PPARs and Xenobiotic-Induced Adverse Effects: Relevance to Human HealthView this Special Issue
Research Article | Open Access
Michael L. Cunningham, Bradley J. Collins, Milton R. Hejtmancik, Ronald A. Herbert, Gregory S. Travlos, Molly K. Vallant, Matthew D. Stout, "Effects of the PPAR Agonist and Widely Used Antihyperlipidemic Drug Gemfibrozil on Hepatic Toxicity and Lipid Metabolism", PPAR Research, vol. 2010, Article ID 681963, 14 pages, 2010. https://doi.org/10.1155/2010/681963
Effects of the PPAR Agonist and Widely Used Antihyperlipidemic Drug Gemfibrozil on Hepatic Toxicity and Lipid Metabolism
Gemfibrozil is a widely prescribed hypolipidemic agent in humans and a peroxisome proliferator and liver carcinogen in rats. Three-month feed studies of gemfibrozil were conducted by the National Toxicology Program (NTP) in male Harlan Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters, primarily to examine mechanisms of hepatocarcinogenicity. There was morphologic evidence of peroxisome proliferation in rats and mice. Increased hepatocyte proliferation was observed in rats, primarily at the earliest time point. Increases in peroxisomal enzyme activities were greatest in rats, intermediate in mice, and least in hamsters. These studies demonstrate that rats are most responsive while hamsters are least responsive. These events are causally related to hepatotoxicity and hepatocarcinogenicity of gemfibrozil in rodents via peroxisome proliferator activated receptor- (PPAR) activation; however, there is widespread evidence that activation of PPAR in humans results in expression of genes involved in lipid metabolism, but not in hepatocellular proliferation.
Gemfibrozil is a nonhalogenated derivative in the class of drugs called fibrates that include clofibrate, fenofibrate, and ciprofibrate. Since its approval by the FDA in 1982, it has been used extensively as a lipid-regulating drug and is an effective treatment of hypertriglyceridemia and hypercholesterolemia. The results of two clinical trials demonstrate that gemfibrozil has proven to be a valuable therapeutic agent in the control of coronary heart disease [1, 2]. It appears that gemfibrozil exerts hypolipidemic effects by decreasing the concentration of triglycerides  and low-density lipoprotein cholesterol (“bad” cholesterol)  and raising the concentration of high-density lipoprotein-cholesterol (“good” cholesterol) [2, 3].
In rodents, gemfibrozil and other fibrates are peroxisome proliferators, inducing a syndrome that includes enlarged livers associated with an increased number and size of hepatic peroxisomes and induction of peroxisomal and microsomal fatty acid-oxidizing enzymes including acyl CoA oxidase, carnitine acetyltransferase, and cytochrome P450 4A [4–7]. In addition to fibrates, peroxisome proliferators include selected herbicides, phthalate ester plasticizers, and endogenous long chain fatty acids [5, 8]. Peroxisome proliferators are associated with hepatocarcinogenicity in rodents. Studies with several peroxisome proliferators, including Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid; the prototype peroxisome proliferator), di (2-ethylhexyl) phthalate and gemfibrozil, and clofibrate have demonstrated carcinogenicity rodents [9–14].
The basis for understanding the biology of peroxisome proliferation in rodents and humans began with the discovery of the peroxisome proliferator activated receptor-α (PPAR) in 1990 . Agonists for the PPAR were found to induce a battery of genes, resulting in peroxisome proliferation in the cytoplasm of rodent liver, which increased lipid catabolism via induction of peroxisomal fatty acyl-CoA -oxidation. In humans, fibrates including gemfibrozil bind PPAR with high affinity, producing reduction in plasma triglycerides and increased HDL concentrations . These effects are thought to result from reducing apoCIII expression and induction of apolipoprotein-AI and AII expression in humans, which are under control of PPAR, and not by proliferation of peroxisomes which occurs in rodents . The molecular basis of differences in response to the hepatic effects of peroxisome proliferators is hypothesized to be a combination of quantitative differences in the hepatic expression of PPAR and qualitative differences in the pattern or functionality of the downstream events that are regulated by the receptor [18, 19].
Although the biochemical and physiologic effects associated with hepatic peroxisome proliferation are thought to play a role in the hepatic toxicity and carcinogenicity in sensitive species of rodents, the mechanism of peroxisome proliferator-induced tumorigenesis and the nature of its species-selectivity are not understood [20–22]. The results of a limited number of published studies suggest that gemfibrozil is not mutagenic [12, 23]. As a result, the observed hepatocarcinogenicity is thought to be the result of indirect mechanisms. Mechanisms of PPAR-induced hepatocarcinogenicity have been recently reviewed . Activation results in increase cell proliferation and decreased apoptosis. PPAR-induced oxidative stress may contribute to cell proliferation via increased signaling or may damage DNA, resulting in the initiation of carcinogenesis; the data for peroxisome proliferator-induced DNA damage are conflicting [25, 26]. Peroxisome proliferator-induced oxidative stress is thought to occur in the rodent because treatment of rodents causes large increases in the activity of the hydrogen peroxide producing peroxisomal -oxidation enzymes while causing only minimal increases in the activity of peroxisomal catalase and decreased activity of glutathione peroxidase [27–29]. One study with Wy-14,643 revealed that hepatocarcinogenicity appears to correlate better with cell proliferation rather than peroxisome proliferation . PPAR null mice have been used to evaluate the role of PPAR in rodent hepatocarcinogenicity. Wy-14,643 hepatocarinogenicity was observed in wild type mice, but not in null mice [31, 32]. In contrast, following exposure to di (2-ethylhexyl) phthalate, more liver tumors were observed in PPARnull mice compared to wild type mice , suggesting that PPAR-independent mechanisms may also be active in the hepatocarcinogenicity of some peroxisome proliferators. Recently, Gonzalez and colleagues have published a series of studies in wild type and humanized PPAR mice [25, 26, 32, 34, 35]. These studies demonstrate that the humanized PPAR mice are resistant to hepatocellular proliferation  and tumors  following exposure to Wy-14,643. In contrast, genes involved in peroxisomal and mitochondrial -oxidation are induced in the wild type and humanized mice. These authors have concluded that the observed differences in the hepatocellular response are the result of differences in the disposition of let-7C microRNA (miRNA) and c-myc expression. In the wild type mice, let-7C miRNA is downregulated, resulting in the increased expression of c-myc, hepatocellular proliferation, and tumors [26, 34, 35]. In contrast, neither downregulation of let-7C miRNA nor increased expression of c-myc occurs in humanized PPARα mice, resulting in a lack of hepatocellular proliferation and tumors. These data may explain the difference in PPAR-mediated effects between rodents and humans.
The National Toxicology Program (NTP) conducted a series of 3-month feed studies in male Harlan Sprague Dawley rats, B6C3F1 mice, and Syrian hamsters to evaluate mechanisms of hepatocarcinogenicity of peroxisome proliferators; Wy-14,643 , gemfibrozil, dibutyl phthalate, and 2,4-dichlorophenoxyacetic acid. Gemfibrozil was included in this initiative because it interacts with the PPAR in rodents and humans as a mechanism of its pharmacological activity, and it induces hepatomegaly, peroxisome proliferation, and hepatocellular tumors in rodents. It was also of interest to evaluate whether these adverse effects were relevant to humans taking this therapeutic agent chronically. Rats and mice are commonly used in studies examining peroxisome proliferators and males are typically more sensitive than females. Hamsters were included because this species, like humans, is believed to be relatively resistant to the hepatotoxicity and carcinogenicity of peroxisome proliferators . In addition to standard endpoints, the studies included assessments of hepatocyte cell proliferation, peroxisomal enzyme analysis, and analysis of lipid levels. Several investigators were awarded RO3 grants to study mechanistic aspects of peroxisome proliferator-induced hepatocarcinogenesis using tissues available from these studies [38–44]. The purpose of this manuscript is to present the effects of gemfibrozil on hepatic toxicity and lipid metabolism following exposure of rats, mice, and hamsters following subchronic exposure in feed, in the context of the NTP studies of Wy-14,643 .
2. Materials and Methods
2.1. Chemical and Dose Formulations
Gemfibrozil was obtained from Sigma Chemical Company (St. Louis, MO) in three lots. Lot 18F0334 was identified as gemfibrozil by infrared spectroscopy (IR) and proton nuclear magnetic resonance spectroscopy (NMR). Purity was determined to be 99% by high performance liquid chromatography (HPLC). Lot 02H0074 was found to be 98.7% pure by HPLC. Lots 18F0334 and 02H0074 were combined prior to the study and renamed as lot S040794. Purity of the combined lot was determined to be 99% by HPLC. A third lot, 104H0551, was identified by IR. Prior to the study the purity of lot S040794 and lot 104H0551 relative to a frozen reference sample of each lot was determined by HPLC to be 103.4% and 99%, respectively. Both of these lots were used in the 90-day studies. To ensure stability, the bulk chemical was stored in amber glass bottles sealed with Teflon-lined lids or sealed buckets lined with double Teflon bags, protected from light, at room temperature. During the studies, periodic reanalyses against frozen reference samples using HPLC revealed no degradation of the bulk chemical. Dose formulations were prepared by mixing gemfibrozil with feed and were stored in plastic buckets at approximately C for up to 3 weeks. Homogeneity of selected dose formulations was confirmed by HPLC. Dose formulations were analyzed at the beginning, midpoint, and end of the studies. Of the dose formulations analyzed for rats, mice, and hamsters, 96% (26/27) were within 10% of the target concentrations.
2.2. Animals and Animal Maintenance
The studies were conducted at Battelle Columbus Laboratories (Columbus, OH) in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). Male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Male B6C3F1 mice were obtained from Taconic Farms, Inc. (Germantown, NY). Male Syrian hamsters were obtained from Frederick Cancer Research and Development Center (Frederick, MD). Study animals were provided NTP-2000 open formula mean diet (Ziegler Brothers, Inc., Gardners, PA) and tap water (via automatic watering system) ad libitum. Animals were quarantined for approximately two weeks prior to the start of the studies and were approximately 8 weeks (rats and mice) or 7 weeks old (hamsters) on the first day of dosing. Study animals were distributed randomly into groups of approximate initial mean body weight and identified by tail tattoo (rats and mice) or ear tag (hamsters). Rats were housed five animals per cage. Mice and hamsters were housed individually. The animal room was maintained at a temperature of , a relative humidity of %, a light/dark cycle of 12 hours (fluorescent light) and 10 air changes per hour.
2.3. Study Design
Core study animals were fed diets containing 0, 10, 100, 1,000, 8,000, or 16,000 ppm (rats), 0, 10, 100, 1,000, 4,000, or 8,000 ppm (mice), or 0, 100, 1,000, 6,000, 12,000, or 24,000 ppm (hamsters) gemfibrozil for 14 weeks (). Additional groups of animals were designated as special study animals () and were fed diets at the same concentrations for up to 13 weeks. For each species, the highest exposure concentration was based on the estimated maximum tolerated dose; in hamsters, the NTP conducted a 14-day study prior to selecting exposure concentrations for the 90-day study. Feed consumption by core study animals was recorded weekly. Core and special study animals were weighed initially, weekly, and at the end of the studies. Clinical findings were recorded weekly for core and special study animals. Other endpoints were determined as indicated below.
2.4. Clinical Chemistry
Blood for clinical chemistry was collected from special study animals on day 34 () and from core study animals at the end of the studies (); animals were not fasted prior to blood collection. The animals were anesthetized with a mixture of carbon dioxide and oxygen, and blood was withdrawn by cardiac puncture and placed in collection tubes devoid of anticoagulant. The samples were allowed to clot and were then centrifuged; the serum was removed and stored at until analysis. The following clinical chemistry endpoints were measured in rats and hamsters: alanine aminotransferase (ALT), alkaline phosphatase (ALP), sorbitol dehydrogenase (SDH), and bile acids; mice were not evaluated for liver biomarkers due to limited serum availability. Cholesterol and triglycerides were measured in rats, mice, and hamsters.
2.5. Liver Histopathology and Weights
Following necropsy of both core and special study animals, the liver was weighed. Livers were then fixed and preserved in 10% neutral buffered formalin, trimmed and processed, embedded in paraffin, sectioned at 5-6 microns, and stained with hematoxylin and eosin for histopathological evaluation. Liver histopathology was conducted on all core study rats, mice (except 10 ppm), and hamsters. The histopathological findings were subjected to a rigorous pathology peer review including an NTP Pathology Working Group (PWG); the final diagnoses represent a consensus of peer review pathologist and the PWG. Details of these review procedures have been described by Maronpot and Boorman  and Boorman et al. .
2.6. Hepatocyte and Peroxisome Proliferation
On study days 1, 29, and 85, five special study rats, mice, and hamsters per group were implanted subcutaneously with osmotic minipumps (Model 2001, Alza Corp., Palo Alto, CA) prefilled with a 30 mg/mL solution of 5-bromo--deoxyuridine (BrDU; Sigma Chemical Company, St. Louis, MO) in 0.01 N sodium hydroxide. The pumps were incubated in phosphate-buffered saline at for at least 4 hours and then implanted between 1300 and 1600 hours in animals anesthetized with 2% isoflurane via inhalation. The exact time of implantation in each animal was recorded. After 5 days ( hours) of BrDU exposure, the livers were evaluated for incorporation of BrDU. Approximately half of the left, right median, and anterior right lobes were fixed in 10% neutral buffered formalin for 48 hours; the remaining tissue was frozen in liquid nitrogen. The formalin-fixed liver samples, as well as a transverse section of duodenum included as an internal control, were embedded in paraffin; tissues not embedded after 48 hours of fixation were transferred to 70% ethanol. Two serial sections of each tissue were made; one slide was used for histopathologic examinations, and the second slide was stained with anti-BrDU antibody. Cell proliferation (labeled hepatocytes as a percentage of total hepatocytes) was measured by examining 2,000 hepatocyte nuclei from the left liver lobe.
A sample of the left liver lobe was collected from the BrDU animals and reserved for peroxisome proliferation analyses; approximately 1 g (rat and hamster) or 0.5 g (mouse) portions of the liver samples were prepared and analyzed for peroxisome proliferation. Peroxisome proliferation was determined in duplicate tissue extractions by measuring -oxidation, catalase activity, and nonspecific carnitine acetyltransferase activity. Peroxisomal -oxidation was estimated by two methods: direct measurement of acyl coenzyme A oxidase activity  and measurement of the -oxidation spiral . Nonspecific carnitine acetyltransferase activity was estimated by the method of Gray et al. [49, 50]. Peroxisomal catalase activity was estimated by a method derived from those of Van Lente and Pepoy  and Yasmineh et al. . Protein concentrations were measured using the bicinchoninic method with bovine serum albumin as the standard ; commercially available reagents were used.
2.7. Statistical Methods
The Fisher exact test , a procedure based on the overall proportion of affected animals, was used to determine the significance of lesion incidence. Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett  and Williams [56, 57]. Clinical chemistry and peroxisomal and hepatocyte proliferation data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley  (as modified by Williams,  ) and Dunn . Jonckheere’s test  was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey  were examined by NTP personnel, and implausible values were eliminated from the analysis.
3. Results and Discussion
3.1. In Life Toxicity
All core study rats, mice, and hamsters survived to the end of the study. Final mean body weight gains of rats, mice, and hamsters were decreased by greater than 10% relative to controls at the highest two concentrations in rats and at the highest concentration in mice and hamsters (Table 1). Although initially reduced at 8,000 and 16,000 ppm (Table 1), feed consumption by exposed rats was similar to that by the controls by the end of the study (consumption was similar after week 2; data not shown). Feed consumption by mice and hamsters was generally similar to those by the controls; however, accurate estimates of food consumption were difficult to obtain due to extensive scattering of feed. Average daily doses that resulted from exposure to gemfibrozil are shown in Table 1. Doses ranged from 0.6–1300 mg/kg in rats, 1.9–2100 mg/kg in mice, and 7–2000 mg/kg in hamsters. No chemical-related clinical findings were observed in rats. Thinness was observed in mice (8,000 ppm) and in hamsters (2,000 and 24,000 ppm). The lack of decreased food consumption or signs of overt toxicity suggests that the decreased weight gains of exposed animals were due to alterations in lipid metabolism; similar findings were reported for Wy-14,643 .
| different () from the control group by Williams’ test; ; of animals surviving at 3 months/number initially in group; error.|
3.2. Clinical Chemistry Analysis
Clinical chemistry data are presented for rats, mice, and hamsters in Table 2; mice were not evaluated for liver biomarkers due to limited serum availability.
*Significantly different () from the control group by Dunn’s or Shirley’s test; **; error, statistical tests were performed on unrounded data; ; ; |
In rats, there was a treatment-related increase (approximately 1.8-fold) in serum alanine aminotransferase activity at the highest concentration on day 34. By week 13, increases (ranging between 1.4- to 2.9-fold) in alanine aminotransferase activity occurred at the top three concentrations. Additionally, increases in sorbitol dehydrogenase activity at the highest three concentrations ranged from 1.8- to 4.7-fold. The increases in serum alanine aminotransferase and sorbitol dehydrogenase activities observed in rats would suggest a treatment-related hepatocellular effect or injury, similar to that observed for the potent peroxisome proliferators Wy-14,643 . Increases in alkaline phosphatase activity and bile salt concentration, suggestive of a cholestatic event, occurred at day 34 and week 13 at the highest three concentrations. For both variables, the increases appeared to be dose-related, ranging between 1.4- to 2.3-fold for alkaline phosphatase and 2.4- to 6.7-fold for bile salts. On day 34, dose-related increases in serum cholesterol concentration occurred at the highest three concentrations; the increases were modest, ranging from 1.3- to 1.7-fold. By week 13, increases in cholesterol concentration (ranging between 1.4- to 1.9-fold) occurred in all but the lowest dose group. Conversely, at week 13, triglyceride concentration decreased by approximately 50% at the highest two concentrations.
In mice exposed for 13 weeks, a slight (20–30%) treatment-related increase in cholesterol concentration occurred at the highest three concentrations. Triglyceride concentrations, however, were decreased at the two highest concentrations; the decrease was dose-related at 35 and 44% in the 4000 and 8000 ppm dose groups, respectively.
In hamsters, increases in bile salt concentration, suggestive of a cholestatic event, occurred on day 34 and at week 13 at the highest three concentrations; the increases appeared to be dose-related, ranging between 1.7- to 7.7-fold. Alkaline phosphatase activity, another marker of cholestasis, however, was decreased at both time points at the highest three concentrations; the decreases were modest ranging between 13 to 28%. At both time points, triglyceride concentration was increased. At day 34, treatment- but not dose-related increases in serum triglyceride concentration occurred in all groups except the lowest concentration; the increases ranged from 1.2- to 1.6-fold. By week 13, triglyceride concentration was increased (1.9-fold) only at the highest concentration. There were no changes in cholesterol concentrations.
There was a clear and interesting difference between the species regarding the serum lipid (triglycerides and cholesterol) lowering effect of gemfibrozil. Rats and mice had decreases in triglycerides but increases in cholesterol concentration whereas hamsters had increases in serum triglycerides and no effect on cholesterol concentrations. The more potent peroxisome proliferator Wy-14,643  had no effect on cholesterol or triglycerides in rats, caused decreases in triglycerides and increases in cholesterol (similar to gemfibrozil in both rats and mice) in mice, and caused decreases in serum cholesterol and triglycerides in hamsters. Hamsters are a better model for human lipoprotein metabolism that rats or mice, as hamsters, like humans, make cholesterol ester transfer protein (CETP) [16, 63]. In addition, hamsters have a similar hepatic sterol synthesis rate to humans; the rate is much higher in rats and mice . It is unclear why lipid-lowering effects were not observed in hamsters following exposure to gemfibrozil in the present study.
3.3. Liver Histopathology and Weights
The incidence of hepatocyte cytoplasmic alteration was significantly increased in all exposed groups of rats and in mice exposed to 1000 ppm or greater (Table 3). The severity of this lesion was increased in rats exposed to 100 ppm or greater and in mice exposed to 4000 or 8000 ppm. A dose-related increase in severity was observed in both rats and mice. Hepatocyte cytoplasmic alteration was characterized by prominently increased cytoplasmic granularity and eosinophilia with some evidence of hepatocyte enlargement in severe cases. This change was generally diffuse but in some cases, the distribution was centrilobular to midlobular and of minimal severity. The granularity observed in the hepatocytes was considered consistent with the known hepatocellular appearance of peroxisome proliferation in the liver. Hepatocyte cytoplasmic alteration was not observed in hamsters, indicating a lack of morphological evidence of peroxisome proliferation; however, hepatic glycogen depletion was significantly increased in all exposed groups and increased in severity at the highest concentration (Table 3). Glycogen depletion was characterized by a decrease or absence of clear vacuoles in the cytoplasm of hepatocytes. The glycogen content of the liver is variable and may fluctuate depending on the physiological state of rodents. While glycogen depletion is commonly seen in animals that have been fasted, it may also be observed due to the pharmacologic or toxic effects of xenobiotic exposure.
| different () from the control group by the Fisher exact test; ; NEnot examined; ; : 1 minimal, 2 mild, 3 moderate, 4 marked; |
Absolute and relative liver weights were recorded in core (data not shown) and special study animals. Table 4 presents the relative liver weight data for special study animals on day 6, day 34, and week 13. In all three species, the maximum increase in relative liver weight was observed at week 13. At all time points, the relative liver weights of rats exposed to 100 ppm or greater were significantly increased. On day 6, the largest increase was observed at 8000 ppm, while the increases at 1000 ppm and 16000 ppm were similar. In mice, relative liver weights were increased at all durations at the highest two exposure concentrations and at all concentrations on day 34. In hamsters, more modest, but significant increases were observed at the highest two concentrations on day 34 and week 13. The largest increases in relative liver weight were observed in rats (up to 2.8-fold) and the smallest increases were in hamsters (up to 1.3-fold); liver weights in mice were increased at up to 1.7-fold.