Investigations of Rodent Urinary Bladder Carcinogens: Collection,Processing,and Evaluation of Urine and Bladders

Abstract

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.

Introduction

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 and Bladder Collection, Processing and Evaluation

Urine

Note: Do not fast the animals for any of the following procedures.

Fresh Void Urine Collection

Rats

Urine can be collected directly into a 1.5 ml microfuge tube or a small, polypropylene funnel can be used to direct the urine into the microfuge tube. Place the animal on the forearm holding the tail with the hand of the same arm. Using the free hand, pick the rat up by gently grasping the skin behind the neck and between the scapula and along the back. Position the rat over the tube or the funnel using the free hand to spread and secure the back legs and to push the rat gently up into the palm of the restraining hand. Firmly and quickly, squeeze the skin along the entire back of the rat to induce the animal to void. If the animal does not void within approximately 15 seconds, discontinue the attempt to collect urine from the animal.

Mice

Secure a large piece of parafilm to a solid surface with a raised edge. Pick the mouse up by gently squeezing the skin behind the ears to secure the head. Place the mouse on the parafilm allowing the mouse to grab the raised edge with the front feet. With the free hand, gently massage the lateral abdomen of the mouse just cranial to the pelvis to induce the mouse to void. Pipet up the voided urine and place in a microfuge tube.

Urinary pH by Microelectrode

A pH meter equipped with a microelectrode (Microelectrodes Inc., Londonderry, NH) capable of measuring pH on volumes as small as 20 ul, should be set up either in the animal room or directly outside of the animal room so pH can be measured immediately after collection of the urine. Once the pH meter is in place, it should be calibrated with at least two commercial buffers. The calibration should be rechecked with the commercial buffers after at least every 15 animals and recalibrated if necessary.

Sediment Collection for Light Microscopic Examination

If other analyses are to be performed on the urine, an aliquot of the well-mixed urine should be removed and processed for light microscopic examination. Urine specimens with a volume of approximately 300 ul or more should be centrifuged for 10 minutes at 3000 rpm. Remove most of the supernatant and resuspend the sediment in the remaining supernatant by gently pipeting the specimen up and down. Place 20–30 ul of the remixed urine on a microscope slide and cover with a cover slip. If the available volume for light microscopic examination is less than 300 ul, do not centrifuge the sample. Mix the urine well by gently pipeting up and down, place a maximum of 30 ul on a microscope slide, and cover with a cover slip.

Sediment Collection for Scanning Electron Microscopy

Remove a 100 ul aliquot of the well-mixed fresh void urine and place in a 1.5 ml microfuge tube. If urinary volume is not sufficient for a 100 ul sample, 50–75 ul may be used, but the decreased volume should be noted. Centrifuge the urine aliquot at 7000 rpm for 10 minutes and remove most of the supernatant. Using needle-nose forceps, place a 0.22 μM nitrocellulose filter (Fisher Scientific, Pittsburgh, PA) on a vacuum apparatus attached to a vacuum source. Gently re-suspend the sediment in the remaining urine by gently pipeting up and down, and place the entire sample on the filter. Allow the vacuum to pull the urine through the filter, and then place the filter on an aluminum SEM stub with pre-applied adhesive.

Scanning Electron Microscopic Examination and Energy Dispersive X-ray Examination (EDS)

The urinary filter should first be examined at a lower magnification (<100×) to obtain an overall view of the urine spot and to determine if any solids are present within that spot. A semiquantitative evaluation of the solids present can be made by estimating the area of the urine spot covered by the particular solid. At higher magnifications, a closer evaluation of morphology can be made and particular solids can be targeted for determination of elemental composition by EDS.

Urinary Solids

Normal rat urine contains varying crystals (struvite) (Figures 1 and 2). These crystals can be identified by their characteristic coffin-shaped morphology and confirmed by an x-ray spectrum showing prominent magnesium and phosphorus peaks. Newer, more sensitive EDS units can also identify the presence of lighter elements, such as oxygen. We have found treatment-related formation of aggregates of normal MgNH₄PO₄ crystals ranging in size from less than 100 μ to greater than 300 μ in size (Figure 3).

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 type of fixative used will be determined by the procedures required to examine the bladder tissue. If light microscopy and/or immunohistochemistry are to be done on the tissue, 10% buffered formalin can be used. Formalin-fixation is not recommended for bladder tissue that will be examined by SEM. The bladder should be allowed to fix in formalin for at least 24 hours prior to bisecting and processing the tissue for paraffin embedding. The fixative of choice for SEM examination of bladder specimens is 2.0% glutaraldehyde with no secondary or postfixation. However, immunohistochemical detection of BrdU cannot be done on glutaraldehyde-fixed tissue. The 2.0% glutaraldehyde should be kept on ice during tissue collection, and tissue fixation should be done at approximately 4°C for at least 24 hours.

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

The tied suture around the neck should be cut off and any excess tissue, including connective tissue, should be trimmed away. Using a sharp, stainless steel razor blade, the bladder should be divided in half longitudinally cutting through the ink dot so that half of the ventral bladder will be processed for light microscopy and the other half of the ventral surface will be processed for SEM. Rinse the tissue with the appropriate solution as specified above, gently blot the bladder halves with filter paper, and weigh. Weighing the tissue after fixation is necessary to prevent any SEM-detectable autolytic changes that might occur if the tissue was weighed prior to fixation.

Light Microscopy

The half of the bladder which will be examined by light microscopy should be cut longitudinally into 2–4 mm wide strips with at least one side of each strip straight for optimal embedding, placed in a tissue cassette between two sponges, and embedded on edge in paraffin. A small section of small or large intestine, stomach (forestomach and glandular stomach), or other epithelial tissue with a high proliferation rate is included in the cassette as a positive control for determination of the labeling index. The slides are stained with hematoxylin and eosin prior to examination by light microscopy.

SEM

The bladder half which will be examined by SEM should be returned to the 70% ethanol after bisection. To dehydrate the tissue, rinse once for 3–5 minutes in an ascending series of 80%, 90%, and 95% ethanol followed by at least 2 rinses for 5 minutes each in 100% ethanol. Add an approximately equal amount of 100% Freon to the last 100% ethanol wash and let the tissue sit for 5 minutes followed by at least 2 rinses for 5 minutes each of 100% Freon. We have found that Freon works best to keep the tissue from curling, which can make it difficult or impossible to observe the surface by SEM. To finish the dehydration process, the bladder tissue is critical point dried. The bladder halves are then mounted on aluminum SEM stubs with double-sided adhesive and coated with a thin layer of gold prior to examination by SEM.

Procedures for Bromodeoxyuridine Injection and Examination

Injection

Animals are injected intraperitoneally with BrdU (100 mg/kg BW) 1 hour ± 1 minute prior to inflation of the bladder. The concentration of the working solution injected into the animals is 20 mg/ml. The injections should be timed so that bladder inflation can be performed over a 2-hour period sometime within the first 6 hours of the lights being turned on in the animal room to avoid diurnal variation in the labeling index (Tiltman and Friedell, 1972). We routinely inflate and collect the stomach at the time of necropsy to use as the positive control but any epithelial tissue with a high proliferation rate can be used.

Immunohistochemistry

Unstained slides of paraffin-embedded tissue sections are deparaffinized in xylene and rehydrated in a series of alcohols including 100%, 95%, and 70% ethanol and endogenous peroxides are blocked by incubation in 3% hydrogen peroxide in 100% methanol. Pretreatment of tissue sections with 2N HCl followed by 0.005% trypsin, both at 37°C, is required for antigen retrieval, and nonspecific binding is blocked by incubation in powdered milk. Tissue sections are incubated overnight at approximately 4°C with the primary antibody (anti-BrdU, mouse monoclonal, Chemicon International, Temecula, CA) diluted 1:200.

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

Only slides with GI or stomach tissue (or other proliferating epithelial tissue) stained positive for BrdU incorporation are evaluated. The bladder sections usually contain 3–5 strips and each strip is counted end to end. In the rat bladder, a minimum of 3000 epithelial cells is counted. Finish counting the strip on which the 3000th cell was counted, to its end. In the mouse bladder, all tissue strips are counted.

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

Immunohistochemistry

Immunohistochemical detection of the nuclear protein Ki-67, elevated during late G1, S, G2, and M phases of the cell cycle, can be used to measure the rate of cell proliferation without the need for injection or administration of exogenous substances to the animal. The procedure for deparaffinization, rehydration, and blocking of endogenous peroxides is the same as that used for detection of BrdU. Pretreatment of tissue sections at high temperature in citrate buffer, pH 6.0 is necessary for antigen retrieval followed by incubation in powdered milk to block nonspecific binding. Tissue sections are incubated overnight at approximately 4°C with the primary antibody (anti-rat Ki-67, mouse monoclonal, or anti-mouse Ki-67, rat monoclonal, Dako, Carpinteria, CA) diluted 1:25. 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 DAB (DAB Substrate kit, Vector Laboratories, Burlingame, CA). Tissue sections are then counterstained with hemotoxylin.

Evaluation

The urinary bladder is evaluated only for those animals with control tissue that is stained positive for Ki-67. In the rat bladder, a minimum of 3000 epithelial cells is counted. In the mouse bladder, all tissue strips are counted. Cells that are positive for Ki-67 incorporation are distinguished from nonproliferating bladder epithelial cells by a dark brown to black-stained nucleus. The number of positive cells is divided by the total number of cells and reported as a percentage.

Light Microscopic Examination and Classification

Most normal rodent bladder epithelium has 3 or less cell layers by light microscopy although the cell thickness in the trigone area of the bladder may increase to 4 or 5, particularly in older animals (Cohen, 1983). There are 4 general categories of proliferative urothelial changes:

1. Simple hyperplasia is an increase in the number of cell layers in the bladder epithelium. It can be characterized as mild (4–5 cell layers), moderate (6–8 cell layers) or severe (9 or more cell layers) simple hyperplasia, and focal or diffuse depending on the extent of the area of the bladder epithelium involved.

2. Papillary or nodular hyperplasia is characterized by the formation of growths within hyperplastic areas of the epithelium that either grow outward (exophytic) or into the lumen (papillary) or grow downward (endophytic) toward the submucosa (nodular). Papillary growths contain a narrow fibrovascular core surrounded by normal or hyper-plastic epithelium. Nodular hyperplasia is generally broad based and lesions may become quite widespread. In both papillary and nodular hyperplasia, the urothelial cells are generally uniform with no nuclear pleomorphism and few mitoses. However, cellular dysplasia has been occasionally noted in nodular lesions.

3. Papillomas are outward growths of the epithelium protruding into the lumen that are generally large enough to be seen macroscopically. Papillomas are composed of a fibrovascular core which is usually wider than that seen in papillary hyperplasia. The number of epithelial cell layers is often normal or only mildly increased (up to 10) in the epithelium covering the core, and the urothelial cells are generally uniform without nuclear pleomorphism and few mitoses. However, if the inciting stimulus is not withdrawn, dysplasia and increased mitoses will develop.

4. Transitional cell carcinoma is characterized by cells with large, pleomorphic nuclei, frequent mitoses, and piling up of cells. It can be papillary, flat, or nodular, and it can be noninvasive or invasive.

SEM Examination and Classification

The SEM classification system we have used for assessing early or slight changes in the bladder epithelium is composed of five classes. Class 1 and Class 2 bladders have an epithelium composed of large flat, polygonal superficial cells with well-developed leafy microridges covering the surface of the cells (Figures 14 and 15). However, the presence and relative number of necrotic and exfoliated cells differs between the two classes. Class 1 bladders may have some instances of necrotic or exfoliated cells due to normal cell death but the underlying cells are almost completely mature. Class 2 bladders have occasional small foci of necrotic or exfoliated cells with underlying cells that are immature (Figure 16).

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: Considerations for Modes of Action

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.