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http://tpx.sagepub.com/ Toxicologic Pathology http://tpx.sagepub.com/content/36/2/218 The online version of this article can be found at: DOI: 10.1177/0192623307311757 2008 36: 218 Toxicol Pathol Gerald G. Long, Vincent L. Reynolds, Alric Lopez-Martinez, Thomas E. Ryan, Sandy L. White and Sandra R. Eldridge Agonist: Lack of Evidence for Urolithiasis as an Inciting Event γ / α -dominant PPAR γ Urothelial Carcinogenesis in the Urinary Bladder of Rats Treated with Naveglitazar, a Published by: http://www.sagepublications.com On behalf of: Society of Toxicologic Pathology can be found at: Toxicologic Pathology Additional services and information for http://tpx.sagepub.com/cgi/alerts Email Alerts: http://tpx.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? - May 12, 2008 Version of Record >> by guest on April 9, 2012 tpx.sagepub.com Downloaded from

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http://tpx.sagepub.com/Toxicologic Pathology

http://tpx.sagepub.com/content/36/2/218The online version of this article can be found at:

 DOI: 10.1177/0192623307311757

2008 36: 218Toxicol PatholGerald G. Long, Vincent L. Reynolds, Alric Lopez-Martinez, Thomas E. Ryan, Sandy L. White and Sandra R. Eldridge

Agonist: Lack of Evidence for Urolithiasis as an Inciting Eventγ/α-dominant PPAR γUrothelial Carcinogenesis in the Urinary Bladder of Rats Treated with Naveglitazar, a

  

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218

Urothelial Carcinogenesis in the Urinary Bladder of Rats Treatedwith Naveglitazar, a γγ-dominant PPAR αα/γγ Agonist: Lack of

Evidence for Urolithiasis as an Inciting Event

GERALD G. LONG,1 VINCENT L. REYNOLDS,1 ALRIC LOPEZ-MARTINEZ,2 THOMAS E. RYAN,2 SANDY L. WHITE,1 AND SANDRA R. ELDRIDGE3

1Lilly Research Laboratories, Greenfield, Indiana, USA, 2Covance Laboratories, Madison, Wisconsin, USA,and 3PAI, Charles River, Frederick, Maryland, USA

ABSTRACT

Naveglitazar, a γ-dominant peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist, was tested for carcinogenicity in F344 rats in a 2-year study. Changes in urine composition and urothelial morphology were characterized in a companion 18-month investigative study. A significantincrease in neoplasms of the bladder occurred only in females of the high-dose group (14/60) in the carcinogenicity study. Trends toward increasedcell proliferation in the urothelium were noted in both sexes at all time points evaluated in the 18-month study. Group means for urothelial mitogen-esis were increased statistically significantly only in high-dose females at 12 and 18 months. Urothelial hyperplasia occurred in high-dose females at18 months. Morphologic changes in the urothelium at earlier time points were limited to hypertrophy and decreased immunolabeling of the superfi-cial cells for cytokeratin 20 (a marker of terminal differentiation in urothelial cells) in both males and females. No treatment-related changes in uri-nary parameters, including urinary sediments, were associated with the occurrence of urothelial proliferation. Urinary pH was unaffected by treatmentin both males and females, but expected diurnal changes were demonstrated. Collectively, these data indicate that naveglitazar was associated withhypertrophic and proliferative effects on the urothelium, but a link with changes in urinary parameters was not demonstrated.

Keywords: PPAR agonist; carcinogenesis; urothelium; urolithiasis; urinary bladder; rats.

carcinogenesis caused by activation of PPAR γ has been seenin a mouse model with inherent abnormalities in cell-cyclecontrol (Lefebvre et al., 1998; Saez et al., 1998).

The immunohistochemical expression of PPAR γ appears tobe increased in human bladder tumor cells compared to normalbladder cells, and the degree of this expression may be associ-ated with tumor grade, recurrence, and/or progression (Possatiet al., 2002; Yoshimura, Matsuyama, Hase, et al., 2003;Yoshimura, Matsuyama, Segawa, et al., 2003; Nakashiro et al.,2001). PPAR γ ligands inhibit the proliferation of normaland neoplastic urothelial cells in vitro (Nakashiro et al., 2001;Yoshimura, Matsuyama, Hase, et al., 2003). With normal urothe-lial cells in culture, PPAR γ agonists cause terminal transitionaldifferentiation, which is maximized when epithelial growthfactor receptor (EGFR) is inhibited (Varley et al., 2003; Varleyet al., 2004; Kawakami et al., 2002; Spencer et al., 2004). Onepotential contrasting effect of PPAR γ ligands on urothelium is anapparent increase in expression of vascular endothelial growthfactor (VEGF) by neoplastic urothelial cells in vitro, comparedto normal urothelial cells (Fauconnet et al., 2002).

PPAR γ is normally expressed in the urothelium of the blad-der and renal pelvis (Guan et al., 1997), and the above-cited invitro studies suggest that PPAR γ agonists should cause decreasedcell proliferation and increased differentiation in the urothelium.Additionally, compounds in this pharmacologic class tend tolack genotoxicity and to be excreted into the urine to only a slightdegree. Taken together, these factors suggest that carcinogenicitycaused by PPAR γ agonists in the rat bladder is not the result

INTRODUCTION

Peroxisome proliferator-activated receptors (PPARs) play acritical role as regulators of lipid metabolism and insulin sen-sitization. Synthetic ligands for PPARs have been developed forthe treatment of diabetes and dyslipidemia (Berger and Moller,2002). Carcinogenicity has been identified as one of the primaryissues in preclinical testing of PPAR γ and α/γ dual agonists.Administration of these PPAR agonists to rodents has been asso-ciated with the development of hemangiosarcomas in mice,liposarcomas and/or fibrosarcomas in rats, and transitional celltumors of the urinary bladder and/or renal pelvis in rats (El Hage,2005a, 2005b).

Mechanistic explanations for the occurrence of neoplasmsrelated to the pharmacologic class are problematic because, withfew exceptions, in vitro and in vivo work indicates that PPARγ agonists generally have antiproliferative effects (Grommeset al., 2004). Differentiation and reversal of malignant changesin colon cancer have been associated with PPAR γ (Sarraf et al.,1998; Sarraf et al., 1999). Conversely, enhancement of colon

Address correspondence to: Gerald G. Long, Eli Lilly and Company, LillyResearch Laboratories, P.O. Box 708, Greenfield, IN 46140; e-mail: [email protected].

Abbreviations: BrdU, bromodeoxyuridine; CK, cytokeratin; EGF, epithelialgrowth factor; EGFR, epithelial growth factor receptor; H&E, hematoxylin andeosin stain; PPAR, peroxisome proliferator-activated receptor; SEM, scanningelectron microscopy; TEM, transmission electron microscopy; TZD, thiazo-lidinedione; ULLI, unit length labeling index; VEGF, vascular endothelialgrowth factor.

Toxicologic Pathology, 36:218-231, 2008Copyright © 2008 by Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1177/0192623307311757

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Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 219

of a direct effect of the agonists but is more likely caused bysome indirect effect. In the rat, the most commonly identifiednongenotoxic mode of carcinogenicity of the lower urinary tractis increased formation of urinary solids, which leads to chronicirritation, proliferation, and eventually, neoplasia (Clayson et al.,1995; Cohen et al., 2002; Cohen, 2005).

The presence of critical components of the indirect modeof urothelial carcinogenesis in the rat was demonstrated withthe PPAR α/γ agonist muraglitazar (Dominick et al., 2006).Muraglitazar caused an increased incidence of urinary bladdertumors in male rats associated with changes in urine composi-tion leading to formation of urinary solids, chronic damage,and resultant proliferative changes. Dietary acidification pre-vented the cytotoxic, proliferative, and tumorigenic responses.Ragaglitazar, another PPAR α/γ agonist that caused urinarybladder neoplasms in long-term rat studies, caused hypertrophyof the urothelial cells in short-term studies (Oleksiewicz et al.,2005). Apparent proliferation of the urothelium was limited to1 male rat treated with ragaglitazar, but the number of animalstested was relatively small, and the duration was limited to 2 to3 weeks. Hypertrophy of the urothelium was not reported withmuraglitazar (Dominick et al., 2006).

Naveglitazar is a nonthiazolidinedione (non-TZD), γ-domi-nant PPAR α/γ dual agonist. In vitro, naveglitazar binds selec-tively to PPAR γ with high affinity (IC50 = 0.024 µM, Ki = 0.022µM) and to PPAR α with lower affinity (IC50 = 1.71 µM, Ki = 1.66µM; Reifel-Miller et al., 2003). Two-year rodent carcinogenicitystudies were conducted to support the development of navegli-tazar as a chronic-use therapeutic agent. Because of the knownpotential for neoplasms of the urinary bladder in rats treated withPPAR γ and α/γ dual agonists, a study to characterize changesin urine composition, the occurrence of urinary solids, and urothe-lial morphology and mitogenesis in rats was conducted at thesame time as the 2-year carcinogenicity study in rats. The testsystem, doses, and dosing routes for the urinalysis study were thesame as those used for the carcinogenicity study. Analyses offreshly voided urine samples and assessments of urothelialhistology were done at periods throughout the study to charac-terize the temporal nature of any changes.

MATERIALS AND METHODS

Test Animals and Treatment

F344/Ntac rats were obtained from Taconic Farms(Germantown, New York). Rats were housed individually instainless steel, wire-bottom cages. Environmental controls forthe animal room were set to maintain a temperature of 19 to25ºC, a relative humidity of 30% to 70%, and a 12-hour light/12-hour dark cycle. The animals were offered certified rodentpellets (#5002C, PMI, Richmond, Indiana) and water ad libitum.Animals were randomly assigned to treatment groups based onweight. Rats (60/sex/group for the carcinogenicity study and50/sex/group for the urinalysis/characterization study) wereassigned to groups receiving 0, 0.3, 1.0, or 3.0 mg naveglitazar/kg body weight/day (males) or 0, 0.1, 0.3, or 1.0 mg naveglitazar/kg body weight/day (females) by oral gavage. These doses resulted

in approximately equal systemic exposures in males and femalesof the respective low-, mid-, and high-dose groups (i.e., AUC0-24hr

in males given 3.0 mg/kg was similar to or slightly in excess ofthat in females given 1.0 mg/kg). All doses, including control,were administered at a dose volume of 1 ml/kg in a vehicle com-posed of 1.0% (w/v) carboxymethylcellulose sodium, 0.5% (w/v)sodium lauryl sulfate, 0.085% (w/v) Povidone, 0.05% (v/v) DowCorning Antifoam 1510-US in reverse osmosis water. Dosingwas conducted in the morning, generally within 4 hours of thebeginning of the light cycle.

Carcinogenicity Study

The rodent carcinogenicity study had a duration of 104 weeks.All rats dying at unscheduled intervals or sacrificed at termina-tion were necropsied, and a standard set of tissues was collected.All collected tissues, including urinary bladder and both kidneysfrom all rats, were examined histologically. The incidence of neo-plasms was analyzed by a survival weighted statistical methodwith significance levels based on relative historical frequencyof specific neoplasms (Lin, 1997; Lin and Rahman, 1998).

Urinalysis/Characterization Study

Urine Collection and Analysis

Freshly voided urine was collected from all surviving animalsfor acclimation to the collection process during weeks 2, 12,25, 38, 51, and 64. Samples were discarded without analysis.

Urinalysis was conducted on freshly voided urine samplescollected within 2 hours after the end of the dark cycle (a.m.)and within 1 to 3 hours before the beginning of the dark cycle(p.m.) during weeks 3, 13, 26, 39, 52, 65, and 78. Rats were notfasted before urine collection, and all a.m. urine samples werecollected within 2 hours of the beginning of the light cycle sothat they would be representative of urine composition of thedark cycle (Cohen et al., 2007). Urine samples collected a.m.were collected before dosing so that rats were not handled forat least 18 hours before urine collection. Rats were sampled until10 animals provided a sample of at least 100 µl from each sex/group; only those samples of at least 100 µl were analyzed. UrinepH was determined within 30 minutes of collection with a pHmicroelectrode. Urine samples of at least 100 but less than 200µl were diluted 1:1 with distilled water after pH measurementand analyzed for urine chemistry. For urine samples of 200 µlor greater, 100 µl of urine was removed after pH measurement,diluted 1:1, and analyzed for urine chemistry; the remainingsample was centrifuged and the sediment examined micro-scopically. Urine chemistry analyses included determinationof concentrations of creatinine, calcium, phosphorus, sodium,potassium, chloride, and magnesium. Ratios of calcium, phos-phorus, sodium, potassium, chloride, and magnesium to creati-nine were calculated.

Urine samples were collected a.m. and p.m. during week 40specifically for light microscopic examination of urine sedi-ments. Freshly voided urine was collected until 10 animals each

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220 LONG ET AL. TOXICOLOGIC PATHOLOGY

provided a sample of at least 100 µl from each sex/group. UrinepH was determined within 30 minutes of collection for samplesof at least 100 µl with a pH microelectrode. After determinationof pH, urine samples were centrifuged and the sediment examinedmicroscopically.

Urine samples were collected a.m. during week 74 for scan-ning electron microscopy evaluation of urine sediments. Freshlyvoided urine was collected from a sufficient number of animalsto provide samples of at least 300 µl from 5 animals/sex/group.Within 1 hour of collection, urine was mixed to resuspend sedi-ments, and each entire urine sample was drawn into a syringe andpushed through a 25-mm diameter 0.22 µm filter (MilliporeCorporation, Billerica, Massachusetts) in a 25-mm filter holder(Structure Probe Inc., West Chester, Pennsylvania). A vacuumwas applied to the underside of each filter holder to pull theremaining liquid through the filter. The filter holders were placedupright in a desiccator chamber with desiccant and allowed toair-dry for at least 1 week. Sections of filter from each control andhigh-dose male and female were excised with a razor, processed,and examined by scanning electron microscopy.

Freshly voided urine samples collected during the acclimationsampling in week 77 were analyzed for calcium concentrationsin acidified and nonacidified subsamples. A subsample of eachurine sample of sufficient volume (60 µl or greater) was acidi-fied with 2 µl of concentrated hydrochloric acid before analy-sis. Urine samples were not centrifuged before subsamplingor analysis.

Urinary Bladder Examination

Ten rats/sex/group were sacrificed during each of weeks 27,53, and 79. Rats selected for sacrifice were given an intraperi-toneal injection of 12 mg bromodeoxyuridine (BrdU, Sigma-Aldrich Corp., St. Louis, Missouri) approximately 24 hoursbefore scheduled necropsy. The BrdU dosing solution was 20 mg/ml in phosphate-buffered saline. At necropsy, rats were anes-thetized with isoflurane, the abdominal cavity was opened, andthe urinary bladder was collected before the animals wereterminated by exsanguination. A clamp was placed on the urethra,and urine was withdrawn from the bladder. The urinary bladderwas inflated with Bouin’s solution (week 27) or 10% neutralbuffered formalin (weeks 53 and 79) until the serosal surface ofthe bladder was smooth. Formalin was used as a fixative for thelater sampling times to provide adequate fixation for transmis-sion electron microscopy (TEM). An additional clamp wasplaced on the neck of the bladder, and the ventral surface of thebladder was marked with a permanent tissue marker. The blad-der and proximal urethra, with both clamps, were removed andplaced in the same fixative as used for inflation. A section ofduodenum from each animal was collected and placed in thesame fixative as used for the bladder.

After 4 hours in fixative, each bladder was transected longi-tudinally through the ventral tissue mark. The right half of eachbladder and respective duodenum were placed in 70% ethanol.The right half of the bladder was then sectioned into 3 longitudi-nal strips, processed, and embedded in paraffin with the respective

section of duodenum. The left half of each bladder was processedfor scanning and TEM. Bladder sections collected week 27 weretransferred to 70% alcohol for processing, and those collectedweeks 53 and 79 were transferred to modified Karnovsky’s fixa-tive (2.5% glutaraldehyde/2% formaldehyde in 0.12M cacody-late buffer [with 0.5 g calcium chloride/liter]).

Four replicate sections of the paraffin block containingurinary bladder were made from each animal sacrificed duringweek 27. Two replicate sections of each block were placed onregular glass slides and stained with hematoxylin and eosin.Two additional replicate sections from each block were placedon positively charged glass slides for immunohistochemicallabeling. Ten replicate sections of the paraffin block containingurinary bladder were made from each animal sacrificed duringweeks 53 and 79. Two replicate sections of each block with uri-nary bladder were placed on regular glass slides and stainedwith hematoxylin and eosin. An additional 8 replicate sectionsfrom each block were placed on positively charged glass slidesfor immunohistochemical labeling.

One slide of urinary bladder and duodenum from each animalon study was immunolabeled for BrdU by standard procedures(Eldridge et al., 1990) and counterstained with hematoxylin.All slides from all animals from each scheduled sacrifice werestained together in a single staining run, and positive and neg-ative controls were included in each staining run. Cell prolifer-ation was quantified for each animal that had evidence of BrdUincorporation in the section of duodenum and if 3 completesections of urinary bladder mucosa were present on the slide.The number of BrdU-labeled epithelial cells within the entirelength of bladder epithelium from each of the 3 bladder sectionspresent on the slide was counted for each rat. BrdU-labeled cellswere not counted in focal proliferative lesions of epitheliumthat projected above and/or below the normal mucosal layer.The unit length labeling index (ULLI) was expressed as thetotal number of labeled cells in all 3 sections evaluated for eachanimal. The Student’s t-test (two-sided, unequal variance) wasused to test for statistical significance in ULLI between controland treatment groups. A p value of less than or equal to .05 wasjudged to be statistically significant.

Immunolabeling for uroplakin and cytokeratins 5, 17, and20 was conducted on sections of urinary bladder collectedduring weeks 53 and 79. Antibodies used were mouse mono-clonal antiuroplakin (Research Diagnostics Inc., Flanders, NewJersey), polyclonal goat antihuman cytokeratin 5 (Santa CruzBiotechnology Inc., Santa Cruz, California), monoclonal mouseantirat cytokeratin 17 (Sigma-Aldrich Corp., St. Louis, Missouri),and monoclonal mouse antihuman cytokeratin 20.8 (SignetLaboratories Inc., Dedham, Massachusetts). Antigen retrievalwas conducted before labeling for cytokeratins 5 and 20, usingDeclere (Cell Marque Corp., Rocklin, California) in a pressurecooker. The targeted expression in the rat urothelium was super-ficial cells for uroplakin and cytokeratin 20, basal cells for cytok-eratin 5, and basal/intermediate cells for cytokeratin 17 (Romihet al., 1998). The distribution and intensity of labeling were eval-uated qualitatively in conjunction with evaluation of hematoxylinand eosin stained slides.

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Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 221

Bladder specimens for electron microscopy were rinsed with2 changes of distilled water for 15 minutes each and transferredto fresh modified Karnovsky’s fixative. The urinary bladder fromcontrol and high-dose animals (3-mg/kg males, 1-mg/kg females)from the week 27, 53, and 79 sacrifices were processed for eval-uation by scanning electron microscopy (SEM). The bladderfrom selected control and high-dose males and females fromthe week 53 and 79 sacrifices (6/sex/group and 5/sex/group,respectively) were evaluated by light microscopy of toluidine-blue–stained plastic-embedded sections and by TEM.

RESULTS

Carcinogenicity Study

Survival of females to week 104 was 46%, 48%, 57%, and39% for the control, low-dose, mid-dose, and high-dose groups,respectively. Survival of males was 10%, 27%, 19%, and 0%,respectively. Much of the mortality in males occurred late inthe study; survival in males at week 80 was 82%, 80%, 74%, and50%, respectively. The incidence of neoplasms of the urinarybladder was increased in naveglitazar-treated female rats andunchanged in male rats. The incidence of transitional cell carci-nomas in the bladder was 0, 0, 2, and 8 (of 60 per group) infemale rats treated with 0, 0.1, 0.3, and 1.0 mg/kg, respectively.The incidence of transitional cell papillomas in the bladder was

0, 0, 0, and 6, respectively, in the female groups. Approximatelyhalf of the urinary bladder neoplasms in females (0, 0, 1, and6, respectively) were grossly visible masses at necropsy. Theincidence of transitional cell papillomas and carcinomas infemales in the 1.0 mg/kg group was statistically greater thanthat in controls. The incidence of hyperplasia of the urotheliumof the bladder was 0, 3, 3, and 28 in the same groups, respectively.Transitional cell neoplasms were not increased in incidence inmale rats, with incidences of 0, 1, 0, and 1 in groups treatedwith 0, 0.3, 1.0, and 3.0 mg/kg, respectively. Hyperplasia of thetransitional epithelium of the urinary bladder occurred in 4, 4,6, and 18 males in the same groups, respectively. Females hadno increases in incidences of neoplasms of other tissues. Malesin all treatment groups had an overall increase in multiple typesof soft-tissue sarcomas. The incidences and descriptions of theseneoplasms and of nonproliferative lesions in other tissues willbe reported elsewhere.

Urine pH and Chemistry

Administration of naveglitazar had no substantive effects onurine pH (Figure 1A, 1B). A few statistically significant differ-ences that occurred in urine pH were minimal and sporadic andhad no consistent effects by sex, time, or direction. There were noapparent differences in urine pH between males and females.

FIGURE 1.—Urinary pH in male and female F344 rats treated with naveglitazar for 18 months. Week of sample collection indicated.Morning and afternoon samples are indicated by “a” and “p,” respectively. Asterisks indicate statistically significant differencesfrom control values.

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222 LONG ET AL. TOXICOLOGIC PATHOLOGY

Normal diurnal variation for urine pH was exhibited by allgroups; pH tended to be approximately 7 in the a.m. samplesand slightly less than 6.5 in the p.m. samples.

Urine chemistry parameters affected by naveglitazar adminis-tration were concentrations of calcium and inorganic phosphorus.These effects were apparent after week 27. Urinary calciumconcentrations were decreased in a poorly dose-responsivemanner for naveglitazar-treated males and females in both a.m.and p.m. samples (Figure 2A, 2B). The differences in femaleswere most prominent in the p.m. samples. Calcium concentra-tions in males tended to be lower than those in females in bothcontrol and naveglitazar-treated rats, and treatment-relateddecreases were more consistently significant in males. Urinaryinorganic phosphorus concentrations were inconsistently lowerfor males and females of the high-dose groups (Figure 3A, 3B).The inorganic phosphorus concentrations showed a strongdiurnal variation; levels in males and females were fairly simi-lar. Urinary creatinine concentrations were not significantlychanged by treatment, and urinary calcium-to-creatinine andphosphorus-to-creatinine concentrations were altered by com-pound similarly to the absolute calcium and phosphorus concen-trations (data not shown).

Results obtained from urine samples collected during week77 indicated that acidification of urine samples had no effect onmeasured calcium concentrations (Table 1). Absolute calcium

concentrations in urine were similar to those measured at othertimes and showed similar treatment-related decreases.

Urine Sediment Examination

There were no apparent differences caused by treatment inurine sediments in samples examined by light microscopyduring weeks 3, 13, 26, 39, 52, 65, and 78. Results for week 3are shown in Table 2. The majority of crystals present wereconsistent morphologically with triple phosphates (MgNH4PO4;Osborne et al., 1990), varied from none to >20 per microscopicfield (semiquantitative) in individual samples, and had noapparent relationship to treatment. Sediments morphologicallyconsistent with amorphous phosphates (amorphous calciumphosphates) were variably present in fewer animals and had noapparent relationship to treatment. Sediments were examinedmicroscopically from samples that were >200 µl. This resultedin examination of 4 to 10 samples/group for most collectiontimes. The numbers examined at week 26 were lower than atother collection times (3 to 9 males/group, 2 to 7 females/group),prompting the more thorough collection and examination forsediments at week 40.

In samples collected during week 40, the majority of crystalspresent were morphologically consistent with triple phosphates.The presence and relative numbers (semiquantitative) of crystals

FIGURE 2.—Urinary calcium in male and female F344 rats treated with naveglitazar for 18 months. Week of sample collectionindicated. Morning and afternoon samples are indicated by “a” and “p,” respectively. Asterisks indicate statistically significantdifferences from control values.

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Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 223

varied from none to >20 per rat in each group. The relativenumbers (based on semiquantitative scores) of triple phosphatecrystals appeared to be increased slightly by dose in both a.m.and p.m. samples in males and in p.m. samples only in females(Table 3), compared to the concurrent controls. The numbers oftriple phosphate crystals in treated rats in p.m. samples weresimilar to the respective control values in the a.m. samples.Sediments morphologically consistent with amorphous phos-phates were present predominantly in females and appeared tobe slightly decreased by treatment. Crystals consistent withcalcium oxalate were observed in low numbers in only 1 sam-ple, a p.m. sample from a male in the mid-dose group.

There were no qualitative morphologic differences in urinesediment composition between control and compound-treatedrats by scanning electron microscopic examination at week 74(Table 4). The urine from both groups contained slight to mod-erate amounts of crystals morphologically consistent with triplephosphates and lesser numbers of crystals consistent with calciumoxalate. There was moderate variation among individual rats inthe amounts of triple phosphate crystals present. The males hadslightly higher incidence and relative numbers of triple phosphatecrystals than females, but there were no apparent differencesbetween control and compound-treated rats within each gender.

Amorphous material and unidentified crystals were presentin both control and compound-treated rats, with no apparentrelationship to treatment.

Histopathology

Microscopic findings in the urinary bladder for the urinalysis/characterization study are listed in Tables 5, 6, and 7 for weeks27, 53, and 79, respectively. The earliest and most consistenttreatment-related change was hypertrophy of the urothelium,predominantly involving the superficial cells (Figure 4). Thesecells appeared more cuboidal, with increased amounts ofeosinophilic cytoplasm. No particular location within the bladderwas noted. The change affected animals in all compound-treatedgroups and generally increased in incidence and severity withdose.

Hyperplasia of the urothelium was present in only 1 high-dose–group male at the 27-week sampling time and 1 high-dose–group female at the 53-week sampling time. Both of theselesions were minimal simple hyperplasia (diagnostic criteria ofFrith et al., 1995). Hyperplasia was more common at week 79,with hyperplasia evident in 2 of 10 males and 6 of 10 femalesin the high-dose groups. Hyperplasia in 1 female was graded

FIGURE 3.—Urinary phosphorus in male and female F344 rats treated with naveglitazar for 18 months. Week of sample collectionindicated. Morning and afternoon samples are indicated by “a” and “p,” respectively. Asterisks indicate statistically significantdifferences from control values.

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224 LONG ET AL. TOXICOLOGIC PATHOLOGY

minimal and was similar to the lesion at week 53. The remai-ning hyperplastic lesions at week 79 were graded as slight. Fourof the 5 slight hyperplastic lesions in females were identified asfocal, multifocal, and/or nodular. One of these 4 was identifiedas being near the ureter. The exact location within the bladderof the other 3 could not be determined from the orientation of

the tissue sections, but it could be determined that 2 of these werenot in the ventral portion of bladder. Neoplastic lesions presentat 79 weeks were a papilloma in a high-dose female and a carci-noma in a high-dose male.

TABLE 1.—Calcium (Ca) concentrations in acidified and nonacidified urine samples collected during week 77.

Male Female

Daily dose (mg/kg) 0.0 0.3 1.0 3.0 0.0 0.1 0.3 1.0

Acidified samplesCa concentration 28.7 21.4* 12.9* 6.5* 43.4 25.6 30.2 22.8*(mean)

Standard deviation 9.7 8.4 3.9 2.1 12.1 12.9 8.8 9.8Number of animals 21 24 18 21 13 14 11 21

Nonacidified samplesCa concentration 28.3 22.1* 13.2* 6.2* 46.5 27.7 28.6 21.9*(mean)

Standard deviation 9.7 9.2 4.6 1.6 13.9 13.1 7.1 8.4Number of animals 18 19 14 18 5 11 7 19

*significant negative trend, p ≤ .05, compared to respective controls.

TABLE 2.—Incidence of crystals by light microscopic examination of sediment in the urine of rats

treated with naveglitazar for 3 weeks.a

Male Female

Daily dose (mg/kg) 0.0 0.3 1.0 3.0 0.0 0.1 0.3 1.0

Number of animalsa.m. sample 9 8 7 8 9 8 9 8p.m. sample 6 7 7 8 10 7 7 7

Triple phosphatesa.m. sample 0.6 1.3 0.9 1.4 2.0 1.8 2.1 2.5p.m. sample 1.5 1.9 2.4 1.5 0.6 0.9 0.9 1.1

Amorphous phosphatesa.m. sample 0.4 1.9 0.4 1.3 0.3 0.5 0.1 0.4p.m. sample 0.5 0.7 0.0 0.3 0.0 0.0 0.0 0.3

aMean scores calculated on semiquantitative scores per rat: 0 = none present; 1 = 1 to 5 perfield; 2 = 6 to 10 per field; 3 = 11 to 20 per field; 4 = >20 per field.

TABLE 3.—Incidence of crystals by light microscopic examination of sediment in the urine of rats

treated with naveglitazar for 40 weeks.a

Male Female

Daily dose (mg/kg) 0.0 0.3 1.0 3.0 0 0.1 0.3 1.0

Number of animalsa.m. sample 10 10 10 10 10 10 10 10p.m. sample 10 10 10 10 10 10 10 10

Triple phosphatesa.m. sample 2.5 2.8 3.8 3.5 2.0 1.9 2.7 1.4p.m. sample 0.7 1.0 1.4 2.0 0.0 1.5 2.6 2.1

Amorphous phosphatesa.m. sample 0.0 0.0 0.0 0.0 0.8 0.4 0.0 0.2p.m. sample 0.0 0.0 0.0 0.5 1.6 0.0 0.4 0.2

aMean scores calculated on semiquantitative scores per rat: 0 = none present; 1 = 1 to 5 perfield; 2 = 6 to 10 per field; 3 = 11 to 20 per field; 4 = >20 per field.

TABLE 4.—Mean incidence of crystals by scanning microscopicexamination of sediment in the urine of rats treated

with naveglitazar for 74 weeks.a

Male Female

Daily dose (mg/kg) 0.0 3.0 0.0 1.0

Number of animals 5 5 5 5Triple phosphates 2.8 2.6 2.0 1.6Calcium oxalates 0.2 0.2 0.8 1.0Amorphous material 0.0 0.0 1.6 0.4

aMean incidence determined by dividing the sum of incidence scores by number of animals/category/group. Scores assigned subjectively per rat: 0 = none present to 4 = moderatenumbers present.

TABLE 5.—Incidence and mean severity scorea of compound-related findings in the urothelium of the bladder in

rats treated with naveglitazar for 27 weeks.

Male Female

Number of animals 10 10 9 10 10 10 10 10

Daily dose (mg/kg) 0.0 0.3 1.0 3.0 0.0 0.1 0.3 1.0Hypertrophy with 0 (0) 2 (0.2) 3 (0.3) 8 (1.3) 0 (0) 6 (0.6) 4 (0.5) 10 (1.7)

cytoplasmic eosinophilic change

Hyperplasia 0 (0) 0 (0) 0 (0) 1 (0.1) 0 (0) 0 (0) 0 (0) 0 (0)

aMean severity ( ) calculated by dividing the sum of severities by number of animals/group.Severity scores assigned to each animal were 0 through 5.

TABLE 6.—Incidence and mean severity scorea of compound-related findings in the urothelium of the bladder in

rats treated with naveglitazar for 53 weeks.

Male Female

Number of animals 10 10 10 10 10 9 10 10

Daily dose (mg/kg) 0.0 0.3 1.0 3.0 0.0 0.1 0.3 1.0Hypertrophy with 0 0 8 (1.1) 8 (1.1) 0 6 (0.7) 9 (1.0) 10 (1.7)

cytoplasmic eosinophilic change

Hyperplasia 0 0 0 0 0 0 0 1(0.1)Cytokeratin 20: 0 0 8 9 0 0 9 7

Immunolabel reduced

Cytokeratin 20: 0 0 0 0 0 0 1 3No significant immunolabel

aMean severity ( ) calculated by dividing the sum of severities by number of animals/group.Severity scores assigned to each animal were 0 through 5. Severity scores assigned tohypertrophy and hyperplasia only.

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Immunolabeling of the urothelium for cytokeratin (CK) 20was reduced in males of the mid- and high-dose groups at53 weeks and in females of the mid- and high-dose groups at53 and 79 weeks. Label for CK 20 in control animals generallyappeared as a granular label in the apical portion of mostsuperficial cells (Figure 4C). A few cells, often in small seg-ments, were not labeled. In the treated rats with reducedlabel, more superficial cells, often in larger segments, were notlabeled (Figure 4D). A few females in the mid- and high-dosegroups at week 53 had no significant CK 20 immunolabel inthe urothelium.

Immunolabeling for CK 17 in controls at 53 and 79 weekswas cytoplasmic, granular, and present on the basal and inter-mediate layers of the transitional epithelium (Figure 4E).Immunolabeling for CK 17 was not changed in naveglitazar-treated rats with hypertrophy of the urothelium (Figure 4F).Immunolabeling for CK 17 was reduced in 1 high-dose maleand 4 high-dose females at week 79. The reduction was usuallysegmental and associated with hyperplastic areas. However, insome hyperplastic areas of urothelium, label for CK 17 wasdistributed normally on the basilar and intermediate cells.

There were no changes in immunolabeling for uroplakin orCK 5 in treated rats compared to controls. Immunolabeling foruroplakin in all rats was cytoplasmic, granular, and present onthe apical transitional epithelium with occasional weak label inthe intermediate layers. Immunolabeling for CK 5 was cytoplas-mic, diffuse, and present throughout the transitional epithelium.

Cell Proliferation

The mean BrdU ULLI in urothelium of control rats tendedto be higher in males than in females and to increase more in

males with increasing age (comparisons not analyzed statistically,Table 8). There were apparent increasing numerical trends forULLI in naveglitazar-treated males and females at all samplingtimes, but mean ULLI was increased based on statistical signifi-cance only in high-dose females at weeks 53 and 79. In thesefemale groups, the ULLI was approximately 3-fold to 6-foldgreater than concurrent controls, and 5 of 9 or 6 of 10 individualrats had an ULLI that was greater than that of the highestconcurrent control value.

In contrast, no significant increases in mean ULLI wereobserved in naveglitazar treated males. However, the mean ULLIfor high-dose males at 79 weeks was approximately 4-fold greaterthan that of concurrent controls, with 4 of 10 animals having aULLI greater than that of the highest control value.

The distribution of labeled cells in most rats was apparentlyrandom across most of the 3 sections of bladder. In some ratswith higher ULLI, the labeled cells were associated with 1 sectionof bladder, but the distribution still tended to be across most ofthe section (e.g., from anterior to posterior). BrdU labeled cellswere noted (subjectively) as being common in focal areas ofhyperplasia in the 7 high-dose rats with diagnoses of slighthyperplasia, but the BrdU-positive cells were not enumeratedwithin proliferative lesions that appeared nodular and/or expan-sile. One of the high-dose males with a relatively high ULLI at79 weeks was the rat with the focal proliferative lesion diagnosedas a carcinoma. The carcinoma had abundant BrdU-positive cells,as did the urothelium in sections of the bladder without the carci-noma, but few BrdU-positive cells were in the epithelium over-lying the carcinoma.

Electron Microscopy

In scanning electron micrographs of the urothelial surface,control animals generally had cells that were flat with indistinctcell borders and limited surface ruffling (Figure 5A). Theurothelial surface of compound-treated animals had bulging ofindividual cells into the lumen, more distinct cell boundaries,more apparent variability in cell size, and more ruffling of thesurface (Figure 5B). These changes were generally diffuse inthe sections examined; the specific location in the bladderof the sections was not identified. The changes in compound-treated animals were similar over time (6 to 18 months). Only1 compound-treated animal, a female at 18 months, had apolypoid lesion consistent with a nodular proliferative lesion ofthe urothelium within the tissues examined by SEM. The exactlocation (e.g., dorsal vs. ventral) of this lesion within the bladderwas not determined.

The evaluation of toluidine-blue–stained sections and TEMsindicated that the superficial urothelial cells of compound-treatedanimals were more cuboidal than those of controls. Superficialcells of both control and naveglitazar-treated rats had variablenumbers of lysosomes, but compound-treated animals had rela-tively more cells with numerous lysosomes compared to controls(Figure 5C, 5D). Minor changes that were inconsistently presentin the superficial urothelial cells of compound-treated animalsincluded increased variability in the shape of fusiform vesicles

Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 225

TABLE 7.—Incidence and mean severity scorea of compound-related findings in the urothelium of the bladder in

rats treated with naveglitazar for 79 weeks.

Male Female

Number of animals 10 10 10 10 10 10 10 10

Daily dose (mg/kg) 0 0.3 1.0 3.0 0 0.1 0.3 1.0Hypertrophy with 0 4 (0.5) 9 (1.4) 9 (1.2) 0 3 (0.3) 7 (1.0) 10 (1.9)

cytoplasmic eosinophilic change

Hyperplasia 0 0 0 2 (0.4) 0 0 0 6 (1.1)Papilloma, 0 0 0 0 0 0 0 1

transitional cellCarcinoma, 0 0 0 1 0 0 0 0

transitional cellCytokeratin 20: 1 1 0 0 0 0 5 8

immunolabel reduced

Cytokeratin 17: 0 0 0 1 0 0 0 4immunolabel reduced

aMean severity ( ) calculated by dividing the sum of severities by number of animals/group.Severity scores assigned to hypertrophy and hyperplasia only.

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226 LONG ET AL. TOXICOLOGIC PATHOLOGY

and intermediate filaments that were less electron-dense and lesswell organized.

DISCUSSION

Neoplasms of the urinary bladder and renal pelvis have beenreported with numerous compounds with PPAR γ or PPAR α/γdual agonist activity (El Hage, 2005a, 2005b; Cohen, 2005).The increased incidence of neoplasms in the urinary bladderassociated with naveglitazar treatment occurred only in females,with an incidence of 23% in the high-dose group. In contrast,the incidence of urinary bladder tumors in male rats treated withmuraglitazar was significantly increased in 3 of 4 compound-treated groups and was 58% in the high-dose group (Tannehill-Gregg et al., 2006). The incidence of urothelial tumors in

naveglitazar-treated female rats in the mid-dose group (2/60)was not significantly different from controls. However, thesetumors are normally rare in female F344 rats, with backgroundincidences ≤0.2% for papillomas and ≤0.1% for carcinomas(Goodman et al., 1979; Haseman et al., 1990). The occurrenceof the neoplasm in 2 rats, with an apparent dose-response inci-dence, in a known target tissue for the pharmacologic classindicates that the urothelial neoplasms in the mid-dose femalesmay also have been compound related. The lack of develop-ment of urothelial neoplasms in male rats in this study mayhave been related to the low survival of male rats, especiallythose of the high-dose group. However, survival of the maleswas sufficient through week 80 to detect a strong carcinogeniceffect in the bladder, had it been present.

Figure 4.—Hypertrophy of urinary bladder mucosa and immunolabeling for cytokeratins 20 and 17 in rats treated with naveglitazarfor 6 or 12 months. (A) Control male rat, 6 months, Bouin’s fixed tissue, H&E, 40×. (B) Male rat treated with 3.0 mg/kg navegli-tazar for 6 months; superficial epithelial cells are more cuboidal than in controls, Bouin’s fixed tissue, H&E, 40×. (C) Controlfemale rat, 12 months, formalin fixation, immunolabeled for cytokeratin 20, labeling of luminal membrane of most superficialcells, 40×. (D) Female rat treated with 1.0 mg/kg naveglitazar for 12 months, formalin fixation, immunolabeled for cytokeratin 20,decreased label on most superficial cells, no label on a few cells, 40×. (E) Control female rat, 12 months, formalin fixation,immunolabeled for cytokeratin 17, labeling of basilar and intermediate cells, 40×. (F) Female rat treated with 1.0 mg/kg naveglitazarfor 12 months, formalin fixation, immunolabeled for cytokeratin 17, labeling of basilar and intermediate cells, 40×. H&E = hema-toxylin and eosin stain.

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Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 227

In the rat, the most commonly identified indirect (non–compound specific) mechanism of carcinogenicity of the lowerurinary tract is increased formation of urinary solids, which leadsto chronic irritation, cell proliferation, and eventually, neopla-sia (Clayson et al., 1995; Cohen et al., 2002; Cohen, 2005).

Because of the previously described association of PPAR α/γdual agonists with neoplasms of the urothelium, we investigatedthe potential for naveglitazar to cause changes in urinary compo-sition, inflammation, and cell proliferation. In the current studies,the increase in cell proliferation in the urothelium identified in

TABLE 8.—Cell proliferation unit length labeling index (ULLI)+ scores for rats treated with naveglitazar for 27, 53, or 79 weeks.

Male Female

Daily dose (mg/kg) 0 0.3 1.0 3.0 0 0.1 0.3 1.0

Week 27Number of animals with urinary bladder 6 8 5 7 7 8 8 10

epithelium evaluatedTotal mean BrdU LI 0.8 1.6 1.8 2.6 0.1 0.4 1.5 1.3Standard deviation 1.2 2.0 1.9 6.0 0.4 0.7 2.7 2.2Number above highest control LI — 1 1 1 — 1 1 2Week 53Number of animals with urinary bladder 10 7 8 8 6 8 8 9

epithelium evaluatedTotal mean BrdU LI 3.7 4.3 6.1 6.4 1.8 2.7 3.0 5.3*Standard deviation 2.0 3.3 4.2 8.6 1.7 5.6 2.1 4.1Number above highest control LI — 2 3 2 — 2 2 5Week 79Number of animals with urinary bladder 9 10 8 10 10 10 10 10

epithelium evaluatedTotal mean BrdU LI 4.6 4.3 6.4 18.1 0.8 2.4 1.5 5.4*Standard deviation 3.2 5.3 4.9 25.3 0.9 2.9 1.9 4.8Number above highest control LI — 2 2 4 — 1 1 6

LI = labeling index.+ULLI represents total number of BrdU-labeled epithelial cells within entire 3 sections of bladder on slide.*Statistically significant at p ≤ .05.

FIGURE 5.—Alterations in surface morphology and lysosome numbers in urinary bladder mucosa of rats treated with naveglitazarfor 12 or 18 months. Scanning electron micrographs of urothelial surface in control male rat (A) and male rat treated with 3.0 mg/kgnaveglitazar for 12 months (B). A 1.2-cm line = 100 microns. Toluidine-blue–stained 1µ plastic embedded sections of controlfemale rat (C) and female rat treated with 1.0 mg/kg naveglitazar for 18 months (D). Arrows indicate cells with visible lysosomesin the toluidine-blue–stained sections. A 1.7-cm line = 25 microns.

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high-dose female rats at 12 and 18 months correlated with theoverall sex and dose occurrence of urothelial neoplasms in thecarcinogenicity study. However, we did not demonstrate anysignificant treatment-related differences in urine composition,especially crystalluria, which could be causally linked to theproliferative changes. Additionally, there were no treatment-related changes in urothelial morphology, such as erosions andinflammation as determined by light microscopy, which wouldsuggest sediment-mediated injury to the urothelium. Since wedid not examine the urothelium histologically during the first 6months of treatment or complete a full evaluation of the urinarybladder mucosa by SEM at any time point, we can not completelyexclude the possibility of crystalluria-like injury of the urothe-lium during the study. However, since the mechanism of actionis based on chronic irritation, one would expect crystalluria and/orurothelial damage to be detected at some point in the study bythe methods we used if the mechanism were causative.

In the naveglitazar-treated rats with increased proliferation inthe urothelium, there was no apparent predilection for a specificsite in the bladder for the increase in cell counts or hyperplasticlesions, suggesting a diffuse stimulus rather than a localized stim-ulus secondary to injury. While the blocking scheme used forsections of bladder did not necessarily maintain the dorsal-ventralorientation of the sections, it did ensure that both dorsal and ven-tral sections were examined. Since no specific lesions indicativeof irritation were noted by light microscopy and the BrdU-labeled cells were generally randomly distributed, a causal rela-tionship to increased crystalluria could not be made. In thisstudy, we expressed the BrdU labeling index as the total numberof labeled cells in all 3 sections of bladder examined per animaland did not normalize the labeling index to the total number ofcells, as has been recommended for urinary bladder mucosa(Cohen et al., 2007). Since the sections were uniformly preparedacross all animals examined, labeling index was normalized toa unit length, which was the total length of urothelium examinedfor each animal. We evaluated the entire length of urothelialmucosa because of the very low background rate of labeled cellsin the urothelium in many animals. The ULLI method is notinfluenced by changes in the total cell population, such as hyper-plasia (Monticello et al., 1990), and thus gives a better indica-tion of total number of replicating cells in a tissue. However,results could be influenced by differences in tissue preparationin an organ such as the urinary bladder, which could complicateinterpretation of apparent effects in individual animals. Despitethe limitations of the methods used, the statistically significantresults for increased cell proliferation and neoplasms of theurothelium of the bladder both occurred in females of the high-dose group.

The earliest morphologic change in the urothelium identi-fied in this study was hypertrophy, primarily of the superficialurothelial cells. This change, which occurred in both male andfemale rats in our studies, is consistent with early changes inthe urothelium described with ragaglitazar, another PPAR α/γdual agonist, in male Sprague-Dawley rats (Oleksiewicz et al.,2005). The urothelial hypertrophy with ragaglitazar was charac-terized as increased cell size by flow cytometry and increased

protein content by protein/DNA measurements, so it was not anartifact of histologic preparation (e.g., differences in degree ofdistention of the bladder). Hypertrophy of the urothelium hasbeen associated with regenerative responses in the urothelium(Molon-Noblot et al., 1992) and with mechanisms not relatedto proliferative events (e.g., alterations in urine volume; Cohen,2005). In the current study, there were no treatment-relatedlesions of urothelial damage and no changes in urinary concen-trations of creatinine to suggest changes in urine volume. Studiesof urothelium from anuric human patients suggest that a lackof urine-derived factors, including mechanical factors, doesnot appear to modulate differentiation in normal urothelium(Stahlschmidt et al., 2005), but this does not rule out the possi-bility that abnormal urine composition or mechanical factorscould modulate urothelial differentiation in the rat. Regardlessof the potential mechanisms causing the hypertrophy, it occurredin both male and female rats and so did not correlate with theincreased incidence of urothelial neoplasms that was limited tofemales.

We did not investigate the potential for decreased apoptosisas a mechanism of carcinogenesis in the urothelium or as apotential cause of the hypertrophy of the superficial cells. The rateof cell turnover in the normal urothelium is very low (Martin,1972), and a decreased rate of apoptosis would be extremelydifficult to quantify. In a study of the effects of muraglitazar onthe urothelium in rats, there were no apparent treatment-relatedchanges in apoptotic rate in the urothelium (Dominick et al.,2006). The reported apoptotic rate in control and treated rats inthe muraglitazar study was <0.1%, which would equate to <1apoptotic cell per the minimum of 500 cells counted per animal.A decreased rate of apoptosis could not reliably or readily bemeasured against this very low background rate.

In our studies with naveglitazar, we looked at expression ofcytokeratins and uroplakin as potential markers of cell differen-tiation. The only change in these markers associated with naveg-litazar treatment was decreased CK 20 expression in superficialcells. This decrease in label occurred in both males and femalesbut appeared to be more severe and occurred in more samplingtimes in females compared to males. CK 20 is normally expressedin terminally differentiated superficial urothelial cells and appearsafter uroplakin is expressed in these cells (Veranic̆ et al., 2004;Harnden et al., 1995; Harnden et al., 1996). This decreasedlabeling for CK 20 could suggest an alteration in terminal dif-ferentiation or increased cell turnover of the superficial cells,but a relationship to possible changes in relative age of the cellsis not known. Alterations in expression of CK 20 in the urothe-lium have been related to some forms of urothelial dysplasia inhumans, but the dysplasia was associated with increased labelingfor CK 20 (Harnden et al., 1996). In contrast, expression of CK20 is associated with low malignancy potential in some urothelialneoplasms (Harnden et al., 1995; Harnden et al., 1999). Thedecreased labeling for CK 20 in rats in the current study may havebeen related to the minor differences in organization of terminalfilaments observed by electron microscopy but was, overall, achange with no apparent relationship to the occurrence of pro-liferative changes in the urothelium. Labeling with CK 17, a

228 LONG ET AL. TOXICOLOGIC PATHOLOGY

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marker for basal and intermediate cells, was reduced in just afew animals, usually in association with hyperplastic areas.This suggested an alteration of differentiation of cells in theseovertly proliferative areas but offered no suggestion for changesin differentiation that might precede the proliferative changes.The lack of changes in immunolabeling for uroplakin in treatedrats compared to controls suggests that the structure of the apicallimiting membrane was not altered and that the barrier functionof the urothelium was intact.

Urothelial cells have potential for rapid cell turnover inresponse to growth factors that are normally present in theurine. However, this normally occurs only in response to breachesin the surface limiting membrane and resultant exposure of thebasilar cells to components of the urine (Messing et al., 1987;Messing, 1992). It is possible but unlikely that the hypertrophyof the superficial urothelial cells associated with naveglitazartreatment was associated with a loss of integrity that allowedincreased exposure of the basilar cells to growth factors in theurine. The superficial cells appeared to have a normal expressionof uroplakin, the protein associated with the asymmetric unitmembrane of the superficial urothelial cells. Additionally, prolif-erative changes in the urothelium were rarely observed at theearlier sampling periods when hypertrophy of the urotheliumwas already established. A possible explanation for increased cellproliferation that we did not investigate is an increased expressionof growth factor receptors in the urothelium. Transgenic micewith increased expression of EGFR have increased cell prolifer-ation in the urothelium (Cheng et al., 2002).

We did not demonstrate any substantive differences in urinaryprecipitates between control and naveglitazar-treated rats. Urinarysediments observed in the routine urine examination at quarterlyintervals were present with some variability but showed noapparent treatment-related effects. There was an apparent minorincrease in incidence of triple phosphates in urine of naveglitazar-treated females, examined in the afternoon at week 40. However,the relative incidences in triple phosphate crystals in the treatedfemales were still within the incidences seen at other times incontrols. This minor difference in relative crystal numbers mayhave been related to differences in crystal formation, sincecontrol rats appeared to have a higher incidence of amorphousphosphates at this time. The overall sediment load was similarbetween control and treated females. Examination of filtratesof entire urine samples by SEM at 74 weeks showed no appar-ent treatment-related differences in incidence or types of urinarysolids. Additionally, analysis of acidified and nonacidified urinesamples at 77 weeks suggested that decreased urinary calciumconcentrations were not caused by precipitation of calciumsalts in the urine. As a critical part of determining the mode ofaction of urothelial carcinogenesis, light microscopic examina-tion of the urothelium showed no evidence of responses to irri-tation (e.g., erosion and inflammation) that would be expectedwith excessive amounts of urinary sediments.

A limitation of this urinary characterization study is that itwas 18 months in duration, and alterations in urinary parame-ters may have occurred after this time. However, naveglitazar-treated female rats had increased incidences of hyperplastic

lesions and increased cell proliferation in the urothelium by 18months, indicating that the causative factors for the prolifera-tive events were present before this time. Another potentiallimitation was the timing of collection of urine, because of thestrong diurnal alterations in urine composition in rats. Althoughwe did not collect urine during the actual ‘dark’ phase in ourstudy, our a.m. collection time was within 2 hours of lights on.This is within the time during which urine composition is stillreflective of the night phase in the rat (Cohen et al., 2007).Additionally, the expected diurnal changes in pH were demon-strated to support the adequacy of sampling.

Decreased urinary calcium concentrations have been linkedto increased rates of cell proliferation in urothelium (Reese andFriedman, 1978). These changes, however, were demonstratedin cell and organ cultures, in which the basilar urothelial cellscould be in contact with the culture medium. In contrast, urothe-lial cells in vivo are segregated from the urine by the asymmetricunit membrane of the superficial cells and the tight junctionsbetween the superficial cells. While urinary calcium concentra-tions were decreased in naveglitazar-treated rats, they were stillhigher than the calcium concentrations (0.3 mM) associatedwith the increased rates of urothelial cell proliferation in vitro.Additionally, urinary calcium concentrations were lowered bytreatment in both male and female rats and were generally loweroverall in male rats. Thus, there was no obvious relationshipbetween decreased urinary calcium concentrations, whichoccurred in both sexes, and the increased incidence of urinarybladder neoplasms, which occurred only in females.

We did not detect any substantive treatment-related changesin urinary pH in the current study. However, we did demonstratethe expected diurnal variation in pH to ensure that we were ade-quately testing for this parameter. The urinary pH in the currentstudy was near the breakpoint (pH ≥ 6.5) that has been identi-fied as supporting the precipitation of calcium and magnesiumsalts in the urine of rats (Cohen, 2005). The urinary pH in bothmales and females was approximately 7 in the morning but wasslightly below 6.5 in the afternoon. Conditions conducive toformation of calcium and magnesium salts may have been pres-ent during the dark phase but may not have been sustained duringthe entire day. Additionally, there were no apparent sex-relateddifferences in urinary pH that could support the sexual dimor-phism in induction of urothelial neoplasms. An additional studyin female rats treated with naveglitazar and dietary acidificationmight answer the question of whether reductions in urinary pHand urinary solids would have prevented urothelial carcinogen-esis. Dietary acidification of male rats treated with muraglitazarinhibited the formation of urinary solids and prevented urothelialcarcinogenesis (Dominick et al., 2006). However, since we didnot demonstrate crystalluria with naveglitazar, a dietary acidifi-cation study was considered unwarranted.

We did not investigate all known potential mechanistic stepsthat have been related to indirect causes of urinary bladdercarcinogenesis in rats. Alterations in urinary citrate and oxalatelevels were demonstrated as being key events associated withincreased urinary precipitates in the urine of rats treated withmuraglitazar (Dominick et al., 2006). Citrate measurements in

Vol. 36, No. 2, 2008 PPAR AGONIST UROTHELIAL CARCINOGENESIS 229

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230 LONG ET AL. TOXICOLOGIC PATHOLOGY

our study might have added to the body of information forchanges associated with PPAR agonists as a class. However,without demonstration of crystalluria for naveglitazar, such datawould not have proven or disproven causation. Alterations inmultiple components of urine, singly or in relationship to eachother, can lead to precipitation of salts in urine (Cohen, 2005),and changes in 1 component may be offset by changes in othercomponents relative to potential for crystalluria.

In the current study, there was no evidence of a direct orindirect compound-related cytotoxic effect on the urothelium.Therefore, proliferative effects independent of cytotoxic effectsshould be considered. Examples have been presented in whichexperimental treatments in rats have led to increased cell prolif-eration in the absence of apparent urothelial damage (Cohenet al., 1994). Possible causes for such effects include alterationsin growth factors in the urine, relative expression of growth-factor receptors on the urothelium, and alterations of cell-cyclecontrol in urothelial cells. Increased amounts of growth factorsin the urine are an unlikely cause because normal rat urinecontains ample amounts of epithelial growth factor to induceincreased cell proliferation. The normal superficial urothelialbarrier prevents the urinary growth factors from reaching thebasilar urothelial cells. Direct effects of PPAR agonists on theurothelium should be considered. All 3 PPAR subtypes (α, γ,and δ) are expressed in the urinary system, and expression ofPPAR γ is greatest in the urothelium (Guan et al., 1997). While theactivity of PPAR γ agonists on urothelium in vitro is generallyantiproliferative or cytostatic (Grommes et al., 2004; Spenceret al., 2004; Kawakami et al., 2002), the possibility exists thatPPAR agonists could cause other changes that could secondarilylead to urothelial proliferation. Simultaneous activation of bothPPAR α and γ has been shown to induce expression of genesin the urothelium that could be related to cell-cycle control(Egerod et al., 2005). PPAR agonists may also act through ligand-independent pathways, as shown by activation of mitogen-activated protein-kinase signaling of EGFR transactivation(Gardner et al., 2003). Additionally, PPAR agonists may causelocalized changes in expression of other growth factors, such asVEGF (Fauconnet et al., 2002).

In summary, the current study could not identify a specificmechanistic cause for the increased cell proliferation and neo-plasms of the urothelium associated with naveglitazar treatmentof female rats.

ACKNOWLEDGMENT

We acknowledge Dr. Samuel M. Cohen for his counsel onmechanistic factors of urothelial carcinogenesis in rats, for hisadvice on study design, and for his review of our experimentalresults.

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Cheng, J., Huang, H., Zhang, Z. T., Shapiro, E., Pellicer, A., Sun, T. T., and Wu,X. R. (2002). Overexpression of epidermal growth factor receptor inurothelium elicits urothelial hyperplasia and promotes bladder tumorgrowth. Cancer Res 62, 4157-63.

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