidences of urinary bladder lesions in rats treated with DMAa
 
As Table IIGo shows, in all groups, the incidences of tumors except for those in the urinary bladder were typical for male F344 rats and did not exceed the historical control incidences/range of the NTP (46). There were no significant differences in the incidences of these tumors at any dose level, although DMA-treated rats tended to have more fibromas than controls.


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Table II. Incidences of tumors other than urinary bladder tumors in F344 male rats treated with DMA
 
Urinary analysis
The urinary pH did not differ significantly among groups. The concentrations of sodium, potassium, chloride and calcium were decreased in the rats treated with DMA in a dose-dependent manner, with statistical significance being reached in the 50 and 200 p.p.m. DMA groups (data not shown). The decrease in urine electrolytes was presumably due to increased urinary volume. These results confirmed previous observations showing the carcinogenic action of DMA in rat urinary bladder is not correlated with urine pH or sodium concentration (15).

The urinary concentrations of arsenic metabolites at week 100 are indicated in Table IIIGo. Arsenic compound levels increased in a dose-dependent manner except for AsBe. Major compounds were DMA itself and TMAO, with small amounts of MMA and TeMa also detected. As described in previous reports, two unidentified metabolites, peak 1 and peak 2 (15,35), were also found in DMA-treated groups but not the controls.


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Table III. Urinary concentrations of arsenic compounds at week 100 in male F344 rats treated with DMA
 
Mutations of the H-ras, p53, K-ras and ß-catenin genes in DMA-induced urinary bladder tumor
Twenty paraffin-embedded DMA-induced rat urinary tumors (18 TCCs and two papillomas) were examined for mutations in exons 1 and 2 of the H- and K-ras oncogenes, in exons 5–8 of the p53 tumor suppressor gene and the ß-catenin gene using PCR-SSCP and direct sequencing techniques. Mutations in exon 1 of H-ras were demonstrated in two TCCs (2 of 20, 10%). Direct sequencing of the two samples indicated one G->T transversion at codon 13 (GGC->GTC) and one G->T transversion and a C->A transversion at the second and third bases of codon 13 (GGC->GTA), respectively. Both mutations resulted in an identical amino acid substitution of glycine for valine. No mutations were found in p53, K-ras, and ß-catenin genes.

MSI analysis
Eighteen microsatellite loci interspersed throughout the rat genome were analyzed in 16 DMA-induced urinary bladder carcinomas, but no alterations were detected.

BrdU labeling index
Urinary bladder epithelial cell proliferation following treatment with DMA were determined by BrdU incorporation. The data are presented as BrdU labeling indices for morphologically normal bladder epithelium. As shown in Figure 1Go, a significant increase was noted for the groups treated with 50 and 200 p.p.m. DMA when compared with controls.



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Fig. 1. BrdU labeling indices for morphologically normal bladder epithelium of rats treated with DMA for 104 weeks. Significantly different from controls (DMA, 0 p.p.m.) at *P < 0.05; **P < 0.01. Bars indicate the SD.

 
Immunohistochemistry
Results of immunohistochemical assessment of p53, p27kip1, cyclin D1 and COX-2 expressions in the rat urinary bladder lesions are shown in Figure 2Go.



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Fig. 2. Immunohistochemical assessment of p53, p27kip1, cyclin D1 and COX-2 expression in rat urinary bladder lesions induced by DMA. Significantly different from PN hyperplasia at *P < 0.05; **P < 0.001. The assessment criteria are described in Materials and methods.

 
Positive p27kip1 staining was noted within the nuclei of the urothelial cells in the control group and in morphologically normal appearing urothelium and simple hyperplasia in DMA-treated groups. Typical positive staining patterns for p27kip1 are shown in Figure 3Go. The majority of PN hyperplasias also stained intensely; in marked contrast, almost all TCC and papillomas demonstrated a heterogeneous pattern of significantly reduced p27kip1 immunoreactivity. Thus 16 of 18 (89%) TTCs, and 3 of 4 (75%) papillomas demonstrated decreased p27kip1 expression. It is worth noting that p27kip1 is significantly downregulated in papillomas and TCCs when compared with PN hyperplasias. In fact, no staining was observed in one papilloma and 10 TCCs.



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Fig. 3. Immunohistochemical assessment of p27kip1 expression. A high level of p27kip1 immunoreactivity is apparent in urothelial cell nuclei of the control group and in morphologically normal appearing epithelium and simple hyperplasia in the DMA-treated groups. (A) Normal bladder mucosa from a control rat; (B) morphologically normal mucosa following 200 p.p.m. DMA; (C) simple and PN hyperplasia; (D) papilloma with nuclear p27kip1 expression >50%; (E) TCC with nuclear p27kip1 expression >50%.

 
All 10 control specimens of the control group did not show positive cyclin D1 nuclear staining in the urothelium (Figure 4AGo). In contrast, all 40 specimens from the DMA-treated group displayed occasional nuclear cyclin D1 staining in small stretches of normal appearing epithelium, including all 10 bladders examined from rats given 12.5 p.p.m. DMA in which no bladder lesion was observed (Figure 4BGo). The cyclin D1 overexpression phenotype, defined as positive immunoreactivity in the nuclei of >5% of neoplastic cells (Figure 4D–FGo), was found in 26 of 47 (55%) PN hyperplasias, 3 of 4 papillomas (75%), and 10 of 18 TCCs (56%). There was no significant difference in incidences of cyclin D1 overexpression between the bladder lesion types.



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Fig. 4. Immunohistochemical detection of cyclin D1 expression in morphologically normal bladder mucosa and neoplastic lesions following DMA treatment. (A) Normal bladder mucosa from a control rat; (B) morphologically normal bladder mucosa following 12.5 p.p.m. DMA; (C) simple hyperplasia from a 200 p.p.m. DMA rat; (D) PN hyperplasia with increased cyclin D1 expression; (E) TCC with increased cyclin D1 expression; (F) higher magnification of the TCC in (E).

 
COX-2 staining was localized to the cytoplasm of tumor or preneoplastic cells but was not present in normal urothelial cells (Figure 5Go). Increased expression was noted in 17 of 18 (94%) TCCs, 4 of 4 (100%) papillomas, and 39 of 47 (83%) PN hyperplasias. Normal epithelial cells in the control group and morphologically normal urothelium of the DMA-treated groups commonly did not show immunoreactivity for COX-2, providing a negative internal control for each specimen. However, positive COX-2 staining also occasionally was noted in the simple hyperplasia and morphologically normal epithelium adjacent to tumors.



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Fig. 5. Increased COX-2 expression in DMA-induced bladder lesions. Note increase in cytoplasm. (A) Normal bladder mucosa from a control rat; (B) nodular hyperplasia; (C) advanced nodular hyperplasia; (D) TCC; (E) higher magnification of the TCC in (C).

 
Only one TCC (6%) demonstrated nuclear accumulation of p53 protein in >5% of the nuclei (Figure 6AGo). We did not find any statistically significant association between p27kip1, cyclin D1 and COX-2 expression in the DMA-induced urinary bladder lesions although most bladder lesions showed decreased p27kip1 expression and increased cyclin D1 and COX-2 expression simultaneously. However, as shown in Figure 6Go, no mutually exclusive distribution pattern was observed.



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Fig. 6. Immunohistochemistry for p53, p27kip1, cyclin D1 and COX-2 in serial sections. Note that multiple alternative expressions existed concurrently in tumors but without a mutually exclusive distribution pattern. (A) The only TCC with p53 overexpression; (B) serial section showed negligible p27kip1 nuclear staining; (C) serial section showing diffusely increased cyclin D1 expression; (D) serial section showed increased COX-2 expression.

 
Increased formation of 8-OHdG in urinary bladder DNA
8-OHdG formation was significantly increased in DMA-treated rats (1.76 ± 0.59/105dG) after treatment with 200 p.p.m. DMA in the drinking water for 2 weeks compared with the controls (1.21 ± 0.13/105dG).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrated that DMA is carcinogenic for the urinary bladder of F344 male rats, but lacks other organ-specific carcinogenic effects, particularly regarding the liver and kidney in which promoting effects on rat carcinogenesis have been shown and arsenic-associated tumors have been reported in humans. We also found that DMA-induced rat urinary bladder tumors had a low rate of H-ras mutations and no mutations in p53, K-ras or ß-catenin genes. Furthermore, we demonstrated that induction of cell proliferation might contribute to the carcinogenicity of DMA via mechanisms involving oxidative stress and/or alterations in cell cycle regulatory protein, p27kip1 and cyclin D1. DMA is a major metabolite of arsenic in most mammals (9–12). Thus, the results from the present study would appear directly relevant to the carcinogenic risk of arsenic.

The bladder-specific carcinogenic effect of DMA in rat may indicate: (i) longer exposure to DMA in the urinary bladder than in other organs due to urinary retention; (ii) promoting activities of DMA on multiple organs need to be considered for assessing carcinogenic effects of arsenic in humans; (iii) humans may be more sensitive than experimental animals to cancer induction by arsenic (5,8); or (iv) DMA may not be the only carcinogenic compound involved in arsenic carcinogenesis. An ongoing 2-year carcinogenicity study of MMA and TMAO in our laboratory should clarify whether this is indeed the case.

p53 is the most frequently mutated tumor suppressor gene described so far in human and experimental animal cancers including urinary bladder cancer (47,48). Examination of the molecular changes in the p53 tumor suppressor gene can contribute to our understanding of the nature of carcinogenic activity. In contrast to our previous finding of frequent p53 mutations in BBN-induced rat urinary bladder cancer (49), no such lesions were found in DMA-induced rat urinary bladder tumors examined in the present study. Our results indicated that DMA differs from BBN, which is genotoxic, and pathways other than the p53 pathway must be involved in the etiology of the DMA-induced rat urinary bladder tumors. Two previous studies reported high frequency of p53 mutations in arsenic-related bladder and skin tumors from the endemic area of black foot disease in Taiwan (50,51). However, the discrepancy might be partly explicable by the fact that development of malignancy in humans is a complex multistep process, and many factors may affect the likelihood that cancer will develop. Therefore, it is reasonable to hypothesize that genetic alterations found in human cancers are results of factors such as smoking and exposure to other arsenicals. DMA is considered to be a clastogenic agent (22) and negative in most mutagenicity studies (19). In light of these actions, mutations of H-ras in two TCCs could occur indirectly by oxidative damage or cytotoxicity of DMA (23,2,52).

It is well established that disruption of the normal cell cycle is a critical step in cancer development (53). Because alterations in the p53 gene were lacking, we therefore focused our attention on the expression of the tumor suppressor gene p27kip1. p27kip1 functions as a p53 independent negative cell cycle regulator involved in G1 arrest, and the reduction in the protein level of p27kip1 have been reported in a variety of human cancers and is likely to provide a selective growth advantage (54). The strong p27kip1 staining in the majority of preneoplastic lesions noted here is consistent with the established role of p27kip1 as a tumor suppressor gene counteracting proliferative signals generated by DMA exposure. The reduced expression of p27kip1 protein in almost all TCCs and papillomas suggests a role in malignant progression in DMA-induced rat bladder tumors.

Overexpression of cyclin D1 in human urinary bladder tumors could be a key regulatory event leading to cell proliferation and tumorigenesis (36). In the present study, we also found that positive cyclin D1 nuclear staining appeared in small stretches of histologically normal appearing epithelium following DMA treatment, even in animals in which no bladder lesions were observed histologically, as well as in most local lesions. The present data strongly suggested that cyclin D1 induction is one of the early events in DMA-induced rat bladder carcinogenesis. However, the fact that failure to induce bladder tumors at a dose of 12.5 p.p.m. DMA suggests that increased cyclin D1 associated with such a low dose may be insufficient for DMA bladder carcinogenesis under the present conditions. In addition, the observed existence of tumors without increased cyclin D1 expression but featuring down-regulation of p27kip1 expression may mean that it is no longer necessary for at least a subset of tumors in the later stages. The actions of cyclin D1 are regulated by CDK inhibitors such as p27kip1, which control its ability to activate CDK4 and CDK6. Therefore, those tumors are likely a result, at least in part, of either increased degradation or transcription of p27kip1. The lack of any exclusive distribution pattern indicates that alterations may occur independently and there might exist other mechanisms by which DMA affects cell cycle regulation. Defects in a cell cycle check point may be responsible for the genomic instability (53). We can conclude that such abnormalities are frequent in DMA bladder carcinogenesis and might induce genomic instability despite the rarity of mutations in the present study.

Increasing evidence supports the hypothesis that ROS may play a role in DMA carcinogenesis. It is reported that metabolism of substances by the P-450 enzyme system can generate oxygen free radicals (55,56). Our recent finding that an increase in hepatic P450 levels, especially in CYP2B1 protein in rat livers after treatment with 100 p.p.m. DMA suggests that DMA are metabolized by P450 in rat liver and could represent a mechanism by which DMA generate ROS (57). Alternatively, the possibility also exists that cytotoxicity of DMA may involve the generation of ROS since xenobiotic chemicals can produce ROS by either direct or indirect means (58,59). Yamanaka et al. (60,61) demonstrated production of oxygen radicals in the metabolism of DMA, such as the superoxide anion radical and the dimethylarsenic peroxyl radical, and might have a role in DNA damage in the lungs of mice and rats. Among ROS-induced forms of DNA damage, 8-OHdG is typical and most commonly used as a marker for quantitative analysis (26,62). The finding of a significant increase in DMA-treated rats in the present study suggests that DMA treatment causes DNA damage via ROS generation, as shown earlier for the rat liver (14).

COX-2 expression, shown to be involved with development of preneoplastic and neoplastic lesions in the human and rat bladder (28,29), was also diffuse in the majority of TCCs, papillomas and PN hyperplasias in the present study. The occasional positive COX-2 staining noted in the morphologically normal epithelium adjacent to tumor, observed also in human invasive TCC of the bladder, may indicate neoplastic cells can exert paracrine effects through the release of cytokines and/or growth factors. ROS are known to play a crucial role in the expression of COX-2, so that this may also be a molecular marker of oxidative stress (63).

Microsatellite instability was absent in the available DMA-induced rat bladder cancers in this study, although this could be due to the low number of markers, only one to three markers for each chromosome. Thus, further study with a larger number of microsatellites is necessary for clarification.

Based on the observations in the present experiment and the results from the literature, potential modes of action for DMA with regard to rat urinary bladder carcinogenesis are given in Figure 7Go. We propose two possible mechanisms by which ROS could be involved in DMA carcinogenesis in rats: (i) DMA-initiated ROS may cause specific molecular changes resulting in the activation of transcription factors such as AP-1 and NF-{kappa}B. Although AP-1 and NF-{kappa}B were not investigated here, ROS has been shown to cause synthesis of AP-1, and activation of AP-1 or NF-{kappa}B promotes carcinogenesis (64,65). DMA induces cell proliferation and gene expression in the bladder epithelium associated with AP-1 in mice (24). It should be remembered that ROS are known to play a crucial role in the expression of COX-2 through activating nuclear factor-{kappa}B. (30,31) (ii) DMA could cause chromosomal abnormalities by generation of ROS and resultant DNA single strand breaks and DNA-protein crosslinks (21,60,66). It is generally accepted that tumor development occurs as the result of accumulation of genetic alterations (67,68). The various genetic alterations induced by DMA may not be the result of independent mechanisms. Some modes may be operating concurrently or sequentially. DMA-initiated defects in cell cycle checkpoints could give rise to genomic instability, while DMA-induced DNA damage would be expected to affect the expression of cell cycle regulators. Moreover, the fact that oxygen radicals may participate in the carcinogenic process, including the stages of initiation, promotion, and progression (69,71), suggest a reasonable mechanism by which DMA may act in all three phases. In addition, an alternative or complementary mechanism suggested by Cohen et al. for rat bladder carcinogenesis, is cytoxicity and regeneration (23,52). The ultimate relationships between oxidative stress, toxic stress and genetic alterations in arsenic carcinogenesis remain to be determined.



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Fig. 7. Potential modes of action underlying DMA carcinogenesis in rat urinary bladder.

 
In conclusion, the present work provides unequivocal evidence that DMA may be a carcinogen for the rat urinary bladder, supporting the epidemiological data that inorganic arsenic is a human bladder carcinogen and suggesting that DMA may be relevant to the carcinogenic risk of inorganic arsenic exposure in humans. DMA-induced urinary bladder tumors occur as the result of an accumulation of diverse genetic alterations. The present elucidation of the cellular and molecular pathways involved in DMA carcinogenesis in rats suggest that particular attention should be paid to oxygen stress in the human populations at risk from arsenic carcinogenesis.


    Notes
 
4 To whom correspondence should be addressed Email: fukuchan{at}med.osaka-cu.ac.jp Back


    Acknowledgments
 
This work was supported in part by Core Research for Evolutional Science and Technology (CREST) grant from Japan Science and Technology Corporation (JST), and Grant in-Aid for Cancer Research of Arsenics from the Environment Agency, Japan, and Fund for Medical Research from Osaka City University Medical Research Foundation. We are grateful to Kawakami Emi and Touma Kaori (Department of Pathology, Osaka City University Medical School, Japan) for assistance in the immunohistochemistry studies.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. IARC (1980) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 23: Some Metals and Metallic Compounds. IARC, Lyon, France, pp. 39–141.
  2. IARC (1987) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 7, Overall Evaluation of the Carcinogenicity: An Updating of IARC. Monographs, Vols. 1–40. IARC, Lyon, France, pp. 100–106.
  3. Chen,C.J., Chen,C.W., Wu,M.M. and Kuo,T.L. (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer, 66, 888–892.[ISI][Medline]
  4. Smith,A.H., Hopenhayn,Rich,C., Bates,M.N., Goeden,H.M., Hertz,P.I., Duggan,H.M., Wood,R., Kosnett,M.J. and Smith,M.T. (1992) Cancer risks from arsenic in drinking water. Environ. Health Perspect., 97, 259–267.[ISI][Medline]
  5. Goering,P.L., Aposhian,H.V., Mass,M.J., Cebrian,M., Beck,B.D. and Waalkes, M.P. (1999) The enigma of arsenic carcinogenesis: role of metabolism. Toxicol. Sci., 49, 5–14.[Abstract]
  6. Ishinishi,N., Mizunoe,M., Inamasu,T. and Hisanaga,A. (1980) [Experimental study on carcinogenicity of beryllium oxide and arsenic trioxide to the lung of rats by an intratracheal instillation (author’s transl)]. Fukuoka Igaku Zasshi, 71, 19–26.[Medline]
  7. Pershagen,G., Nordberg,G. and Bjorklund,N.E. (1984) Carcinomas of the respiratory tract in hamsters given arsenic trioxide and/or benzo[a]pyrene by the pulmonary route. Environ. Res., 34, 227–241.[ISI][Medline]
  8. Huff,J., Chan,P. and Nyska,A. (2000) Is the human carcinogen arsenic carcinogenic to laboratory animals? Toxicol. Sci., 55, 17–23.[Free Full Text]
  9. Bertolero,F., Marafante,E., Rade,J.E., Pietra,R. and Sabbioni,E. (1981) Biotransformation and intracellular binding of arsenic in tissues of rabbits after intraperitoneal administration of 74As labelled arsenite. Toxicology, 20, 35–44.[ISI][Medline]
  10. Buchet,J.P., Lauwerys,R. and Roels,H. (1980) Comparison of several methods for the determination of arsenic compounds in water and in urine. Their application for the study of arsenic metabolism and for the monitoring of workers exposed to arsenic. Int. Arch. Occup. Environ. Health., 46, 11–29.[ISI][Medline]
  11. Tam,G.K., Charbonneau,S.M., Bryce,F., Pomroy,C. and Sandi,E. (1979) Metabolism of inorganic arsenic (74As) in humans following oral ingestion. Toxicol. Appl. Pharmacol., 50, 319–322.[ISI][Medline]
  12. Vahter,M. (1981) Biotransformation of trivalent and pentavalent inorganic arsenic in mice and rats. Environ. Res., 25, 286–293.[ISI][Medline]
  13. Yamamoto,S., Konishi,Y., Matsuda,T., et al. (1995) Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens. Cancer Res., 55, 1271–1276.[Abstract]
  14. Wanibuchi,H., Hori,T., Meenakshi,V., et al. (1997) Promotion of rat hepatocarcinogenesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver. Jpn. J. Cancer Res., 88, 1149–1154.[ISI][Medline]
  15. Wanibuchi,H., Yamamoto,S., Chen,H., Yoshida,K., Endo,G., Hori,T. and Fukushima,S. (1996) Promoting effects of dimethylarsinic acid on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in rats. Carcinogenesis, 17, 2435–2439.[Abstract]
  16. Wei,M., Wanibuchi,H., Yamamoto,S., Li,W. and Fukushima,S. (1999) Urinary bladder carcinogenicity of dimethylarsinic acid in male F344 rats. Carcinogenesis, 20, 1873–1876.[Abstract/Free Full Text]
  17. van Gemert,M. and Eldan,M. (1998) Chronic carcinogenicity assessment of cacodylic acid. 3 rd International Conference on Arsenic Exposure and Health Effects, Book of Abstracts, 113 pp.
  18. Fukushima,S., Wanibuchi,H., Wei,M., Salim,E.I., (2000) Carcinogenicity of dimethylarsinic acid in rats and mice. 3 rd International Conference on Arsenic Exposure and Health Effects, Book of Abstracts, 110pp.
  19. U.S. Environmental Protection Agency. (1997) Report on the Expert Panel on Arsenic Carcinogenicity. National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington D.C.
  20. Dong,J.T. and Luo,X.M. (1993) Arsenic-induced DNA-strand breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts. Mutat. Res., 302, 97–102.[ISI][Medline]
  21. Yamanaka,K., Hasegawa,A., Sawamura,R. and Okada,S. (1989) DNA strand breaks in mammalian tissues induced by methylarsenics. Biol. Trace. Elem. Res., 21, 413–417.[ISI][Medline]
  22. ATSDR (1999) Toxicological profile for arsenic (update). Agency for Toxic Substances and Disease Registry, Atlanta, GA.
  23. Arnold,L.L., Cano,M., St John,M., Eldan,M., van Gemert,M. and Cohen,S.M. (1999) Effects of dietary dimethylarsinic acid on the urine and urothelium of rats. Carcinogenesis, 20, 2171–2179.[Abstract/Free Full Text]
  24. Simeonova,P.P., Wang,S.Y., Toriuma,W., et al. (2000) Arsenic mediates cell proliferation and gene expression in the bladder epithelium: association with activating protein-1 transactivation. Cancer Res, 60, 3445–3453.[Abstract/Free Full Text]
  25. Hei,T.K., Liu,S.X. and Waldren,C. (1998) Mutagenicity of arsenic in mammalian cells: Role of reactive oxygen species. Proc Natl Acad Sci. USA, 95, 8103–8107.[Abstract/Free Full Text]
  26. Floyd,R.A. (1990) The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis, 11, 1447–1450.[ISI][Medline]
  27. Nakae,D., Kobayashi,Y., Akai,H., Andoh,N., Satoh,H., Ohashi,K., Tsutsumi,M. and Konishi,Y. (1997) Involvement of 8-hydroxyguanine formation in the initiation of rat liver carcinogenesis by low dose levels of N-nitrosodiethylamine. Cancer Res., 57, 1281–1287.[Abstract]
  28. Mohammed,S.I., Knapp,D.W., Bostwick,D.G., Foster,R.S., Khan,K.N., Masferrer,J.L., Woerner,B.M., Snyder,P.W. and Koki,A.T. (1999) Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res., 59, 5647–5650.[Abstract/Free Full Text]
  29. Kitayama,W., Denda,A., Yoshida,J., Sasaki,Y., Takahama,M., Murakawa,K., Tsujiuchi,T., Tsutsumi,M. and Konishi,Y. (2000) Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine. Cancer Lett., 148, 145–152.[ISI][Medline]
  30. Kosaka,T., Miyata,A., Ihara,H., Hara,S., Sugimoto,T., Takeda,O., Takahashi,E. and Tanabe,T. (1994) Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2. Eur. J. Biochem., 221, 889–897.[Abstract]
  31. Sen,C.K. and Packer,L. (1996) Antioxidant and redox regulation of gene transcription [see comments]. FASEB J., 10, 709–720.[Abstract/Free Full Text]
  32. Gonzalez Zulueta,M., Ruppert,J. M., Tokino,K., et al. (1993) Microsatellite instability in bladder cancer. Cancer Res., 53, 5620–5623.[Abstract]
  33. Honchel,R., Halling,K.C. and Thibodeau,S.N. (1995) Genomic instability in neoplasia. Semin. Cell Biol., 6, 45–52.[ISI][Medline]
  34. Mao,L., Schoenberg,M.P., Scicchitano,M., Erozan,Y.S., Merlo,A., Schwab,D. and Sidransky,D. (1996) Molecular detection of primary bladder cancer by microsatellite analysis. Science, 271, 659–62.[Abstract]
  35. Yoshida,K., Inoue,Y., Kuroda,K., Chen,H., Wanibuchi,H., Fukushima,S. and Endo,G. (1998) Urinary excretion of arsenic metabolites after long-term oral administration of various arsenic compounds to rats. J. Toxicol. Environ. Health Part A, 54, 179–192.[ISI][Medline]
  36. Lee,C.C., Yamamoto,S., Morimura,K., et al. (1997) Significance of cyclin D1 overexpression in transitional cell carcinomas of the urinary bladder and its correlation with histopathologic features. Cancer, 79, 780–789.[ISI][Medline]
  37. Dashwood,R.H., Suzui,M., Nakagama,H., Sugimura,T. and Nagao,M. (1998) High frequency of beta-catenin (ctnnb1) mutations in the colon tumors induced by two heterocyclic amines in the F344 rat. Cancer Res., 58, 1127–1129.[Abstract]
  38. Jacob,H.J., Brown,D.M., Bunker,R.K., et al. (1995) A genetic linkage map of the laboratory rat, Rattus norvegicus. Nat. Genet., 9, 63–69.[ISI][Medline]
  39. Toyota,M., Ushijima,T., Weisburger,J.H., Hosoya,Y., Canzian,F., Rivenson,A., Imai,K., Sugimura,T. and Nagao,M. (1996) Microsatellite instability and loss of heterozygosity on chromosome 10 in rat mammary tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Mol. Carcinog., 15, 176–182.[ISI][Medline]
  40. Lee,C.C., Yamamoto,S., Wanibuchi,H., Wada,S., Sugimur,K., Kishimoto,T. and Fukushima,S. (1997) Cyclin D1 overexpression in rat two-stage bladder carcinogenesis and its relationship with oncogenes, tumor suppressor genes, and cell proliferation. Cancer Res., 57, 4765–4776.[Abstract]
  41. Lee,T.C., Tanaka,N., Lamb,P.W., Gilmer,T.M. and Barrett,J.C. (1988) Induction of gene amplification by arsenic. Science, 241, 79–81.[ISI][Medline]
  42. Porter,P.L., Malone,K.E., Heagerty,P.J., Alexander,G.M., Gatti,L.A., Firpo,E.J., Daling,J.R. and Roberts,J.M. (1997) Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients [see comments]. Nat. Med., 3, 222–225.[ISI][Medline]
  43. Esposito,V., Baldi,A., De Luca,A., et al. (1997) Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer. Cancer Res., 57, 3381–3385.[Abstract]
  44. Helbock,H.J., Beckman,K.B., Shigenaga,M.K., Walter,P.B., Woodall,A.A., Yeo,H.C. and Ames,B.N. (1998) DNA oxidation matters: The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. USA, 95, 288–293.[Abstract/Free Full Text]
  45. Helbock,H.J., Beckman,K.B. and Ames,B.N. (1999) 8-hydroxydeoxyguanosine and 8-hydroxyguanine as biomarkers of oxidative DNA damage. Oxid. Antioxid. Part B, 300, 156–166.
  46. Haseman,J.K., Arnold,J., and Eustis,S. L. (1990) Tumor Incidences in Fischer 344 Rats: NTP Historical Data. In: Boorman,G.A., Eustis,S.L., Elwell,M.R., Montgomery,C.A. and Mackenzie,W. F., (eds) Pathology of the Fischer Rat, Reference and Atlas. Academic Press, San Diego, CA., pp. 555–564.
  47. Sidransky,D., Von Eschenbach,A., Tsai,Y.C., et al. (1991) Identification of p53 gene mutations in bladder cancers and urine samples. Science, 252, 706–709.[ISI][Medline]
  48. Hollstein,M., Sidransky,D., Vogelstein,B. and Harris,C.C. (1991) p53 mutations in human cancers. Science, 253, 49–53.[ISI][Medline]
  49. Masui,T., Dong,Y., Yamamoto,S., Takada,N., Nakanishi,H., Inada,K., Fukushima,S. and Tatematsu,M. (1996) p53 mutations in transitional cell carcinomas of the urinary bladder in rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine. Cancer Lett., 105, 105–12.[ISI][Medline]
  50. Hsu,C.H., Yang,S.A., Wang,J.Y., Yu,H.S. and Lin,S.R. (1999) Mutational spectrum of p53 gene in arsenic-related skin cancers from the blackfoot disease endemic area of Taiwan. Br. J. Cancer, 80, 1080–1086.[ISI][Medline]
  51. Shibata,A., Ohneseit,P.F., Tsai,Y.C., Spruck,C.H., 3rd, Nichols,P.W., Chiang,H.S., Lai,M.K. and Jones,P.A. (1994) Mutational spectrum in the p53 gene in bladder tumors from the endemic area of black foot disease in Taiwan. Carcinogenesis, 15, 1085–1087.[Abstract]
  52. Cohen,S.M., Yamamoto,S., Cano,M. and Arnold,L.L. (2001) Urothelial cytotoxicity and regeneration induced by dimethylarsinic acid in rats. Toxicol. Sci., 59, 68–74.[Abstract/Free Full Text]
  53. Hartwell,L. (1992) Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell, 71, 543–546.[ISI][Medline]
  54. Steeg,P.S. and Abrams,J.S. (1997) Cancer prognostics: past, present and p27 [news; comment]. Nat. Med., 3, 152–154.[ISI][Medline]
  55. Parke,D.V. and Ioannides,C. (1990) Role of cytochromes P-450 in mouse liver tumor production. Prog. Clin. Biol. Res., 331, 215–230.[Medline]
  56. Klaunig,J.E., Xu,Y., Isenberg,J.S., Bachowski,S., Kolaja,K.L., Jiang,J., Stevenson,D.E. and Walborg,E.F., Jr. (1998) The role of oxidative stress in chemical carcinogenesis. Environ. Health. Perspect., 106 (Suppl. 1), 289–295.[ISI][Medline]
  57. Nishikawa,T., Wanibuchi,H., Ogawa,M., et al. (2002) Promoting effects of monomethylarsonic acid, dimethylarsinic acid and trimethylarsine oxide on induction of rat liver preneoplastic glutathione S-transferase placental form positive foci: A possible reactive oxygen species mechanism. Int. J. Cancer (In press).
  58. Trush,M.A. and Kensler,T.W. (1991) An overview of the relationship between oxidative stress and chemical carcinogenesis. Free Radic. Biol. Med., 10, 201–209.[ISI][Medline]
  59. Halliwell,B. (1996) Mechanisms involved in the generation of free radicals. Pathol Biol (Paris), 44, 6–13.[ISI][Medline]
  60. Yamanaka,K., Hasegawa,A., Sawamura,R. and Okada,S. (1989) Dimethylated arsenics induce DNA strand breaks in lung via the production of active oxygen in mice. Biochem. Biophys. Res. Commun., 165, 43–50.[ISI][Medline]
  61. Yamanaka,K., Hoshino,M., Okamoto,M., Sawamura,R., Hasegawa,A. and Okada,S. (1990) Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biochem. Biophys. Res. Commun., 168, 58–64.[ISI][Medline]
  62. Dizdaroglu,M. (1991) Chemical determination of free radical-induced damage to DNA. Free. Radic. Biol. Med., 10, 225–242.[ISI][Medline]
  63. Romanenko,A., Morimura,K., Wanibuchi,H., Salim,E.I., Kinoshita,A., Kaneko,M., Vozianov,A. and Fukushima,S. (2000) Increased oxidative stress with gene alteration in urinary bladder urothelium after the Chernobyl accident. Int. J. Cancer, 86, 790–798.[ISI][Medline]
  64. Schenk,H., Klein,M., Erdbrugger,W., Droge,W. and Schulze,O.K. (1994) Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1. Proc. Natl Acad. Sci. USA, 91, 1672–1676.[Abstract]
  65. Kerr,L.D., Inoue,J. and Verma,I.M. (1992) Signal transduction: the nuclear target. Curr. Opin. Cell. Biol., 4, 496–501.[Medline]
  66. Rin,K., Kawaguchi,K., Yamanaka,K., Tezuka,M., Oku,N. and Okada,S. (1995) DNA-strand breaks induced by dimethylarsinic acid, a metabolite of inorganic arsenics, are strongly enhanced by superoxide anion radicals. Biol. Pharm. Bull., 18, 45–48.[ISI][Medline]
  67. Weinberg,R.A. (1991) Tumor suppressor genes. Science, 254, 1138–1146.[ISI][Medline]
  68. Bishop,J.M. (1987) The molecular genetics of cancer. Science, 235, 305–311.[ISI][Medline]
  69. Copeland,E.S. (1983) A National Institutes of Health Workshop report. Free radicals in promotion: a chemical pathology study section workshop. Cancer Res., 43, 5631–5637.[ISI][Medline]
  70. O’Connell,J.F., Klein Szanto,A.J., DiGiovanni,D.M., Fries,J.W. and Slaga,T.J. (1986) Enhanced malignant progression of mouse skin tumors by the free-radical generator benzoyl peroxide. Cancer Res., 46, 2863–2865.[Abstract]
  71. Slaga,T.J. (1983) Overview of tumor promotion in animals. Environ. Health Perspect., 50, 3–14.[ISI][Medline]
Received December 21, 2001; revised April 16, 2002; accepted May 1, 2002.