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Investigations of Rodent Urinary Bladder Carcinogens: Collection, Processing, and Evaluation of Urine and BladdersDepartment of Pathology and Microbiology, and the Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198-3135, USA Correspondence: Address correspondence to: Samuel M. Cohen, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135, USA; e-mail:scohen{at}unmc.edu
Examination of the urine and the bladder epithelium are essential to the investigation of mechanisms of urinary bladder carcinogens in rodents. However, urine and bladder tissue specimens must be collected and processed properly if accurate data are to be derived. The optimum specimen for analysis of urinary constituents is fresh void urine collected from nonfasting animals. Fasting the animal prior to urine collection changes the normal composition, including pH. Many of the normal urinary constituents can influence the mode of action of bladder carcinogens, especially for non-genotoxic agents. Light microscopy is routinely used to examine the bladder epithelium. However, it is often necessary to use more sensitive techniques, such as scanning electron microscopy (SEM) to detect subtle cytotoxic changes in the superficial cell layer of the urothelium, and bromodeoxyuridine (BrdU) incorporation, PCNA, or Ki-67 immunohistochemistry to determine the labeling index for cell proliferation. The urinary bladder must be handled gently and inflated with fixative in situ before the animal dies to avoid artifactual autolytic damage to the bladder epithelium that is visible by SEM and may be mistaken for treatment-related changes. The purpose of this paper is to provide information for the proper collection and examination of urine and the urinary bladder.
Key Words: Urinary bladder • urothelium • urine • scanning electron microscopy • cell proliferation • hyperplasia • urine collection Abbreviations: BrdU, bromodeoxyuridine • DAB, diaminobenzidine tetrahydrochloride • DNA, deoxyribonucleic acid • ED01, 1% effective dose • EDS, energy dispersive X-ray spectroscopy • HCl, hydrochloric acid • MgNH4PO4, magnesium ammonium phosphate • PCNA, proliferating cell nuclear antigen • SEM, scanning electron microscopy
Urine is a readily accessible body fluid that is fundamental to investigations concerning metabolism, kinetics, renal function, and alterations of the lower urinary tract. Methods for its collection in rodent studies vary widely, depending on the purpose of the investigation. Unfortunately, many procedures are routinely utilized for investigational purposes for which they were not developed or intended. This has lead to significant misinformation. Urine is a dynamic fluid that reflects not only urinary tract function, but also food and water intake and systemic metabolic malfunctions and alterations, and is a major route for the excretion of exogenously administered and endogenously generated substances with potential toxic manifestations. Because of these dynamic variations in the organism as a whole, the composition of urine can vary markedly during a 24-hour period (Fisher et al., 1989; Cohen, 1995). Since urine composition reflects food and water consumption most closely, it is not surprising that there is a distinct diurnal variation in its composition. The lower urinary tract is lined by an urothelial epithelium that normally is a slowly proliferating tissue. However, it has tremendous potential for regeneration if injured due to chemicals, physical trauma, or infectious organisms. Cancer of the urothelium is observed, in response to exposure to a variety of agents, either administered exogenously or generated endogenously. Exposure of the urothelium to carcinogenic stimuli has become accepted as being through the urine rather than hematogenously (Clayson and Cooper, 1970). Several specific agents have been identified as urinary bladder carcinogens in humans, and many of these have proven to be carcinogenic in various animal models, particularly in rodents (Cohen, 1998). Classes of chemical agents that are associated with bladder cancer include aromatic amines and amides, phosphoramide mustards, nitrosamines, arsenicals, and various alkylating and cancer chemotherapeutic agents. In addition, chronic bacterial cystitis and schistosomiasis have been associated with an increased risk of bladder cancer, and there is some evidence that long-standing lower urinary tract calculi can lead to an increased risk of bladder cancer (Cohen and Johansson, 1992). In addition, several occupational and environmental exposures have been associated with an increased risk of bladder cancer as identified by a variety of epidemiologic investigations (Cohen et al., 2000). Examination of the urine is critical to investigation of the mechanisms of action of bladder carcinogenesis. During the past three decades, much has been learned about normal variations in urinary composition, as well as variations between sexes and species and the influences of exogenous and endogenous manipulations. Urinary composition is expressed in a variety of ways, including total amount in urine over a given period of time, amount per unit of creatinine, and absolute concentrations of urinary constituents on a molar basis. For the most part, it is the latter that is critical for the evaluation of the carcinogenicity of either the agent and/or its metabolite or as an indication of the composition of normal constituents of the urine, such as pH, calcium, phosphate, magnesium, protein, or creatinine. It has become routine to examine urine collected from rodents housed in special cages, referred to as metabolism cages, which are designed for the collection of urine with as much separation from fecal contamination as possible (Cohen, 1995). A variety of cages have been manufactured with several different designs. It has also become common practice to withhold food and water while the animal is in the metabolism cage during the urine collection procedure, and, in many cases, the animals are also fasted prior to placement in the metabolism cage. An acclimation period to adjust for the stress of changing environment is frequently not included. This is a reflection of the focus of urine collection on pharmacokinetic and metabolism purposes and evaluation of renal function, especially urine concentrating capability. However, for evaluation of urothelial carcinogenesis, these procedures are not appropriate and frequently lead to misleading results. Rodents are nocturnal animals, eating and drinking when it is dark. They discontinue eating and drinking when lights come on in the animal room in the morning and sleep during the daylight hours (Fisher et al., 1989; Cohen, 1995). These dietary habits produce dramatic changes in the urinary composition. During the night, when they are eating, the concentration of many urinary components increases, as a reflection of the ingestion and generation of these components. The concentration of these components rapidly decreases shortly after the lights come on in the morning and remains low until the lights are turned off in the evening, when the animals again begin eating. The abrupt changes in the urine composition that occur when the animal stops eating result in urine composition which is markedly different within 3 to 4 hours. It is apparent, therefore, that fasting of the animal will lead to marked distortion of the normal composition of urine, and an accurate profiling of the normal constituents of urine in the nonfasted state is essential for the evaluation of mode of action of urothelial carcinogenesis. This is particularly true for agents that are not DNA reactive; however, chemical influences of these agents on urine parameters, especially pH, can affect formation and structure of various reactive carcinogenic metabolites (Cohen, 1995). It is therefore essential that the urine is collected from nonfasted animals at appropriate times during the day to reflect the various compositions of urine over a 24-hour period (Dominick et al., 2006). For most purposes, this requires fresh void urine collected either at night or shortly after the lights go on in the morning. We have found that generally it is best to complete collection of urine within two to four hours from the time the lights go on. Reasonable amounts of urine can be obtained in a fresh void specimen from rats, although this may be difficult for studies in mice. If fresh void urines are not obtainable, or more urine volume is necessary for specific measurements, collection in metabolism cages for brief periods of time, two to four hours, can be performed. If this is necessary, it is essential that a period of acclimation be provided to the animals by placing them in the metabolism cages at least 48 hours prior to the collection of urine. It is also essential that they have food and water available during the entire time that they are in the metabolism cages. Collecting urine from animals immediately after placing them in the metabolism cage can frequently lead to misleading results since the animals do not behave in a normal fashion, especially with respect to eating and drinking, as well as having changes in the urine in response to stress (Cohen et al., 1996). Collection of urine over a period of time is further complicated by the necessity of either collecting it over ice or with an antibiotic present in the urine to prevent contamination. Obviously, collection of the specimen over ice changes the solubility characteristics of the solution. Collection of fresh void urine can only be done periodically, since handling a rat or mouse on a frequent basis can alter urine composition as well as produce changes in the urothelium (Cohen et al., 1996). There is evidence of mild urothelial cytotoxicity and regeneration in rodents that are handled at least once daily; this is unavoidable in studies involving daily gavage. Such changes need to be taken into account when planning and evaluating urinalysis investigations. Fresh void urines can be utilized for nearly any type of analysis, including sediment analyses, metabolism and chemical kinetic studies, and several chemical analyses. The specific studies to be performed on a given collection will be prioritized to take into account the amount of urine that is collected. This will vary not only with respect to the amount the animal is eating and drinking, but also as a reflection of the treatment that they are receiving. In rats, it is usually possible to collect at least 100 microliters of urine in a fresh void specimen. This provides an ample amount of sample for a variety of analyses. We routinely include a creatinine determination as a way of providing some standardization with respect to variations in fluid amount. The creatinine can give some indication of the fluid intake of the animal as well as the output. Nevertheless, it is important to express urinary chemical components in their actual concentrations as well as expressing the ratio with creatinine. It is essential to have absolute concentrations available for evaluation when analyzing parameters such as solubility and chemical interactions. It has become common practice to evaluate urine composition utilizing dipstick technologies. Although this information can be of some use in gross screening procedures, it is not useful for detailed analyses regarding mode of action. It is best to analyze urinary pH immediately upon voiding, with use of a pH meter with a microelectrode. This requires literally only a drop of urine. Automated analyzers found in most chemistry laboratories today can be utilized to determine several parameters on small amounts of urine including creatinine, protein, calcium, phosphate, magnesium and other electrolytes. If the quantity of urine is sufficient, it is also useful to determine osmolality since it is extremely high in rodents compared to humans (Cohen, 1995). However, creatinine can be used as a surrogate marker for osmolality. Manual procedures are also available for several urinary parameters. For protein analysis, we have found that the Bradford procedure is most reliable for rodent urine. This is a reflection of the large amounts and unique nature of proteins present in rodent urine, particularly in males (Hard, 1995) Urinary sediment analysis is essential. For many purposes, including observation of urinary solids (precipitate, crystals, and/or calculi), cells, or casts, this can be accomplished by using light microscopy in the usual manner. Seminal plugs can be identified occasionally in males. If detection of cells is important, the sediment must be examined immediately or placed in a fixative, since cells do not survive for long in the extremely dense rodent urine (osmolality of 1500–2500 mOsm/L). We have found that immediately processing the urine sediment by ultrafiltration and examination by scanning electron microscopy (SEM) to be best for analysis of urinary solids, especially if an energy dispersive x-ray spectroscopy (EDS) unit is attached to the SEM for elemental analysis of any solids. SEM is more sensitive than light microscopy, both qualitatively and quantitively, and with EDS can be used to evaluate elemental analysis of any solid detected. It is essential that urine be processed immediately after collection for any examinations that are required. Reactive metabolites will disappear upon setting, normal components, such as pH, can drift, and urinary solids can rapidly dissolve. Proper collection of urine is essential for evaluating mode of action, but collection of the urothelium in a proper manner is also essential. Partial inflation of the bladder in situ provides the best sample for evaluations of subtle changes. Care must be taken to avoid overdistention and tearing of the mucosa. For routine purposes, light microscopic evaluation of the bladder is generally adequate, but increasingly, it is becoming necessary to provide a measure of cell proliferation. This can be accomplished either by injecting the rat intraperitoneally with bromodeoxyuridine (BrdU) followed by immunohistochemical detection of BrdU in the urothelial cells as an indication of S-phase, DNA synthesis, or by immunohistochemical detection of endogenous PCNA or Ki-67. 3H-Thymidine detection by autoradiography is no longer used because immunohistochemistry is simpler and faster and does not require the use of radioactive material. Subtle signs of cytotoxicity and necrosis of the superficial cell layer frequently cannot be detected by light microscopy reliably, partly because of the thinness of the superficial cell layer, and partly due to artifacts that can occur during processing, including tearing of the superficial cells. Scanning electron microscopy provides a more reliable means of evaluating the luminal surface of the urothelium, and also provides a means of evaluating a larger surface area in comparison to light microscopic sections. Transmission electron microscopy is not useful, primarily because of the sampling problems. The portion of the bladder being evaluated can be critical. In rodents, the urine settles more toward the ventral portion of the dome, and it is important to be able to identify this in the tissue collection procedure. This can be readily accomplished by placing a small dot of India ink (or other permanent colored ink) on the ventral surface before the bladder is removed. To be sure that we examine the dome and the ventral portion of the bladder, we routinely slice the bladder longitudinally, processing one half for scanning electron microscopy if necessary, and dividing the other half into several slices longitudinally for embedding on edge. If a labeling index is to be performed, it is essential that a high cell turnover tissue, such as a portion of the gastrointestinal tract, be included in the histology cassette due to the low cell turnover normally found in the urothelium. This will verify that the immunostaining procedure was successful and, in the case of exogenously administered agents such as BrdU, it will help verify that the injected agent was absorbed and that a bladder with no labeled cells is due to a lack of proliferation and not due to a misinjection. We have found that administration of exogenous agents via an osmotic minipump is problematic due to the increase in proliferation that occurs in the bladder epithelium in response to any stress (unpublished observations). Autolysis is a particular problem for the bladder epithelium, similar to many other tissues. A delay of minutes in fixation is of little consequence if the bladder is only to be examined by light microscopy. However, if it is to be examined by scanning electron microscopy, the bladder must be obtained from the animal while it is under deep anesthesia, inflating the bladder with fixative while the animal is still alive. Autolytic changes are morphologically detectable by scanning electron microscopy within 60 seconds of the time of death of the animal (Takayama et al., 1998 and unpublished data). This is true not only for rodents, but in monkeys and presumably for other species as well. The fixative to be used for preserving the bladder will depend on the purposes of the examination. Formalin is adequate for light microscopy and BrdU labeling index or other immunohistochemical analyses, but does not provide adequate fixation for SEM examination. For these purposes, we utilize Bouin’s fixative, which gives adequate preservation for SEM examination as well as providing adequate preservation for light microscopy and immunohistochemical analyses. Methods need to be developed for any of these analyses based on the fixative that is used. This is especially true for immunohistochemical evaluations, since antigenic sites may only be recoverable on tissue fixed with certain fixatives. It is the purpose of this paper to provide a guide for actual procedures used in the collection and examination of urine and the collection and examination of the urinary bladder.
Urine Note: Do not fast the animals for any of the following procedures.
Fresh Void Urine Collection
Mice
Urinary pH by Microelectrode
Sediment Collection for Light Microscopic Examination
Sediment Collection for Scanning Electron Microscopy
Scanning Electron Microscopic Examination and Energy Dispersive X-ray Examination (EDS)
Urinary Solids
Calcium oxalate crystals are normally not found in rat urine but they may form due to treatment-related changes in the pH of the urine or changes in the chemical composition. Calcium oxalate crystals commonly have an octahedral shape (Figure 4), and the X-ray spectrum shows prominent calcium and oxygen peaks. A variant form of calcium oxalate crystals with a thin rod-like shape (Figure 5) has been found in the urine of rats treated with certain drugs. Calcium and phosphate-containing crystals have also been found in urine from treated rats. These crystals do not have a characteristic morphology other than sharp, jagged edges (Figure 6). Amorphous calcium phosphate-containing precipitate (Figure 7) often occurs in treated rats or in rats that are frequently handled for purposes such as oral gavage. The amounts of this precipitate can vary widely from a light coating covering only a few areas on the filter to heavy amounts that are mounded up on the filter. Treatment-related changes in the urine can also lead to the formation of calculi of varying sizes and composition (Figure 8).
X-ray examination of calculi found on the filter can be used to help determine elemental composition. If further chemical analysis of the calculi is necessary, the calculi must be collected from the bladder at necropsy prior to placing the bladder in fixative. In addition to crystals and calculi composed of normal urinary constituents, SEM with EDS is useful in identifying crystals and calculi composed of exogenously administered chemicals such as silicates (Figure 9) or sulfosulfuron (Figure 10).
Urinary Bladder Removal from Rodent If proliferation of the urothelium is being evaluated by detection of BrdU incorporation into DNA, the animal must be injected with BrdU one hour prior to bladder removal (see below for details). As mentioned previously, the removal of the bladder should occur while the animal is under deep anesthesia, as indicated by the absence of a pedal reflex, but prior to the animal’s death to avoid degenerative changes in the bladder epithelium due to autolysis (Figure 11), which are indistinguishable from treatment-related necrosis.
Such changes include intercellular separation, loss of cellular surface detail, focal cellular exfoliation, and fragmentation of cells. Lay the animal on its back, and taking care not to damage any abdominal contents, open the abdomen by a mid-line incision extending from the xiphoid through the diaphragm to the pubis. Expose the bladder by reflecting the intestines and dissecting the attached ligaments, connective tissue, and prostate from the bladder tissue. If it is necessary to hold the bladder, use only toothless forceps to avoid artifacts that are visible by SEM (Figure 12). Gently squeeze the bladder with the fingers to remove any urine from the bladder (or remove the urine through the needle inserted for inflation with fixative) and blot the ventral surface of the bladder to dry it prior to marking it with permanent ink.
In situ Bladder Inflation and Removal Loop a length of suture over the bladder and around the bladder neck. Insert a needle attached to a syringe containing fixative through the bladder neck into the bladder through the loop of suture. Care should be taken to make sure the needle is inserted through the neck and into the lumen of the bladder. Insertion of the needle through the bladder wall into the lumen will cause an artifactual injury to the bladder epithelium that is visible when the bladder is examined by SEM (Figure 13).
Inflate the bladder with fixative until the bladder is just smooth. This requires approximately 200 ul of fixative in mice and 300–500 ul in adult rats. Pull the suture tight as the needle is removed. Remove the bladder by transection across the bladder neck while holding the bladder by the suture, and place in a container of the same fixative used to inflate the bladder. A section of tissue with a highly proliferating epithelium such as the gastrointestinal tract should be collected and placed in the fixative with the bladder. If stomach tissue is to be used instead of a section of GI tract, the stomach should be inflated in situ.
Fixation The bladder should be bisected and rinsed a minimum of 3 times in 0.1 M phosphate buffer before processing for SEM. In our laboratory, the fixative of choice for examination of the bladder tissue by light microscopy and SEM and immunohistochemical detection of BrdU on the same bladder is Bouin’s fixative. The tissue should be fixed for a minimum of 1 hour and a maximum of 4 hours in Bouin’s fixative, bisected, and rinsed in 70% ethanol before processing for paraffin embedding.
Processing Bladder for Light Microscopy and Scanning Electron Microscopy
Light Microscopy
SEM
Procedures for Bromodeoxyuridine Injection and Examination
Immunohistochemistry The sections are then incubated with biotinylated second antibody followed by incubation with a peroxidase-conjugated biotin-avidin complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA). The presence of the peroxidase is detected by staining with diaminobenzidine tetrahydrochloride (DAB). Tissue sections are then counterstained with hemotoxylin.
Evaluation When counting the strips, first locate the basement membrane that separates the epithelium from the lamina propria. A cell that sits on the epithelial side of the basement membrane or is predominantly inside the epithelial layer but protrudes into the lamina propria is considered an epithelial cell. Cells that are positive for BrdU incorporation are distinguished from nonproliferating bladder epithelial cells by a dark brown to black-stained nucleus. Cells in the lamina propria such as the endothelial cells lining the blood vessels also stain positive but care should be taken to count only the positive epithelial cells. The number of positive cells is divided by the total number of cells and reported as a percentage.
Procedure for Ki-67 Determination
Evaluation
Light Microscopic Examination and Classification
SEM Examination and Classification
Class 3 bladders have pleomorphic polygonal superficial cells with numerous small foci of necrotic or exfoliated cells especially in the dome of the bladder and thickening and folding of the epithelium may be present (Figure 17). Normal rodent bladders are usually class 1 or 2, and occasionally class 3. Class 4 bladders have extensive superficial urothelial necrosis of the large, polygonal, superficial cells especially in the dome of the bladder (Figure 18). Small, pleomorphic, polygonal and round cells are present and there may be extensive folding of the epithelium in the bladder dome. Class 5 bladders have extensive superficial necrosis and exfoliation with piling up of small, round urothelial cells (Figures 19 and 20). These small, round cells generally have uniform microvilli but pleomorphic microvilli may be present. Again, the changes are the most severe in the dome of the bladder.
Urothelial carcinogenesis can be produced in rodents by a variety of agents (Cohen, 1998). Fundamentally, the modes of action involve either DNA reactivity or an increase in cell proliferation. DNA reactive carcinogens usually involve metabolic activation of the parent chemical to a reactive intermediate. For urothelial carcinogenesis, it is essential that the reactive intermediate be generated or is present in the urine, or the precursor chemical must be able to be incorporated into the urothelial cells and metabolically activated there. Several components of the urine can modify the availability of a potential reactive carcinogen in the urine, most notably urinary pH and protein. pH can alter the ionization of a specific chemical, and thus alter its potential for incorporation into the urothelial cells. Significant acidification or alkalinization can lead to hydrolysis of various precursor chemicals, such as glucuronides, to yield an active metabolite in the urine. Binding to proteins, especially albumin, a common phenomenon in blood, can also occur in urine. Thus, it is not only important to know the urinary metabolites of a given agent, but it is essential to know the conditions in which it is occurring in the urine. Most DNA reactive carcinogens also produce increased cell proliferation and hyperplasia, usually due to cytotoxicity, and consequent regeneration if the administered dose is sufficiently high. This will greatly modify the dose response for the chemical carcinogen, as was demonstrated in the megamouse experiment performed with 2-acetylaminofluorene in mice (Cohen and Ellwein, 1990). For non-DNA reactive agents, carcinogenesis occurs due to an increase in cell proliferation. Increased cell proliferation can occur either due to an increase in cell births or due to a decrease in cell deaths. Increased cell births can occur either by direct mitogenesis, usually involving hormonal and/or growth factor alterations, or as a result of cytotoxicity and regenerative proliferation. A decrease in cell deaths will lead to an accumulation of more cells, and even if they are proliferating at background rates, it represents an increase in the number of cell replications. A decrease in cell deaths can occur either by inhibiting apoptosis or by inhibiting cell differentiation. In the urothelium, non-DNA reactive agents have been demonstrated to act either by direct mitogenesis (only one example, propoxur, is known) or by cellular toxicity, necrosis, and regeneration. No examples of decreased cell deaths as a mode of action have been identified in urinary bladder carcinogenesis to date. Cytotoxicity can be produced either by generation of a reactive chemical in the urine or by alteration of the urinary composition leading to formation of urinary solids (precipitate, crystals, and/or calculi). Urine composition, such as pH, calcium, magnesium, phosphate, oxalate, urate, citrate, or protein, can alter the structure of a reactive chemical (e.g., ionization) or alter the potential for formation of urinary solids. There have been some claims that alterations in volume or in the normal constituents of the urine, such as pH, sodium, potassium, or calcium, by themselves can produce cytotoxicity and regenerative proliferation. However, when these examples are examined in closer detail, they are usually only one component of a more complex reaction that frequently involves formation of urinary solids or reactive metabolites or both. Nevertheless, on theoretical grounds, it is worthwhile examining these parameters in urine as a possible mode of action for an exogenously administered chemical. For all bladder carcinogens in rodents that have been examined to date, an increase in cell proliferation can be detected in a 13-week (or less) bioassay. However, a labeling index for DNA synthesis may be required to detect this increase, since it might not be evident by light microscopy in the form of hyperplasia. Thus, for examination of a chemical for bladder carcinogenicity, caused either by DNA reactivity or by an increase in cell proliferation, we recommend utilization of either BrdU labeling index, requiring administration of BrdU in either the drinking water or by injection (we prefer ip injection) at the time of sacrifice, or determination of a Ki-67 or PCNA labeling index. It may be that Ki-67 will become routinely acceptable, but presently there is insufficient evidence supporting this marker as a reliable endpoint for the assessment of urothelial proliferation in rodents. Determination of PCNA does not appear to be as reliable as the BrdU labeling index. Thus, in a 13-week bioassay, the bladder is removed at the time of autopsy and evaluated by light microscopy and for a labeling index. It is generally not necessary to include scanning electron microscopy. If a proliferative response is detected in the initial experiment, a separate experiment should be performed to determine mode of action. In parallel with the animal experiment, the chemical should be evaluated for DNA reactivity, using analyses such as an Ames assay, and also by structure-activity relationships. If a more rigorous evaluation is required, P32 postlabeling evaluation or some other method directly targeted for the urothelium is suggested. Evaluation of mode of action involves a second thirteen-week experiment (it may require shorter periods of time, but we suggest 13 weeks based on the extensive experience using that treatment duration). Several doses are evaluated to provide a detailed dose response analysis. During the course of this experiment, fresh void urine should be collected from the animals at least at 2 time points; we recommend utilizing four weeks and eleven weeks of the experiment as the time points. Additional time points can be included if further details are being sought. It also may be necessary to collect fresh void urine at multiple time points during a 24-hour period due to diurnal variations in the urine composition (Dominick et al., 2006). Fresh void specimens should be examined for urinary pH, a variety of analytes, especially including calcium, phosphate and magnesium, and filters for examination by scanning electron microscopy for the presence of urinary solids should be prepared. Citrate, a key chelator of calcium, oxalate, and uric acid are also helpful parameters for evaluation. Urinary solids can be formed from high urinary excretion of an administered substance or metabolite, calcium phosphate, calcium oxalate, uric acid, or other solids that readily form from endogenous urinary constituents, from treatment mediated changes in urinary composition, or by a combination of normal urinary constituents and the administered chemical (or metabolite). If urinary solids are detected, a dietary acidification experiment can be conducted to determine if inhibiting the formation of urinary solids by adding ammonium chloride or a carbonate to the diet to reduce urinary pH to below 6.5 inhibits the urothelial cytotoxic and proliferative response. Examination of the urothelium by light microscopy, labeling index, and SEM is essential for identification, localization and classification of cytotoxic and proliferative changes in the bladder. Observation of cage bedding material for hematuria can occasionally provide evidence of cytotoxicity, but for this to occur, damage must involve the full thickness of the urothelium and a break in the basement membrane. Cytotoxicity sometimes can be completely repaired by 13 weeks. Thus, if no evidence of cytotoxicity is present at thirteen weeks in the presence of increased proliferation, it may be necessary to repeat the experiment with a shorter time period (1 week and 4 weeks) to completely eliminate the possibility of urothelial cytotoxicity as the inciting process. Based on these experiments, one can readily identify all agents with a potential for acting as a rodent urothelial carcinogen as well as identify the mode of action as either DNA reactivity, cytotoxicity and regeneration, or direct mitogenicity. Furthermore, if cytotoxicity is involved, the cause can be narrowed to either formation of urinary solids, presence of a reactive metabolite in the urine, alteration in urine composition of a normal urinary component, or a combination of these. However, to come to a proper conclusion, it is essential that the urine and the urothelium be collected and examined properly and thoroughly.
We gratefully acknowledge the valuable assistance of Laurie Bruck in the preparation of this manuscript.
* Samuel M. Cohen is the Havlik-Wall Professor of Oncology, University of Nebraska Medical Center. 
Arnold, LL, Cano, M, St John, MK, Healy, CE, & Cohen, SM. (2001). Effect of sulfosulfuron on the urine and urothelium of male rats. Toxicol Pathol, 29, 344-52 Clayson, DB, & Cooper, EH. (1970). Cancer of the urinary tract. Adv Cancer Res, 13, 271-381[Medline] [Order article via Infotrieve] Cohen, SM. In Cohen, SM, & Bryan, GT (Eds.). (1983). Pathology of experimental bladder cancer in rodents. The Pathology of Bladder Cancer, II, 1-40). Boca Raton, FL: CRC Press Cohen, SM. (1995). Role of urinary physiology and chemistry in bladder carcinogenesis. Fd Chem Toxicol, 33, 715-30[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Cohen, SM. (1998). Urinary bladder carcinogenesis. Toxicol Pathol, 26, 121-7 Cohen, SM, & Ellwein, LB. (1990). Proliferative and genotoxic cellular effects in 2-acetylaminofluorene bladder and liver carcinogenesis: biological modeling of the ED01 study. Toxicol Appl Pharmacol, 104, 79-93[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Cohen, SM, & Johansson, SL. In Lamm, D (Ed.). (1992). Epidemiology and etiology of bladder cancer. Urologic Clinics of North America, 19, 421-8). Philadelphia, PA: W. B. Saunders Co[Web of Science][Medline] [Order article via Infotrieve] Cohen, SM, Fisher, MJ, Sakata, T, Cano, M, Schoenig, GP, Chappel, CI, & Garland, EM. (1990). Comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography. Scanning Microsc, 4, 135-42[Web of Science][Medline] [Order article via Infotrieve] Cohen, SM, Cano, M, Earl, RA, Carson, SD, & Garland, EM. (1991). A proposed role for silicates and protein in the proliferative effects of saccharin on the male rat urothelium. Carcinogenesis, 12, 1551-5 Cohen, SM, Cano, M, Anderson, T, & Garland, EM. (1996). Extensive handling of rats leads to mild urinary bladder hyperplasia. Toxicol Pathol, 24, 251-7 Cohen, SM, Shirai, T, & Steineck, G. (2000). Epidemiology and etiology of premalignant and malignant urothelial changes. Scand J Urol Nephrol, 205, 105-15 Cohen, SM, Yamamoto, S, Cano, M, & Arnold, LL. (2001). Urothelial cytotoxicity and regeneration induced by dimethylarsinic acid in rats. Toxicol Sci, 59, 68-74 Dominick, MA, White, MR, Sanderson, TP, Van Vleet, T, Cohen, SM, Arnold, LL, Cano, M, Tannehill-Gregg, S, Moehlenkamp, JD, Waites, CR, & Schilling, BE. (2006). Urothelial carcinogenesis in the urinary bladder of male rats treated with muraglitazar, a PPAR Fisher, MJ, Sakata, T, Tibbels, TS, Smith, RA, Patil, K, Khachab, M, Johansson, SL, & Cohen, SM. (1989). Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet. Fd Chem Toxicol, 27, 1-9[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Hard, GC. (1995). Species comparison of the content and composition of urinary proteins. Fd Chem Toxicol, 33, 731-46[CrossRef][Web of Science][Medline] [Order article via Infotrieve] Takayama, S, Sieber, SM, Adamson, RH, Thorgeirsson, UP, Dalgard, DW, Arnold, LL, Cano, M, Eklund, S, & Cohen, SM. (1998). Long-term feeding of sodium saccharin to nonhuman primates: implications for urinary tract cancer. J Natl Cancer Inst, 90, 19-25 Tiltman, AJ, & Friedell, GH. (1972). Effect of feeding N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide on mitotic activity of rat urinary-bladder epithelium. J Natl Cancer Inst, 48, 125-9[Web of Science][Medline] [Order article via Infotrieve]
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Abstract
Introduction
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agonist: evidence for urolithiasis as the inciting event in the mode of action. Toxicol Pathol, 34, 903-20
