Department of Pathology/Microbiology and the Eppley Cancer Center, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, Nebraska 681983135
Received July 27, 2000; accepted September 11, 2000
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ABSTRACT |
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Key Words: arsenic; urinary bladder; dimethylarsinic acid; cell proliferation; necrosis.
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INTRODUCTION |
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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, 1998) or drinking water (Wei et al., 1999
) 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 (25100 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, 1998
).
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., 1996). 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., 1995
). 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, 1980; Jacobson-Kram and Montalbano, 1985
; Leonard and Lauwerys, 1980
; Rossman, 1998
; Wang and Rossman, 1996
) 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., 1996
; Gurr et al., 1993
; Kochhar et al., 1996
; Lerda, 1994
; Oya-Ohta et al., 1996
), 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., 1999; Germolec et al., 1998
; Simeonova et al., 1999
; Trouba et al., 1999
). 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., 1999). 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.
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MATERIALS AND METHODS |
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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., 1984) 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 1.
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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., 1996). 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, 1972). 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., 1990
). 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, 1983; Cohen et al., 1990
). 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., 1993
).
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RESULTS |
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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 2). 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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Bladder changes.
Examination of the bladder epithelium by light microscopy showed no incidence of hyperplasia after 2 weeks of treatment with DMA (Table 4). 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. 1
) were detectable by SEM (Table 4
) after 2 weeks of treatment and included clear evidence of cytotoxicity and necrosis (Fig. 2
). 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. 3
).
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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 5). 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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DISCUSSION |
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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., 1999). 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, 1998
). 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., 1996
; Wei et al., 1999
).
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, 1997). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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