Evaluation of the Carcinogenic Potential of Clofibrate in the FVB/Tg.AC Mouse After Dermal Application—Part II

Abstract

This study was conducted as part of the International Life Sciences Institute (ILSI) Alternatives to Carcinogenicity Testing program and evaluated the carcinogenic potential of clofibrate, a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) α agonist following dermal application to transgenic Tg.AC and nontransgenic FVB mice for a minimum of 26 weeks. Clofibrate doses of 12, 28, or 36 mg/200 μl/day were used. Positive controls for papilloma formation were benzene (174.8 mg/200 μl), and 12-o-tetradecanoylphorbol-13-acetate (TPA [0.00250 mg/200 μl]). Clofibrate was tolerated at doses up to 36 mg/200 μl. In Tg.AC mice, clofibrate produced a dose-related increase in the incidence of mice with cutaneous papillomas; and dose-related decreases in mean time to first tumor, mean multiplicity of tumors per mouse, and mean weeks to maximal yield, as well as numerous nonneoplastic microscopic lesions in the liver, kidney, spleen, and skin. Benzene and TPA induced both neoplastic and/or non-neoplastic proliferative lesions in Tg.AC mice. Clofibrate did not increase the incidence or multiplicity of papillomas, or any other tumors in FVB mice. These data show that the Tg.AC dermal model has increased sensitivity in detecting skin papillomas caused by the nongenotoxic rodent carcinogen, clofibrate, compared to wild type FVB mice, at systemic exposures that are 3× higher than the systemic exposure observed in humans taking clofibrate (AUC = 1100 μg·h/ml) at the recommended maximum therapeutic dose of 500 mg. In addition, this study supports the proposed concept that Tg.AC model may detect compounds with nongenotoxic carcinogenic potential in a shorter timeframe than conventional mouse carcinogenicity bioassays.

Clofibrate is a nongenotoxic, peroxisome proliferator-activated receptor (PPAR) α agonist (NDA 1993), and a member of a diverse class of compounds that have therapeutic indications, including dyslipidemia (Yki-Jarvinen 2004). These chemicals bind to peroxisome proliferator-activated receptor α (PPARα) receptor, which then forms a heterodimer with the retinoid X receptor (RXR) and interacts with DNA to affect transcription (Yki-Jarvinen 2004). Chronic administration of PPARα ligands, such as fenofibrate and clofibrate, typically lead 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 mice after 6 months of oral exposure (Nesfield et al. 2005a), but 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), and in Tg.AC after 6 months of oral exposure (Torrey et al. 2005b). Recent literature suggests that structural differences between the human and rodent PPARα receptor or tissue/species-specific coactivators/corepressors may be responsible for the species-specific difference in hepatocarcinogenesis (Cheung et al. 2004). Based on its mechanism of action and extensive human safety and efficacy experience, 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 mice 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 the ILSI program have demonstrated a strong concordance between positive responses for mutagenic carcinogens in the alternative models for carcinogenicity and wild-type strains (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] [Torrey et al. 2005b] and part II [dermal]) 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. Subsidiary objectives included comparison of the variability in drug plasma levels between strains and sexes, and comparison of micronucleus production between Tg.AC and other strains with clofibrate and the positive controls chemicals, benzene and 12-o-tetradecanoylphorbol-13-acetate (TPA).

MATERIALS AND METHODS

Animals

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

Mice were housed in polycarbonate caging containing Bed-O’Cobs bedding. 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, 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 acetone at concentrations ranging from 12 to 365 mg/200 μl and stored refrigerated (2–8°C); benzene was prepared neat at 874 mg/ml and stored frozen (–4°C), or prepared in high-performance liquid chromatography (HPLC)-grade acetone at 374 mg/ml in corn oil, and stored at refrigerated (2–8°C); TPA was prepared in acetone at 0.0625 and 0.1250 mg/ml and stored at refrigerated (2–8°C).

Dose concentration and homogeneity testing (clofibrate only) demonstrated that the actual clofibrate dosing suspension concentrations were within 10% of the target concentrations and were homogeneous (data not shown).

Male and female Tg.AC mice (toxicology and toxicokinetic groups) were administered daily doses of clofibrate at 12, 28, or 36 mg/200 μl/day in acetone dermally for at least 26 weeks. FVB mice in the toxicology and toxicokinetic groups received only the high dose of clofibrate (36 mg/200 μl) 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 36 mg/200 μl were studied. At 36 mg/200 μl/day, no mortality was noted in males; however, 1 female out of 50 died at each dose of 20, 28, or 36 mg/200 μl/day. No clinical signs were noted in the unscheduled death on day 5 at 20 mg/200 μl/day. Clinical signs prior to death on days 3 and 4 at 28 or 36 mg/200 μl/day included decreased activity, dehydration, and loss of righting reflex. These signs were also noted in other female mice treated with 36 mg/200 μl/day on day 2, only. Histological changes of thickening of the epidermis at the skin application site and hepatocellular hypertrophy were noted at ≥20 mg/200 μl/day. In addition, a comparative toxicity 14-day dermal range finding study of FVB and Tg.AC mice demonstrated no differences in mortality or clinical signs at 36 mg/200 μl/day. Thus, the high dose of 36 mg/200 μl/day was anticipated to produce some toxicity with perhaps some mortality, and some hepatocellular hyperplasia and changes to the skin at the site of application, during 26 weeks of treatment. The low dose of 12 mg/200 μl/day was anticipated to be a no-effect dose. The mid dose (28 mg/200 μl/day) was anticipated to produce some toxicity, and to provide comparisons of drug plasma concentrations to those obtained in the range-finding study. Vehicle-control groups received acetone daily at a dosing volume of 200 μl. All doses were applied to a shaved 2 × 4-cm area on the back of each mouse, and allowed to dry before the mouse was returned to its cage. The environmental control groups did not receive any treatment.

Because this animal model has been proposed for use in detecting both genotoxic and nongenotoxic carcinogens, benzene, and TPA were selected as genotoxic and nongenotoxic positive controls for papilloma formation. TPA doses were chosen as specified in the ILSI protocols (Robinson and MacDonald 2001). The benzene dose was selected based on published data in male ICR Swiss mice (Legator and Harper 1988). Male and female Tg.AC and/or FVB mice were dermally administered 87.4 (micronucleus groups only) or 174.8 mg/200 μl (toxicology and micronucleus groups) of benzene using a 3 times a week dosing regimen. Male and female Tg.AC and/or FVB mice were administered 0.00125 (micronucleus groups only) or 0.00250 mg/200 μl (toxicology and micronucleus groups) of TPA using a 3 times a week dosing regimen. Vehicle control groups received acetone using a 3 times a week dosing regimen at a dosing volume of 200 μl.

In-life data collected for up to 26 weeks included clinical observations, body weight, food consumption, and mass palpation. The number (up to a maximum of 20 per mouse) and location of papillomas detected by visual inspection were recorded weekly.

Toxicokinetic samples from 3/sex/group/timepoint were collected in tubes containing EDTA from the vena cava after administration of CO₂ (terminal sample) on days 1, 30 and 178 at 0 (predose), 1, 3, 6, and 24 h post dosing. At 0 (predose) and at 24 h post dosing on days 1, 30, and 178, samples were similarly collected from mice in the vehicle control group. Samples were also taken for unscheduled sacrifices. Samples were placed on wet ice immediately after collection, and then centrifuged at approximately 3500 rpm for 10 to 20 min at 3°C to 6°C. Plasma was transferred to labeled sample tubes and stored frozen at approximately −20°C. Samples were analyzed for clofibric acid.

Mice were anesthetized with CO₂ then euthanized by exsanguination via transection of the caudal vena cava. Postmortem data collection included the following: macroscopic and microscopic observations, organ weights, hematology measurements, genotyping, tumor sampling, and micronuclei assessment. 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, 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 at application site, 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’s gland. All tissues from Tg.AC mice in group 6 (high-dose of clofibrate), group 2 (benzene), and group 3 (TPA) and the livers from all animals in groups 1 (acetone control), 4 (low dose of clofibrate), and 5 (mid dose of clofibrate) were examined microscopically. Tissues with neoplastic or nonneoplastic proliferative lesions found in group 6 were also examined in groups 1, 17 (acetone control, FVB mice), and 18 (high dose of clofibrate, FVB mice). Additionally, tissues with any lesion considered treatment-related in group 6 were examined in groups 1, 4, and 5. Also, tissues with neoplastic or nonneoplastic proliferative lesions seen in groups 2 and 3 were examined in group 1.

Genotyping

Approximately 1 cm of the tail was collected from all animals, 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 conducted by Charles River Therion, Troy, NY, USA confirmed that >98% of mice were the intended genotype (data not shown), in agreement with other Tg.AC studies.

Micronucleus Analysis

Micronucleus sampling conditions are given in Table 2. The blood samples were placed into test tubes containing sodium heparin solution and diluted 1:4 with fetal bovine serum. Slides with blood smear preparations were air dried, fixed with methanol, and stained with acridine range solution (12 μg/ml in pH 6.8 phosphate buffer) for 1 min just prior to analysis. The polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio was established to assess bone marrow cytotoxicity using >1000 cells per mouse. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from >1000 PCEs per animal.

Statistical Methods and Data Representation

Data are expressed as mean ± standard deviation of the mean. Tables 4 through 14 only include data for Tg.AC mice with the correct genotype. Nonresponders (i.e., those not expressing the v-Ha-ras trangene) were excluded. Micronucleus determination used a one-way analysis of variance followed by the Dunnett’s multiple comparison test (Dunnett 1955). Data described in this report as different implies significantly different, p < .05. Statistical analyses were carried out using SAS JMP software (Cary, North Carolina, USA).

RESULTS AND DISCUSSION

Toxicokinetics

Toxicokinetic data are presented in Table 3. No marked differences in AUC, C _max or T _½ (data not shown) were noted between the FVB and the Tg.AC mice. Female mice generally had higher AUC and Cmax values than males. Exposure decreased between days 1 and 178. Exposure of FVB mice at day 30 was similar to that at the end of the 28-day range-finding study, showing that these results were quite reproducible. Day 30 exposures (AUC_0–24) in the high-dose Tg.AC animals were 3174 and 2542 μg·h/ml for males and females, respectively. On day 30, these values are approximately 3× times 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× higher than that observed in the oral study in Tg.AC mice, where no tumors were noted (Torrey et al. 2005b). Plasma concentrations on day 178 in the high-dose Tg.AC animals were 141 and 173 μg/ml for males and females, respectively, which were approximately 2× lower than the plasma concentrations on days 182/183 in the oral study in rasH2 mice, where hepatic tumors were observed (Nesfield et al. 2005a).

In-Life Findings

The incidence of mortality for all mice is summarized in Table 4. No significant differences in mortality were noted between the two animal strains after dermal administration of clofibrate. Unscheduled deaths in the clofibrate-treated groups were similar to those observed in the vehicle control groups. Tg.AC mice administered TPA had a high incidence of early deaths in males (93%) and females (87%) due to tumor formation. Clinical signs (data not shown) related to the growth of odontomas were noted in the clofibrate and vehicle control Tg.AC groups. Prolapsed penes were noted in the majority of males given benzene beginning week 8 and in males given TPA beginning week 4. Leather-like skin and erythema at the application site was noted in the majority of males and females given TPA beginning week 3. Stiffened hindlimbs were noted in a minority of males and females from both strains given 36 mg/200 μl of clofibrate on day 3. Other clinical signs noted in all treatment animals were random in occurrence and included ataxia, urogenital staining, distended abdomen, or decreased activity. Stiffened hindlimbs noted in this study were also noted in the Tg.AC mice after oral administration of clofibrate (Torrey et al. 2005b).

With regard to papilloma formation in the target area after dermal application (Table 5), clofibrate-related responses were noted in male and female Tg.AC mice and included decreased time to onset and increased numbers of mice affected compared to mice receiving the vehicle control material. In general, clofibrate produced a dose-related increase in the incidence of mice with papillomas, and dose-related decreases in mean time to first tumor, and decreased mean multiplicity of tumors per mouse and mean weeks to maximal yield than acetone-treated mice. Conversely, no papillomas were noted in FVB mice treated dermally with clofibrate. The positive controls, benzene and TPA, also showed decreased time to onset of papillomas and increased numbers of mice affected in Tg.AC mice of both sexes compared to mice receiving the vehicle alone. However, compared to benzene, TPA produced a greater incidence of mice with papillomas, a shorter time of onset, a shorter mean time to maximal tumor yield and a lower mean multiplicity of tumors per mouse.

No treatment-related changes in body weight or food consumption were noted in either strain given clofibrate (data not shown). TPA caused slight increases in body weight and food consumption in both sexes (data not shown). Benzene caused a slight decrease in body weight in both, although food consumption was unaffected (data not shown).

Hematology

Hematology changes in Tg.AC and FVB mice are illustrated in Table 6. Mean white blood cell counts were increased in male and female Tg.AC and male FVB mice given 36 mg/200 μl of clofibrate. Mean hematocrit, hemoglobin, and red blood cell counts were lower in male and female Tg.AC mice administered clofibrate, especially at the high dose (36 mg/200 μl). MCV and mean corpuscular hemoglobin (MCH) were slightly higher in the Tg.AC clofibrate groups given 12 or 28 mg/200 μl, possibly related to anemia. In addition, the mean red cell distribution widths (RDWs) were higher in the male and female Tg.AC mice given 36 mg/200 μl of clofibrate, which is consistent with erythrocyte size changes in response to anemia. Some of these same red blood cell parameters also appeared slightly altered in FVB mice treated with 36 mg/200 μl or clofibrate, but less dramatically than the effects seen in the Tg.AC mice. Benzene produced hematologic changes consistent with benzene-associated regenerative anemia and leukocytosis.

Organ Weights

Organ weight changes in Tg.Ac and FVB mice are illustrated in Table 7. Increases in absolute and relative liver weights were noted in male and female Tg.AC mice given 12, 28, and/or 36 mg/200 μl of clofibrate, and female Tg.AC mice treated with TPA. The increases may have correlated with enlarged livers noted at necropsy and/or hepatocellular hypertrophy. Absolute and relative spleen weights were significantly greater in female Tg.AC mice given 36 mg/200 μl of clofibrate compared to controls, as well as male and females treated with TPA. This finding may be related to the increased extramedullary hematopoiesis in the spleen, which was likely due to demand associated with anemia, and/or enlarged tissue noted at necropsy.

Kidney weights were increased in female Tg.AC mice given 36 mg/200 μl of clofibrate and male and female Tg.AC mice given TPA. This finding may have correlated with the tubular cell hypertrophy noted microscopically, and/or enlarged tissue noted at necropsy.

Splenic and kidney weights were increased in benzene-treated females. These mice also had higher incidences of increased extramedullary hematopoiesis in the spleen and tubular cell hypertrophy in the kidney.

Micronucleus Analysis

Dermal administration of clofibrate, benzene, or TPA did not produce a genotoxic response in the micronucleus assay in male Tg.AC mice (Table 8), even at doses that clearly produced skin papillomas. Although anemia was noted in the benzene-treated group, the lack of genotoxicity of benzene and TPA is likely due to reduced systemic exposure at the site of dermal application. Other expected benzene-induced effects were noted, and included mild anemia and leukocytosis with histological findings of myeloid hyperplasia and extramedullar hematopoiesis.

Macroscopic Lesions

Macroscopic findings in the dermal application study are illustrated in Tables 9 through 13. In unscheduled deaths of clofibrate-treated Tg.AC mice, liver enlargement was present in high-dose (36 mg/200 μl) females, and papillary masses of the treated skin were present in high-dose (36 mg/200 μl) females and a male and a female at mid dose (28 mg/200 μl). Macroscopic findings with TPA included skin papillary masses, spleen enlargement, liver enlargement, and heart lesions. These heart lesions (enlargement, mass, focus, and discoloration) generally correlated with atrial thrombi noted microscopically. The splenic and liver enlargement noted in the TPA-treated mice correlated with increased extramedullary hematopoiesis and myelodysplasia (data not shown). In terminal sacrifice animals (Tables 11 and 12), all doses of clofibrate produced papillary skin papillomas at the skin application site and remote sites, and an increased incidence of liver and kidney enlargement, as well as, spleen enlargement in the high-dose group only. Benzene produced papillary skin papillomas, spleen and liver enlargement in Tg.AC mice. In FVB mice, clofibrate did not produce macroscopic papillary masses of the skin (Table 13), but did produce, an increased incidence of liver and kidney enlargement.

Microscopic Pathology—Unscheduled Deaths

At 36 mg/200 μl, the cause of death in the majority of animals treated with clofibrate was undetermined by microscopic examination (data not shown). Erytholeukemia, noted in females at this dose, is a frequent background finding in Tg.AC transgenic mice (Mahler et al. 1998). No consistent cause of death was determined in the benzene group. In the TPA group, myelodysplasia and atrial thrombosis, often seen in the same animal, were a frequent cause of death.

Microscopic Pathology

Neoplasms and Proliferative Non-Neoplastic Findings

Neoplastic findings and selected proliferative changes in Tg.AC mice are shown in Table 14. Squamous cell papillomas occurred at a high incidence at the skin application site, and to a slightly lesser extent at the remote skin site, in males and females in Tg.AC clofibrate-treated mice. The incidences of papillomas were less in the low-dose males and females; however, a no-effect level was not established. No skin papillomas occurred in the clofibrate-treated or vehicle-control FVB mice. Squamous cell papillomas of treated and nontreated skin occurred at a high incidence in the positive-control mice treated with benzene or TPA. Thus, the microscopic findings correlated with the visual scoring of papillomas. A cutaneous squamous cell carcinoma was diagnosed in a benzene-treated male and in a high-dose clofibrate-treated male. Similarly, a sarcoma of the skin was diagnosed in a mid-dose clofibrate-treated male and in a benzene-treated male. No neoplasms were noted in any FVB treatment group. Several neoplasms recognized as common background tumors in Tg.AC 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. Otherwise, neoplasia and proliferative lesions were confined to the skin.

Non-Neoplastic, Nonproliferative Findings

Clofibrate increased the incidence of several non-neoplastic microscopic findings in Tg.AC mice (Table 15). The incidence of hepatocellular hypertrophy was increased in the mid-dose and high-dose mice and was more severe in the high-dose animals. Clofibrate is a PPARα ligand, and hepatocellular hypertrophy is an expected histopathological effect (Tomaszewski, Derks, and Melnick 1987; Marsman et al. 1988; Khaliq and Srivastava 1993; Isenberg et al. 2001; Nesfield et al. 2005a). Additionally, hypertrophy of proximal tubule epithelium in the kidney was increased in high-dose male and female Tg.AC mice. This proximal tubule hypertrophy appeared to involve segment 3 of the proximal tubules, and was characterized microscopically as enlarged with finely granular eosinophilic cytoplasm. Thus, this was a zone of tubules with enlarged epithelial cells along the corticomedullary junction. Kidney proximal tubular epithelium contains PPARα and peroxisomes and it is probable that the hypertrophy is similar to the clofibrate-induced hepatocellular hypertrophy seen in the liver.

Extramedullary hematopoiesis was increased in incidence and severity in males and females Tg.AC mice administered clofibrate. This was characterized microscopically as red pulp areas filled with erythroid, myeloid, and occasionally megakaryocytoid precursors. The increased extramedullary hematopoiesis correlated with increased white blood cell counts in affected treatment groups. Increased extramedullary hematopoiesis was also seen in mice treated with benzene and TPA.

Minimal to mild acanthosis, thickening of the epidermis caused by epithelial cell hyperplasia, occurred at the skin application site in clofibrate-treated Tg.AC and FVB mice. This was seen in conjunction with squamous cell papillomas, but also occurred in animals without papillomas. In Tg.AC mice, the severity was less in the low-dose groups, but the severity of acanthosis in the mid-dose and high-dose mice was similar. Myelodysplasia was diagnosed only in the TPA-treated Tg.AC mice (data not shown). This condition has been reported in Tg.AC mice previously (Mahler et al. 1998). Myelodysplasia typically affects multiple tissues, especially liver, spleen, lymph nodes, bone marrow and lung, and is characterized by space-occupying infiltration of immature hematopoietic cells and mature granulocytes and mononuclear cells. It resembles a vigorous hematopoietic response and is difficult to differentiate from extramedullary hematopoiesis, especially in the spleen.

Diffuse retinal degeneration (atrophy) was present in the eyes of all Tg.AC mice for which this tissue was examined in treated and control animals (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).

CONCLUSION

The dermal application of clofibrate at 12, 28, or 36 mg/200 μl to Tg.AC male and female mice for 6 months resulted in the formation of squamous cell papillomas at the skin application site. No papillomas were visually present or microscopically diagnosed in vehicle control Tg.AC mice or in FVB mice given 36 mg/200 μl of clofibrate. Non-neoplastic findings in Tg.AC associated with clofibrate application included hematological changes, hepatocellular hypertrophy in mice given 28 or 36 mg/200 μl, renal proximal tubular epithelial hypertrophy in mice given 36 mg/200 μl, increased splenic hematopoiesis in mice given 12, 28, or 36 mg/200 μl, and acanthosis at the skin application site in mice given 12, 28, or 36 mg/200 μl. Plasma levels of clofibric acid were similar in Tg.AC and FVB mice, decreased up to two fold from day 1 to day 178, and were greater in females. The benzene and TPA groups exhibited a slightly stronger squamous cell papilloma effect at the skin application site than clofibrate. Additionally, the TPA group exhibited high unscheduled mortality (>87%) and had a high incidence of systemic myelodysplasia, whereas benzene caused decreased body weight, regenerative anemia and leukocytosis, increased extramedullary hematopoiesis in the spleen, and tubular cell hypertrophy in the kidney. Neither clofibrate, benzene, nor TPA caused micronuclei in bone marrow after dermal application in males of either strain. These data show that the Tg.AC dermal model has increased sensitivity in detecting skin papillomas caused by the nongenotoxic rodent carcinogen, clofibrate, compared to wild-type FVB mice, at systemic exposures that are 3× higher than the systemic exposure observed in humans taking clofibrate (AUC = 1100 μg·h/ml) at the recommended maximum therapeutic dose of 500 mg. In addition, this study supports the proposed concept that Tg.AC model may detect compounds with nongenotoxic carcinogenic potential in a shorter timeframe than conventional mouse carcinogenicity bioassays.