Urothelial Cytotoxicity and Regeneration Induced by Dimethylarsinic Acid in Rats

Samuel M. Cohen1, Shinji Yamamoto, Marty Cano and Lora L. Arnold

Department of Pathology/Microbiology and the Eppley Cancer Center, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, Nebraska 68198–3135

Received July 27, 2000; accepted September 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic arsenic is a known human carcinogen of the skin and respiratory tract. Epidemiologic evidence indicates that it is also carcinogenic to the urinary bladder and other internal organs. Lack of an animal model has limited progress on understanding the mechanism of arsenic carcinogenesis. It was recently reported that high doses of an organic arsenical, dimethylarsinic acid (DMA), increased urinary bladder tumors in rats when administered in the diet or in the drinking water for 2 years, with the female being more sensitive than the male. We previously showed that high doses of DMA (40 or 100 ppm of the diet) fed for 10 weeks increased urothelial cell proliferation in the rat. Treatment with DMA also increased renal calcification and increased urinary calcium concentration. In 2 experiments, we examined the urothelial proliferative effects of treatment with 100 ppm DMA in the diet in female F344 rats for 2 and 10 weeks and for 6 and 24 h, and 3, 7, and 14 days. Cytotoxic changes in the urothelium were evident by SEM as early as 6 h after treatment was begun. Foci of cellular necrosis were detected after 3 days of treatment, followed by widespread necrosis of the urothelium after 7 days of treatment. The bromodeoxyuridine (BrdU) labeling index was not increased until after 7 days of treatment, suggesting that administration of DMA results in cytotoxicity with necrosis, followed by regenerative hyperplasia of the bladder epithelium. Although the rat provides an animal model to study the urothelial effects of DMA, the relevance of this finding to inorganic arsenic carcinogenesis in humans must be extrapolated cautiously, due to the high doses of DMA necessary to produce these changes in the rat and the differences in metabolism of arsenicals in rodents, especially rats, compared to humans.

Key Words: arsenic; urinary bladder; dimethylarsinic acid; cell proliferation; necrosis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic arsenic is a known human carcinogen, producing tumors principally in the skin following oral ingestion, but also leading to the induction of respiratory tract tumors following inhalation (IARC, 1980Go). More recently, there is epidemiologic evidence that ingestion of high levels of inorganic arsenic in the drinking water can produce an increased incidence of bladder cancer and possibly tumors of other internal organs (Bates et al., 1992Go). Although inorganic arsenic is considered to be the likely form of arsenic that produces these neoplastic effects, little is actually known concerning the mechanism of arsenic carcinogenesis. This has primarily been due to the lack of an animal model for the induction of tumors of any organ by any arsenical. This difference between rodents and man may be related to the striking differences in the metabolism of arsenicals.

During the past few years, it has been reported that an organic arsenical, dimethylarsinic acid (DMA), administered to rats at high doses in either the diet (van Gemert and Eldan, 1998Go) or drinking water (Wei et al., 1999Go) produced an increased incidence of urinary bladder tumors. The effect appears to be stronger in female than in male rats, in contrast to a male-dominant effect for nearly all previously reported bladder carcinogens in animal models. The tumorigenicity appears only after extraordinarily high doses (25–100 ppm), in contrast to human exposures to arsenic species. In contrast to the effects in rats, administration of comparable levels in the diet to mice were without a carcinogenic effect to the urinary bladder or to other tissues (van Gemert and Eldan, 1998Go).

Administration of DMA in the drinking water has also been shown to increase the incidences of bladder tumors when administered following a known genotoxic bladder carcinogen such as N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) (Wanibuchi et al., 1996Go). Increased incidences of tumors of other organs, such as liver, thyroid, and kidney, have also been observed when DMA is administered in the drinking water following the administration of known carcinogenic agents specific for these organs (Yamamoto et al., 1995Go). However, the latter experiment involved extremely high doses that were in excess of a maximally tolerated dose.

The mechanism by which DMA or other arsenicals, organic or inorganic, might produce a carcinogenic effect is unknown. It is clear that none of these arsenicals lead to metabolic products which bind to DNA, as no DNA adducts have been identified (IARC, 1980Go; Jacobson-Kram and Montalbano, 1985Go; Leonard and Lauwerys, 1980Go; Rossman, 1998Go; Wang and Rossman, 1996Go) and the chemistry of these compounds suggests that DNA adducts would not be expected to form. Indirect effects on DNA have been suggested, such as positive results in chromosome aberration assays (Dulout et al., 1996Go; Gurr et al., 1993Go; Kochhar et al., 1996Go; Lerda, 1994Go; Oya-Ohta et al., 1996Go), but whether these aberrations are due to effects on the DNA or are secondary to cytotoxic effects is unknown.

In contrast to a DNA-reactive mechanism, it has been suggested that the carcinogenicity of these agents is more likely due to an increase in cellular proliferation (Arnold et al., 1999Go; Germolec et al., 1998Go; Simeonova et al., 1999Go; Trouba et al., 1999Go). Increased cell proliferation could occur either as a result of a direct mitogenic effect, such as seen following administration of certain hormones, or it can be produced by cytotoxicity with consequent regeneration, as is frequently seen with numerous non-DNA-reactive chemical carcinogens.

We have previously demonstrated that administration of DMA at high doses, either 40 or 100 ppm of the diet, for 10 weeks produces an increase in urothelial proliferation in female rats (Arnold et al., 1999Go). The proliferative effect is reversible if the chemical is withdrawn from the diet. By scanning electron microscopy (SEM), there was also evidence of cellular necrosis of the superficial layer of the urothelium. The goal of the present study was to clarify whether the increased proliferation, as detected by the presence of hyperplasia by light microscopy, piling up of cells as viewed by SEM, and an increase in bromodeoxyuridine (BrdU) labeling index by immunohistochemistry, was due to a direct mitogenic effect on the bladder or whether it was strictly due to cytotoxicity and regeneration, as was suggested by the presence of necrosis. The following experiments were designed to evaluate the time course of events of the cytotoxic and proliferative effect on the urothelium in female rats administered a high dose of DMA to determine which came first, the proliferation or the necrosis. Significant cytotoxic and proliferative effects were observed at 2 weeks in the first experiment. We therefore performed the second experiment to examine changes at earlier times. We also observed an increase in urinary calcium excretion along with calcium deposition in the kidneys. The relationship of these calcium changes to the toxicity and proliferation that was observed in the bladder was not clear. A further evaluation of the urinary calcium changes was also included in the experiments.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemical and Diets
The DMA was provided by Luxembourg Industries (Pamol; Tel-Aviv, Israel). The purity of the test article was documented by Luxembourg Industries and confirmed by NMR at our facility at the University of Nebraska Medical Center. The level of DMA fed in the diet was 100 ppm. The diets were prepared at Dyets, Inc. (Bethlehem, PA) and stored at –20°C until fed to the animals. Diets were fed within 10 weeks of pelleting, and fresh diet was provided to the animals at least weekly.

Test Animals and Experimental Design
Experiment 1.
Forty female F344 rats, 4 weeks old, were purchased from Charles River Breeding Laboratories (Raleigh, NC) and quarantined for 7 days. On arrival, they were housed in polycarbonate cages (5/cage) on dry corncob bedding in a room with a targeted temperature of 22°C, humidity of 50%, and a 12-h light/dark cycle. They were fed pelleted Purina Mills Certified Lab Chow 5002 (St. Louis, MO). Food and water were available ad libitum throughout the study. After quarantine, the rats were randomized into 2 groups of 20 rats each using a weight stratification method (Martin et al., 1984Go) and started on the appropriate diet (0 ppm DMA in Group 1 or 100 ppm DMA in Group 2). The last 10 animals in each group were sacrificed after 2 weeks of treatment. The remaining 10 rats in each group were sacrificed after 10 weeks of treatment.

Experiment 2.
Sixty-three female F344 rats, 4 weeks old, were obtained from Charles River Breeding Laboratories and maintained under the same conditions as above. After a 12-day quarantine, the rats were randomized into 9 groups of 7 rats each and treated as shown in Table 1Go.


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TABLE 1 Study Design for Experiment 2
 
Experimental Procedures
Water and food consumption were measured at various time points over a 7-day period. Water and food consumptions were measured over the entire treatment periods for rats in Experiment 2 that were treated for less than 7 days. Body weights of all rats were measured on the day after arrival, on study day 0, on the last day of each 7-day consumption period, before and after placement in metabolism cages if urine was collected, and on the day of sacrifice. Detailed clinical observations of each animal were conducted on day 0 and just prior to sacrifice in both experiments and at the end of each 7-day consumption period in Experiment 1.

Fresh-void urine was collected from the remaining 10 animals in each group in Experiment 1, between the h of 0700 and 0900 during study weeks 4 and 9. Urinary pH was determined by microelectrode (Microelectrodes, Inc., Londonderry, NH). The same animals were acclimated to metabolism cages for 48 h before collection of 24-h urine during study weeks 4 and 8. Fresh-voided urine and 24-h urine were collected during different study weeks to avoid excessive handling of the rats on any given day. Excessive handling produces urothelial hyperplasia by itself (Cohen et al., 1996Go). Food and water were available ad libitum while the rats were in the metabolism cages. The specimens were collected over nylon netting to prevent contamination of the urine with diet or feces. Total volume was determined and calcium and creatinine were measured on the Vitros 250 Chemistry Analyzer (Johnson and Johnson Clinical Diagnostics, Rochester, NY). Urine was collected by aspiration from the bladder, just prior to inflation of the bladder with fixative at necropsy (see below), from all rats sacrificed at 3, 7, and 14 days. In Experiment 2, urine was analyzed for calcium and creatinine concentrations.

One h before sacrifice, the rats were injected ip with bromodeoxyuridine (BrdU), 100 mg/kg body weight. All rats in each experiment were sacrificed by an overdose of Nembutal (50 mg/kg of body weight). Rats treated for 6 and 24 h were sacrificed between 0800 and 1000 h. All other rats were sacrificed between 1000 and 1200 h to avoid diurnal variations in the labeling index (Tiltman and Friedell, 1972Go). In Experiment 1, the urinary bladder and stomach were inflated in situ with Bouin`s fixative, removed and placed in the same fixative. In Experiment 2, only the urinary bladder was removed in the manner described. The kidneys were removed from all animals in each experiment, weighed, and placed in formalin. A section of intestine was removed from the animals in Experiment 2 and placed in Bouin`s fixative as a positive control instead of using a section of stomach. One half of the bladder from each animal was processed for examination by SEM and classified as previously described (Cohen et al., 1990Go). Briefly, Class 1 bladders show flat, polygonal superficial urothelial cells; Class 2 show occasional small foci of urothelial necrosis; Class 3 bladders show numerous small foci of superficial urothelial necrosis; Class 4 bladders show extensive superficial urothelial necrosis, especially in the dome of bladder; and Class 5 bladders show necrosis and also piling up of rounded urothelial cells. Normal bladders are usually Class 1 or 2, but occasionally Class 3.

The other half of the bladder was cut longitudinally into strips and with a slice of stomach (Experiment 1) or with a section of intestine (Experiment 2), was embedded in paraffin, stained with hematoxylin and eosin (H&E) and examined histopathologically (Cohen, 1983Go; Cohen et al., 1990Go). The stomach or intestine served as a positive control for determination of the BrdU labeling index. The kidneys were also embedded in paraffin, stained with H&E and examined histopathologically. The BrdU labeling index was determined by immunohistochemistry, using anti-BrdU (Chemicon International, Temecula, CA) at a dilution of 1:50 and methodology previously described from our laboratory (Garland et al., 1993Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1
Body weights and food and water consumption.
Treatment with DMA had no effect on body weights or food consumption. There was a significant increase in water consumption in the DMA-treated group by week 9 (Control-19.8 g/rat/day; 100 ppm DMA-23.9 g/rat/day, p < 0.05).

Urine pH, volume, and chemistries.
The pH of fresh-void urine was significantly decreased in the DMA-treated group at week 4, but by week 9 the urinary pH was comparable to controls (Table 2Go). Treatment with DMA significantly increased the volume of 24-h urine, resulting in a statistically significant decrease in creatinine at weeks 4 and 8. The calcium concentration was significantly increased at week 4 and significantly decreased at week 8, compared to the control group. When the calcium concentration was normalized for creatinine concentration, the calcium in the DMA-treated group was two times that found in the control group at week 4 but was significantly decreased in the treated group compared to controls at week 8.


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TABLE 2 Treatment with DMA on Urinary Parameters in Female Rats in Experiment 1
 
Tissue weights and non-bladder tissues.
Kidney and bladder weights were comparable in the control and treated groups after 2 weeks of treatment (Tables 3 and 4GoGo). There was a significant increase in actual and relative weights of bladders and kidneys following 10 weeks of treatment with DMA.


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TABLE 3 Effects of DMA on the Kidney in Female Rats in Experiment 1
 

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TABLE 4 Effects of DMA on the Urinary Bladder in Female Rats in Experiment 1
 
There was a slight increase in calcification in the kidneys at the corticomedullary junction after 2 weeks of treatment (Table 3Go). The increase in calcification of the kidneys in the DMA-treated group was statistically significant following 10 weeks of treatment. There were no changes detected in the stomach at either time point.

Bladder changes.
Examination of the bladder epithelium by light microscopy showed no incidence of hyperplasia after 2 weeks of treatment with DMA (Table 4Go). There was a significant increase in the occurrence of simple hyperplasia by light microscopy after 10 weeks of treatment with DMA. Changes in the bladder epithelium (Fig. 1Go) were detectable by SEM (Table 4Go) after 2 weeks of treatment and included clear evidence of cytotoxicity and necrosis (Fig. 2Go). Following 10 weeks of treatment with DMA, there was a significant increase in the number of class-5 bladders in the treated group, indicating not only extensive cellular necrosis but evidence of hyperplasia (Fig. 3Go).



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FIG. 1. Normal bladder epithelium observed by scanning electron microscopy, showing large, flat, polygonal cells (original magnification x570).

 


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FIG. 2. Bladder epithelium (Class 5) from a rat treated with DMA for 2 weeks showing extensive cellular necrosis, rounding up of cells, and the presence of ropy microridges and uniform microvilli on the cell surface, indicating decreased differentiation (original magnification x960).

 


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FIG. 3. Bladder epithelium (Class 5) from a rat treated with DMA for 10 weeks showing piling up of rounded cells indicative of epithelial hyperplasia (original magnification x350).

 
The labeling index was increased 6-fold in the DMA-treated group when compared to the controls after 2 weeks of DMA treatment (Table 4Go). After 10 weeks of treatment, the labeling index in the treated group was decreased by half compared to the week-2 results, but it was significantly higher than that seen in the control group.

Experiment 2
Body weights, food and water consumption, and non-bladder tissues.
Body weights and food consumption of the DMA-treated groups were similar to their respective control groups. Water consumption was increased in the DMA-treated groups at all time points except at 3 days (data not shown). There was no treatment-related increase in calcification of the kidneys, and tissue from the small intestine was normal.

Urine chemistries.
The creatinine concentration of urine collected at the time of necropsy was decreased at day 3 of treatment but was increased at day 7 and day 14 when compared to the control group at each time point (Table 5Go). The urinary calcium concentration in the DMA-treated groups was comparable to controls at all time points measured except for day 14 when it was significantly decreased compared to controls, even when normalized for the creatinine concentration.


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TABLE 5 Effects of Treatment with DMA on Urinary Calcium and Creatinine Concentrations in Female Rats in Experiment 2
 
Urinary bladder changes.
The light microscopic histological diagnosis of the bladder epithelium was normal at all time points in the control and treated groups (Table 6Go). Changes in the bladder epithelium detected by SEM (Table 6Go) were noted as early as six h after treatment was begun, including small, focal, punctate lesions formed on the luminal surface of the urothelial superficial cells (Fig. 4Go). By 24 h, there was clear evidence of focal cellular necrosis and exfoliation (Fig. 5Go). By day 7, all bladders in the DMA-treated group exhibited ropy microvilli on the luminal surface of the urothelial cells and there was pitting, necrosis, and exfoliation of the cells (Fig. 6Go). The urothelial changes appeared earlier and were more severe in the dome of the bladder.


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TABLE 6 Effects of DMA on the Urinary Bladder in Female Rats in Experiment 2
 


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FIG. 4. Bladder epithelium (Class 3) from a rat administered DMA for 6 h showing extensive pitting of the cell surface (arrows) and increased separation of cells (arrowheads) indicating a loosening of cell junctions (original magnification x1620).

 


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FIG. 5. Bladder epithelium (Class 3) from a rat treated with DMA for 24 h showing cellular necrosis and cells in the process of exfoliation (original magnification x520).

 


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FIG. 6. Bladder epithelium (Class 4) from a rat treated with DMA for 7 days showing extensive areas of cellular necrosis (original magnification x85).

 
The labeling index was significantly decreased in the DMA-treated group at 6 h and 24 h when compared to the control group (Table 6Go). At 3 days the labeling index was similar in the control and treated groups, but by day 7 the labeling index in the treated group was greater than twice the labeling index in the control group. There was a 5-fold increase in the DMA-treated group compared to controls at day 14, similar to the results in Experiment 1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of Experiment 1 demonstrated that there was increased cell proliferation in the bladder epithelium by 10 weeks following administration to female rats of 100 ppm of DMA in the diet. Simple hyperplasia was detected by light microscopy, piling up of cells was detected by scanning electron microscopy, and an increased labeling index was determined by BrdU injection and immunohistochemical analysis. An increase in SEM changes was also evident at two weeks of administration, as was an increase in the BrdU labeling index which was higher than that seen after 10 weeks of DMA administration. Similar to our previous experiment, an increase in urinary calcium was present at 4 weeks but measurement of urinary calcium at a second, later time point (8 weeks) in this experiment showed a decreased concentration. Renal calcification was again evident at 10 weeks, but was not evident at earlier time periods of the experiment. The observation that the cytotoxic and proliferative changes were already present in the bladder by two weeks of administration suggests that the previously reported changes in urinary calcium and renal calcification observed after 10 weeks of feeding are not critical for the initial development of the urothelial proliferative changes or the cytotoxicity. Whether they contribute to the continued presence of these effects is unknown.

In the second experiment, it was evident that cytotoxic changes, as manifested by pitting of the luminal surface of the superficial cells detected by SEM, were present by as early as 6 h after administration of DMA had begun. The cytotoxic effect became more severe by 24 h with focal cellular necrosis, and the necrosis was more widespread by 3 and 7 days. In contrast, the BrdU labeling index did not increase above control levels until after 7 days of administration of DMA. These results strongly suggest that the sequence of events in the bladder urothelium following DMA administration is cytotoxicity, necrosis, and subsequent regeneration rather than a direct mitogenic effect. Urinary solids, such as calculi, microcystalluria, or precipitate formation were not detected in previous studies in our laboratory (Arnold et al., 1999Go). The effects were altered only slightly by significant changes in urinary pH. The short-term effects were greater in female than in male rats, similar to the sex differences in the carcinogenicity of DMA in rats reported previously (van Gemert and Eldan, 1998Go). The actual mechanism by which these cytotoxic effects are produced in the urothelium by feeding DMA is not clear. Although some of the administered DMA is excreted unchanged in the urine in the rat, significant amounts are metabolized to inorganic and various other organic arsenic metabolites (Wanibuchi et al., 1996Go; Wei et al., 1999Go).

Although this presently is the only example of arsenic-compound carcinogenesis in animals, there are several limitations with respect to extrapolation to humans. To begin with, this is an organic arsenic compound, rather than inorganic arsenic, which appears to be the carcinogenic form(s) in humans. In addition, there are extensive quantitative differences in the toxicokinetics of arsenical species in humans compared to rodents in general, and especially compared to rats (Aposhian, 1997Go). Rats appear to have the least comparable relationship to human arsenical toxicokinetics compared to other rodent species that have been studied. This is due in part to the retention of arsenic in red blood cells in rats so that the half-life of arsenic in rats is related to red-cell survival. In addition, the doses that are used to produce the carcinogenic effect with DMA in rats are significantly higher than those experienced in human exposures, even in areas where arsenic levels in the drinking water are considered to pose a significant risk to human health.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert technical assistance of Traci Anderson and Margaret St. John with the performance of these experiments, and the assistance of Kainette Jones and Denise Miller with the preparation of this manuscript. This research was supported in part by grants CA32513 and CA36727 from the National Cancer Institute.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (402) 559-9297. E-mail: scohen{at}unmc.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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