Evaluation of the Carcinogenic Potential of Clofibrate in FVB/Tg.AC Mouse After Oral Administration—Part I

Abstract

This study was conducted as part of the International Life Sciences Institute (ILSI) program to evaluate the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) α agonist following oral administration to Tg.AC (transgenic) and wild-type FVB (nontransgenic) mice for a minimum for 6 months. Clofibrate was well tolerated at doses up to 500 (males) and 650 (females) mg/kg/day. Oral administration of clofibrate to Tg.AC or FVB (wild-type) male and female mice for 6 months did not result in the increased formation of neoplastic lesions. Epithelial hyperplasia in the urinary bladder (Tg.AC and FVB) and prostate gland (Tg.AC only), and interstitial-cell hyperplasia in the testes (Tg.AC) were noted at 500 mg/kg/day. Non-neoplastic nonproliferative findings included hepatic hypertrophy and hematopoietic changes (myeloid hyperplasia, myelodysplasia, lymphoid depletion, and erythropoiesis) in Tg.AC and FVB mice of both sexes; reproductive (cystic degeneration and dilatation, hypospermia, spermatocele, dilated inspissated protein) and urogenital (tubular-cell hypertrophy, degenerative/regenerative nephropathy, necrosis/fibrosis) changes in Tg.AC and FVB male mice; congestion in the lung in male Tg.AC mice; gall bladder dilatation in female Tg.AC mice; and adrenal (intracellular lipofuscinosis and atrophy) and heart (eosinophillic myofibers) findings in Tg.AC mice of both sexes and in female FVB mice. The results of this study indicate that the clofibrate is not carcinogenic when administered to Tg.AC mice by oral gavage for 6 months at doses up to 500 (males) and 650 (females) mg/kg/day, which did produce liver hypertrophy.

Clofibrate is a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) α agonist (NDA 1993), which comprises a diverse class of compounds that have therapeutic indications, including dyslipidemia (Yki-Jarvinen 2004). These chemicals bind to the PPARα, which forms a heterodimer with the retinoid X receptor (RXR) and interacts with DNA to regulate genes by affecting transcription (Yki-Jarvinen 2004). Chronic administration of PPARα ligands, such as fenofibrate and clofibrate, typically leads to hepatocarcinogenesis in rodents (Kluwe et al. 1982; NDA 1993; Reddy, Rao, and Moody 1976). However, humans are resistant to peroxisomal proliferation and the development of liver tumors after exposure to PPARα ligands (Klaunig et al. 2003). Recent data from our laboratory indicate that clofibrate is hepatocarcinogenic in rasH2 male mice after 6 months of exposure (Nesfield et al. 2005b) and produced cutaneous papillomas in Tg.AC mice after dermal application (Torrey et al. 2005b), but is noncarcinogenic in p53^+/– mice after 6 months of exposure (Torrey et al. 2005a) or in neonatal mice up to 1 year after treatment on litter days 9 and 16 (Nesfield et al. 2005b). Recently published data suggest that structural differences between the human and rodent PPARα receptor and tissue/species-specific coactivators/corepressors may be responsible for the differential response noted amongst mouse strains/models (Cheung et al. 2004). Based on its mechanism of action and extensive human experience, indicating safety and efficacy, clofibrate was selected for inclusion in the International Life Sciences Institute (ILSI) program in alternative carcinogenicity models (Robinson and MacDonald 2001). These models included the rasH2 transgenic mouse model, Tg.AC transgenic mouse model, the p53^{+ / –}knockout mouse model, the XPA^{– / –} knockout mouse model, the XPA^{– / –}/p53^{+ / –} double-knockout mouse model, and the neonatal mouse model (Robinson and MacDonald 2001).

The Tg.AC transgenic mouse alternative carcinogenicity model was created by fusing the v-Ha-ras gene into the FVB/N mouse strain. Genetic lesions in the Ha-ras are common occurrences contributing to human cancers (Tamaoki 2001). The transgene is transcriptionally dormant until activated by specific chemicals (Eastin et al. 2001), ultraviolet (UV) light (Trempus et al. 1998), or trauma (Cannon et al. 1997). This Tg.AC transgenic mouse model generates epidermal or forestomach squamous cell papillomas/carcinomas in response to carcinogens after topical application or oral administration, respectively (Eastin et al. 2001). Moreover, the hemizygous Tg.AC mouse exhibits a higher average tumor burden and shorter latency period to tumor multiplicity compared to the homozygous strain. These data suggest that a rapid onset and a higher incidence of malignant tumors can be expected after treatment with various carcinogens in the Tg.AC mouse due to increased oncogene or tumor suppressor gene expression.

The Tg.AC transgenic mouse detects both genotoxic and nongenotoxic carcinogens (Tennant et al. 2001). Although the National Toxicology Program (NTP) and ILSI have demonstrated a strong concordance between positive responses for mutagenic carcinogens in the alternative models for carcinogenicity (Storer et al. 2001; Tennant, French, and Spalding 1995; Tennant, Spalding, and French 1996), there is still some concern regarding the limited amount of published data available in transgenic models related to their replacement of the 2-year rodent bioassays (Blain 2003). These studies (part I [oral] and part II [dermal; Torrey et al. 2005b]) were conducted as part of the ILSI program and evaluated the carcinogenic potential of clofibrate following oral or dermal administration to Tg.AC transgenic heterozygous mice for a minimum of 26 weeks. Because this animal model has been proposed for use in detecting both genotoxic and nongenotoxic carcinogens, dimethylvinyl chloride (DMVC) and N-methyl-N-nitrosourea (NMU) were selected as positive controls for tumor incidence. Subsidiary objectives of this oral gavage study included comparison of clofibric acid plasma levels between strains and sexes and investigations of the carcinogenicity of a single oral dose of NMU in mice sacrificed at 3 and 6 months after treatment.

MATERIALS AND METHODS

Animals

Male and female FVB/N-[Tg]v-Ha-ras hemizygous mice (transgenic) and wild-type FVB (nontransgenic) parental strain mice (nontransgenic), approximately 8 to 10 weeks old, were obtained from Taconic (Germantown, NY, USA).

Mice were housed in suspended, stainlesssteel, wire-bottom cages through day 65, then transferred to polycarbonate caging containing Bed-O’Cobs bedding (The Andersons, Maumee, OH, USA) for the remainder of the study. Environmental controls for the animal rooms were set to maintain a temperature of 64°C to 79°C, a relative humidity of 30% to 70%, and a 12-h light/12-h dark cycle. Certified Purina 5002 pelleted diet (PMI Feeds, Inc., Richmond, IN, USA) and municipal tap water treated by reverse osmosis were supplied ad libitum throughout the duration of the studies. Animals were randomized to treatment groups by random number generation.

Treatments and Data Collection

Treatment groups are illustrated in Table 1. Clofibrate (2-(4-chlorophenoxy)-2-methylpropanoic acid, ethyl ester; batch number 514) was obtained from Zeneca Pharmaceuticals (Cheshire, UK), prepared in 0.5% methylcellulose at concentrations ranging from 5 to 65 mg/ml, and stored at room temperature (15–25°C); DMVC was prepared in corn oil at 10 mg/ml and stored at room temperature (15–25°C); NMU was obtained from Sigma (St. Louis, MO, USA), prepared in citrate-buffered saline at pH 4.5 at 9 mg/ml, and stored refrigerated (2–8°C).

Analysis of clofibrate dosing suspensions showed they were homogeneous and within 10% of the target concentrations (data not shown).

Tg.AC mice (carcinogenicity assay and toxicokinetic groups) and FVB mice (toxicokinetic group only) were administered daily doses of clofibrate at 50, 300, or 500 mg/kg/day (males) or 50, 300, or 650 mg/kg/day (females) in 0.5% methylcellulose in water by oral gavage (10 ml/kg) for at least 26 weeks. FVB mice in the carcinogenicity assay group received only the high dose of clofibrate (500 or 650 mg/kg/day) for at least 26 weeks. The doses of clofibrate were based on a 1-month dose range–finding study in FVB mice in which doses of up to 500 (males) or 650 mg/kg/day (females) were well tolerated with no evidence of treatment-related mortality. In addition, a comparative FVB and Tg.AC 14-day oral dose range–finding study was conducted at 300 and 500 mg/kg/day with no evidence of mortality. Clinical signs noted in both the 14-day and 1-month studies at ≥500 mg/kg/day included ataxia and decreased activity. Thus, the high doses of 500 (males) and 650 (females) mg/kg/day were anticipated to produce some toxicity with perhaps some mortality during 26 weeks of treatment. The low dose of 50 mg/kg/day was anticipated to be a no-effect dose. The 300 mg/kg/day dose was anticipated to produce some toxicity with no mortality based on clinical signs (rough coat, tip-toe gate, and limited use of hindlimbs) noted in the 14-day study. Various vehicle control groups (0.5% methylcellulose, citrate-buffered saline, reverse-osmosis water, or corn oil) corresponded to vehicles used for the different test articles. The environmental control group did not receive any treatment.

DMVC doses were chosen as specified in the ILSI protocols (Robinson and MacDonald 2001). Male and female Tg.AC mice (carcinogenicity assay group) received oral doses of DMVC at 100 mg/kg/dose using a 3 times/week (10 ml/kg) dosing regimen. NMU was administered as a single oral dose at 90 mg/kg (10 ml/kg) as per the ILSI rasH2 protocol (Robinson and MacDonald 2001); mice were necropsied 3 and 6 months later.

In-life data collected for up to 26 weeks included clinical observations, body weight, food consumption, and mass palpation. Cutaneous papilloma examination was conducted on a 2 × 4 cm shaved area of dorsal skin.

Toxicokinetics samples at 1–3/sex/group/time point were collected from the vena cava in tubes containing EDTA after administration of CO₂ (terminal sample) on days 1 and 30 at 0 (predose), 1, 3, 6, and 24 h post dosing. On day 182, a sample was also collected 1 h post dosing. Samples were also collected from mice treated with the vehicle control material at 0 (pre-dose) and 24 h post dosing on days 1 and 30, or 1 h post dosing on day 182. Samples were placed on wet ice immediately after collection and centrifuged at approximately 3500 rpm for 10 to 20 min at 3° to 6°C. Plasma was stored frozen at approximately –20°C. Samples were analyzed for clofibric acid. Briefly, 50 μl of mouse plasma, calibration standard, or quality control was mixed with 150 μl of water and 50 μl of 10% perchloric acid. After the mixing by vortex and centrifugation 30 μl of the supernatant was injected into the high-performance liquid chromatography (HPLC) system. The mobile phase consisting of 23% acetonitrile, 23% methanol in 0.1% phosphoric acid was pumped at 1.5 ml/min. The column used for separation was BDS Hypersil C8, 150 × 4.6 mm, 5 μ particle size. Detection system used was UV at 220 nm. Each batch contained calibration standards at 0.5, 1.0, 10, 50, 200, and 500 μg/ml in duplicate, as well as quality control samples at 1.5, 75, and 350 μg/ml, also in duplicate. The coefficient of variance ranged from 4% to 55%. The variability in the C _max estimates is similar across groups and the standard deviation of each value is provided. Because of the small volume of blood available, a destructive sampling design is utilized in which each animal provides one point within each time cohort. Thus, a single AUC value for each group is constructed by using the mean concentration value of each time cohort within the group. Although an estimation of the variance of each group AUC estimate could be made by using the method of Bailer (Bailer 1988) and later extended by Yuan (Yuan 1993), which incorporates the variability at each time point, an a priori design to provide the power to detect a specific difference was not made. Thus a strict statistical evaluation of any difference between groups was considered to be not appropriate. However, trends and any apparent differences among the groups can be made by inspection of the data.

Mice were anesthetized with CO₂, then euthanized by exsanguination via the caudal vena. At termination, parameters evaluated included macroscopic and microscopic observations, organ weights, clinical laboratory measurements, genotyping, tumor sampling, and micronuclei. Hematology parameters evaluated included hemoglobin concentration, hematocrit, red and white blood cells, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelets, red blood cell distribution width, and differential leukocytes (absolute and relative). At termination, the following organs were weighed: adrenal glands, brain, heart, liver with gallbladder, lungs, kidneys, testes, pituitary gland, prostate gland, spleen, thymus, thyroid gland, and ovaries (paired organs weighed together).

Histopathological Evaluation

Tissues collected for histopathological evaluation were fixed in 10% phosphate-buffered formalin, except for the eyes and optic nerves and the testes and epididymides, which were fixed in Bouin’s solution. Tissues were subsequently embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin for histopathological examination. The following tissues were examined in one or more treatment groups, as indicated below: adrenal glands, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, eyes and optic nerves, femur/joint/bone marrow, gross lesions, Harderian glands, heart, ileum, jejunum, jaw, gallbladder, kidneys, liver, lungs with bronchi, lymph nodes (mesenteric and mandibular), ovaries, pancreas, peripheral nerve, pituitary gland, prostate gland, rectum, salivary glands, seminal vesicles, skeletal muscle, skin with mammary glands, skin dorsal area, spinal cord (cervical, thoracic, and lumbar), spleen, sternum/bone marrow, stomach, testes, thymus (thymic area), thyroid with parathyroid gland, tongue, trachea, tumors/masses, urinary bladder, uterus with cervix vagina, and zymbal gland. All tissues from Tg.AC mice treated with DMVC, high dose of clofibrate, citrate-buffered saline, or NMU, and FVB mice treated with methlycellulose or a high dose of clofibrate were examined. The liver, strain specific tumors (skin, thymus, and bone), and targets identified in the high dose clofibrate- or DMVC-treated Tg.AC mice were also examined in the control Tg.AC mice treated with methylcellulose. The liver and target tissues identified in the high-dose group of clofibrate were also examined in Tg.AC mice treated with the lower doses of clofibrate.

Genotyping

Approximately 1 cm of the tail was collected from all animals and frozen in liquid nitrogen, transported frozen on dry ice, and stored at or below approximately −70°C. Genotyping analysis for heterozygous and wild-type strains was conducted by Charles River Therion, Troy, NY, USA. Analysis of the tail tissue confirmed that the 96% of mice were the intended genotype (data not shown).

Statistical Methods and Data Representation

Data are expressed as the mean (with or without standard deviations). Tables 3 through 9 only include data for Tg.AC mice with the correct genotypes. Statistical analysis of nonresponders (i.e., those not expressing the v-Ha-ras transgene) were excluded. The general approach was to assess the statistical significance of pairwise contrasts between control and treated groups. For body weights, food consumption, and absolute and relative organ weights, analysis of variance was performed, followed by application of Dunnett’s (Dunnett 1955) or Williams’ (Williams 1971) multiple-comparison procedure, as determined by the significance of trend (i.e., p < .01) assessed using Jonckheere’s test (Jonckheere 1954). For hematology, nonparametric multiple-comparison procedures were utilized. Shirley’s (1977) or Dunn’s (1955) test was used, based on an assessment of dose-related trends using Jonckheere’s test (Jonckheere 1954). For extreme values, the outlier test of Dixon and Massey (Dixon and Massey 1951) was employed. Values were identified by this test as being extreme if they were at least three times as large as the next largest value or three times smaller than the next smallest value. Statistical analysis of the toxicology data was performed by Analytical Sciences Inc., Durham, NC, USA, using their Subchronic Data Analysis System (SDAS) software. This system is implemented in the SAS system language (Version 6.12) using existing SAS procedures and specialized DATA step programming.

RESULTS AND DISCUSSION

Toxicokinetics

Toxicokinetic data are presented in Table 2. Generally Tg.AC and FVB mice had similar Cmax values; AUC values showed more variability. Exposure was generally reduced (up to 55% for AUC and 44% for Cmax) on day 30 compared to day 1 at the two highest dose levels. Exposure in FVB mice of both sexes was generally higher than in the 31-day range-finding study at a similar dose. On day 30, these values are approximately 2× higher than the the systemic exposure observed in humans (DeSante et al. 1979) taking clofibrate (AUC = 1100 μg · h/ml) at the recommended maximum therapeutic dose of 500 mg, and approximately 1.3× lower than that observed in the dermal study in Tg.AC mice, where skin papillomas were noted (Torrey et al. 2005b). After 6 months (day 182), mean plasma concentrations ranged from 64 to 307 μg/ml, generally increased with increasing dose, and were similar to those concentrations obtained on day 30 (data not shown).

In-Life Findings

The incidence of mortality for all mice is summarized in Table 3. Unscheduled mortality was slightly higher in the Tg.AC strain than in the FVB strain. Within the Tg.AC groups, unscheduled mortality in the 300 and 500/650 mg/kg/day clofibrate groups was higher than in the vehicle control groups. Similarly, FVB mice administered 500/650 mg/kg/day of clofibrate had a higher incidence of unscheduled mortality compared to the FVB controls (data not shown). Clinical observations of decreased activity, dehydration, hypothermia, or hunched posture generally preceded the majority of deaths. In addition, prolapsed penes were noted in most clofibrate- and NMU-treated males that had unscheduled deaths. Additional signs related to treatment with clofibrate included decreased activity, stiffened hindlimbs, or hypersensitivity to touch. The frequency and incidence of these signs decreased over time. One papilloma was observed in a single Tg.AC control female weeks 16 through 26. No other papillomas were noted in the shaved skin areas of either strain for any test materials.

No treatment-related changes in body weight or food consumption were noted between Tg.AC and FVB strains given clofibrate (data not shown).

Hematology

Hematology changes in Tg.AC and FVB mice are illustrated in Table 4. Statistically significant decreased mean corpuscular hemoglobin concentration (MCHC) was noted in Tg.AC males given 500 mg/kg/day clofibrate and FVB females given 650 mg/kg/day clofibrate. Statistically significant increased mean corpuscular volume (MCV) was also noted in the 650 mg/kg/day clofibrate FVB female group. All of these values were not markedly different from those obtained for the environmental control groups. No changes were noted in mice receiving other doses of clofibrate, or DMVC.

Organ Weights

Organ weight changes in Tg.AC and FVB mice are illustrated in Table 5. Increases in absolute and relative liver weights were noted in Tg.AC females administered 300 or 650 mg/kg/day of clofibrate or FVB males and females given 500 or 650 mg/kg/day of clofibrate compared with vehicle controls. Liver weights for Tg.AC males were not increased compared to the corresponding vehicle control; however, it should be noted that mean values for liver weights for this vehicle control were much higher than those for the other three vehicle controls for male mice used in this study, and thus may have obfuscated increases after clofibrate treatment in Tg.AC males. High dose Tg.AC and FVB mice of both sexes had enlarged livers at necropsy and hepatocellular hypertrophy was detected microscopically. Previous studies also showed increases in liver weight and hepatocellular hypertrophy after clofibrate treatment, well known effects of clofibrate and other PPARα agonists (Tomaszewski, Derks, and Melnick 1987; Marsman et al. 1988; Khaliq and Srivastava 1993; Isenberg et al. 2001; Nesfield et al. 2005a). Tg.AC females given 300 mg/kg/day of clofibrate had significantly lower absolute and relative adrenal gland weights than the vehicle control mice. Absolute and relative testicular weights were slightly lower in males given 500 mg/kg/day of clofibrate compared to FVB controls, which is consistent with previous studies with other PPAR ligands (Argarwal et al. 1989).

The Tg.AC female mice given NMU had significantly increased absolute liver weights compared to the Tg.AC citrate vehicle-control group.

Macroscopic Pathology and Lesions

The most common finding for Tg.AC mice treated with 500 mg/kg/day of clofibrate and NMU-treated males was penis prolapse. Enlarged liver and discoloration of the kidney were seen in female Tg.AC mice treated with 650 mg/kg/day of clofibrate and were noted previously in rasH2 mice (Nesfield et al. 2005a). For unscheduled and scheduled sacrifice FVB males, macroscopic findings attributable to clofibrate included an increased incidence of prolapses in the penis; distended, enlarged, and thickened urinary bladder; and enlarged liver. In unscheduled and scheduled sacrifice FVB females, macroscopic findings included a very slight increase in the incidence of enlarged liver and white discoloration of adipose tissue surrounding the uterus. Macroscopic findings in terminal sacrifice animals attributable to oral administration of clofibrate also included a low incidence of papillary masses of the skin (including lip or gum, thorax, axilla, or urogenital areas) compared to those masses noted at the treated site in the dermal study (Torrey et al. 2005b). In females, liver enlargement was also noted macroscopically. DMVC treatment caused an increase in stomach forestomach masses. Distention of the urinary bladder was commonly found in NMU-treated males.

Microscopic Pathology—Unscheduled Deaths

At 500/650 mg/kg/day, the cause of death in the majority of Tg.AC mice treated with clofibrate was undetermined by microscopic examination (data not shown). In the female group receiving 650 mg/kg/day, myelodysplasia was diagnosed in three mice and lymphoma in another three. There was no consistent cause of death attributable to DMVC. In the vehicle-control group, three females were diagnosed with myelodysplasia, which is a frequent background finding in Tg.AC transgenic mice (Mahler et al. 1998). In NMU-treated animals, lymphomas were commonly diagnosed in males and females, as was observed previously in other strains (Hoivik et al. 2004; Urano et al. 2001).

Microscopic Pathology—Terminal

Several neoplasms, recognized as common background tumors in Tg.AC transgenic mice (Mahler et al. 1998), were diagnosed: forestomach papillomas, odontogenic tumors of the mandible or maxilla, erythroleukemia, lymphoma, ovarian teratoma, alveolar/bronchiolar adenoma of the lung, and squamous-cell carcinoma of the salivary gland.

Neoplasms and Non-Neoplastic Proliferative Lesions

Neoplastic findings are shown in Tables 6 and 7 for males and females, respectively. Previous studies have shown that oral administration of clofibrate is hepatocarcinogenic in rasH2 mice after 6 months of exposure (Nesfield et al. 2005a) and produced papillomas in Tg.AC mice after dermal application (Torrey et al. 2005b), but is noncarcinogenic in p53^{+ / –} mice after 6 months of exposure (Torrey et al. 2005a) or in neonatal mice up to 1 year after treatment on litter days 9 and 16 (Nesfield et al., 2005b). Oral administration of 50, 300, or 500 (male) and 50, 300, or 650 (female) mg/kg/day of clofibrate to Tg.AC mice for 6 months did not result in the increased formation of neoplastic lesions. In contrast, doses ≥12 mg/200 μl/day of clofibrate induced squamous cell papillomas at the skin application site, and numerous non-neoplastic microscopic lesions in the liver, kidney, spleen, and skin after dermal application (Torrey et al. 2005b). At 36 mg/200 μl/day of clofibrate, squamous cell papillomas at the skin application site were noted in 10/15 males, and 14/15 females. The difference between carcinogenic potential of clofibrate between the dermal and oral studies may be related to a slightly lower systemic exposure (approximately 1.3×) noted in the oral study. DMVC caused stomach papillomas in male and female Tg.AC mice and hematopoietic lymphomas in male mice. A single dose of NMU caused stomach papillomas, glandular stomach carcinomas, and hematopoietic lymphomas in Tg.AC mice of both sexes, observable after 3 or 6 months. For mice necropsied after 3 months, incidences were 15/15 and 13/13 for lymphoma, 0/15 and 1/13 for stomach carcinoma, and 8/15 and 6/13 for stomach papillomas for males and females, respectively, in NMU-treated animals. Thus a single oral dose of NMU produces detectable carcinogenicity after 3 months in Tg.AC, but not FVB mice. These findings were observed previously in other strains (Hoivik et al. 2004, 2005; Urano et al. 2001). Importantly, FVB males and females showed no increase in tumor incidence in any organ.

Proliferative non-neoplastic findings are shown for males in Table 8. A mild clofibrate effect was detected for some of the proliferative diagnoses. In male Tg.AC mice, the ≥300 mg/kg/day clofibrate treatment group had a greater number of epithelial hyperplastic lesions of the prostate compared to animals treated with the vehicle control material. Similarly, a greater number of the 500 mg/kg/day clofibrate treatment animals showed interstitial-cell hyperplasia in the testes compared to the vehicle control, and epithelial hyperplasia of the urinary bladder corresponding to increased urinary bladder thickness noted at necropsy at ≥300 mg/kg/day. In male FVB mice, the 500 mg/kg/day clofibrate treatment group did not appear to have a greater incidence of proliferative changes compared to the vehicle control animals except for epithelial hyperplasia of the urinary bladder. Urinary epithelial hyperplasia was associated with cystitis/inflammation (data not shown). Although, urinary bladder transitional cell carcinomas were not observed in the clofibrate-treated groups, epithelial hyperplasia was noted in both Tg.AC and FVB mice. Collectively, the findings in the urinary bladder and testes are consistent with the early proliferative changes in rodents induced by other PPAR ligands (Klaunig et al. 2003; Kennedy et al. 2004; Storer 2004). In addition, PPARα is expressed in human prostate cancer, prostatic intraepithelial neoplasia, benign prostatic hyperplasia, and normal prostate tissues (Segawa et al. 2002). These data suggest that PPAR ligands may mediate proliferative changes in prostate tissues. No clofibrate effect was noted for any of the proliferative lesions diagnosed in female Tg.AC or FVB mice (data not shown).

In summary, the oral administration of 50, 300, or 500 (male) and 50, 300, or 650 (female) mg/kg/day of clofibrate to Tg.AC for 6 months did not result in the increased formation of neoplastic lesions. In addition, clofibrate is noncarcinogenic in p53^{+ / –}mice after 6 months of exposure (Torrey et al. 2005a) or in neonatal mice up to 1 year after treatment on litter days 9 and 16 (Nesfield et al., 2005b). In contrast, clofibrate is hepatocarcinogenic in rasH2 male mice after 6 months of exposure (Nesfield et al. 2005a) and in Tg.AC mice after dermal application (Torrey et al. 2005b). The difference between carcinogenic potential of clofibrate between the dermal and oral studies may be related to a slightly lower systemic exposure (approximately 1.3×) noted in the oral study. The difference in hepatocarcinogenic responses seen between rasH2 mice and Tg.AC mice could not be explained on the basis of exposure. On day 30, there were no marked differences in drug plasma parameters between Tg.AC and FVB mice. Recently published data suggest that structural differences between the human and rodent PPARα receptor and/or tissue/species-specific coactivators/corepressors may be responsible for the differential response noted amongst mouse strains/models (Cheung et al. 2004).

Non-Neoplastic, Nonproliferative Findings

Non-neoplastic nonproliferatve findings are shown in Table 9 for males. In general, similar findings were noted in females as described below (data not shown). Clofibrate increased the incidence of findings in the adrenal glands, epididymis, heart, kidney, liver, lung, prostate, seminal vesicles, spleen, testis, urinary bladder, and Zymbal’s gland in Tg.AC mice and in the bone, epididymis, liver, spleen, nonglandular stomach, and urinary bladder in FVB mice.

Diffuse retinal degeneration (atrophy) was present in the eyes of all Tg.AC mice for which this tissue was examined (data not shown). The outer nuclear and receptor layers were absent in the retinas. These retinal lesions have previously been recognized in these genetically modified mice (Colitz et al. 2000).

Clofibrate at 500 mg/kg/day increased the incidence of adrenal gland intracellular lipofuscinosis and adrenal medulla atrophy in male Tg.AC mice. In Tg.AC females, an increased incidence of adrenocortical intracellular lipofuscinosis was induced by treatment with DMVC (data not shown). An increased incidence of eosinophilic myofibers in the heart was noted in the 500 (males) and 650 mg/kg/day (females and data not shown) clofibrate-treated Tg.AC groups. Cystic dilatation of the Zymbal’s gland was increased in male Tg.AC mice receiving 500 mg/kg/day of clofibrate. In contrast to male Tg.AC mice, dilatation of the gallbladder could be attributed to clofibrate-treatment in females (data not shown). Compared to the vehicle controls, erythropoiesis and/or myelodysplasia was observed in the spleen at the high dose (500 or 650 mg/kg/day) of clofibrate (data not shown) in Tg.AC and FVB mice. In male Tg.AC mice, the 500 mg/kg/day group also had changes in the epididymis, prostate, seminal vesicles, and testes.

The incidence of enlarged (hepatocellular hypertrophy with karyomegaly) and discolored (basophilic) hepatocytes in 500 mg/kg/day clofibrate-treated Tg.AC and FVB male mice was greater than in vehicle-control animals. Clofibrate, as with other PPARα ligands, causes hepatocellular hypertrophy and so is an expected histopathological effect (Tomaszewski, Derks, and Melnick 1987; Marsman et al. 1988; Khalig and Srivastava 1993; Isenberg et al. 2001, Nesfield et al. 2005a). The incidences of liver inflammation and Kupffer-cell proliferation in 650 mg/kg/day clofibrate-treated female Tg.AC and FVB mice were greater than in vehicle control animals (data not shown).

In both males and females, the lymph nodes (cervical and mesenteric) showed lymphoid depletion in the NMU-treated group compared to the other groups.

Some changes for DMVC (Cannon et al. 1997) and for NMU (Ando et al. 1992) have been described previously.

At 500 mg/kg/day, male FVB mice had a higher incidence of myelodysplasia of the femur and sternum compared to the FVB vehicle control group. As in the Tg.AC males, hepatic, reproductive, and urogenital changes compared to FVB controls were of greatest incidence in this group. At 650 mg/kg/day, female Tg.AC mice had a higher incidence of adrenal, hepatic, and hematopoietic tissue changes than the vehicle control group (data not shown).

CONCLUSION

The oral administration of 50, 300, or 500 (male) and 50, 300, or 650 (female) mg/kg/day of clofibrate to Tg.AC mice for 6 months did not result in the increased formation of neoplastic lesions. Both DMVC and a single dose of NMU were carcinogenic to Tg.AC, but not FVB mice. The results of this study indicate that clofibrate is not carcinogenic when administered to Tg.AC mice by oral administration for 26 weeks, but causes some of the other expected liver effects such as increased liver weight and hepatocellular hypertrophy, in addition to non-neoplastic effects in urinary and reproductive tracts. Proliferative lesions of epithelial hyperplasia in the urinary bladder (Tg.AC and FVB) and prostate (Tg.AC, only), and interstitial-cell hyperplasia in the testes (Tg.AC) were noted at 300 and/or 500 mg/kg/day. Although, urinary bladder transitional cell carcinomas were not observed in the clofibrate-treated groups, epithelial hyperplasia was noted in both Tg.AC and FVB mice. FVB mice showed no increase in tumor incidence after oral administration of clofibrate. In contrast, topical doses ≥12 mg/200 μl/day of clofibrate induced squamous-cell papillomas at the skin application site and numerous non-neoplastic microscopic lesions in the liver, kidney, spleen, and skin of Tg.AC mice (Torrey et al. 2005b). Clofibrate increases renal lesions (tubular-cell hypertrophy, degenerative/regenerative nephropathy) in both Tg.AC males and females, testicular and sex gland lesions in Tg.AC males, and uterine atrophy in Tg.AC females after oral administration. We conclude that the nongenotoxic PPARα ligand, clofibrate, is not carcinogenic when administered to Tg.AC mice by oral gavage for 6 months at doses up to 500 (males) and 650 (females) mg/kg/day.