GlaxoSmithKline Pharmaceuticals, 1 Five Moore Drive, Research Triangle Park, North Carolina, USA and 2 Upper Merion, Pennsylvania, USA
3 To whom correspondence should be addressed Email: debie.j.hoivik{at}gsk.com
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: ACOX, acyl CoA oxidase; CAT, catalase; CDKN1A, cyclin-dependent kinase inhibitor 1A; PCNA, proliferating cell nuclear antigen; PEX11A, peroxisomal biogenesis factor 11 alpha; RB, retinoblastoma protein.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is probable that the mechanism of PPAR agonist-induced hepatocarcinogenesis is multifactorial. Hypothesized contributing factors have centered on peroxisome proliferation (5,13), cell proliferation (apoptosis and mitogenesis) (7,14) and oxidative stress (5,13,1517). A number of specific cellular and molecular mechanisms have been proposed recently as they may relate to oxidative stress and/or cell proliferation caused by PPAR agonists in rodents (reviewed in refs 18,19). These recently proposed mechanisms include alterations of tumor necrosis factor alpha (20,21), interleukins 1 and 6 (22), NF-
B (23), transforming growth factor ß (20,24), Kupffer cells (21), thyroid hormone homeostasis (25) and inhibition of gap junction intracellular communication (26). Many of the cell proliferative effects are thought to be regulated through the p38 MAP kinase pathway (19,20).
In rodents, PPAR agonists increase peroxisome number and volume in conjunction with an increase in peroxisomal ß-oxidation enzymatic activities, in addition to
-oxidation activities by CYP4A enzymes in the smooth endoplasmic reticulum. Increased ß- and
-oxidation can result in increased H2O2 production (5,13,1517) and the shift to a pro-oxidant state is thought to result in oxidative stress and may contribute to carcinogenesis.
Administration of PPAR agonists results in increased hepatocellular replication in rodents and this increase has been detected indirectly and directly by numerous modalities [e.g. [3H]thymidine uptake, proliferating cell nuclear antigen (PCNA) expression, mitotic activity, BrdU incorporation, flow cytometry] (7,24,27). Marsman et al. (14) demonstrated that the hepatocarcinogenic potency of two PPAR
agonists correlated with the ability of these agents to cause sustained cell proliferation. In addition, other hepatocarcinogenic PPAR
agonists have been shown to induce hepatocellular replication (2830) The data are the basis for the hypothesis that the PPAR
agonists that cause hepatocarcinogenesis do so, in part, by promoting pre-neoplastic foci within the liver. This is supported by the observations that the foci and tumors regress upon cessation of exposure and that PPAR
ligands given to older rats induce a greater incidence of hepatocellular carcinomas than in young rats. Presumably, older rats have a higher number of spontaneous foci than young rats and other species (31). Consistent with this increase in cell proliferation, the gene expression changes for immediate response genes c-myc, c-Ha-ras gene, c-jun, jun-B and jun-D, c-fos, erg-1, NUP475 have been noted in the rodent (13,3234). Some peroxisome proliferators can reduce hepatocellular apoptosis (35), and increase TGF ß-1 gene expression (36). Collectively the data suggest that an alteration in the cellular balance between proliferation and apoptosis may be important for tumor formation. Causal relationships between these modulators of cell cycle control and PPAR
agonist-induced tumorigenesis have not been established since linkages with PPAR
pathways have not been determined, nor have tumorigenic responses in mice lacking these gene products been evaluated. However, the preponderance of data suggests that dysregulated cell cycle control is the major contributor to tumorigenesis but a role for oxidative stress cannot be excluded.
Epidemiology studies have indicated that humans exposed chronically to PPAR agonists such as fibrates are not at increased risk for liver tumor development. However, the robustness of these studies has been debated. Low levels of peroxisome proliferation (<2-fold) have been observed in patients taking hypolipidemic agents (37) and peroxisome proliferation has been reported in non-human primates, although without knowledge of exposure (13,3840). Furthermore, a comprehensive body of literature evaluating the expression of genes in the liver of humans following therapeutic doses is lacking. The few reports that are available are consistent with the notion that PPAR-responsive genes implicated in the development of carcinogenesis are differentially regulated, depending on the species evaluated. In general, the magnitude for change in the rodent is greater than that for humans (4143).
Primates are relatively refractory to PPAR agonist-induced peroxisome proliferation and presumably hepatocarcinogenesis (2,4447). This species difference is one of the lynch-pin arguments put forth regarding the postulated low risk of liver tumors for humans exposed to rodent peroxisome proliferating agents (48). The reason for an apparent species-related difference in peroxisome proliferation susceptibility has not been clearly established. The level of the PPAR
receptor in the liver is greater in rodents than non-rodents (49,50), supporting the proposal that in non-rodents there is less receptor available to transactivate the PPAR-responsive genes that may contribute to the development of hepatocarcinogenesis. However, over-expression of human PPAR
in primary rat and human hepatocyte cells did not confer greater induction of peroxisome proliferation-associated genes (51), suggesting that receptor level alone is not sufficient to explain relative susceptibility to the effects of PPAR
agonists. It has been suggested that the response elements in critical PPAR
target genes within the primate are hypo- or non-functional (52,53), which limits the spectrum of PPAR
target genes that are activated. Similarly, peroxisomal ß-oxidation activity as determined by AcylCoA oxidase activity is significantly increased in rats and mice (54) and only moderately increased in non-rodents (55,56) and the human AcylCoA peroxisome proliferator responsive element (PPRE) has been shown to differ from the rat by one base. Site-directed mutagenesis of the rat PPRE renders it inactive (52). A further hypothesis for the apparent general unresponsiveness of primates to peroxisome proliferation is that humans express a dominant negative form of PPAR
(49).
Interpretation of previous studies of PPAR-induced liver effects in primates has been hampered by lack of concordant effects (e.g. peroxisome proliferation), and/or lack of relevant exposure data. Given the paucity of primate risk factor data in the literature evaluating the relationship between proposed mechanisms of PPAR agonist-induced hepatocarcinogenesis (peroxisome proliferation, cell replication and oxidative stress) at clinically relevant therapeutic exposures of PPAR
agonists, we conducted studies to characterize the hepatic effects of two PPAR
agonists, fenofibrate and ciprofibrate, in the cynomolgus monkey.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies were conducted in two phases, a dose-range finding study followed by the definitive study. In the dose range finding study, monkeys (2/group) were dosed with fenofibrate (4, 20, 400 or 1000 mg/kg/day) or ciprofibrate (2, 50 or 400 mg/kg/day) for 4 consecutive days and in the definitive study, monkeys (4/group) were dosed with fenofibrate (250, 1250 or 2500 mg/kg/day; 2500 mg/kg/day was achieved by administering two doses of 1250 mg/kg 12 h apart) or ciprofibrate (3, 30, 150 or 400 mg/kg/day) for 15 consecutive days. After the indicated treatment period, animals were anesthetized with sodium pentobarbital, weighed, exsanguinated and necropsied. All animals were fasted overnight before necropsy. All dosing was by oral gavage (5 ml/kg) and vehicle control monkeys were similarly given 0.5% hydroxypropyl methylcellulose vehicle only. Data collected in both studies included clinical observations, body weight, food consumption, toxicokinetic analysis, liver weight, histopathology, immunohistochemical staining for Ki-67 antigen and gene expression; clinical pathology parameters were also evaluated following 15 days of dosing. Results presented and discussed are from the 15th day (definitive phase) of the study unless otherwise identified.
Ciprofibrate analytical method
A Waters Oasis HLB extraction cartridge was conditioned with methanol followed by water. 100 µl of serum was loaded onto the column followed by water wash. The analyte was eluted with methanol containing fenofibric acid as internal standard at 1000 ng/ml. The eluate was diluted with acetonitrile and analyzed by HPLC using a multi-step water (0.1% formic acid)/acetonitrile (0.1% formic acid) gradient with flow at 0.3 ml/min. The analyte was separated on a Waters XTerra MS C8 column. Detection was by MS/MS on a Sciex 4000 mass spectrometer with a turbo ion-spray interface. Detection was in positive ion mode with a parent/daughter transition of 287/85 m/z. The internal standard transition was 317/231 m/z. The standard curve had a range of 500200 000 ng/ml serum.
Fenofibric acid analytical method
Fenofibric acid was extracted from 100 µl of serum by protein precipitation with 400 µl of 50:50 acetonitrile:isopropyl alcohol. The samples were centrifuged and an aliquot removed for analysis by HPLC with UV detection (280 nm). The processed samples were chromatographed using a multi-step water (0.1% formic acid)/acetonitrile (0.1% formic acid) solvent gradient with flow at 0.5 ml/min. The analyte was separated on a Waters XTerra MS C8 column. The range of the standard curve was 5050 000 ng/ml. No internal standard was used.
Toxicokinetic analysis
Each individual concentrationtime profile was analyzed with model independent analysis using WinNonlin, version 3.0 (Pharsight, Mountain View, CA) to determine area under the curve [area under the curve (AUC) and AUC24] and half-life (t1/2) values. Cmax and Tmax were obtained by data inspection.
Real-time quantitative polymerase chain reaction
Real-time quantitative polymerase chain reaction (RTPCR) was performed as described previously (28). Primers and probes were preferentially designed toward the 5' regions of the corresponding human genes, which tend to show less sequence divergence between species. The ability to use rhesus mRNA with a human Affymetrix GeneChip has been demonstrated and a large fraction of the probe sets were effective at cross-species hybridization (57). Two methods of RTPCR were used in the present study: (i) TaqMan, which utilizes an internal fluorescent probe in addition to the forward and reverse primers and (ii) Sybr Green, which uses only forward and reverse primers. If the TaqMan amplification was successful, it was assumed that the cynomolgus homolog had been amplified, the use of three specific primers based on the human sequence made this assumption justifiable. The melting temperature of the PCR product was determined for all Sybr Green amplifications, and in all cases, the calculated Tm based on the human sequence was in agreement with the actual Tm for the cynomolgus PCR product. Thus, it is assumed that in all cases the desired homologus monkey mRNA had been amplified. No sequence verification of the amplification product was conducted. Affymetrix GeneChips were also used to examine liver gene expression and generally confirmed the RTPCR results, however, the microarray data are not presented in this manuscript. Genes were selected for examination using RTPCR based on (i) literature reports of genes known to be modulated by PPAR agonists, (ii) mechanisms from the literature regarding PPAR-induced rodent hepatocarcinogenesis and (iii) preliminary examination of the microarray analysis.
Histopathological evaluation
Sections of liver were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned at 5 mm and stained with hematoxylin and eosin. Liver sections were examined by light microscopy by a pathologist and alterations were recorded.
Transmission electron microscopy
Liver for transmission electron microscopy was fixed in 4°C phosphate-buffered modified Karnovsky's fixative pH 7.4. One-millimeter cubes of liver were processed and embedded in Spurr's resin for examination by transmission electron microscopy. Multiple sections were cut at 0.5 mm and stained with toluidine blue. An appropriate section was selected and trimmed to limit thin sections for transmission electron microscopic examination of Zone 1 (periportal hepatocytes). Thin sections (90 nm) were mounted on 150-mesh copper grids, stained with 5% methanolic uranyl acetate and Reynold's lead citrate, then examined on a Zeiss EM10C transmission electron microscope. Five random representative ultrastructural photomicrographs taken on the basis of a complete hepatocyte cross section with crisp well-defined membranes were taken per monkey in each group. In all cases, the final print magnification of the photomicrographs was 10 000x. Significant features for each liver sample were described and recorded for each animal. Peroxisomes and mitochondria per print were manually counted and then an average for each animal was calculated. Each electron micrograph was digitized. The image file was then opened within the image analysis software (Image-Pro Plus, Media Cybernetics, Carlsbad, CA) and a point grid was superimposed over the image. The main (or largest) hepatocyte within the image was chosen, and each grid point overlying this hepatocyte was tagged based upon the organelle over which the grid point was superimposed (nucleus, peroxisome or mitochondria). If the grid point was not superimposed over one of these three organelles, then it was tagged as non-mitochondrial non-peroxisomal cytoplasm. The relative area for nuclei, peroxisomes, mitochondria and non-peroxisome non-mitochondria cytoplasm for the central hepatocyte in each print were calculated. Calibration of the grid was not necessary for measurement of relative areas as reported here. Based on Weibel (58,59), the volumetric density of a component in a tissue can be estimated by measuring the area fraction of a random section occupied in a trans-section. Therefore, the fraction of points to be counted and superimposed over an image is an estimate of its volume. This procedure is the most efficient to use, particularly in electron microscopy, since the measurement consists merely of counting. Superimposed point counts were measured for nucleus, mitochondria, peroxisomes and non-peroxisome non-mitochondria cytoplasm. The cellular area occupied by each type of organelle was defined as the percentage of the total tag points that were represented by each type of tag. Relative to each micrograph, the total number of grid point counts superimposed over a hepatocyte provides for an estimate of the size of that hepatocyte (if the entire hepatocyte is represented and measured in each micrograph). Thus, by correcting for the total number of grid points overlying the hepatocyte, the volumetric area of the cytoplasm occupied by mitochondria or peroxisomes can be evaluated. The data do not provide an indication of hepatocyte shape, size or number.
Ki-67 staining
Livers were evaluated for cell proliferation based on Ki-67 immunohistochemistry (60). Liver was sectioned at 6 µ. Each slide evaluated contained a section of small intestine that served as a positive control of staining quality. Antigen retrieval was performed in a pressure cooker using 1 M citrate buffer. Slides were stained with an automated immunohistochemical stainer (DAKO, Carpenteria, CA). Sections were blocked with 3% hydrogen peroxide and DAKO protein block (DAKO). Sections were stained with a monoclonal anti-human antibody Ki-67 (MIB-1) (DAKO Carpenteria). A negative control was run for each slide by omitting the primary antibody. A negative control in which the primary antibody was replaced with an isotype matched mouse IgG control (Southern Biotechnology, Birmingham, AL) was performed with each run. Primary antibody was labeled with mouse envision plus polymer (DAKO, Carpenteria). 3,3-Diaminobenzidine was used as the detection system. Slides were counter stained with hematoxylin. Random digital images of the Ki-67 stained liver sections were captured using Image-Pro Plus, Media Cybernetics. From the captured digital images 1000 hepatocytes from each of two sections (2000 from each animal) were tagged using Image-Pro Plus for the presence (positive) or absence (negative) nuclear expression of Ki-67. Numbers were recorded and percent positive and percent negative cells were determined.
Enzymatic analysis
Frozen liver samples, stored previously at 70°C, were thawed quickly in a water bath at room temperature and transferred to tared 30-ml Potter-Elvehjem homogenizers containing five volumes of ice cold 50 mM TrisHCl154 mM KCl buffer (pH 7.2) at 4°C. All subsequent postnuclear supernatant (PNS) preparation procedures were conducted at 4°C. The liver was homogenized and homogenates were centrifuged at 2500 g for 10 min. The resulting PNS was divided into 2-ml portions, flash frozen in liquid nitrogen and stored at 70°C. Protein was determined by a modified Bradford method (61) by using a DC Protein Assay kit purchased from Bio-Rad (Hercules, CA) with bovine serum albumin as a standard. Carnitine acetyltransferase (CPT-1) activity was determined using the method of Markwell et al. (62), catalase (CAT) activity was determined using the method of Aebi (63), enoyl-CoA hydratase activity was determined by the method of Steinman and Hill (64) and palmitoyl-CoA oxidase activity was determined by the method of Lazarow (65).
Statistical methods
Data are expressed as mean ± SD of the mean. For the enzymatic activities, statistical differences between treated groups and vehicle control were determined using one-way analysis of variance (ANOVA) followed by Dunnett's test (SAS/STAT® Version 8.0). For the transmission electron microscopy, ANOVA was performed using Proc GLM in SAS using the mean of five prints from one animal as a single value. The residual versus predicted values were plotted and found to be satisfactory with respect to homogeneity of variance across treatment groups. Dunnett's test was used to evaluate the statistical significance of the treated groups when compared with the vehicle control. All statistical tests were performed at a minimum of 5% level of statistical significance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Transmission electron microscopy
At the level of light microscopy, the hypertrophied hepatocytes tended to have increased granular eosinophilic cytoplasm. In fibrate-treated rats, an increase in granular eosinophilic cytoplasm has been associated with peroxisome proliferation as determined by an increase in the volume or number of peroxisomes (44,74). To determine if the described morphological change was due to peroxisome proliferation, liver sections were assessed for ultrastructural changes by transmission electron microscopy. Representative photomicrographs from a control and treated monkey are provided in Figure 2.
Mild to moderate hepatocellular hypertrophy was observed at some of the doses with both fenofibrate and ciprofibrate by transmission electron microscopy, consistent with the finding noted by light microscopy. Qualitative assessment indicated that the individual hepatocytes in ciprofibrate-treated monkeys given doses 150 mg/kg/day were considered the largest compared with fenofibrate-treated monkeys and the controls. The increase in hepatocyte size was due mostly to an increase in smooth endoplasmic reticulum (SER), although the increase in other organelles (peroxisomes and mitochondria) also contributed to the increased individual hepatocyte size.
The average number of peroxisomes and mitochondria per treatment group are summarized in Figure 3. There was a trend for a dose-related increase in the number of peroxisomes, which was statistically significant for monkeys treated with 2500 mg/kg/day fenofibrate and doses 150 mg/kg/day ciprofibrate when compared with controls. Specifically, the increase in peroxisome number when compared with controls was 2.5-fold for 2500 mg/kg/day fenofibrate and for ciprofibrate there was a 1.6-, 2.5- and 2.8-fold increase for doses of 30, 150 and 400 mg/kg/day, respectively. Similarly there was a trend for a dose-related increase in the number of mitochondria for the fenofibrate and ciprofibrate groups compared with the controls with a statistically significant increase in the number of mitochondria for monkeys treated with doses
1250 mg/kg/day fenofibrate and doses
30 mg/kg/day ciprofibrate. Specifically, the increase in mitochondria number when compared with controls was 1.8- and 2-fold for doses of 1250 and 2500 mg/kg/day fenofibrate, respectively, and 1.8-, 2.3- and 2.5-fold for doses of 30, 150 and 400 mg/kg/day ciprofibrate, respectively.
|
Ki-67 staining
Cell proliferation, occurring in conjunction with peroxisome proliferation, has been implicated as a major contributing factor to the generation of a carcinogenic response with multiple agents, including fibrates (7,14). Evaluation of hematoxylin and eosin stained sections by light microscopy indicated there was no treatment-related increase in cell proliferation as determined qualitatively by the presence of mitotic figures. To further evaluate if the administered doses of fenofibrate and ciprofibrate resulted in an increase in cell proliferation, liver samples were evaluated immunohistochemically using an MIB 1 antibody against the Ki-67 antigen. Ki-67 protein is a nuclear protein preferentially expressed during all active phases of the cell cycle (G1, S1 and G2) and mitosis, but is absent in quiescent or resting cells in G0 (75). The number of Ki-67 positive hepatocytes was very low in all groups with the highest number of Ki-67 positive hepatocytes in two of the control monkeys. In a study with another agent, cell proliferation was detectable in monkey liver by Ki-67 staining, indicating the method functions as anticipated (unpublished results). Consistent with the results from the light microscopy, there was no indication of increased cell proliferation in hepatocytes of monkeys treated with doses of fenofibrate up to 2500 mg/kg/day or with ciprofibrate at doses up to 400 mg/kg/day. Results of individual counts are summarized in Table III. Labeling indices from the 4-day exposure were similar with no increase compared with controls and again with the highest individual result being from one control animal with the same labeling index in one of the 50 mg/kg/day ciprofibrate-treated animals (data not shown).
|
|
Similar to that noted for palmitoyl CoA oxidase activity, there was a trend towards a mild, dose-related increase in carnitine acetyltransferase activity for fenofibrate-treated monkeys and a dose-independent increase in carinitine acetyltransferase activity for ciprofibrate-treated monkeys. A statistically significant increase when compared with controls was noted at 2500 mg/kg/day fenofibrate, which was 1.5-fold over control values and at 150 and 400 mg/kg/day ciprofibrate,
2.1- and 1.6-fold, respectively, over control values. In contrast, there was no appreciable increase in enoyl-CoA hydratase activity for fenofibrate-treated monkeys. Enoyl-CoA hydratase activity was statistically significantly increased over controls at doses
150 mg/kg/day with the magnitude of change (1.7-fold over controls) similar for both doses.
Liver gene expression
To further evaluate the hepatic effects of fenofibrate and ciprofibrate treatment, RTPCR studies were undertaken to evaluate the expression level for a number of genes in the primate liver after treatment with fenofibrate and ciprofibrate. Figure 5 shows a hierarchical clustering of the data and illustrates some salient features of the gene expression.
|
The expression levels for genes known to respond to the presence of oxidative stress were also evaluated, namely, CAT, glutathione peroxidase 1, heme oxygenase 1, manganese superoxide dismutase and thioredoxin reductase (THXRD1). The changes in the genes related to oxidative stress were either of a small magnitude, or independent of dose, with the exception of THXRD1, which was up-regulated after 4-days of treatment with both fenofibrate at 2500 mg/kg and ciprofibrate at 400 mg/kg and after 15 days of treatment with ciprofibrate at 400 mg/kg.
To further determine if cells were experiencing oxidative stress and subsequent DNA damage, the expression of two DNA repair genes, growth arrest and DNA damage inducible gene 153 (GADD153) and growth arrest and DNA damage inducible 45 (GADD45) were evaluated. Small changes in these genes occurred but the changes were not consistent in terms of direction, nor were they dose-responsive. Rusyn et al. recently reported a dose-responsive increase in the expression of DNA repair genes in the liver of rats exposed to a hepatocarcinogenic PPAR agonist, which may occur in response to indirect DNA damage, including that imparted by oxidative stress (76). Therefore, genes that respond to various types of DNA damage were also evaluated, including flap endonuclease I (FEN1), O-6-methylguanine DNA methyltransferase (MGMT), DNA polymerase delta (POLD2) and DNA excision repair controlling gene Xpac (XPA). There were little changes in the expression levels for these genes. FEN1 was consistently down-regulated at all time-points and dose levels and the expression of MGMT was not consistently up-regulated and was often down-regulated at the highest dose levels. The gene expression of XPA was not dose-related and did not show an indication of up-regulation and although POLD2 showed a slight trend towards an increase in expression, the magnitude was only over 2-fold at one dose level. Furthermore, there was no notable increase in the expression of P21/Waf 1/cyclin-dependent kinase inhibitor 1A (CDKN1A) a gene shown to be induced in association with diethylmaleate-induced oxidative stress in HeLa cells (77).
PPAR agonists up-regulate cell cycle associated genes in rodent liver, including PCNA, cyclin D and C-myc (22,25,76,78,79). To evaluate the effect of fenofibrate and ciprofibrate on hepatocyte growth control in primates, the expression of genes that influence cell proliferation and apoptosis were examined. Specifically, P21/Waf 1/CDKN1A, c-myc (CMYC), cyclin D1 (CYCLIN_D1), PCNA, RB and phosphoprotein p53 gene exon 11 (TP53). In general, all of the cell cycle/apoptosis genes were down-regulated. CMYC and CYCLIN_D1 showed down-regulation at nearly all dose levels and the down-regulation of CMYC was particularly evident. Generally, the expression levels for P21/Waf 1/CDKN1A, PCNA, RB and TP53 were weakly reduced, although not nearly as consistently or strongly as the down-regulation of CMYC and CYCLIN_D1. For example, the maximum decrease in PCNA expression at any dose level was about 1.5. No increase in PCNA transcription was observed.
To determine if the increase in peroxisome number noted by transmission electron microscopy would have corresponding changes at the mRNA level for peroxisome-related genes, the expression of acyl CoA oxidase (ACOX), peroxisomal biogenesis factor 3 (PEX3), peroxisomal biogenesis factor 11 alpha (PEX11A) and peroxisomal thioesterase IA (PTE1) was examined. Generally, PEX11A was the only gene that appeared to be substantially up-regulated. ACOX, PEX3 and PTE1 generally showed small and inconsistent changes.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There were no treatment-related increases in biochemical markers of hepatotoxicity nor were there treatment-related effects on thyroid function. Studies in the rat have shown that treatment with WY-14,643 resulted in a decrease in total T3 and T4 levels and associated with this was an increase in mitogenic activity, which could contribute to the hepatocarcinogenic response of WY-14,643 in rats and could contribute to the sensitivity of rodents to the hepatic effects of peroxisome proliferators (25). In this study, there was no doseresponsive relationship for effects of fibrates on TSH, T3 or T4 levels. Briefly, the percent change for TSH levels in fenofibrate or ciprofibrate treated monkeys ranged from 3 to +97% of that for the mean control value of 0.525 ng/ml. Similarly, the percent change relative to controls for T3 levels ranged from 13 to 45% (mean control value was 149.75 ng/dl) and the percent change relative to controls for T4 levels ranged from 25% to +18% (mean control value was 5.6 mg/dl).
At the molecular level, the majority of the changes noted were related to the pharmacological activity ascribed to the compounds. There was an increase in the expression level of genes related to fatty acid metabolism and ß-oxidation, especially mitochondrial ß-oxidation. The magnitude of change for some genes was up to 5-fold higher than controls with the strongest response at the higher dose levels of fenofibrate (2500 mg/kg/day) and ciprofibrate (150 mg/kg/day). The genes evaluated included FADS1, FABP1, SCD, ACADM, HADHA, HMGSC2, CPT1 and AACA2.
Administration of fenofibrate and ciprofibrate for 15 days to monkeys resulted in an increase in liver weight up to 1.3- and 1.9-fold that observed for controls for fenofibrate and ciprofibrate, respectively. The magnitude of hepatomegaly was similar to that reported following 2-week feeding studies in cynomolgus monkeys with ciprofibrate (13) and in the rat with di-2-ethylhexyl phthalate (26).
The increase in liver weight was primarily due to an increase in smooth endoplasmic reticulum with increases in peroxisomes and mitochondria contributing. The increase in peroxisome number (up to 2.5- and 2.8-fold compared with controls for fenofibrate and ciprofibrate, respectively) occurred in the absence of an increase in peroxisome volume, which indicates that the peroxisomes were more numerous but smaller in size. The total cross-sectional peroxisomal area per cell was not increased by treatment. In agreement with the increase in peroxisome number but not volume there was an increase in PEX11A gene expression but minimal increases in ß-oxidation. Similarly, ciprofibrate treatment in mice resulted in a significant increase in PEX11A mRNA expression (80). However, the mRNA levels for other peroxisome specific proteins were increased only to a small extent and the change was not consistently dose-related (e.g. ACOX, PEX3 and PTE1). The lack of effect on ACOX did not correlate with the increase in palmitoyl CoA enzymatic activity noted at doses 250 mg/kg/day for fenofibrate and
3 mg/kg/day for ciprofibrate. ACOX mRNA expression is tightly correlated with enzyme levels in the rat (81), no such correlation has been determined with primates. Interestingly, in the absence of PPAR
agonist exposure, mice lacking AcylCoA oxidase activity develop liver tumors to a greater extent than do wild-type mice counterparts (81).
The magnitude of change for the mitochondria number was similar to that of the peroxisomes. Mitochondrial numbers increased up to 2-fold with fenofibrate and 2.5-fold with ciprofibrate, but in contrast to the peroxisomes, the increase in mitochondria number paralleled an increase in the percent of the cellular area occupied by mitochondria. The magnitude of increase in the peroxisome and mitochondria numbers did not parallel the magnitude of the dose-dependent increase in exposure, suggesting a maximal response was achieved.
In agreement with the results from a group of studies conducted by Gray and de la Iglesia (38) in primates treated with gemfibrozil, the magnitude of change in peroxisome number and volume is modest. Administration of 300 mg/kg gemfibrozil to monkeys resulted in a 35-fold increase in the number of peroxisomes and a 13-fold increase in peroxisome volume. Similarly, Lalwani et al. (82) reported a 5-fold increase in peroxisome volume in rhesus monkeys following the administration of DL-040 (4-{[(1,3-benzodioxol)-5-yl]methyl}amino-benzoic acid). In the present study, there was an 3-fold increase in peroxisome number. Gray and de la Iglesia also reported a decrease in peroxisome size, which mirrors the results obtained in this study.
The magnitude for change in peroxisome number and volume in the present study is modest when compared with that reported for the rat. Administration of 300 mg/kg gemfibrozil to the rat resulted in a 7-fold increase in peroxisome number compared with controls and over a 20-fold increase in peroxisome volume (38). Likewise, other investigators have reported a marked increase in peroxisome number and/or volume after treating rats and mice with a PPAR ligand (14).
The results from this study are similar to that reported for a study in humans where liver biopsies were collected from patients before and after treatment with a lipid lowering PPAR agonist, p-chlorophenoxyisobutyric acid (83). There was a concordant increase in the numerical and volume density of peroxisomes and mitochondria and the magnitude of the response was consistent to that noted in this study. p-Chlorophenoxyisobutyric acid treatment for four weeks resulted in a 38 and 50% increase in mitochondria and peroxisome numbers, respectively, and a 34 and 23% increase in mitochondria and peroxisome volume, respectively (37). In a previous study, Hanefeld et al. (83) reported a greater increase in mitochondria numbers compared with peroxisome numbers. This is in contrast with the rat where PPAR agonists consistently increase peroxisome numbers and volume significantly (38) with little or no effect on mitochondria numbers. It has been suggested that this differential response, a parallel increase in mitochondria and peroxisome numbers in the primate and an increase in only peroxisome numbers in the rat, provides evidence for the hypothesis that there are species-related differences in the overall mechanisms by which hepatic cells respond to and metabolize PPAR agonists. However, the nature of these differences are not clearly defined at a cellular or molecular level and their contribution to hepatocarcinogenesis has not been established.
Administration of PPAR agonists to rats results in hepatocellular hypertrophy, hyperplasia and hepatomegaly. Di-2-ethylhexyl phthalate has been shown to increase DNA synthesis 4.8-fold over that of controls after 2 weeks of treatment (26). Similar effects have been shown in other studies (14). In the present study, cell proliferation was assessed qualitatively on H&E sections of liver by evaluating the frequency of mitotic figures and quantitatively by counting cells positive for the Ki-67 antigen. Ki-67 is a nuclear protein expressed during the G1 phase of the cell cycle. In the primate, neither fenofibrate nor ciprofibrate resulted in an increase in cell proliferation at doses up to 4- and 9.4-fold, respectively, the clinical therapeutic exposure. Consistent with the lack of changes in cell proliferation noted at the cellular level, there was no evidence of cell proliferation or suppression of apoptosis at the gene expression level. In general, and in contrast to the response seen in rodent liver, all of the cell cycle genes/apoptosis genes were down-regulated.
CMYC and CYCLIN_D1 showed down-regulation at nearly all dose levels, with a strong down-regulation for CMYC. An increase in CYMC transcription has been established as a factor that promotes cell cycle progression (84,85), and conversely, inhibition of CYMC transcription is associated with cell cycle arrest (86). Likewise, there is considerable evidence that increased expression of D-type cyclins, including CYCLIN_D1, is associated with entry into the cell cycle and that blocking D-type cyclins will prevent cells from entering S phase (87).
Generally, the expression levels of P21/Waf 1/CDKN1A, PCNA, RB and TP53 were weakly reduced, although not nearly as consistently or strongly as the down-regulation of CMYC and CYCLIN_D1. For example, the maximum decrease in PCNA expression at any dose level was 1.5-fold when compared with controls. It is well-established that increased expression of PCNA is associated with increased cell replication and DNA synthesis and increased PCNA expression is seen in rodent liver upon exposure to PPAR
agonists (76,79). No increase in PCNA transcription was observed in the present study. CDKN1A encodes a protein, which binds to several cyclin-dependent kinases, thereby inhibiting their activities and preventing entry into S phase (88). The role of CDKN1A as a negative regulator of cell growth has been well-established (89). Therefore, the down-regulation of CDKN1A could be consistent with increased cell proliferation; however, the magnitude of the decrease was small, never exceeding 1.4-fold.
The shift in the cellular balance to a pro-oxidant state is considered by some to be a critical contributing factor to the resultant hepatocarcinogenesis for PPAR agonists in rats and mice. There was a slight increase in enzymes responsible for cellular ß-oxidation (acyl CoA hydroxylase, enoyl CoA hydratase and carnatine transferase) but no increase in CAT, the primary enzyme responsible for degrading reactive oxygen species in the peroxisomes. The greatest increase, noted for palmitoyl CoA, was
4-fold higher than that noted for controls, which was a relatively mild response when compared with that reported for the rat where increases in ß-oxidative enzymes have been shown to be increased >10-fold with PPAR
administration (26,54,90). Induction of ß-oxidation enzymes of the magnitude similar to the rat has been reported for rhesus monkeys however the authors considered the doses to be well in excess of therapeutic dose levels. Consistent with the marked increase in ß-oxidation enzymes there was a significant increase in peroxisome number (82). The slight increase in cellular ß-oxidation enzymatic activity in the present study is in agreement with the lack of an increase in peroxisome numbers and slight increase in mitochondria numbers. There has been a report of peroxisome proliferation in the absence of a concomitant increase in ß-oxidation enzymatic activity (91) demonstrating that enhanced ß-oxidation and peroxisome proliferation are not necessarily inter-dependent.
In rats, a significant increase in ß-oxidation enzymes has been associated with an increase in the generation of reactive oxygen species. Several lines of evidence from the current study suggest that substantial oxidative damage is not occurring, namely (i) relatively small increase in ß-oxidation enzymes, (ii) lack of response of genes known to be associated with oxidative stress, (iii) lack of response of genes known to be associated with DNA damage induced by oxidative stress and (iv) lack of lipofuscin accumulation.
We are currently examining the liver gene expression from the animals in this publication using Affymetrix GeneChipsTM. In addition, we are also examining the liver gene expression in fenofibrate-treated rats with Affymetrix GeneChipsTM. Not surprisingly, the magnitude of the response in the rat, both in terms of the number of genes changed and the fold-change, is considerably greater than in the primate. We have seen changes in some specific cell cycle associated kinases in the rat, which are not responding in the primate; this may reflect the activation of signaling pathways in the rat but not the monkey (E.Romach, personal communication).
Administration of a PPAR agonist to monkeys at doses that achieve exposures up to
49.4 times that of a therapeutic exposure results in a modest increase in peroxisome and mitochondrial number and mitochondrial area, without noteworthy increases in cellular ß-oxidation, or uncompensated oxidative stress or cell replication at the molecular and cellular level. Collectively, the data from these studies suggest that the primate responds to PPAR
agonists in a manner that is different from the rat and the mouse, and the nature of these differences suggest that the primate may be refractory to PPAR
-induced hepatocarcinogenesis.
![]() |
Supplementary material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Conflict of interest statement |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|