Effects of Di-isononyl Phthalate, Di-2-ethylhexyl Phthalate, and Clofibrate in Cynomolgus Monkeys

George Pugh, Jr.*, Jason S. Isenberg{dagger}, Lisa M. Kamendulis{dagger}, David C. Ackley{dagger}, Lisa J. Clare{ddagger}, Ray Brown§, Arthur W. Lington*,1, Jacqueline H. Smith* and James E. Klaunig{dagger},2

* Exxon Biomedical Sciences, Inc., East Millstone, New Jersey 08875; {dagger} Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202; {ddagger} T.P.S., Inc., Mount Vernon, Indiana 47620; and § Research Pathology Services, Inc., New Britain, Pennsylvania 18901

Received January 5, 2000; accepted February 8, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of the peroxisome proliferators di-isononyl phthalate (DINP) and di-2-ethylhexyl phthalate (DEHP) were evaluated in young adult male cynomolgus monkeys after 14 days of treatment, with emphasis on detecting hepatic and other effects seen in rats and mice after treatment with high doses of phthalates. Groups of 4 monkeys received DINP (500 mg/kg/day), DEHP (500 mg/kg/day), or vehicle (0.5% methyl cellulose, 10 ml/kg) by intragastric intubation for 14 consecutive days. Clofibrate (250 mg/kg/day), a hypolipidemic drug used for cholesterol reduction in human patients was used as a reference substance. None of the test substances had any effect on body weight or liver weights. Histopathological examination of tissues from these animals revealed no distinctive treatment-related effects in the liver, kidney, or testes. There were also no changes in any of the hepatic markers for peroxisomal proliferation, including peroxisomal beta-oxidation (PBOX) or replicative DNA synthesis. Additionally, in situ dye transfer studies using fresh liver slices revealed that DINP, DEHP, and clofibrate had no effect on gap junctional intercellular communication (GJIC). None of the test substances produced any toxicologically important changes in urinalysis, hematology, or clinical chemistry; however, clofibrate produced some emesis, small increases in serum triglyceride, decreased calcium, and decreased weights of testes/epididymides and thyroid/parathyroid. The toxicological significance of these small changes is questionable. The absence of observable hepatic effects in monkeys at doses that produce hepatic effects in rodents suggests that DINP, DEHP, and clofibrate would also not elicit in primates other effects such as liver cancer. These data, along with results from in vitro hepatocyte studies, indicate that rodents are not good animal models for predicting the hepatic effects of phthalates in primates, including humans.

Key Words: tumor promotion; nongenotoxic carcinogens; phthalate esters; gap junctions; intercellular communication; hepatocytes; peroxisomes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxisomal proliferators are a chemically and structurally diverse class of chemicals that have been utilized in a wide spectrum of uses for a number of decades. This class of chemicals induces hepatic tumors in rats and mice and peroxisome proliferation in the liver (Ashby et al., 1994Go; IARC, 1995Go). While a mechanistic relationship between peroxisomal proliferation and hepatic cancer induction by this class of chemicals has not been confirmed, associated cellular changes, including inhibition of gap junctional intercellular communication, inhibition of apoptosis, and stimulation of cell proliferation by these agents appear to correlate with hepatic carcinogenicity. However, humans appear refractory to the induction of peroxisomal proliferation by chemicals that are peroxisome proliferators in rodents. Thus, the relevance of the various rodent liver effects, including hepatocellular carcinoma to humans, remains a topic of debate (Cattley et al., 1998Go; Doull et al., 1999Go). In the present investigation, the hepatic effects of two commercial phthalate plasticizers, di-2-ethylhexyl phthalate (DEHP) and di-isononyl-phthalate (DINP), were examined in male cynomolgus monkeys, since primates are a closer surrogate for predicting the human response than are rodents. The pharmaceutical agent clofibrate was included as a reference substance.

Clofibrate, a hypolipidemic drug developed and marketed in the early 1960s for the treatment of high cholesterol in humans (IARC, 1996Go; Tucker and Orton, 1993Go) is still used in the treatment of hypercholesterolemia. For therapeutic purposes, it is administered orally at a total daily dose of 2 g, or ~ 20–30 mg per kilogram body weight per day (mg/kg/day) (Brown and Goldstein, 1990Go; IARC, 1996Go). Clofibrate was used as a reference substance because it appears to be a more potent inducer of peroxisomal-proliferation-related hepatic effects in rats and mice than the phthalate esters (Ashby et al., 1994Go; Cohen and Grasso, 1981Go; Doull et al., 1999Go; Lake et al., 1984Go). Additionally, because there are nearly 40 years of human clinical experience with this drug (Brown and Goldstein, 1990Go; Tucker and Orton, 1993Go), the clofibrate data affords the opportunity to relate the primate data to humans.

IARC reviewed the comprehensive human data and found no evidence for cancer associated with the use of clofibrate when used by humans as a cholesterol-lowering drug (IARC, 1996Go). Likewise, numerous high-dose studies in rhesus monkeys and marmosets treated with clofibrate for up to 6.5 years at doses exceeding the therapeutic dose have shown no evidence of peroxisome proliferation, liver tumors, or related hepatic effects (Tucker and Orton, 1993Go). However, at a dietary dose of 5000 ppm (~250 mg/kg/day), clofibrate induced peroxisomes and produced liver tumors in 20 to 91% of chronically treated rats (Ashby et al., 1994Go; Doull et al., 1999Go; Hartig et al., 1982Go; Reddy and Qureshi, 1979Go; Svoboda and Azarnoff, 1979Go).

There are no comparable epidemiology data available for the phthalate esters, but there are extensive animal data. Chronic feeding of DEHP to male Fischer 344 rats produced an increased incidence of liver tumors (hepatocellular carcinoma or neoplastic nodules) at dietary doses of 12,000 ppm (~670 mg/kg/day) but not at 6000 ppm (~320 mg/kg/day) (Kluwe et al., 1982Go). More recent studies using lower doses of DEHP reported an increase in hepatocellular tumors in male F344 rats following treatment with 2500 ppm (estimated daily intake ~140 mg/kg/day), but not at 500 ppm (estimated as ~30 mg/kg/day) (David et al., 1999Go; Doull et al., 1999Go). DINP produced no increase in hepatic tumors in male F344 rats at lifetime dietary doses of 6000 ppm (>360 mg/kg/day) (Butala et al., 1996Go; Lington et al., 1997Go). However, the incidence of combined hepatic adenomas and carcinomas increased to 26% of rats on lifetime dietary doses of 12,000 ppm DINP (>730 mg/kg/day) (Butala et al., 1996Go). There are comparable results in chronic feeding studies in mice (Kluwe et al., 1982Go; Butala et al., 1997Go; David et al., 1999Go; Doull et al., 1999Go). Estimates of human exposure to phthalates range from approximately 10–50 µg/kg/day (ATSDR, 1993Go; Doull et al., 1999Go; Peijnenburg et al., 1991Go) but can be higher for some specific applications such as medical devices (Doull et al., 1999Go; Huber et al., 1996Go).

Previous studies of DINP and DEHP in non-human primates focused on hepatic and metabolic effects occurring within 14 to 21 days of treatment. Treatment of cynomolgus monkeys with up to 500 mg/kg/day of DEHP by oral gavage produced no histological (light and electron microscopy) or biochemical evidence of peroxisome proliferation as assessed by determination of peroxisomal beta-oxidation (PBOX) activity and lauric acid hydroxylation (Astill, 1989Go; Short et al., 1987Go). Likewise, marmosets treated with up to 200 mg/kg/day DEHP by oral gavage for 14 consecutive days did not exhibit morphological or biochemical changes in the liver (Rhodes et al., 1986Go). In several in vitro studies, rat and primate hepatocytes were exposed to the active monoester metabolite of DINP and/or DEHP. The dramatic differences in response provided additional evidence that primates, including humans, are insensitive to the hepatic effects of peroxisomal proliferators in contrast to rodents (Baker et al., 1996; Benford et al., 1986; Bichet et al., 1990; Dirven et al., 1993; Elcombe and Mitchell, 1986; Kamendulis et al., 1999 [unpublished]). In vitro studies with clofibrate, ciprofibrate, and derivatives show similar results (Bichet et al., 1990Go; Blaauboer et al., 1990Go; Elcombe et al., 1997; Perrone et al., 1998Go).

Short-term (2–4 weeks) exposure of DINP and DEHP to rats and mice inhibits gap junctional intercellular communication (GJIC) and increases PBOX activity and replicative DNA synthesis (Isenberg et al., 1999; Smith et al., 2000Go). Compounds that block GJIC and increase replicative DNA synthesis appear to function at the tumor promotion phase of the chemical carcinogenesis process. Modulation of these endpoints has been implicated in peroxisome proliferator-induced hepatocarcinogenesis in rodents. The purpose of this study was to determine whether there were any effects on these more sensitive, additional hepatic endpoints, as well as other systemic effects, in cynomolgus monkeys treated with DINP and DEHP in comparison to those treated with clofibrate. The rationale was that observations in a non-human primate, such as the cynomolgus monkey, would provide more meaningful information on the human health response to phthalates and other peroxisome proliferators. The dose level of 500-mg/kg/day DINP and DEHP chosen for these studies produced hepatic effects in rats. Further, the administration of DEHP to cynomolgus monkeys at doses greater than 500 mg/kg/day will not necessarily increase the absorbed dose in this species (Short et al., 1987Go). Clofibrate was administered at a dose of 250 mg/kg/day, which is comparable to the dietary dose in rats that induces peroxisomes and produces liver tumors (5000 ppm, ~250 mg/kg/day) (Reddy and Qureshi, 1979Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
JAYFLEX® DINP Plasticizer (di-isononyl phthalate, DINP, >98% purity, CAS RN 68515–48–0) was obtained from Exxon Chemical Company (Houston, TX). Phthalates are produced by esterification of phthalic anhydride with various alcohols in a closed system. The alcohol used to make DINP is prepared by oligimerization of propylene and mixed butenes resulting in a C9-rich mixture consisting of roughly equivalent amounts of 3,4-, 4,6-, 3,6-, 4,5-, and 5,6-dimethyl heptanol, with smaller amounts of methyl octanol, and iso-decanol. Di-2-ethylhexyl phthalate (DEHP, >98% purity, CAS RN 117–81–7) and phthalic acid (PA, 99+% purity) were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). Clofibrate (2-[4-chlorophenoxy]-2-methylpropionic acid, ethyl ester, >99% purity) and methyl cellulose were purchased from Sigma Chemical Company, Inc. (St. Louis, MO). Mono-isononyl phthalate (MINP) was synthesized by Aldrich Chemical from the same type of alcohol feedstock used to make the DINP, and mono-2-ethylhexyl phthalate (MEHP) was a gift from Dr. Heindel (NIEHS, Research Triangle Park, NC).

The use of methylcellulose (0.5%) as the vehicle control was based on preliminary studies that indicated that the use of corn oil as a vehicle reduced appetite and produced gastrointestinal upset. Dosing solutions were prepared once weekly by dissolving the appropriate amounts of test articles in 0.5% methylcellulose to give the specified dose in a volume of 10 ml/kg.

Animals and treatment.
Captive-bred, colony-raised, naïve young adult (~2-year-old) male cynomolgus monkeys (Macaca fascicularis) were obtained from HRP-Texas Primate Center (Covance Research Products, Inc., Alice, TX) and acclimated for at least 30 days prior to treatment initiation. Monkeys were housed individually in stainless steel cages suspended over a flush pan in an isolated-temperature (66–78°F) and humidity-controlled (63–88%) animal room with filtered air supply (10–15 air changes/h) and cycled lighting (12 h of light daily). All monkeys were shown to be free of internal parasites, SRV, SIV, Herpes B virus, HSV-1, hepatitis B, and tuberculosis.

At study initiation, monkeys weighing 1977 to 2921 grams were randomly assigned to one of the following treatment groups (4/group): vehicle (0.5% methyl cellulose), DEHP (500 mg/kg/day), DINP (500 mg/kg/day), or clofibrate (250 mg/kg/day). Test materials were administered in a constant volume of 10 ml/kg once a day for 14 consecutive days using an adult/pediatric nasogastric feeding tube.

Animal observations.
All monkeys were observed twice daily for mortality, morbidity, and toxicological or other clinical signs, including behavioral changes, appetite, and excreta. Standard and acceptable veterinary health care practices were used during this evaluation to ensure good health of the animals. Individual body weights were obtained upon receipt, weekly during the pretest and study periods, and prior to necropsy. Monkeys were given 6 biscuits of PMI® Monkey Diet No. 5038 each morning and late afternoon and monitored for food consumption except when overnight deprivation was required for clinical pathology collections and prior to necropsy.

Hematology and serum chemistry.
Blood samples were collected from the femoral vein of each monkey after an overnight fast, during the second and fourth weeks of the pretest period and prior to necropsy. Hematologic measurements and calculations were by standard methods and included leukocyte count (total and differential), erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet count. Clinical chemistry parameters were assessed by standard methods and included sodium, potassium, chloride, blood urea nitrogen, glucose, creatinine, cholesterol, alanine aminotransferase, aspartate aminotransferase, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, inorganic phosphorus, calcium, triglycerides, and alkaline phosphatase.

Urinalysis.
Urine was collected from each monkey during the overnight fasting period (16–18 h) prior to the collection of blood samples. Urine evaluations were performed by standard methods, which included specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, occult blood, leukocytes, nitrite, and a microscopic examination.

Necropsy.
Monkeys were sacrificed on the day following the last dose (day 15). Each animal was anesthetized with ~10 mg/kg Ketamine HCl (im), euthanized with pentobarbital (iv, 325 mg/ml at 1 ml/4.5 kg), and then exsanguinated. A gross necropsy was performed including a thorough visual examination of all organs and body tissues. Organ weights were obtained for the liver, kidneys, testes/epididymides, adrenals, brain, heart, lung, spleen, and thyroid/parathyroid, and organ to body weight ratios were calculated. Sections of liver, kidney, and testes, were fixed in 10% formalin and embedded in paraffin for subsequent histopathological evaluation, after staining with hematoxylin and eosin. Sections of liver were also examined for replicative DNA synthesis by immunohistochemical detection, using proliferating cell nuclear antigen (PCNA). Additional slices of fresh liver were used for evaluation of GJIC. The remaining sections were flash frozen in liquid nitrogen for assessment of peroxisomal activity and determination of phthalate and metabolite concentrations.

Replicative DNA synthesis assay.
Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed as previously described by Foley et al. (1993). Briefly, hepatocytes that expressed PCNA were visualized by the accumulation of red pigment within the nuclei compared to the counterstained blue nuclei of non-labeled cells. The labeling index was determined by dividing the total number of labeled hepatocytes by the total number of hepatocytes counted and multiplying by 100. A total of at least 1000 hepatocytes were examined for each animal in each treatment group.

Gap Junctional Intercellular Communication (GJIC).
Immediately upon sacrifice, a representative strip of liver was used for determination of GJIC by the direct measure of dye flow into liver slices using incision loading dye transfer as described by Isenberg et al. (1999).

Peroxisomal Beta-Oxidation (PBOX) activity.
Hepatic PBOX activity was measured by the method of Lazarow and DeDuve (1976) as described by Isenberg et al. (1999). All test samples were stored at –80°C until measurement.

Analysis of DEHP and DINP and metabolites in liver.
Extraction and high pressure liquid chromatography (HPLC) analysis of phthalates and their corresponding monoester metabolites (MEHP and MINP) and phthalic acid (PA) in the liver were performed as previously described by Isenberg et al. (1999) and Smith et al. (1999).

Statistical evaluation.
Statistical differences (p < 0.05) from control values for all data were determined by ANOVA followed by a Dunnett's test (Gad and Weil, 1988Go). The least-squares means post-hoc test was used for analysis of the dosimetry data. Data are expressed as the mean ± standard deviation (SD).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal Observations
There were no overt changes in the general health or behavior of the monkeys following 14 days of dosing. With the possible exception of occasional emesis in two animals treated with clofibrate, following dosing, no adverse treatment-related effects were observed.

Body and Organ Weights
Treatment with DINP and DEHP for 14 days had no effect on body weights (Table 1Go), food consumption (data not shown), or relative weights of any organs assessed, including liver, kidney, thyroid/parathyroid, and testes/epididymides (Table 1Go). Statistically significant decreases in relative thyroid/parathyroid and testes/epididymides weights were observed in monkeys treated with clofibrate for 14 days (Table 1Go). Treatment with DINP, DEHP, and clofibrate for 14 days had no effect on absolute or relative weights of adrenals, brain, heart, lung, or spleen (data not shown).


View this table:
[in this window]
[in a new window]
 
TABLE 1 Effects of DEHP, DINP and Clofibrate on Body and Organ Weights in Cynomolgus Monkeys
 
Serum Chemistry and Urinalysis
Treatment of monkeys with 500 mg/kg/day DINP and DEHP for 14 days did not produce any changes in serum chemistry that were significantly different from control-treated monkeys. Statistically significant changes seen only in serum triglycerides and calcium levels were observed in monkeys treated with clofibrate when compared to controls (Table 2Go). Urinalysis was unremarkable (data not shown).


View this table:
[in this window]
[in a new window]
 
TABLE 2 Effects of DEP, DINP, and Clofibrate on Serum Chemistry in Cynomolgus Monkeys
 
Hematology
There were no important changes in hematological parameters associated with the administration of DEHP, DINP, or clofibrate. Statistically significant increases in neutrophil count and decreases in lymphocyte count were observed in monkeys treated with DINP for 14 days (Table 3Go). These values were similarly affected in other treatment groups, but changes were not statistically significant.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Effects of DEHP, DINP, and Clofibrate on Hematology in Cynomolgus Monkeys
 
Peroxisomal Beta-Oxidation (PBOX)
There were no statistically significant increases in PBOX activity in cynomolgus monkeys treated with DEHP, DINP, and clofibrate for 14 days (Table 4Go).


View this table:
[in this window]
[in a new window]
 
TABLE 4 Effects of DEHP, DINP and Clofibrate on Gap Junctional Intracellular Communication (GJIC) and Indicators of Perosixomal Proliferation
 
Gap Junction Intercellular Communication (GJIC). DEHP, DINP, and clofibrate had no effect on GJIC (Table 4Go). In the control group, the distance of dye transfer was 0.26 ± 0.03 mm. In the treated groups, the distance of dye transfer for DEHP, DINP and clofibrate was 0.28 ± 0.01 mm, 0.27 ± 0.02 mm, and 0.27 ± 0.03 mm, respectively.

Replicative DNA Synthesis
There were no statistically significant changes in total hepatic DNA synthesis in animals treated with DEHP, DINP, or clofibrate (Table 4Go).

Histopathology
Microscopic evaluations were performed on tissues collected from the liver, kidney and testes of control and treated monkeys. Diffuse hepatocellular vacuolation was observed in one animal in the DEHP treatment group and in one animal in the clofibrate treatment group. There were no distinctive treatment-related effects observed in the kidneys or testes following DEHP, DINP, or clofibrate administration (Table 4Go). Furthermore, no signs of inflammation or necrosis were seen in any of the tissues evaluated.

Analysis of DINP, DEHP, and Metabolites in Liver and Serum
Low levels of DINP and DEHP were detected in the livers of monkeys treated with the methyl cellulose vehicle control. Due to the ubiquitous nature of the phthalate esters, the low level of phthalate esters measured in control samples may have resulted from leeching during storage of the samples. In the DINP and DEHP treatment groups, the parent diester, monoester metabolite, and phthalic acid (PA) were detected. DEHP (500 mg/kg/day) resulted in MEHP levels of 17.0 µmol/g compared with MINP levels of 2.2 µmol/g in monkeys treated with DINP (500 mg/kg/day). Higher levels of DEHP were found in the liver as compared to DINP; however, this finding was small and not statistically significant. The levels of PA were similar for monkeys treated with DEHP and DINP (Table 5Go).


View this table:
[in this window]
[in a new window]
 
TABLE 5 Analysis of DEHP, DINP and Metabolites in Cynomolgus Monkey Liver
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The overall objective of this study was to assess the effects of DEHP, DINP, and clofibrate on peroxisomal proliferation in the cynomolgus monkey. The specific parameters measured included weights and histologic changes in target organs (liver, kidneys, and testes), induction of hepatic peroxisomal enzymes, hepatic replicative DNA synthesis, and hepatic gap junctional intracellular communication (GJIC). Recent evidence suggests that while induction of peroxisomal proliferation per se does not correlate with hepatic carcinogenesis, other cellular changes that are frequently seen with the peroxisome-proliferating compounds (blockage of GJIC, decrease apoptosis, increased cell proliferation) have been implicated in nongenotoxic/tumor-promotion aspects of the cancer process. Increased hepatic peroxisome proliferation is among the most prominent cellular effect observed with phthalate treatment in rodents. This change, along with inhibition of GJIC, is believed to be involved in rodent liver carcinogenesis. The absence of changes in the liver is in agreement with studies that have shown primates unresponsive to the hepatic effects of peroxisome proliferators (Foxworthy et al., 1990Go; Gray and De la Iglesia, 1984Go; Hall et al., 1999Go; Holloway, 1982; Kurata et al., 1997). On the other hand, these results differ significantly from rodent studies in which phthalates alter GJIC, PBOX and replicative DNA synthesis at doses similar to those used in the present study (Isenberg et al., 1999; Smith et al., 1999). The lack of modulation of these endpoints in primates by peroxisome proliferators provides additional evidence for species-specificity of phthalates in the induction of carcinogenesis.

Increases in liver and kidney weight have been shown to occur with peroxisomal proliferators. In the present study, all of the test compound studies failed to elicit an increase in liver or kidney weight. Relative weight of testes/epididymis was unaffected by DEHP or DIDP treatment but was significantly decreased in clofibrate-treated animals. This finding was likely due to a decreased absolute weight in one of the animals (data not shown), and does not appear to be treatment-related. We have also observed that the relative thyroid/parathyroid weight was decreased in cynomolgus monkeys treated with clofibrate. Although this decrease was statistically significant, there were no obvious signs of toxicity associated with this change. In studies conducted in marmosets, a non-human primates species, Hall et al. (1999) demonstrated that neither DINP (2500 mg/kg/day) nor clofibrate had any effect on the relative liver weights over a 13-week treatment period. In similar studies, Kurata et al. (1998) showed that neither DEHP (2500 mg/kg/day) nor clofibrate (250 mg/kg/day) had any effect on relative liver, kidney, or testes weights.

There were no statistically significant changes in serum chemistry values in monkeys treated with DEHP or DINP in the present study. These results are in agreement with previous reports in which treatment with DEHP or DINP for 13 weeks in marmosets produced no effect on serum lipid measurements (Hall et al., 1999Go; Kurata et al., 1998Go). In the present study, clofibrate induced slight changes in blood triglyceride and calcium levels. No changes in cholesterol were observed following any of the treatments. The dose of clofibrate used in this study (250 mg/kg/day) exceeded the therapeutic dose for humans (20–30 mg/kg/day), (Brown and Goldstein, 1990Go; IARC, 1996Go). Clofibrate at 250 mg/kg/day produces marked effects on hepatic peroxisome proliferation in rodents (Ashby et al., 1994Go; Cohen and Grasso, 1981Go; Doull et al., 1999Go; Lake et al., 1984Go). It might therefore be expected that clofibrate treatment would alter the serum lipid balance in cynomolgus monkeys. Since the serum chemistry measurements in the present study examined total cholesterol and did not separate sub-fractions of cholesterol, clofibrate may have affected the overall lipid balance. Additionally, the efficacy of clofibrate on non hyperlipidemic rodents and/or primates is not known. These results are consistent with those reported previously for the marmoset that showed no change in cholesterol or trigliceride levels following clofibrate treatment for 13 weeks (Hall et al., 1999Go; Kurata et al., 1998Go).

The induction of peroxisome proliferation has been implicated as a mechanism in rodent hepatocarcinogenesis. It has been hypothesized that peroxisome proliferators increase the activity of peroxisomal peroxide-producing enzymes in the liver without elevating the levels of H2O2 scavenging enzymes (Rao and Reddy, 1991Go; Reddy and Rao, 1986Go). Following this line of reasoning, the level of oxidative stress to the cell would be expected to increase dramatically. The resulting oxidative stress could result in oxidative DNA adducts (OH8dG) and/or could modulate normal cellular processes. In the former, oxidative adducts may produce mutation, and/or modify gene expression resulting in increased cell proliferation. In the latter case, oxidative modification of cellular processes including GJIC, second messengers, gene transcription elements, and mitochondrial function may result in aberrant cell proliferation. In the present studies, DEHP, DINP, and clofibrate showed no evidence for inducing PBOX activity in cynomolgus monkeys. Similar observations were made in marmosets treated for 90 days with DINP (2500 mg/kg/day) (Hall et al., 1999Go) and DEHP (2500 mg/kg/day) (Kurata et al., 1998Go).

In order to relate the results of the present work to humans, we chose clofibrate as a reference material, because its potency as a peroxisome proliferator is greater than the phthalates and also because it is used therapeutically for long periods of time by human patients with altered cholesterol metabolism. Liver biopsies from patients receiving hypolipidemic drugs such as clofibrate or gemfibrozil for long-term therapy did not reveal any evidence of increased peroxisomal enzyme activity (De La Iglesia et al., 1982Go; Hanefeld et al., 1980Go). Thus, the results of the primate studies of markers of peroxisome proliferation are consistent with human experience.

It has been hypothesized that elevated rates of cellular DNA synthesis represent an important mechanism by which DEHP and related phthalates induce tumors in rodents (Cattley et al., 1990Go; Chen et al., 1994Go; Roberts et al., 1995Go). In contrast DEHP, DINP, and clofibrate had no effect on DNA synthesis in cynomolgus monkeys. Similar to the species-specific response observed in the GJIC and PBOX assays, replicative DNA synthesis appears to be selectively modulated in rodents. Potent induction of replicative DNA synthesis has been reported in the rat following treatment with the hepatocarcinogen Wy-14,623 (Wada, 1992); however, only a slight increase was observed in hamsters (Durnford et al., 1995Go; Lake et al., 1993Go). Species differences have been reported in other studies where ciprofibrate produced an acute increase in replicative DNA synthesis in rats, but resulted in a small decrease in DNA synthesis in human hepatocytes (Perrone et al., 1998Go).

There was no evidence of inhibition of GJIC in the primate studies. Similarly, in vitro studies using hepatocytes isolated from primates failed to demonstrate an inhibition of GJIC following exposure to peroxisome proliferators (Kamendulis et al., 1999, unpublished). The strongest evidence supporting a causal relationship between GJIC inhibition and cancer has been shown in rodents where GJIC is inhibited in vivo in rats and mice at tumorigenic doses of phthalates, but not at lower, non-tumorigenic doses (Isenberg et al., 1999; Smith et al., 1999). In studies using primary cultured rat and mouse hepatocytes, Kamendulis et al. (1999, unpublished) have shown that the major monoester metabolite of several phthalates inhibits GJIC; however no effects are observed in hepatocytes isolated from hamsters or humans. Based on these results, it appears that inhibition of GJIC by phthalates such as peroxisomal proliferation is species-specific and dose-dependent.

In an attempt to quantitate the actual levels of monoester metabolites present in the livers of monkeys, following DEHP and DINP treatment, HPLC analysis was performed on liver samples collected at the time of sacrifice. Consistent with published studies indicating that the monoester is the putative hepatotoxic metabolite, we observed significantly higher levels of MEHP in the liver compared to levels of the parent diester, DEHP. Interestingly, higher levels of MINP were not associated with DINP administration. This finding is possibly due to problems encountered in the extraction-recovery procedure rather than to incomplete hydrolysis of DINP to MINP. Following metabolism to the monoester metabolite, the phthalate monoesters are subsequently metabolized to phthalic acid. Similar levels of this metabolite were found in both DEHP- and DINP-treated animals and it does not appear to play a significant role in phthalate-induced liver toxicity. Although the monoester metabolites were detected in the livers of monkeys treated with DEHP and DINP, the levels of these metabolites were much lower than the levels reported in rodents (Isenberg et al., 2000Go; Smith et al., 1999). This finding may be attributed to the fact that the rate of DEHP hydrolysis in the gut of primates is much less than the rate observed in rats (Rhodes et al., 1986Go; Short et al., 1987Go).

In conclusion, the present findings showed primates to be unresponsive to the induction of DNA synthesis and peroxisomal ß-oxidation, and to inhibition of GJIC following short-term treatment with these established rodent peroxisome proliferators and liver carcinogens. Furthermore, these data, in combination with previous research, lend additional support to the hypothesis that the hepatotoxicity of peroxisome proliferators is species-specific and that rodents may not be an appropriate model to predict the carcinogenic risk for peroxisome proliferating compounds in humans. Based on the present study and previous findings, phthalate esters do not appear to produce hepatic effects associated with peroxisome proliferation and hepatic carcinogenicity in humans.


    ACKNOWLEDGMENTS
 
This manuscript is dedicated to the memory of our deceased colleague, Arthur W. Lington, for his enthusiasm and dedication to the mechanistic understanding of hepatocarcinogenesis induced by peroxisome proliferators. We thank Dr. Richard H. McKee for his critical review of and input on this manuscript.


    NOTES
 
1 Deceased. Back

2 To whom correspondence should be addressed at Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 625 Barnhill Drive, MS-1021, Indianapolis, IN 46202–5120. Fax: (317) 274-7787. E-mail: jklauni{at}iupui.edu. Back

Presented in part at the 38th annual meeting of the Society of Toxicology in New Orleans, LA, March 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ashby, J., Brady, A., Elcombe, C. R., Elliot, B. M., Ishmael, J., Odum, J., Tugwood, J. D., Kettle, S., and Purchase, I. F. (1994). Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Hum. Exp. Toxicol. 13(Suppl. 2), S1–S117.

Astill, B. D. (1989). Metabolism of DEHP: Effects of prefeeding and dose variation, and comparative studies in rodents and the cynomolgus monkey (CMA studies). Drug Metab. Rev. 21, 35–53.[ISI][Medline]

ATSDR (1993). Toxicological profile for di(2-ethylhexyl) phthalate. US Dept. Health & Human Services, TP-92/05, 147 pages.

Baker, T. K., Kalimi, G. H., Lington, A. W., Isenberg, J. S., Klaunig, J. E., and Nikiforov, A. I. (1996). Gap junctional intercellular communication (GJIC) studies on 5 phthalate monoesters in hepatocytes of four species: Implications for cancer risk assessment. Toxicologist 30, 208.

Benford, D. J., Patel, S., Reavy, H. J., Mitchell, A., and Sarginson, N. J. (1986). Species differences in the response of cultured hepatocytes to phthalate esters. Food Chem. Toxicol. 24, 799–800.[ISI]

Bichet, N., Cahard, D., Fabre, G., Remandet, B., Gouy, D., and Cano, J.-P. (1990). Toxicological studies on a benzofuran derivative: III. Comparison of peroxisome proliferation in rat and human hepatocytes in primary culture. Toxicol. Appl. Pharmacol. 106, 509–517.[ISI][Medline]

Blaauboer, B. J., van Hosteijn, C. W., Bleumink, R., Mennes, W. C., van Pelt, F. N., Yap, S. H., van Pelt, J. F., Van Iersel, A. A., Timmerman, A., and Schmid, B. P. (1990). The effect of beclobric acid and clofibric acid on peroxisomal oxidation and peroxisome proliferation in primary cultures of rat, monkey, and human hepatocytes. Biochem. Pharmacol. 40, 521–528.[ISI][Medline]

Brown, M. S., and Goldstein, J. L. (1990). Drugs used in the treatment of hyperlipoproteinemias. In The Pharmacological Basis of Therapeutics, 8th Ed. (A. G. Gilman, T. W. Rall, A. S. Nies, and P. Taylor, Eds.), pp. 874–896. Pergamon Press, New York.

Butala, J. H., Moore, M. R., Cifone, M. A., Bankston, J. R., and Astill, B. (1996). Oncogenicity study of di (isononyl) phthalate in rats. Toxicologist 30, 202.

Butala, J. H., Moore, M. R., Cifone, M. A., Bankston, J. R., and Astill, B. (1997). Oncogenicity study of di (isononyl) phthalate in mice. Toxicologist 36, 173.

Cattley, R. C., DeLuca, J., Elcombe, C., Fenner-Crisp, P., Lake, B. G., Marsman, D. S., Pastoor, T. A., Popp, J. A., Robinson, D. E., Schwetz, B., Tugwood, J., and Wahli, W. (1998). Do peroxisome proliferating compounds pose a hepatocarcinogenic hazard to humans? Regul. Toxicol. Pharmacol. 27, 47–60.[ISI]

Cattley, R. C., Marsman, G., and Popp, J. A. (1990). Cell proliferation and promotion in the hepatocarcinogenicity of peroxisome proliferating chemicals. In Mutation and the Environment, Part D (M. L. Mendelsohn, Ed.), pp. 1123–1132. Wiley-Liss, New York.

Chen, H., Huang, C., Wilson, M. W., Lay, L. T., Robertson, L. W., Chow, C. K., and Glauert, H. P. (1994). Effect of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cell proliferation and toxicity in Sprague-Dawley rats. Carcinogenesis 15, 2847–2850.[Abstract]

Cohen, A. J., and Grasso, P. (1981). Review of the hepatic response to hypolipidaemic drugs in rodents and assessment of its toxicological significance to man. Food Cosmet. Toxicol. 19, 585–605.[ISI][Medline]

David, R. M., Moore, M. R., Cifone, M. A., Finney, D. C., and Guest, D. (1999). Chronic peroxisome proliferation and hepatomegaly associated with the hepatocellular tumorigenesis of di(2-ethylhexyl)phthalate and the effects of recovery. Toxicol. Sci. 50, 195–205.[Abstract]

De La Iglesia, F. A., Lewis, J. E., Buchanan, R. A., Marcus, E. L., and McMahon, G. (1982). Light and electron microscopy of liver in hyperlipoproteinemic patients under long-term gemfibrozil treatment. Atherosclerosis 43, 19–37.[ISI][Medline]

Dirven, H. A., van den Broek, P. H., Peeters, M. C., Peters, J. G., Mennes, W. C., Blaauboer, B. J., Noordhoek, J., and Jongeneelen, F. J. (1993). Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey. Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies. Biochem. Pharmacol. 45, 2425–2434.[ISI][Medline]

Doull, J., Cattley, R., Elcombe, C., Lake, B. G., Swenberg, J., Wilkinson, C., Williams, G., and van Gemert, M. (1999). A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new U.S. EPA risk assessment guidelines. Regul. Toxicol. Pharmacol. 29, 327–357.[ISI][Medline]

Durnford, J. M., Brodey, R. S., Hejtmancik, M. R., Kurtz, P. J., Renne, R. A., Gideon, K. M., and Marsman, D. S. (1995). Differential enzyme-inducing activity of peroxisome proliferators in the male Syrian hamster. Int. Toxicol. VII 47, 4.

Elcombe, C. R., Bell, D. R., Elias, E., Hasmall, S. C., and Plant, N. J. (1996). Peroxisome proliferators: species differences in response of primary hepatocyte cultures. Ann. N Y Acad. Sci. 804, 628–635.[ISI][Medline]

Elcombe, C. R., and Mitchell, A. M. (1986). Peroxisome proliferation due to di(2-ethylhexyl) phthalate (DEHP): species differences and possible mechanisms. Environ. Health Perspect. 70, 211–219.[ISI][Medline]

Foley, J., Ton, T., Maronpot, R., Butterworth, B., and Goldsworthy, T. L. (1993). Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ. Health Perspect. 101(Suppl. 5), 199–205.

Foxworthy, P. S., White, S. L., Hoover, D. M., and Eacho, P. I. (1990). Effect of ciprofibrate, bezafibrate and LY171883 on peroxisomal beta-oxidation in cultured rat, dog, and rhesus monkey hepatocytes. Toxicol. Appl. Pharmacol. 104, 386–394.[ISI][Medline]

Gad, S., and Weil, C. S. (1988). Statistics and Experimental Design for Toxicologists. Telford Press, New Jersey.

Gray, R. H., and de la Iglesia, F. A. (1984). Quantitative microscopy comparison of peroxisome proliferation by the lipid-regulating agent gemfibrozil in several species. Hepatology 4, 520–530.[ISI][Medline]

Hall, M., Matthews, A., Webley, L., and Harling, R. (1999). Effects of di-isononyl phthalate (DINP) on peroxisomal markers in the marmoset-DINP is not a peroxisome proliferator. Toxicol. Sci. 24, 237–244.

Hanefeld, M., Kemmer, C., Leonhardt, W., Kunze, K. D., Jaross, W., and Haller, H. (1980). Effects of p-chlorophenoxyisobutyric acid (CPIB) on the human liver. Atherosclerosis 36, 159–172.[ISI][Medline]

Hartig, F. Stegmeir, K., and Hebold, G. (1982). Study of liver enzymes: Peroxisome proliferation and tumour rates in rats at the end of carcinogenicity studies with bezafibrate and clofibrate. Ann. N.Y. Acad. Sci. 386, 464–467.[ISI]

Holloway, B. R., Thorp, J. M., Smith, G. D., and Peters, T. J. (1982). Analytical subcellular fractionation and enzymic analysis of liver homogenates from control and clofibrate-treated rats, mice, and monkeys with reference to the fatty acid-oxidizing enzymes. Ann. N.Y. Acad. Sci. 386, 453–454.[ISI]

Huber, W. W., Grasl-Kraupp, B., Schulte-Hermann, R. (1996). Hepatocarcinogenic potential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk. Crit. Rev. Toxicol. 26, 365–481.[ISI][Medline]

International Agency for Research on Cancer (IARC) (1995). Peroxisome proliferation and its role in carcinogenesis. IARC Technical Report No. 24. IARC, Lyon, France.

International Agency for Research on Cancer (IARC) (1996). Clofibrate, In IARC Monographs, Some Pharmaceutical Drugs, Vol. 66, pp. 391–426, IARC, Lyon, France.

Isenberg, J. S., Kamendulis, L. M., Smith, J. H. Ackley, D. C., Pugh, G., Jr., Lington, A. W., and Klaunig, J. E. (2000). Effects of di-2-ethylhexyl phthalate (DEHP) on gap junction intercellular communication (GJIC), DNA synthesis and peroxisomal beta-oxidation in rat, mouse, and hamster liver. Toxicol. Sci. (in press).

Kluwe, W. M., Haseman, J. K., Douglas, J. F., and Huff, J. E. (1982). The carcinogenicity of dietary di(ethylhexyl)phthalate (DEHP) in Fischer 344 rats and B6C3F1 mice. J. Toxicol. Environ. Health 10, 797–815.[ISI][Medline]

Kurata, Y., Kidachi, F., Yokoyama, M., Toyota, N., Tsuchitani, M., and Katoh, M. (1998). Subchronic toxicity of di(2-ethylhexyl)phthalate (DEHP) in common marmosets: lack of hepatic peroxisome proliferation, testicular atrophy, or pancreatic acinar cell hyperplasia. Toxicol. Sci. 42, 49–56.[Abstract]

Lake, B. G., Evans, J. G., Cunninghame, M. E., and Price, R. J. (1993). Comparison of the hepatic effects of nafenopin and WY-14,643 on peroxisome proliferation and cell replication in the rat and Syrian hamster. Environ. Health Perspect. 101(Suppl. 5), 241–247.

Lake, B. G., Rijcken, W. R., Gray, T. J., Foster, J. R., and Gangolli, S. D. (1984). Comparative studies of the hepatic effects of di- and mono-n-octyl phthalates, di-(2-ethylhexyl) phthalate and clofibrate in the rat. Acta. Pharmacol. Toxicol. 54, 167–176.[Medline]

Lazarow, P. B., and De Duve, C. (1976). A fatty acyl-CoA oxidizing system in rat liver peroxisomes: enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. U S A 73, 2043–2046.[Abstract]

Lington, A. W., Bird, M. G., Plutnick, R. T., Stubblefield, W. A., and Scala, R. A. (1997). Chronic toxicity and carcinogenic evaluation of diisononyl phthalate in rats. Fundam. Appl. Toxicol. 36, 79–89.[ISI][Medline]

Peijnenburg, W. J. G. M., Ewijk, M, de Hann, M. W. A., Janus, J. A., Ros, J. P. M., Slooff, W., and Velde, E. G. (1991). Update of the exploratory report: Phthalates. Report no. 710401008. National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.

Perrone, C. E., Shao, L., and Williams, G. M. (1998). Effect of rodent hepatocarcinogenic peroxisome proliferators on fatty acyl-CoA oxidase, DNA synthesis, and apoptosis in cultured human and rat hepatocytes. Toxicol. Appl. Pharmacol. 150, 277–286.[ISI][Medline]

Rao, M. S., and Reddy, J. K. (1991). An overview of peroxisome proliferator-induced hepatocarcinogenesis. Environ. Health Perspect. 93, 205–209.[ISI][Medline]

Reddy, J. K., and Qureshi, S. A. (1979). Tumorigenicity of the hypolipidaemic peroxisome proliferator ethyl-a-p-chlorophenoxyisobutyrate (clofibrate) in rats. Br. J. Cancer 40, 476–482.[ISI][Medline]

Reddy, J. K., and Rao, M.S. (1986). Peroxisome proliferators and cancer: mechanisms and implications. Trends Pharmacol. Sci. 7, 438–443.[ISI]

Rhodes, C., Orton, T. C., Pratt, I. S., Batten, P. L., Bratt, H., Jackson, S. J., and Elcombe, C. R. (1986). Comparative pharmacokinetics and subacute toxicity of di-(2-ethylhexyl) phthalate (DEHP) in rats and marmosets: Extrapolation of effects in rodents to man. Environ. Health Perspect. 65, 299–307.[ISI][Medline]

Roberts, R. A., Soames, A. R., Gill, J. H., James, N. H., and Wheeldon, E. B. (1995). Non-genotoxic hepatocarcinogens stimulate DNA synthesis and their withdrawal induces apoptosis, but in different hepatocyte populations. Carcinogenesis 16, 1693–1698.[Abstract]

Short, R. D., Robinson, E. C., Lington, A. W., and Chin, A. E. (1987). Metabolic and peroxisome proliferation studies with di(2-ethylhexyl)phthalate in rats and monkeys. Toxicol. Ind. Health 3, 185–195.[ISI][Medline]

Smith, J. H., Isenberg, J. S., Pugh, G., Jr., Kamendulis, L. M., Ackley, D., Lington, A. W., and Klaunig, J. E. (2000). Comparative in vivo hepatic effects of di-isononyl phthalate (DINP) and related C7–C11 dialkyl phthalates on gap junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX), and DNA synthesis in rat and mouse liver. Toxicol. Sci. 54, 312–321.[Abstract/Free Full Text]

Svoboda, D. J., and Azarnoff, D. L. (1979). Tumors in male rats fed ethyl chlorophenoxyisobutyrate, a hypolipidemic drug. Cancer Res. 39, 3419–3428.[Abstract]

Tucker, M. J., and Orton, T. C. (1993). Toxicological studies in primates with three fibrates. In Peroxisomes: Biology and Importance in Toxicology and Medicine (G. Gibson and B. Lake, Eds.), pp. 425–447. Taylor and Francis, Washington, DC.

Wada, N., Marsman, D. S., and Popp, J. A. (1992). Dose-related effects of the hepatocarcinogen, WY-14,643, on peroxisomes and cell proliferation. Fundam. Appl. Toxicol. 18, 149–154.[ISI][Medline]