Evaluation of the Carcinogenic Potential of Clofibrate in the Neonatal Mouse

Abstract

This study was conducted in support of the International Life Sciences Institute (ILSI) alternative carcinogenicity models initiative to evaluate the carcinogenic potential of clofibrate, a nongenotoxic peroxisome proliferator-activated receptor (PPAR) α agonist, following oral administration to neonatal mice. Male and female neonatal CD-1 mice were dosed with clofibrate at doses of 100, 250, and 500 mg/kg or with the positive control, diethyl-nitrosamine (DEN), at 2 mg/kg by oral gavage on days 9 and 16 post birth and observed for approximately 1 year for the development of tumors. Plasma levels of clofibric acid after the second administration increased with dose, but were not dose proportional. Clofibrate administered by gavage on litter days 9 and 16 to neonatal mice at doses of 100, 250, or 500 mg/kg did not produce a carcinogenic effect. The positive control DEN did produce tumors in the liver and lung (single and multiple adenomas and carcinomas) and harderian gland (adenoma) of both sexes. Non-neoplastic lesions related to DEN treatment were confined to myocardial degeneration/fibrosis and testicular interstitial hyperplasia in males, and to glomerulonephrosis and gastritis in both sexes.

The International Life Sciences Institute (ILSI) and Health and Environmental Science Institute (HESI) acknowledged the utility of alternative carcinogenicity models for use in human cancer risk assessment by proposing that 1 of the 2-year rodent bioassays might be complemented with a study in one of the recently developed alternative mouse carcinogenicity models or the neonatal mouse bioassay (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). Compounds from diverse modes of action were selected to rigorously test the models and included immunosuppressive, genotoxic, enzyme-inducing, and peroxisome proliferator agents. An important component of the ILSI efforts was the cross-site validation of the studies. Chemicals were evaluated at multiple sites under similar study designs that were approved by a core committee. GlaxoSmithKline, a participant in this effort, tested clofibrate, a nongenotoxic nuclear receptor peroxisome proliferator-activated receptor (PPAR) α agonist (NDA 1993), in multiple alternative carcinogenicity models. Chronic administration of PPARα ligands, such as fenofibrate WY-14643, and clofibrate, typically lead to hepatocarcinogenesis in rodents, as well as tumors in other tissues (Kluwe et al. 1982; NDA 1993; Reddy, Rao, and Moddy 1976). However, humans and nonhuman primates are considered resistant to peroxisomal proliferation and the development of liver tumors after exposure to PPARα ligands (Klaunig et al. 2003).

The neonatal mouse assay for tumorigencity has been used since 1959 (Flammang et al. 1997) and is believed to be sensitive to a wide range of genotoxic chemicals. The neonatal mouse model is reported to have a sensitivity of 85% and a positive prediction rate of 91% with respect to data generated in the 2-year mouse bioassay (Fujii 1991). In addition, neonatal mice have a decreased time-to-tumor formation due to their developmental stage at the time of dosing (Fujii 1991) and are highly sensitive to structurally different genotoxic carcinogens inducing tumors after a short latency period (McClain et al. 2001). Developmental changes in metabolic capacities between adult and neonatal mice may provide insight into the apparent differential sensitivities of the these models to chemically induced carcinogenesis (Flammang et al. 1997). For example, glutathione depletion in adult mice provides a protective effect by increasing hepatic ascorbic acid synthesis, which reduces reactive metabolites capable of eliciting eye and tissue damage. In contrast, newborn mice cannot make ascorbate and develop cataracts (Meister 1995). 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 are still limited data available in transgenic models concerning their replacement of the 2-year rodent bioassays (Blain 2003). The neonatal mouse model offers a high sensitivity for genotoxic chemicals inducing tumors in a short period (McClain et al. 2001). This study supports the evaluation program for alternatives to the mouse carcinogenicity study sponsored by ILSI and was designed using a standardized ILSI protocol to evaluate the carcinogenic potential of clofibrate following oral administration to neonatal mice for a minimum of 2 days (days 9 and 16) post birth with observation for approximately 1 year for the development of tumors. Recent data from our laboratory indicate that clofibrate is hepatocarcinogenic in rasH2 mice after 6 months of exposure (Nesfield et al. 2005) and produced papillomas in Tg.AC mice after dermal application (Torrey et al. 2005a) but is noncarcinogenic in p53^{+ / −} mice after 6 months of exposure (Torrey et al. 2005b).

MATERIALS AND METHODS

Animals

Male and female (n = 24–28/sex/group in the toxicology groups and 4–16/sex/group in the toxicokinetics groups; Table 1) CD-1 mice were produced by mating male and female mice obtained from Charles River (Margate, Kent, United Kingdom). Litters were randomized to treatment groups, and animals were weaned on litter day 21. In general, after weaning mice were housed singly (males) or in groups of 4 (females) in plastic solid-bottom cages. Environmental controls for the animal rooms were set to maintain a temperature of 19°C–22°C, a relative humidity of 45%–70%, and a 12-h light/12-h dark cycle. Either Rat and Mouse No. 1 or No. 3 Expanded Diets (Special Diets Services, Witham, Essex, United Kingdom) and domestic tap water were supplied ad libitum throughout the duration of the study. The use of animals in these experiments performed in the United Kingdom was in accordance with the requirements of the Animals (Scientific Procedures) Act 1986.

Treatments and Data Collection

Clofibrate (2-(4-Chlorophenoxy)-2-methylpropanoic acid, ethyl ester; batch number 514) was obtained from Zeneca Pharmaceuticals (Cheshire, UK), prepared in 0.5% hydroxypropyl methylcellulose in sterile water for irrigation at concentrations ranging from 5 to 32 mg/g (weight/weight), and stored refrigerated (2–8°C). Diethylnitrosamine (DEN) was obtained from Sigma Chemical (Dorset, UK), prepared in sterile water for irrigation at 2 mg/ml (weight/volume), and stored refrigerated (2–8°C).

Design of the study is shown in Table 1. Mice were dosed by oral gavage on litter days 9 and 16 post birth (litter day 1 was equal to the day of littering) with clofibrate at 100, 250, or 500 mg/kg (using dose volumes of 100 and 200 μl on day 9 and day 16, respectively) and observed for approximately 1 year for the development of tumors. Positive control mice were administered 2 mg/kg of DEN. Vehicle-control animals received 0.5% methylcellulose only. In a 1-month dose range–finding study in neonatal mice, doses of 10, 250, 375, or 500 mg/kg were given on days 9 and 16. No mortality was noted at 500 mg/kg. However, 500 mg/kg caused a reduction in body weight in males and females on days 21 or 27. In addition, males and females had an increase in mitotic activity in the liver. The maximum tolerated dose (MTD) was considered to be 500 mg/kg. Thus, the clofibrate high dose (500 mg/kg) was selected based according to ILSI study design (McClain et al. 2001) as the maximum dose tested should correspond to the MTD in the dose range–finding study. This dose was anticipated to produce some toxicity with perhaps some mortality during 1 year of treatment. The low dose (100 mg/kg) was the lowest used in the range-finding study and anticipated to be the no-effect dose, and the intermediate dosage is the approximate geometric mean of the low and high doses. The positive control dose of DEN used the standard dose for this type of study as recommended by ILSI guidelines (Robinson and MacDonald 2001), which was based on the finding that male and female mice treated subcutaneously with DEN (2 mg/kg) 24 h after birth and observed for 1 year showed treatment-related tumors in the liver and lung (Fujii 1991). The vehicle control (0.5% methylcellulose in sterile water) was the vehicle used to prepare the clofibrate dosing suspensions.

In-life data collected up to litter day 369 included clinical observations, body weight, food consumption, and mass palpation. Toxicokinetics was assessed after mice received the final dose of clofibrate given in 200 μl on day 16 (day 1 of final dose). At 1, 2, 4, 10, and 24 h post dosing, toxicokinetic samples from 2 mice/sex/group/time point were collected in tubes containing EDTA via cardiac puncture after administration of CO₂. At 1 or 4 h post dosing, samples were collected from mice treated with the vehicle control material. Samples were placed on wet ice immediately after collection, then centrifuged at approximately 3500 or 4500 rpm for 15 min at 4°C to 57°C. Plasma was transferred to labeled sample tubes and stored frozen at approximately −20°C. Samples were analyzed for clofibric acid. The coefficient of variance ranged from 2% to 60%.

Mice were euthanized by administration of CO₂. At termination (litter days 366 to 399 for toxicology groups), observations included macroscopic and microscopic observations.

Histopathological Evaluation

Tissues collected for histopathological evaluation were fixed in 10% phosphate-buffered formalin. 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 all the toxicology treatment groups: eyes and optic nerves, gallbladder, harderian glands, heart, kidneys, liver, lungs, and testes. The following tissues were examined from the vehicle control, DEN, and the 500 mg/kg clofibrate treatment groups only: adrenal glands, aorta, brain, cecum, colon, duodenum, epididymides, esophagus, femur/stifle joint/bone marrow, ileum, jejunum, lymph nodes (cervical and mesenteric), nasal chambers, ovaries, pancreas, peripheral nerve, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin with mammary glands, spinal cord, spleen, sternum/bone marrow, stomach, thymus (thymic area), thyroids with parathyroid, tongue, trachea, tumors/masses, urinary bladder, uterus, and vagina. In addition, gross lesions were similarly collected, processed, and examined by light microscopy.

Statistical Methods

Data are expressed as mean ± standard deviation of the mean.

RESULTS AND DISCUSSION

In-Life Findings

There were 52 animals found dead, killed for humane reasons, or lost during the study as shown in the Table 2. Twenty-three deaths occurred during the first 4 weeks of the study. Generally, these animals were either found dead or killed due to ill health in the days following dosing. The incidence of deaths was slightly higher for both sexes treated with clofibrate at 500 mg/kg. One pup was lost, presumed to have been eaten by its dam on litter day 9. Following litter day 28, animals were killed for humane reasons following ill health or due to the size of their masses (i.e., greater than 15 mm). The highest mortality was for mice treated with the positive control or with clofibrate at 500 mg/kg. Nevertheless, adequate numbers of mice survived to provide meaningful analysis of neoplastic and non-neoplastic lesions (see Tables 4 and 6, respectively).

During weeks 1 to 4, the most frequent clinical observations were subdued behavior, decreased activity, unsteady gait, dehydration, patchy coat, and runt in litter. These observations were generally confined to animals treated with clofibrate at 500 mg/kg or seen only at a low incidence in other groups.

Following week 4, there were few clinical observations that could be unequivocally related to treatment. Most common findings included circling, hair loss, scabs, piloerection, and hunched posture. These were generally noted at a low incidence.

A total of 49 animals were noted as having mass(es) following palpation (Table 3). The majority of these were males (n = 41) with masses in the urogenital region. Otherwise, the most palpable masses were noted in the ventral abdomen, especially for males treated with DEN.

Males and females treated with clofibrate at 500 mg/kg showed a reduction in body weight gain over the dosing period between litter days 9 and 28. On litter day 35, males and females treated with 500 mg/kg of clofibrate had an approximate 10% decrease in body weight change compared to controls. At 500 mg/kg, females had slightly less body weight gain than males at all timepoints. Positive-control males showed body weight losses or reduced body weight gain between weeks 39 and 52 (data not shown).

Toxicokinetics

Plasma concentration of clofibric acid increased with dose, but less than proportionally (Figure 1). No clofibric acid was detected in plasma from control mice (data not shown). No marked differences were noted between the sexes (data not shown).

Macroscopic Pathology and Lesions

There were no notable treatment-related macroscopic findings for clofibrate-treated animals (data not shown). Treatment-related macroscopic findings, consistent with previous studies (McClain et al. 2001), were present in the liver and lungs of mice treated with the positive control, DEN (data not shown). One or more variable-sized masses, foci, pale areas, or nodules were seen in the liver for the majority of mice or lungs for a few mice treated with DEN. More males than females were affected.

Forty-five chemicals belonging to structurally distinct chemical classes, including aromatic hydrocarbons, aromatic amines, azo dyes, nitroso compounds, steroids, pryrolysates of amino acids, and tryptophan metabolites, were examined in the newborn mouse model for carcinogenic activity and conformance to the 2-year bioassay (Fujii 1991). In this study, the neonatal mouse model had a positive prediction rate of 91% with respect to data generated in the 2-year mouse bioassay. In addition, McClain and coworkers (2001) examined the data from the ILSI studies in the neonatal mouse with human carcinogens, nongenotoxic rodent carcinogens, and nongenotoxic non-carcinogens. This study also demonstrated strong concordance between the newborn mouse model and the 2-year bioassay. Chronic administration of PPARα ligands, such as fenofibrate, WY-14643, and clofibrate, typically lead to hepatocarcinogenesis in rodents, as well as tumors in other tissues (Kluwe et al. 1982; NDA 1993; Reddy, Rao, and Moody 1976). However, humans and nonhuman primates are considered resistant to peroxisomal proliferation and the development of liver tumors after exposure to PPARα ligands (Klaunig et al. 2003). Treatment-related neoplastic lesions for mice given clofibrate and or DEN are summarized in Tables 4 and 5, respectively. There were no treatment-related neoplastic findings in clofibrate-treated mice. A low incidence of tumors was seen for both clofibrate-treated mice and vehicle controls. Adenoma of the harderian gland was noted in 2 females treated with clofibrate at 500 mg/kg and 1 male treated with clofibrate at 100 mg/kg. Bronchoalveolar adenoma in the lung was noted in both sexes in most clofibrate-treated groups and in the vehicle control groups. A similar incidence of hepatocellular adenoma of the liver was seen in all male groups including the vehicle control. The low incidence and lack of dose response of neoplasms did not indicate any correlation with clofibrate treatment.

Clofibrate and peroxisome proliferation have been associated with the development of hepatic neoplasia in conventional mouse lifetime carcinogenicity studies (Greaves 1990). However, no neoplastic response was observed 12 months after clofibrate administration by gavage to neonatal mice at doses up to 500 mg/kg. Recent studies indicate that oral administration of the nongenotoxic rodent carcinogen clofibrate is hepatocarcinogenic in rasH2 mice after 6 months of exposure (Nesfield et al. 2005) and produced papillomas in Tg.AC mice after dermal application (Torrey et al. 2005a). These findings may be related to an increased incidence of peroxisome proliferation and/or other PPARα-related effects such as inhibition of apoptosis, oxidative stress, alteration of cell signaling molecules or gap communications (Klaunig et al. 2003). However, is noncarcinogenic in p53^{+ / −} mice after 6 months of exposure (Torrey et al. 2005a) or in Tg.AC mice after 6 months of oral exposure (Torrey et al. 2005c), or as indicted in these data, neonatal mice up to 1-year after treatment on litter days 9 and 16. The induction of cell proliferation and liver enlargement, are thought to be key to hepatocarcinogenesis of PPARα agonists, such as clofibrate, as well as suppression of hepatic apoptosis (James et al. 1990). Production of cell proliferation in the liver by nongenotoxic promoting agents generally requires repeated exposure. Therefore, it is not unexpected that clofibrate treatment does not result in neoplasms after only two doses to neonatal 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).

Administration of DEN resulted in a treatment-related increase in the incidence of neoplastic changes in the liver, lungs, and harderian gland of both sexes (Table 5) which was consistent with previous reports (McClain et al. 2001). Developmental changes in metabolic capacities between adult and neonatal mice may provide insight into the apparent differential sensitivities of the these models to chemically induced carcinogenesis (Flammang et al. 1997). Thus, a similar pattern of tumors is noted after only one or two doses.

Treatment-related nonproliferative lesions for clofibrate- and DEN-treated animals are summarized in Tables 6 and 7, respectively. Non-neoplastic treatment-related findings were present in the eyes and heart of clofibrate-treated male mice at 500 mg/kg and comprised increased incidences of retinal atrophy and myocardial degeneration/fibrosis. However, these changes were also noted at a lower incidence in control animals, and similar changes were not reported during the toxicology program for clofibrate (Tucker and Orton 1995). Therefore, the significance of these findings are unknown.

In general, PPARα has been implicated as a regulator of cardiac energetics. PPARα overexpression mimics the cardiac effects seen with diabetes mellitus (Finck et al. 2002). Diabetic hearts are predisposed to death and contractile dysfunction (Zhou et al. 2000). In addition, the aged PPARα knockout mice demonstrate the development of cardiac fibrosis (Kersten et al. 1999; Leone, Weinheimer, and Kelly 1999).

Non-neoplastic changes considered to be related to treatment with DEN were seen in the heart, kidney, stomach, and testes. In the heart, an increased incidence of myocardial degeneration/fibrosis in male mice treated with DEN was noted. The incidence and severity of glomerulonephrosis was markedly increased in the kidneys of male mice treated with DEN. A slight increase in incidence of glomerulonephrosis was noted for DEN-treated females. DEN is excreted via the kidney, which may contribute to the higher incidence of glomerulonephrosis in these animals (Table 7). There was increased incidence and severity of gastritis in the stomach of both sexes treated with DEN. This change was considerably more marked in males. An increased incidence of interstitial-cell hyperplasia occurred in the testes of males. The mechanism causing gastritis in the stomach and the interstitial-cell hyperplasia in the testis is unclear.

CONCLUSION

Clofibrate is a nongenotoxic PPARα agonist (NDA 1993). PPAR ligands represent a diverse class of compounds which have potential therapeutic indications including dyslipidemia (Yki-Jarvinen 2004). In general, chronic administration of PPARα ligands induce hepatocarcinogenesis in rats and mice (Kluwe et al. 1982; Reddy, Rao, and Moody 1976). However, humans and nonhuman primates are resistant to hepatic effects, including peroxisomal proliferation and the development of hepatocarcinomas, after exposure to PPAR ligands (Ashby et al. 1994; Hoivik et al. 2004). Clofibrate administered by gavage on litter days 9 and 16 to neonatal mice at doses of up to 500 mg/kg did not produce a carcinogenic effect in the liver or any other organ. The positive control DEN did produce tumors in the liver and lung (single and multiple adenomas and carcinomas) and harderian gland (adenoma). Recent studies indicate that oral administration of nongenotoxic rodent carcinogen, clofibrate, is hepatocarcinogenic in rasH2 mice after 6 months of exposure (Nesfield et al. 2005) and produced papillomas in Tg.AC mice after dermal application (Torrey et al. 2005b). However, is non-carcinogenic in p53^{+ / −} mice after 6 months of exposure (Torrey et al. 2005a) or in Tg.AC mice after 6 months of oral exposure (Torrey et al. 2005c), or as indicted in these data, neonatal mice up to 1 year after treatment on litter days 9 and 16. The induction of cell proliferation and liver enlargement, are thought to be key to hepatocarcinogenesis of PPARα agonists, such as clofibrate, as well as suppression of hepatic apoptosis (James et al. 1990). Production of cell proliferation in the liver by nongenotoxic promoting agents generally requires repeated exposure. Therefore, it is not unexpected that clofibrate treatment does not result in neoplasms after only two doses to neonatal mice. Non-neoplastic lesions related to DEN treatment were confined to myocardial degeneration/fibrosis and testicular interstitial hyperplasia in males, and to glomerulonephrosis and gastritis in both sexes.