ToxSci Advance Access originally published online on August 26, 2008
Toxicological Sciences 2008 106(2):350-363; doi:10.1093/toxsci/kfn184
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Effects of Inorganic Arsenic on the Rat and Mouse Urinary Bladder
Department of Pathology and Microbiology and Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, Nebraska 68198-3135
3 To whom correspondence should be addressed at Nishi-Kobe Medical Center, Pathology Division, 5-7-1 Koujidai, Kobe 651-2273, Japan. Fax: (402) 559-9297. E-mail: scohen{at}unmc.edu.
Received June 23, 2008; accepted August 21, 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Inorganic arsenic (arsenate and arsenite) is a known human carcinogen, inducing tumors of the skin, urinary bladder, and lung. Understanding the mechanism of inorganic arsenic carcinogenesis has been hampered by a lack of animal models. To define the urothelial effects of inorganic arsenic, we administered arsenate and arsenite in the diet or drinking water to rats and mice in several short-term experiments (2–10 weeks). Treatment with arsenate or arsenite in the drinking water or diet induced cytotoxicity and necrosis of the urothelial superficial layer and hyperplasia in rats and mice. Arsenate-induced changes occurred later in mice compared with arsenite-induced changes, but not in the rat. Hyperplasia in rats was evident by light microscopy at an earlier time point (2 weeks) than previously observed after treatment with dimethylarsinic acid (DMAV). The bromodeoxyuridine labeling index was increased in treated rats. We were unable to determine the bromodeoxyuridine labeling index in mice. The effects of inorganic arsenicals on the bladder were greater when administered in the drinking water than in the diet in rats and mice, but so was the overall toxicity to the animal. The female rat appeared more sensitive to the effects of inorganic arsenic than the male rat, but effects were similar in female and male mice. The mode of action of inorganic arsenic in rats and mice appears to involve urothelial cytotoxicity, increased cell proliferation and ultimately tumors. Cytotoxicity is likely due to the generation of reactive trivalent arsenicals excreted in the urine.
Key Words: inorganic arsenic; urinary bladder; cytotoxicity; cell proliferation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Inorganic arsenic (arsenate and arsenite) is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung in individuals exposed to high concentrations, primarily through drinking water (National Research Council Subcommittee on Arsenic in Drinking Water, 1999
, 2001). Exposure occurs also through the diet and can occur by inhalation in certain occupational exposure scenarios, such as mining. The mechanism by which inorganic arsenic induces tumors is unknown. Progress in the understanding of inorganic arsenic carcinogenesis has been hampered by the lack of an animal model. Sodium arsenate and sodium arsenite administered in rat diet at doses up to 400 and 250 µg/g arsenic, respectively, were negative in 2-year bioassays performed in rats in the 1960's (Byron et al., 1967). However, from that publication, it is unclear whether tissue examinations in those studies were performed adequately or if sufficient numbers of animals were tested to detect weak carcinogenic activity. During the past decade, models of arsenical-induced carcinogenesis have been developed, specifically for the dimethylarsinic acid (DMAV)–induced bladder carcinogenesis in the rat (Arnold et al., 2006; Wei et al., 1999) and transplacental arsenite-induced tumors of several tissues in mice (Waalkes et al., 2003, 2007). These models of arsenical-induced carcinogenesis have provided data regarding metabolic, kinetic, and dynamic aspects of exposure to arsenic compounds. These data are being used to delineate the mechanism of inorganic arsenic carcinogenesis and its dose response, providing information critical to determining safe levels of inorganic arsenic in the drinking water (EPA-SAB-http://www.epa.gov/sab/pdf/sab-07-008.pdf). However, both of these models have significant limitations with extrapolation to humans.
Waalkes et al. (2003) observed that inorganic arsenic administered as sodium arsenite to mouse dams during days 8 through 18 of gestation at extremely high doses (42.5 or 85 µg arsenic per ml of the drinking water) induced tumors of the liver, ovary, lung and adrenal in the offspring. High exposures were also required to produce cancer in the rat urinary bladder with DMAV in 2-year bioassays where weak tumorigenic effects were only seen at dietary concentrations of 40 and 100 µg/g (Arnold et al., 2006) and at levels of 25, 50, and 250 µg/ml in the drinking water (Wei et al., 1999). No effects were seen by light microscopy at lower exposures, that is, dietary doses of 10 µg/g (Arnold et al., 2006; Cohen et al., 2006, 2007a) or at 12.5 µg/ml in drinking water (Wei et al., 1999). In contrast, DMAV at similarly high doses did not induce tumors of the urinary bladder or any other tissue when administered to mice in a 2-year bioassay (Arnold et al., 2006). The tumorigenic response of DMAV in rats appeared to be somewhat greater in females than in males (Arnold et al., 2006), and was affected to some extent by the composition of the diet (Arnold et al., 1999). DMAV also enhanced bladder carcinogenesis when administered in the drinking water after four weeks of treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine, a known DNA-reactive bladder carcinogen (Wanibuchi et al., 1996). There was no detectable effect on the hamster's urinary tract when DMAV was administered at high doses in the diet for 10 weeks (Cano et al., 2001). Another pentavalent organic arsenical, monomethylarsonic acid (MMAV) did not produce tumors in rats or mice when administered in the diet for 2 years (Arnold et al., 2003b).
The mode of action for DMAV-induced rat urinary bladder tumors involves cytotoxicity of the urothelium, which occurs within six hours after oral exposure to DMAV and is followed by regenerative proliferation, hyperplasia, and ultimately urothelial tumors at relatively low incidences (Cohen et al., 2001, 2007a). In a previous publication, we demonstrated that the cytotoxicity is not caused by major alterations in urinary composition or by formation of urinary solids (Arnold et al., 1999). We have hypothesized that the cytotoxicity must be due to excretion of DMAV and/or a cytotoxic, reactive metabolite and demonstrated that the reactive metabolite is likely to be dimethylarsinous acid (DMAIII) (Cohen et al., 2002).
Trivalent arsenicals are considerably more cytotoxic that their corresponding pentavalent arsenicals (Cohen et al., 2002; Petrick et al., 2000; Styblo et al., 1999), and methylated trivalent arsenicals, such as DMAIII and monomethylarsonous acid (MMAIII), are more cytotoxic than trivalent inorganic arsenic (AsIII) (Cohen et al., 2002; Petrick et al., 2000; Styblo et al., 1999). In rat and human urothelial cell lines, the trivalent arsenicals are cytotoxic at concentrations of 1µM or less, whereas arsenate, the pentavalent inorganic arsenical (AsV), is cytotoxic at concentrations of 5–10µM and the methylated pentavalent arsenicals, MMAV, DMAV, and trimethylarsenic oxide (TMAO), are cytotoxic at millimolar concentrations (Cohen et al., 2002). In the urine of rats administered high doses of DMAV in the diet or drinking water, concentrations of DMAV and TMAO are in the 50–100µM range but never at concentrations of 1mM or higher (Cohen et al., 2002). These concentrations are significantly lower than the concentrations of DMAV and TMAO found to be cytotoxic in vitro. In contrast, the concentrations of DMAIII that are produced in the urine are in the 1–10µM range which are at or above the range found to be cytotoxic in vitro. In addition, the levels of DMAIII measured in the urine are probably lower than the actual concentration due to the instability of DMAIII. There is also a clear dose response for the formation of DMAIII in the urine following treatment with different concentrations of DMAV in the diet, with undetectable levels (detection limit, 0.026µM) in the urine of rats administered DMAV as 2 µg/g of the diet (Arnold et al., 2003a).
The sequence of events for the occurrence of DMAV-induced rat bladder tumors appears to be absorption of sufficient amounts of DMAV to yield adequate concentrations of DMAIII as a reactive metabolite in the urine. DMAIII produces cytotoxicity in the rat bladder urothelium, followed by regenerative proliferation, hyperplasia and eventually tumors. When the formation of DMAIII is inhibited, by concurrent administration of a chelating agent, such as dimethylpropanesulfonate together with DMAV, this cascade of events is inhibited and neither cytotoxicity nor increased proliferation occurs (Cohen et al., 2002).
Extrapolating the rat model to humans requires an understanding of the differences between the metabolism and toxicokinetics of arsenicals in these species. In most mammalian species, arsenic is metabolized by a sequence of reductions and irreversible oxidative methylations through which the administered arsenate produces arsenite, MMAV, MMAIII, DMAV, DMAIII, and TMAO (Aposhian, 1997). When DMAV is administered to the rat, it is excreted primarily as unchanged DMAV with similar concentrations of TMAO (Cohen et al., 2002; Wanibuchi et al., 1996) and considerably smaller amounts of DMAIII (Cohen et al., 2002). In contrast, little or no TMAO is produced in humans exposed to arsenicals, including DMAV (Cohen et al., 2006). A second metabolic difference in rats compared with other mammals is the longer retention time of arsenic in the blood (Buchet et al., 1981; Lerman and Clarkson, 1983; Vahter et al., 1984), primarily in the rat red blood cells (RBCs). Lu et al. (2007) demonstrated that the retention in the rat RBCs was because arsenic binds to rat hemoglobin as DMAIII regardless of whether arsenate, MMAV, or DMAV is administered. Unlike other mammals, rat hemoglobin has an extra cysteine on the 
-globin chain of the hemoglobin, which has a strong affinity to trivalent arsenic. Therefore, in rats exposed to arsenicals a substantial amount of the arsenic binds to hemoglobin. It is unknown if this binding occurs in humans. In vitro, it has been demonstrated that human hemoglobin has a much lower affinity for binding trivalent arsenic compared with rat hemoglobin indicating that if binding doses occur it would be to a much lesser extent than in rats.
Lu et al. (2007) also reported that hemoglobin-DMAIII levels in the RBCs and plasma of the DMAV-treated animals corresponded to the urothelial cytotoxicity and regenerative proliferation observed in rats administered DMAV. Somewhat surprisingly, rats administered sodium arsenate in the drinking water also demonstrated extremely high levels of DMAIII bound to hemoglobin in the RBCs and plasma, as well as significant hyperplasia of the urinary bladder epithelium. Previously, Simeonova et al. (2000) demonstrated urothelial hyperplasia in mice administered sodium arsenite in the drinking water for four weeks at a concentration of 100 µg/g arsenic (approximately 170 µg/g sodium arsenite). In this experiment, rats were also administered 100 µg/ml arsenic as sodium arsenate (416 µg/ml) in the drinking water and the effects were evaluated at several time points from 1 to 15 weeks.
Based on similar urothelial changes found in arsenate-treated rats compared with DMAV-treated rats, the present series of experiments was performed in an effort to develop a rodent model for inorganic arsenic similar to the DMAV model, and use that model to define the urothelial effects of both sodium arsenate and arsenite administered to rats and to mice.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals and Route of Administration
Sodium arsenate and sodium arsenite were obtained from Sigma Chemical (St Louis, MO). The purity of the sodium arsenate and sodium arsenite was determined by the supplier to be 98 and 94%, respectively and was accepted without further testing by our laboratory. Sigma also determined that the sodium arsenite contained 0.14% antimony but no further testing to identify other impurities was done. Although the purity of the sodium arsenite used in the current studies (94%) was slightly lower than that of the product we normally use, arsenite doses were not adjusted. Sodium arsenate was administered in the drinking water only and sodium arsenite was administered in the drinking water or in the diet. In Experiment 1, arsenic (100 µg/ml) was added to tap water as sodium arsenate (416 µg/ml). In Experiment 2, arsenic (100 µg/ml) was added to the drinking water as sodium arsenite (173 µg/ml). The arsenic-supplemented drinking water was prepared twice weekly. Analysis of the tap water by the Metropolitan Utilities District (Omaha, NE) showed an arsenic level of less than 0.1 µg/l. In Experiment 3, Certified Purina 5002 diet was supplemented with 200 µg/g arsenic as sodium arsenite (350 µg/g) and in Experiment 4, Certified Purina 5002 diet, was supplemented with 50, 100, 200, or 400 µg/g arsenic as sodium arsenite (87, 170, 350, or 690 µg/g, respectively). In Experiment 5, drinking water or Certified Purina 5002 diet was supplemented with 100 µg/g arsenic as sodium arsenite (173 µg/g). All diets were prepared and pelleted by Dyets, Inc. (Bethlehem, PA) and stored at –20°C until fed. Fresh diets were provided at least weekly. Analysis of the basal diet by PMI Nutrition International (Richmond, IN) showed an arsenic level of < 0.2 ppm in the diet.
Test Animals
F344 rats for all experiments and C57Bl/6 mice for Experiment 1 were purchased from Charles River Breeding Laboratories, Kingston, NY. C57BL/6 mice for Experiments 2–4 were purchased from Charles River Breeding Laboratories, Portage, MI. F344 rats were used based on the responsiveness of the strain to the urothelial effects of arsenate (Lu et al., 2007) and DMAV (Arnold et al., 1999, 2006; Cohen et al., 2001, 2002) in previous experiments. C57BL/6 mice were used because the only previous report of urothelial hyperplasia induced by inorganic arsenic in mice was in this strain (Simeonova et al., 2000). On arrival, all animals were placed in a level four barrier facility accredited by the American Association for Accreditation of Laboratory Animal Care. The level of care provided to the animals met or exceeded the basic requirements outlined in the Guide for the Care and Use of Laboratory Animals (NIH publication #86-23, revised 1986). The animals were group housed (five per cage) in polycarbonate cages with dry corn cob bedding. The cages housing the mice were equipped with microfiltration tops. Nestlets (Ancare Bellmore, NY) were added to the mouse cages and Nylabones (Nylabone Products, Neptune, NJ) were added to the rat cages for environmental enrichment. The animal rooms were on a 12-h light/dark cycle at a targeted temperature of 22 ± 2°C and humidity of 50 ± 5%. All animals were fed Certified Purina 5002 diet during quarantine. Food and drinking water were available ad libitum at all times during the study. Animals were quarantined for at least 7 days prior to starting treatment. Animals were randomized into treatment groups using a weight stratification method (Martin et al., 1984).
Experimental Design
Table 1 briefly summarizes the experimental designs for the various studies in this report.
|
Experiment 1—100 µg/ml arsenic as sodium arsenate (416 µg/ml) in the drinking water for 2 or 10 weeks (Tables 1
–4).The objectives of this study were to verify the effects of treatment with AsV in the drinking water on the female rat urothelium reported in Lu et al. (2007)
, determine the effects of treatment with AsV in the drinking water on the male rat and female and male mouse urothelium, and to compare the effects between males and females. Female and male F344 rats and C57Bl/6 mice, approximately 5 weeks old, were randomized based on species and sex into groups as follows: two groups of five female rats, two groups of 10 male rats and two groups each of 10 female and 10 male mice. One group of each sex and species was given 100 µg/ml arsenic as sodium arsenate (416 µg/ml) in tap water and the other group was given only tap water and served as control. All female rats and five animals from each of the male rat and female and male mouse groups were sacrificed after treatment. All remaining animals were sacrificed after treatment for 10 weeks. (A 10-week study of sodium arsenate administration to female rats was previously performed and the report published by Lu et al., 2007).
|
|
|
Experiment 2—100 µg/ml arsenic as sodium arsenite (173 µg/ml) in the drinking water for 2 or 10 weeks (Tables 1–
3, 5–7).The objective of this experiment was to determine the effects of administration of arsenic as sodium arsenite in the drinking water on the urinary bladder epithelium of female and male rats and mice. Twenty each female and male rats and female and male mice, approximately 5 weeks old, were randomized by species and sex into two groups of 10 animals per group. One group of each sex and species was given 100 µg/ml arsenic as sodium arsenite (173 µg/ml) in tap water and the other group was given only tap water and served as control. Five animals from each group were sacrificed after treatment for 2 weeks. Three of the five remaining AsIII-treated male rats were sacrificed during study week 6 due to an ongoing failure to gain weight and severe morbidity. The dose of arsenic in the treated male rat group was decreased to 75 µg/ml (130 µg/ml sodium arsenite) on study day 39. The remaining animals from each group were sacrificed after treatment for 10 weeks.
|
|
|
Experiment 3—200 µg/g arsenic as sodium arsenite (350 µg/g) in the diet for 2 weeks (Tables 1
–3, 5, 7).The objective of this experiment was to determine if treatment with sodium arsenite in the diet instead of in the drinking water caused the same urothelial effects in male and female rats and mice without the accompanying weight loss and dehydration. Ten each female and male rats and male mice, approximately 6 weeks old, were randomized by species and sex into two groups of five animals per group. Twenty female mice were randomized into two groups of 10 animals per group. One group of each sex and species was fed Certified Purina 5002 diet containing 200 µg/g arsenic as sodium arsenite (350 µg/g) and the other group was fed Certified Purina 5002 diet only. All animals from each group were sacrificed after treatment for 2 weeks.
Experiment 4—50, 100, 200, 400 µg/g arsenic as sodium arsenite (87, 170, 350, 690 µg/g) in the diet for 2 weeks (Tables 1–3, 5, 7).
The objective of this experiment was to determine the maximum tolerated dose of sodium arsenite in the diet. Twenty-five each female rats and female mice, 7 weeks old, were randomized by species into five groups of five animals each. Groups were fed Purina 5002 diet supplemented with 0, 50, 100, 200, or 400 ppm arsenic as sodium arsenite (87, 170, 350, or 690 µg/g, respectively) for 2 weeks. All animals were sacrificed after treatment for 2 weeks.
Experiment 5—100 µg/g arsenic as sodium arsenite (173 µg/g) in the drinking water or 100 µg/g arsenic as sodium arsenite (173 µg/g) in the diet to 5- or 8-week-old rats for 2 weeks (Tables 6).
The objective of this experiment was to determine the route of administration of arsenic (drinking water or diet) and the age (5 weeks or 8 weeks) at which to start treatment to study the urothelial effects of arsenic treatment with the minimum amount of toxicity to the animal. Fifteen female rats, 8 weeks old, and 15 female rats 5 weeks old, were randomized by age into three groups of five animals each. One group each of 8-week-old and 5-week-old rats was treated for 2 weeks with no arsenic, 100 µg/ml arsenic as sodium arsenite (173 µg/ml) in the drinking water, or 100 µg/g arsenic as sodium arsenite (173 µg/g) in Purina 5002.
Experimental Procedures
Body weights were measured and detailed clinical observations were conducted for all animals the day after arrival, just prior to randomization, on the last day of water and food consumption measurements, and just prior to sacrifice. Water and food consumptions were measured over a 7-day period at various time points during each experiment.
One hour prior to sacrifice, all animals were injected intraperitoneally with bromodeoxyuridine (BrdU), 100 mg/kg of body weight (Cohen et al., 2007b) except for the mice in Experiment 4 which were injected with 250 mg/kg of body weight. The higher dose of BrdU was used for the mouse injections because of difficulty labeling BrdU in mouse urinary bladder epithelium tissue in previous experiments using our standard rat tissue procedure. All animals were sacrificed by an overdose of Nembutal (150 mg/kg of body weight) between 1000 and 1200 hours to avoid diurnal variations in the labeling index (Tiltman and Friedell, 1972).
In Experiment 3, the urinary bladders from two control and two treated female rats and mice were inflated with 4% paraformaldehyde and one half of the bladders were processed for examination by transmission electron microscopy (TEM) for the purpose of studying intracytoplasmic granules observed by light microscopy in the bladder epithelium of arsenic-treated mice but not in arsenic-treated rats. The urinary bladders from all other male and female rats, three of the remaining eight female mice, and all male mice were inflated in situ with Bouin's fixative, removed and placed in the same fixative (Cohen et al., 2007b). The bladders were rinsed in 70% ethanol, bisected, and weighed. The bladders from five female mice were fixed in 4% paraformaldehyde to determine the effects of the tissue fixative on determination of the BrdU labeling index. Half of the bladders from each animal were processed for examination by scanning electron microscopy (SEM) and classified as previously described (Cohen et al., 1990). Mouse bladders were classified similarly, although there was more variation in size of the superficial cells than seen in the rat. The other half of the bladder from all other animals (except animals used for TEM) was cut longitudinally into strips and with a slice of intestinal tissue removed at the time of necropsy, was embedded in paraffin, stained with hemotoxylin and eosin (H&E), and examined histopathologically (Cohen, 1983; Cohen et al., 1990, 2007b). A diagnosis of mild simple hyperplasia was made when there were four to five cell layers in the bladder epithelium, and a diagnosis of moderate simple hyperplasia was made when six to eight cell layers were present.
Unstained slides of the bladder and intestinal tissue were used for immunohistochemical detection of BrdU (Cohen et al., 2007b). The intestinal tissue served as the positive control. Anti-BrdU (Chemicon International, Temecula, CA) was used at a dilution of 1:200. The number of BrdU-labeled cells in at least 3000 urothelial cells in the rat was counted to determine a labeling index. We were unable to detect BrdU labeling in the mouse tissues even though modifications of our standard procedure for rat tissue were made, including increasing the dose of BrdU injected, shortening the tissue fixation time, enzyme and heat-based retrieval methods, and various concentrations of anti-BrdU were tried.
Statistics
Group means for body weights, consumptions, tissue weights, and labeling indices were evaluated using analysis of variance followed by Duncan's multiple range test for group-wise comparisons. Histopathology was compared using the two tail, Fisher's exact test. SEM data were analyzed using one-way nonparametric procedures followed by a chi square test. p values less than 0.05 were considered significant. All statistical analyses were performed using SAS for Windows (Version 9.1; Cary, NC).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Rats
Body weights, water consumption, and food consumption.
Body weight gain was severely depressed in the female and male rats treated with 100 µg/ml AsIII (173 µg/ml sodium arsenite) or AsV (416 µg/ml sodium arsenate) in the drinking water (Tables 4–
6). The decrease was most severe in the AsIII-treated male rat group in which three animals were sacrificed during study week 6 after which the dose of AsIII administered to the male rat group was decreased to 75 µg/ml AsIII (130 µg/ml sodium arsenite). Increasing the age at which treatment was started from 5 to 8 weeks did decrease the toxicity of administering 100 µg/g AsIII in the drinking water to female rats, but the mean body weight in the 8-week-old, AsIII-treated female rat group after treatment with AsIII for 2 weeks was still decreased by approximately 16% compared with control (26% decrease in AsIII-treated 5-week-old rats). Administration of AsIII in the diet to female rats at doses of 200 µg/g (as 350 µg/g sodium arsenite) or lower for 2 weeks caused little or no depression in body weight gain (less than 7%). However, the mean body weight in the male rat group fed 200 µg/g AsIII in the diet for 2 weeks was decreased by more than 15% when compared with the control group. Administration of 100 µg/ml AsIII or AsV in the drinking water resulted in a severe decrease in water consumption in the female and male rats when measured during week 2 (Table 2) with the decrease more severe in the female rat compared with the male rat and when AsIII was administered compared with AsV. When AsIII was administered in the diet to female rats at 100 µg/g or 200 µg/g, water consumption during week 2 was decreased but the extent of the decrease was variable. Administration of 400 µg/g (690 µg/g sodium arsenite) resulted in a severe decrease. Food consumption measured during week 2 in female and male rats administered AsV in the drinking water was decreased although the decrease was not as severe as that seen in the water consumption of treated rats (Table 2). Administration of AsIII in the drinking water had no effect on food consumption of the female and male rats during week 2. Administration of 50 or 100 µg/g AsIII in the diet for 2 weeks had no effect on food consumption in the female rats, but at doses of 200 or 400 µg/g, food consumption was decreased. Administration of 200 µg/g AsIII in the diet had no effect on food consumption in the male rats.
Urothelial effects.
Treatment with 100 µg/ml AsIII or AsV in the drinking water for 2 weeks caused a statistically significant increase in the relative bladder weight of male rats, and treatment with 200 µg/ml AsIII in the diet for 2 weeks caused a statistically significant decrease in the absolute bladder weight of female rats (data not shown). Simple hyperplasia was observed in the bladder epithelium of all but one female rat (three studies, n = 20) and all male rats (2 studies, n = 10) treated with 100 µg/ml AsIII or AsV in the drinking water for 2 weeks (Tables 4–6). The severity of the simple hyperplasia (mild vs. moderate) induced by AsIII or AsV was less in the male rats compared with the female rats. The incidence and the severity of simple hyperplasia was somewhat less after treatment of female and male rats with AsIII or treatment of male rats with AsV in the drinking water for 10 weeks. Treatment of female rats with AsIII in the diet at doses of 100 µg/ml and higher in the diet and treatment of male rats at a dose of 200 µg/ml in the diet for 2 weeks caused an increase in the incidence of simple hyperplasia although the incidence was less than that induced by treatment with AsIII in the drinking water. The incidence of simple hyperplasia was similar regardless of whether treatment with 100 µg/ml AsIII in the drinking water or diet was started at 8 weeks or 5 weeks of age (Table 6). The SEM classification of bladders from female and male rats treated with 100 µg/ml AsIII or AsV in the drinking water for 2 weeks was significantly different when compared with their respective control groups (Tables 4–6) (Fig. 1). Immature small round urothelial cells were present in the bladders of all female rats treated with AsIII or AsV in the drinking water. In addition, piling up of these small round cells was observed in the majority of bladders of AsIII-treated and AsV-treated females indicative of hyperplasia (class 5) (Figs. 2 and 3). Cytotoxicity with areas of necrosis and exfoliation of the large polygonal superficial cells that normally line the surface of the bladder were observed in the bladders of AsIII-treated and AsV-treated male rats (class 4). Immature small round urothelial cells were also observed in several AsIII-treated and AsV-treated male rat bladders, but piling up of the small round cells was observed in only one AsIII-treated male rat bladder. The changes observed by SEM were less severe in the bladders from female rats treated with AsIII and from male rats treated with AsV in the drinking water for 10 weeks. When AsIII was administered to female rats in the diet, the SEM classification for bladders was significantly different from the control rats only in the group treated for 2 weeks with 100 µg/g AsIII (Table 5). Although bladders from female rats treated with 200 µg/g AsIII in the first dietary experiment showed areas of cytotoxicity with necrosis and exfoliation of superficial cells, the areas were not extensive and all bladders were classified as class 2 or 3 (normal). In the second dietary experiment, the effects of treatment with 200 µg/g AsIII on the female rat bladder were somewhat more extensive, but the SEM classification still was not significantly different from the control group. Some cytotoxicity with necrosis and exfoliation of the superficial cells was also observed at the lowest (50 µg/g) and highest (400 µg/g) doses of AsIII administered in the diet to female rats for 2 weeks. All bladders from male rats treated with 200 µg/g AsIII in the diet for 2 weeks were classified as normal. SEM examination showed extensive necrosis and exfoliation in the bladder epithelium of female rats in the 8- and 5-week-old groups administered 100 µg/ml AsIII in the drinking water for 2 weeks and four of five bladders in each group were classified as class 5 (Table 6). There were also extensive changes in the 8-week-old group treated with dietary AsIII for 2 weeks (Fig. 4) with three bladders classified as class 4 and 1 bladder as class 5. However, in the 5-week-old group treated with dietary AsIII, only one of five bladders was classified as class 5. The remaining bladders were classes 1 and 3 which are seen in control rats. The BrdU labeling index was significantly increased in the female and male rat groups treated with 100 µg/ml AsIII or AsV in the drinking water for 2 weeks compared with the respective control group. After treatment for 10 weeks, the labeling index was also increased in all treatment groups but the increase was statistically significant only in the AsV-treated male rat group (Tables 4–6). In the first dietary experiment, treatment with 200 µg/g AsIII for 2 weeks had no effect on the BrdU labeling index in the female or male rats (Table 5). In the second dietary experiment, treatment with AsIII for 2 weeks increased the BrdU labeling index at all doses (50, 100, 200, and 400 µg/g AsIII) in the female rats but the increase was only significant at doses of 50 and 100 µg/g AsIII. There was a statistically significant increase in the BrdU labeling index compared with the respective control group in the 8- and 5-week-old groups treated with 100 µg/ml AsIII in the drinking water for 2 weeks (Table 6). However, when AsIII was administered in the diet, the BrdU labeling index was increased only in the 8-week-old group although the increase was not statistically significant.
|
|
|
|
Mice
Body weights, water consumption, and food consumption.
Treatment with 100 µg/ml AsV (416 µg/ml sodium arsenate) in the drinking water had no effect on body weight gain in female or male mice (Table 4). However, female mice treated for 2 or 10 weeks and male mice treated for 10 weeks with 100 µg/ml AsIII (173 µg/ml sodium arsenite) in the drinking water had significantly decreased body weights compared with their respective control groups (Table 7). When mice were treated with AsIII in the diet for 2 weeks, a significant decrease in body weight was seen at doses of 100 µg/g and higher in the female and at a dose of 200 µg/g (350 µg/ml sodium arsenite) in the male. The most severe effects on body weight were seen in the female mouse groups treated with 200 or 400 (690 µg/g sodium arsenite) µg/g AsIII in the diet where animals were sacrificed prior to the end of the study due to severe weight loss. There was a severe decrease in water consumption measured during week 2 in female and male mice treated with 100 µg/ml AsV in the drinking water and during week 7 in female and male mice treated with 100 µg/ml AsIII in the drinking water (Table 3). The decrease was greater in the female mice than in the male mice. Treatment of female mice with doses of AsIII in the diet of 100 µg/g or less had no effect on water consumption. Food consumption was significantly decreased when female mice were treated with 100 µg/ml AsV in the drinking water during week 2 and with 100 µg/ml AsIII in the drinking water during week 7, but the decrease was greater in the AsIII-treated female mice (Table 3). Treatment at doses of 50 µg/g AsIII and higher in the diet caused a dose dependent decrease in food consumption. Neither arsenical regardless of the route of administration had an effect on food consumption in the male mice.
Urothelial effects.
The only effects on tissue weights in arsenic-treated mice was a statistically significant increase in relative bladder tissue weights in male mice treated with 100 µg/ml AsIII in the drinking water for 10 weeks and with 200 µg/g AsIII in the diet for 2 weeks (data not shown). Treatment with 100 µg/ml AsV in the drinking water caused an increase in the incidence of simple hyperplasia in male mice after treatment for 10 weeks (Table 4), and treatment with 100 µg/ml AsIII in the drinking water caused a significant increase in the incidence of simple hyperplasia in male mice after treatment for 2 weeks (Table 7). Treatment with 100 µg/ml AsV in the drinking water had no effect on the incidence of simple hyperplasia in female mice and treatment with 100 µg/ml AsIII in the drinking water had a minimal effect. No changes were detected by SEM in the bladder epithelium of AsV-treated female or male mice after 2 or 10 weeks of treatment (Table 4). SEM examination showed extensive necrosis and exfoliation of superficial cells in the bladders from two of five female mice (Table 7) (Fig. 5) and one of five male mice treated with 100 µg/ml AsIII in the drinking water for 2 weeks. The remaining bladders in each group were classified as normal. All bladders from female and male mice treated for 10 weeks with 100 µg/ml AsIII in the drinking water were classified as normal. There was a significant increase in the number of class 5 bladders in the female (9/10) and male mice (3/5) treated with 200 µg/g AsIII in the first dietary experiment. In the second dietary experiment, cytotoxicity with extensive necrosis and exfoliation and piling up of small round cells indicative of hyperplasia was observed in the bladder epithelium of a few female mice treated with 50 or 100 µg/g AsIII in the diet but the SEM classification in these two groups was not significantly different from control.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Arsenate and arsenite produced a proliferative effect in both the rat and the mouse urinary bladder urothelium, unlike DMAv which produced urothelial effects only in the rat. However, arsenate-induced urothelial changes in the mouse were limited occurring only after 10 weeks of treatment and only in the male mouse. Similar to the DMAV-induced urothelial changes in the rat, high doses were required to produce the effect. A greater response was observed in the female rat than in the male rat. A no observed effect level for arsenite in the rat and mouse has yet to be determined. Also, similar to the DMAV rat model, there appears to be a rapid induction of cytotoxicity and subsequent regenerative proliferation. In the present experiments with inorganic arsenic, hyperplasia was apparent by light microscopy by 2 weeks after the beginning of administration. In a previous study, hyperplasia was apparent by light microscopy 1 week after beginning administration of arsenate (Lu et al., 2007
), which is a much earlier time point than we were able to observe hyperplasia by light microscopy in rats treated with DMAV (Cohen et al., 2001, 2002). The urothelial effects of shorter periods of treatment with inorganic arsenic have not been examined.
Urothelial cytotoxicity is not easily detected by light microscopy, especially if it involves only the superficial layer of the urothelium (Cohen et al., 1990). This is because urothelial superficial cells are thin and not always readily evident by light microscopy. Furthermore, artifactual tears of the surface frequently occur during processing of bladder tissue for light microscopic examination. These tears are not easily distinguished from actual cytotoxicity and necrosis. SEM readily demonstrates cytotoxicity, urothelial necrosis, and exfoliation of the surface cells, and in addition, a larger surface area is available for examination compared with that examined by light microscopy.
In the rat, treatment with arsenate and arsenite caused an increase in the labeling index similar to the results found in rats treated with DMAV. Unlike the rat, an increase in labeling index was not observed in the mouse despite the presence of hyperplasia by light microscopy. This could be due to an accumulation of cells (decreased rate of death) that are proliferating at a rate that is not much different from controls, thereby not causing an increase in the labeling index but which would give the appearance of hyperplasia or could be due to technical issues with the assay in mice. Although we have investigated several modifications to our standard procedure (including changes in the dose of BrdU injected, the tissue fixative, the concentration of anti-BrdU used for immunostaining, the retrieval method, and processing times), we have not been able to determine the basis for the lack of BrdU labeling of the mouse urothelial cells and this remains an area for further investigation.
Intracytoplasmic eosinophilic granules were observed by light microscopy in the urothelium of mice treated with arsenite or arsenate via the drinking water or via the diet. These granules occurred earlier in mice exposed to arsenite than in those exposed to arsenate. By TEM, the granules were identified as being intramitochondrial and composed of an amorphous-staining material (Suzuki et al., in press). Isolation of the urothelial cell mitochondria and analysis of the arsenic content demonstrated the presence of high levels of arsenic in the granules, mainly in the form of arsenite. These granules do not appear to have any toxic effect on the urothelial cells and do not appear to influence proliferation. Similar urothelial cytotoxicity was seen in the rat without the presence of such granules. The morphology and composition of these granules will be described in a separate publication. The cause for these granules in the mouse urothelium following arsenite or arsenate administration cannot be explained at this time.
There are a limited number of modes of action possible for a chemical producing an effect on the epithelium of the urinary bladder. The most common mode of action in both animal models and in humans is that a chemical is DNA reactive (Cohen et al., 2000). DNA-reactive chemicals include a variety of aromatic amines, nitroaromatics, nitrosamines, and cytotoxic chemotherapeutic agents, such as cyclophosphamide. However, it has been clearly demonstrated that arsenicals do not react directly with DNA (Nesnow et al., 2002). There is some in vitro evidence that arsenicals can inhibit DNA repair, inhibit the mitotic apparatus by binding to tubulin, or produce oxidative damage (Cohen et al., 2006; Kitchin, 2001). However, the evidence for these cellular effects occurring in vivo is limited. Arsenic-induced oxidative damage, for example, is limited to exposures that are well in excess of those required for a cytotoxic or tumorigenic effect (EPA-SAB-http://www.epa.gov/sab/pdf/sab-07-008.pdf). Similarly, the concentrations required to produce oxidative damage in vitro are significantly greater, frequently by orders of magnitude, than the concentrations required to produce cytotoxicity (Cohen et al., 2006; Kligerman et al., 2003; Nesnow et al., 2002). Furthermore, exposure to a variety of antioxidants in vivo and in vitro has a limited effect on the cytotoxic and proliferative effects induced by arsenicals (Wei et al., 2005). In summary, arsenicals are not DNA reactive. However, they do have some indirect genotoxic properties, but these are generally only at doses greater than required to produce cytotoxicity, suggesting that the indirect genotoxic effects are secondary to cytotoxicity.
As previously demonstrated for DMAV (Cohen et al., 2001, 2006) and in this study for arsenate and arsenite, the mode of action clearly involves cytotoxicity and regeneration, possibly also by DMAIII. For administration of DMAV, accumulating evidence supports the hypothesis that the urothelial cytotoxicity is due to production of DMAIII and excretion in the urine at concentrations sufficiently high to produce a cytotoxic affect (Cohen et al., 2006). The mode of action of cytotoxicity and regeneration generally implies a threshold process. It is true also for other chemicals involving other tissues, such as chloroform's effects on liver and kidney (Cohen et al., 2006). This proposed mode of action is consistent with the observation that DMAV is not cytotoxic at low levels of exposure. A certain minimal amount of DMAIII must be generated to attain a sufficiently high urinary concentration that will produce the cytotoxic effects. If these urinary concentrations of DMAIII are not attained, then cytotoxicity would not occur and a proliferative or tumorigenic effect would not follow.
Lu et al. (2007) have demonstrated that DMAIII is found at significant levels in the RBCs and plasma of rats administered arsenate in the drinking water. It is not yet clear whether the urinary concentrations of DMAIII produced in rats and mice following exposure to arsenate and arsenite are similar to the levels observed following DMAV administration in rats (Cohen et al., 2006). Considerably more research is necessary to further delineate the metabolism and toxicokinetics of these arsenicals and to determine the mechanism by which cytotoxicity is produced.
Further support for the hypothesis of cytotoxicity as the mechanism/mode of action for inorganic arsenic bladder carcinogenicity lies in the effects following route of exposure. Regardless of whether inorganic arsenic was administered in the drinking water or the diet, we observed urothelial cytotoxicity and increased cell proliferation. However, administration of the arsenicals resulted in significant toxicity in the rats and mice as evidenced by the decreased body weight gains in the arsenic-treated groups. Inorganic arsenicals administered in the drinking water were most toxic to the male rats, although significant decreases in body weight were seen in the female rats and male and female mice. Arsenite administered in the diet was most toxic to male and female mice compared with rats. Based on water consumption data, it appears the rodents were unwilling to drink the water containing the arsenical when it was present at such high concentrations. The lower water consumption caused a significantly lower urinary output, likely resulting in a higher concentration of the arsenicals in the urine, even if the total amount of arsenic excreted is the same compared with that which follows dietary administration. Because the urinary concentration, not the total amount of arsenic present in the urine, is the key parameter (Cohen, 1995), it is not surprising that we observed a greater effect in animals administered arsenate or arsenite in the drinking water compared with the diet.
Based on a previous report showing that DMA administered in the drinking water was less toxic to older animals (Wei et al., 1999), we examined whether the toxicity might be decreased by beginning administration to higher weight animals that were past the maximum growth phase rather than administering the arsenicals to younger, lighter weight animals during the period where normally the maximum weight gain would occur. To do this, we compared the effects in 5-week-old animals of administration of arsenite in the diet or drinking water to the effects in 8-week-old animals to determine if there was a difference in toxicity and response. At both ages, there was significant toxicity associated with administration in the drinking water. There was no toxicity when arsenite was administered in the diet regardless of the age when treatment began. We observed an effect on the urothelium when arsenic (100 µg/ml AsIII as sodium arsenite) was administered to either the 5-week-old rats or the 8-week-old rats. However, the response was somewhat greater when arsenite was administered beginning at 8 weeks of age compared with 5 weeks of age based on the SEM classification and the BrdU labeling index.
In summary, high doses of inorganic arsenic administered as sodium arsenite or sodium arsenate in the diet or drinking water to rats or mice induced a hyperplastic response in the bladder epithelium. The mode of action appears to involve generation of a reactive metabolite (possibly DMAIII; Cohen et al., 2002; Lu et al., 2003), leading to urothelial cytotoxicity, increased cell proliferation (hyperplasia), and ultimately tumors. A similar mode of action could be operative in humans because there is evidence that exposure to extremely high levels of inorganic arsenic in the drinking water can lead to excretion of cytotoxic levels of DMAIII in the urine (Wang et al., 2004). In addition, cytotoxicity of the lower urinary tract is evident in the form of hematuria in individuals exposed to large amounts of inorganic arsenic (Xu et al., 2008). It is not clear if the hematuria is from the bladder or the kidney, but because there is no other evidence of renal damage (i.e. no urinary casts or effects on serum urea or creatinine) reported, the hematuria is most likely from the bladder.
|
NOTES |
|---|
1 Current address: Nishi-Kobe Medical Center, Pathology Division, 5-7-1 Koujidai, Kobe 651-2273, Japan.
2 Havlik-Wall Professor of Oncology.
|
ACKNOWLEDGMENTS |
|---|
We gratefully acknowledge the technical contributions of Nicole Clark, Jun He, Karen Pennington, and Satoko Kiyota to this research, and Connie Winters for her assistance with the preparation of this manuscript. We would also like to thank Dr Michal Eldan for her critical review of our manuscript. This research was presented in part at the 2007 annual meeting of the Society of Toxicology (The Toxicologist, 96, 762, 2007).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aposhian HV. Enzymatic methylation of arsenic species and other new approaches to arsenic toxicity. Annu. Rev. Pharmacol. Toxicol. (1997) 37:397–419.[CrossRef][Web of Science][Medline]
Arnold LL, Cano M, Cohen SM, Le XC, Lu X. Effects of the dose of dimethylarsinic acid (DMA) on the urinary concentration of dimethylarsinous acid (DMAIII). The Toxicologist (2003a) 72:232. (Abstract).
Arnold LL, Cano M, St John M, Eldan M, van Gemert M, Cohen SM. Effects of dietary dimethylarsinic acid on the urine and urothelium of rats. Carcinogenesis (1999) 20:101–109.
Arnold LL, Eldan M, van Gemert M, Capen CC, Cohen SM. Chronic studies evaluating the carcinogenicity of monomethylarsonic acid in rats and mice. Toxicology (2003b) 190:197–219.[CrossRef][Web of Science][Medline]
Arnold LL, Nyska A, Eldan M, van Gemert M, Cohen SM. Dimethylarsinic acid: Results of chronic toxicity/oncogenicity studies in F344 rats and B6C3F1 mice. Toxicology (2006) 223:82–100.[CrossRef][Web of Science][Medline]
Buchet JP, Lauwerys R, Roels H. Comparison of the urinary excretion of arsenic metabolites after a single oral dose of sodium arsenite, monomethylarsonate, or dimethylarsinate in man. Int. Arch. Occup. Environ. Health (1981) 48:71–79.[CrossRef][Web of Science][Medline]
Byron WR, Bierbower BW, Brouwer JB, Hansen WH. Pathologic changes in rats and dogs from two-year feeding of sodium arsenite or sodium arsenate. Toxicol. Appl. Pharmacol. (1967) 10:132–147.[CrossRef][Web of Science][Medline]
Cano M, Arnold LL, Cohen SM. Evaluation of diet and dimethylarsinic acid on the urothelium of Syrian golden hamsters. Toxicol. Pathol. (2001) 29:600–606.
Cohen SM. Pathology of experimental bladder cancer in rodents. In: The Pathology of Bladder Cancer—Cohen SM, Bryan GT, eds. (1983) Vol. 2. Boca Raton, FL: CRC Press. 1–40.
Cohen SM. Role of urinary physiology and chemistry in bladder carcinogenesis. Food Chem. Toxicol. (1995) 33:715–730.[CrossRef][Web of Science][Medline]
Cohen SM, Arnold LL, Eldan M, Schoen AS, Beck BD. Methylated arsenicals: The implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit. Rev. Toxicol. (2006) 36:99–133.[CrossRef][Web of Science][Medline]
Cohen SM, Arnold LL, Uzvolgyi E, Cano M, St John M, Yamamoto S, Lu X, Le XC. Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat. Chem. Res. Toxicol. (2002) 15:1150–1157.[CrossRef][Web of Science][Medline]
Cohen SM, Fisher MJ, Sakata T, Cano M, Schoenig GP, Chappel CI, Garland EM. Comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography. Scan. Microsc. (1990) 4:135–142.
Cohen SM, Ohnishi T, Arnold LL, Le XC. Arsenic-induced bladder cancer in an animal model. Toxicol. Appl. Pharmacol. (2007a) 222:258–263.[CrossRef][Web of Science][Medline]
Cohen SM, Ohnishi T, Clark NM, He J, Arnold LL. Investigations of rodent urinary bladder carcinogens: collection, processing, and evaluation of urine and bladders. Toxicol. Pathol. (2007b) 35:337–347.
Cohen SM, Shirai T, Steineck G. Epidemiology and etiology of premalignant and malignant urothelial changes. Scand. J. Urol. Nephrol. (2000) 205(Suppl.):105–115.
Cohen SM, Yamamoto S, Cano M, Arnold LL. Urothelial cytotoxicity and regeneration induced by dimethylarsinic acid in rats. Toxicol. Sci. (2001) 59:68–74.
Kitchin KT. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Toxicol. (2001) 172:249–261.
Kligerman AD, Doerr CL, Tennant AH, Harrington-Brock K, Allen JW, Winkfield E, Poorman-Allen P, Kundu B, Funasaka K, Roop BC, et al. Methylated trivalent arsenicals as candidate ultimate genotoxic forms of arsenic: Induction of chromosomal mutations but not gene mutations. Environ. Mol. Mutagen. (2003) 42:192–205.[CrossRef][Web of Science][Medline]
Lerman SA, Clarkson TW. The metabolism of arsenite and arsenate by the rat. Fundam. Appl. Toxicol. (1983) 3:309–314.[CrossRef][Web of Science][Medline]
Lu X, Arnold LL, Cohen SM, Cullen WR, Le C. Speciation of dimethylarsinous acid (DMAIII) and trimethylarsine oxide in rats fed with dimethylarsinic acid (DMAV) in the diet. Anal. Chem. (2003) 75:6463–6468.[Medline]
Lu M, Wang H, Arnold LL, Cohen SM, Li X-F, Lu X, Cullen WR, Le XC. Binding of dimethylarsinous acid to cys-13 of rat hemoglobin is responsible for the retention of arsenic in rat blood. Chem. Res. Toxicol. (2007) 20:27–37.[CrossRef][Web of Science][Medline]
Martin RA, Daly AM, DiFonzo AJ, de la Iglesia FA. Randomization of animals by computer program for toxicology studies. J. Am. Coll. Toxicol. (1984) 3:1–11.
National Research Council Subcommittee on Arsenic in Drinking Water. Arsenic in Drinking Water (1999) Washington, DC: National Academy Press.
National Research Council Subcommittee on Arsenic in Drinking Water. Arsenic in Drinking Water. 2001 Update (2001) Washington, DC: National Academy Press.
Nesnow S, Roop BC, Lambert G, Kadiiska M, Mason RP, Cullen WR, Mass MJ. DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. (2002) 15:1627–1634.[CrossRef][Web of Science][Medline]
Petrick JS, Ayala-Fierro F, Cullen WR, Carter DE, Aposhian H. Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes. Toxicol. Appl. Pharmacol. (2000) 163:203–207.[CrossRef][Web of Science][Medline]
Simeonova PP, Wang S, Toriumi W, Kommineni C, Matheson J, Unimye N, Kayama F, Harki D, Ding M, Vallyathan V, et al. Arsenic mediates cell proliferation and gene expression in the bladder epithelium: Association with AP-1 transactivation. Cancer Res. (2000) 60:3445–3453.
Styblo M, Del Razo LM, LeCluyse EL, Hamilton GA, Wang C, Cullen WR, Thomas DJ. Metabolism of arsenic in primary cultures of human and rat hepatocytes. Chem. Res. Toxicol. (1999) 12:560–565.[CrossRef][Web of Science][Medline]
Suzuki S, Suzuki S, Arnold LL, Muirhead D, Lu X, Le CX, Bjork JA, Wallace KB, Ohnishi T, Kakiuchi-Kiyota S, et al. Inorganic arsenic intramitochondrial granules in mouse urothelium. Toxicol. Pathol. in press.
Tiltman AJ, Friedell GH. Effect of feeding N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide on mitotic activity of rat urinary bladder epithelium. J. Natl. Cancer Inst. (1972) 48:125–129.[Web of Science][Medline]
Vahter M, Marafante E, Dencker L. Tissue distribution and retention of As-74-dimethylarsenic acid in mice and rats. Arch. Environ. Contam. Toxicol. (1984) 13:259–264.[CrossRef][Web of Science][Medline]
Waalkes MP, Liu J, Diwan BA. Transplacental arsenic carcinogenesis in mice. Toxicol. Appl. Pharmacol. (2007) 222:271–280.[CrossRef][Web of Science][Medline]
Waalkes MP, Ward JM, Liu J, Diwan BA. Transplacental carcinogenicity of inorganic arsenic in the drinking water: Induction of hepatic, ovarian, pulmonary, and adrenal tumors in mice. Toxicol. Appl. Pharmacol. (2003) 186:7–17.[CrossRef][Web of Science][Medline]
Wang Z, Zhou J, Lu X, Gong Z, Le XC. Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment. Chem. Res. Toxicol. (2004) 17:95–103.[CrossRef][Web of Science][Medline]
Wanibuchi H, Yamamoto S, Chen H, Yoshida K, Endo G, Hori T, Fukushima S. Promoting effects of dimethylarsinic acid on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in rats. Carcinogenesis (1996) 17:2435–2439.
Wei M, Arnold L, Cano M, Cohen SM. Effects of co-administration of antioxidants and arsenicals on the rat urinary bladder epithelium. Toxicol. Sci. (2005) 83:237–245.
Wei M, Wanibuchi H, Yamamoto S, Wei L, Fukushima S. Urinary bladder carcinogenicity of dimethylarsinic acid in male F344 rats. Carcinogenesis (1999) 20:1873–1876.
Xu Y, Wang Y, Zheng Q, Li B, Li X, Jin Y, Lv X, Qu G, Sun G. Clinical manifestations and arsenic methylation after a rare subacute arsenic poisoning accident. Toxicol. Sci. (2008) 103:278–284.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||