Toxicological Sciences 54, 312-321 (2000)
Copyright © 2000 by the Society of Toxicology
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
,1
* Exxon Biomedical Sciences, Inc., East Millstone, New Jersey 08875; and
Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received September 13, 1999; accepted October 18, 1999
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ABSTRACT |
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The short-term hepatic effects of DINP (CAS 68515-48-0, designated DINP-1) in rats and mice were evaluated at tumorigenic and nontumorigenic doses from previous chronic studies. Groups of male F344 rats were fed diets with DINP-1 at concentrations of 0, 1000, or 12,000 ppm and male B6C3F1 mice at 0, 500, or 6000 ppm DINP-1. After 2 or 4 weeks of treatment, changes in liver weight, gap junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX), and replicative DNA synthesis were examined. In addition, hepatic and serum concentrations of the parent compound and major metabolites were determined. Relative to controls in both species, increased liver weight and PBOX at the high dose of DINP-1 were consistent with peroxisomal proliferation. Hepatic GJIC was inhibited and DNA synthesis was increased at the high dose of DINP-1, which is also consistent with the tumorigenic response in rats and mice reported in other chronic studies at these doses. These hepatic effects were not observed at the low doses of DINP-1. At comparable low doses of DINP-1 in other chronic studies, no liver tumors were observed in rats and mice. The monoester metabolite (MINP-1) was detected in the liver at greater concentrations in mice than rats. This result is also consistent with the dose-response observations in rat and mouse chronic studies. Additionally, other structurally similar dialkyl phthalate esters ranging from C7 to C11 were evaluated using a similar protocol for comparison to DINP-1; these included an alternative isomeric form of DINP (DINP-A), di-isodecyl phthalate (DIDP), di-isoheptyl phthalate (DIHP), di-heptyl, nonyl undecyl phthalate (D711P), and di-n-octyl phthalate (DNOP). Collectively, these data indicate that in rats and mice, DINP-1 and other C7–C11 phthalates exhibit a threshold for inducing hepatic cellular events. Further, where previous chronic data were available for these compounds, these phthalates elicited hepatic effects at doses that correlated with the tumorigenic response. Overall, these studies suggest a good correlation between the inhibition of GJIC when compared with the data on production of liver tumors in chronic studies.
Key Words: di-isononyl phthalate (DINP); di-isodecyl phthalate (DIDP); di-isoheptyl phthalate (DIHP); di-n-octyl phthalate (DNOP); di-heptyl, nonyl, undecyl phthalate (D711P); DNA synthesis; gap junctional intercellular communication (GJIC); in situ dye transfer (ISDT); peroxisome proliferation; phthalate esters; rodent liver.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dialkyl ortho phthalate esters with carbon numbers ranging from C6 to C13 are widely used in plasticizer applications to add flexibility to a wide variety of materials including polyvinyl chloride (PVC). Phthalates are produced by esterification of phthalic anhydride with various C6–C13 alcohols in a closed system. The observation that di-2-ethylhexyl phthalate (DEHP) produced liver tumors in rats and mice following chronic dietary exposures (Kluwe et al., 1982
) prompted the evaluation other dialkyl phthalate esters. Several studies have indicated that some phthalates induce hepatic tumors in rats and mice by a process that appears to involve peroxisome proliferation. The absence of evidence of peroxisomal proliferation in primates and in cultured human cells has led to questions of the relevance of peroxisome proliferation and other pleiotropic effects to humans and other species (Ashby et al., 1994; IARC, 1995).
The present study examined the potential for a range of phthalates to produce early hepatic changes that may be markers for the tumorigenic response of phthalate esters in rats and mice. These changes included relative liver weight, inhibition of gap junctional intercellular communication (GJIC), induction of peroxisomal beta-oxidation (PBOX) activity, and changes in replicative DNA synthesis. Groups of male rats and mice were fed diets with a representative low and high concentration of each phthalate (representing tumorigenic and nontumorigenic doses observed in previous chronic studies). The study design employed here used information obtained, in part, from more extensive previous dose- and time-response studies conducted with DEHP in rats (Isenberg et al., 2000). Additionally, the study by Isenberg et al. (2000) showed that the species-specific and dose-related responses of liver tumors induced by dietary DEHP were best correlated with inhibition of GJIC and increased DNA synthesis in rats, mice, and hamsters. However, the increased DNA synthesis was a transient effect that occurred only within the early weeks of treatment, whereas GJIC was inhibited throughout treatment as long as 24 months (Isenberg et al., 2000; Kamendulis et al., 1999). As the loss of controlled cell growth mediated by GJIC is a response associated with rapidly proliferating cells and tissues, and because this event may play an important role in the development of neoplasms (Klaunig and Ruch, 1990; Yamasaki, 1990), further studies on other phthalates were conducted.
The present study compared the hepatic effects in rats of mice of di-isononyl phthalate (designated here as DINP-1) to five other phthalate plasticizers that include linear, mixed, and branched alkyl chains ranging from C7 to C11. The series included a second isomeric form of di-isononyl phthalate (designated here as DINP-A), di-isodecyl phthalate (DIDP), di-isoheptyl phthalate (DIHP), di-(heptyl, nonyl, undecyl) phthalate (D711P, mixture of ~2/3 linear and 1/3 branched C7,C9,and C11 alkyls), and the linear di-n-octyl phthalate (DNOP). The chemical differences between the two forms of DINP were related to differences in preparing the corresponding C9 branched alcohol feedstocks that make up the dialkyl carbon chains. The main alcohol component of DINP-1 was prepared by oligimerization of propylene and mixed butenes (also known as polygas) to form a C9-rich mixture consisting of roughly equivalent amounts of 3,4-, 4,6-, 3,6-, 4,5-, and 5,6-dimethyl heptanol. This was designated as DINP-1 to be consistent with the terminology used in other publications on different isomeric forms of DINP (Hellwig et al., 1997). The term DINP-A was used to designate DINP-alternative for an isomer that was never produced commercially. The main alcohol component of DINP-A is less branched and was prepared from a C9-rich isononyl alcohol mixture consisting mainly of monomethyl-1-octanol, dimethyl-1-heptanol, and normal nonanol.
The liver is a target organ for many phthalates in rodents, but the intensity of the observed effects appears to be compound, dose, and species dependent. The observation of liver tumors in rats in chronic feeding studies at high dietary doses, but not at lower doses, indicates a threshold for phthalate-induced hepatocellular tumors in rats and mice. DINP-1 demonstrated no increase in hepatic tumors in male F344 rats at lifetime dietary doses of 6000 ppm (> 360 mg phthalate per kilogram body weight per day [mg/kg/day]) (Butala et al., 1996; Lington et al., 1997). However, the incidence of combined hepatic adenomas and carcinomas increased to 26% rats at lifetime dietary doses of 12,000 ppm DINP-1 (> 730 mg/kg/day) (Butala et al., 1996; EU, 1997). In male B6C3F1 mice treated with lifetime dietary doses of DINP-1, the no-observed-effect-level (NOEL) for liver tumors was 500 ppm (90 mg/kg/day) and the low-observed-effect-level (LOEL) was 1500 ppm (275 mg/kg/day) (Butala et al., 1997; EU, 1997). A non-commercially available DINP, with a similar synthetic composition to DINP-A, demonstrated a NOEL for hepatic tumors in male Sprague-Dawley rats of 500 ppm (estimated as 27 mg/kg/day) and a LOEL of 5000 ppm (estimated as 271 mg/kg/day) (Monsanto, 1986a, as cited in EU, 1997). Dietary administration of 3000 ppm D711P revealed no increases of hepatic tumors in male rats (Monsanto, 1986b, as reported by Hirzy, 1989), but data for the higher doses comparable to those evaluated for the other phthalates (i.e., DINP, DEHP) are not available for D711P. No bioassay data were available for DIDP, DIHP, or DNOP. However, two separate studies indicated that DNOP acted as a tumor promoter in rats; dietary administration of 10,000 ppm DNOP for 10 or 26 weeks promoted the development of preneoplastic hepatic lesions in male Sprague-Dawley and Fischer rats initiated with diethyl nitrosamine (Carter et al., 1992; DeAngelo et al., 1986, 1988).
Comparison of the responses for linear versus branched phthalates was of interest in this study because some data suggest that linear phthalates may have different mechanisms of action than branched phthalates in rodents. Mechanistic studies on the linear phthalate DNOP in rats indicated minimal effects on relative liver weight and peroxisomal proliferation when compared to DEHP and other branched phthalates (Barber et al., 1987; Lake et al., 1984; Mann et al., 1985; Poon et al., 1997; Shellenberger et al., 1983). When compared with DEHP, repeated exposure to high doses of linear phthalates that included DNOP, D711P, di-n-hexyl phthalate, and di-n-heptyl, nonyl phthalate (D79P) showed a different histopathologic profile and affected the centrilobular region of rat liver (DeAngelo et al., 1988; Hinton et al., 1986; Mangham et al., 1981; Mann et al., 1985; Shellenberger et al., 1983). In contrast to DEHP, these linear phthalates did not show histopathologic evidence for peroxisome proliferation. However, linear phthalates were reported to produce a slow developing accumulation of fat in centrilobular hepatocytes and some evidence of fatty necrosis that was accompanied by acute inflammation (Hinton et al., 1986; Mangham et al., 1981; Mann et al., 1985). Evaluations of repeated dose exposures to linear phthalates in mice are limited to observations from continuous breeding studies. Exposure of male mice to 5.0% (50,000 ppm) DNOP (7.5 g/kg /day) for 15 weeks increased absolute (and relative) liver weights as compared to control groups (Heindel et al., 1989). Examination of linear versus branched phthalate monoesters indicated that only branched chain phthalates inhibited GJIC in primary culture mouse hepatocytes, and that this may be a predictor for carcinogenicity (Klaunig et al., 1988). Furthermore, in vitro studies comparing branched to linear phthalate monoesters indicated that the branched phthalates were more active in inducing peroxisomal enzymes than linear chain phthalates (Benford et al., 1986; Gray et al., 1983; Lake et al., 1986).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Di-isoheptyl phthalate (DIHP, purity > 98%, CAS 71888-89-6), mono-isoheptyl phthalate (MIHP), di-n-octyl phthalate (DNOP, purity > 99%, CAS 117-84-0), two isomeric forms of di-isononyl phthalate (designated as DINP-1 [CAS 68515-48-0] and DINP-A [CAS 71549-78-5], both with purity > 98%), di-isodecyl phthalate (DIDP, purity > 98%, CAS 68515-49-1), mono-isodecyl phthalate (MIDP), di-(heptyl, nonyl, undecyl)phthalate (D711P, purity > 98%, a mixture with no CAS ) were provided by Exxon Chemical Company (Baton Rouge, LA). Mono-isononyl phthalate (two isomeric forms designated as MINP-1 and MINP-A), mono-(heptyl, nonyl, undecyl) phthalate (M711P) were synthesized by Aldrich Chemical (Milwaukee, WI). Mono-n-octyl phthalate (MNOP) was a gift from Dr. J. Heindel of NIEHS (Research Triangle Park, NC). Phthalic acid (PA, 99+% purity), was purchased from Aldrich Chemical (Milwaukee, WI). All of the mono-ester phthalates were custom synthesized by reacting the corresponding alcohol with phthalic anhydride. There are chemical differences between the two forms of DINP that are related to differences preparing the corresponding C9 branched alcohol feedstocks. The main alcohol component of MINP-1 was prepared by oligimerization of propylene and mixed butenes to form a C9-rich mixture consisting of roughly equivalent amounts of 3,4-, 4,6-, 3,6-, 4,5-, and 5,6-dimethyl heptanol, which corresponds to DINP-1 (CAS 68515-48-0). The main alcohol component of MINP-A was prepared from a C9-rich isononyl alcohol mixture consisting mainly of monomethyl-1-octanol, dimethyl-1-heptanol, and normal nonanol. DINP-A corresponds to DINP CAS 71549-78-5. M711P was prepared from an alcohol that contained predominantly linear isomers (around 67% of heptanol-1, nonanol-1, and undecanol-1) plus approximately 33% 2-methyl or 2-ethyl branched isomers. MIDP was formed from an alcohol that was a di-C9, C10, C11 branched alkyl ester, C10-rich.
Animals and treatment.
Male B6C3F1 mice and male Fischer 344 (F344) rats (6–8 weeks of age) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed under standard conditions at the AALAC-accredited laboratory animal research center (LARC) at the Indiana University School of Medicine (Indianapolis, IN). Males were used because of previous reports that the severities of hepatotoxic effects were generally greater in males than females (Barber et al., 1987; Mangham et al., 1981). All animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals (US DHEW, 1978) with a 12-h light/dark cycle. Animals were housed in polycarbonate cages with microbarrier isolation tops (five mice/cage and two rats/cage), bedding, and a water bottle. During a 1-week acclimation period, all animals received NIH-07 pelletized diet and deionized water ad libitum. Animals were randomly placed into treatment groups of five animals per group. NIH-07 diets containing individual di-alkyl phthalates at 500, 1000, 6000, 10,000, and 12,000 ppm (mg/kg) were formulated and verified by Dyets, Inc. (Bethlehem, PA). Treatment groups and durations of exposure included the following:
- Rats: 0, 1000, and 12,000 mg/kg NIH-07 diet of DINP-1, DINP-A, DIDP, DIHP, and D711P, groups treated for 2 and 4 weeks. For DNOP only, dose groups were 0, 1000 and 10,000 mg/kg NIH-07 diet treated for 2 and 4 weeks.
- Mice: 0, 500,and 6000 mg/kg NIH-07 diet of DINP-1, DINP-A, DIDP, DIHP, and D711P, groups treated for 2 and 4 weeks. For DNOP only, dose groups were 0, 500 and 10,000 mg/kg NIH-07 diet treated for 2 and 4 weeks.
The in-life portions of these studies were conducted between January 1996 and September 1997. Because these experiments were not conducted concurrently, an untreated control group of animals was included for each study.
Assessment of hepatic effects.
Osmotic minipumps (model 2001 in mice, model 2ML1 in rats, Alza Company, Palo Alto, CA) containing 5-bromo-2'-deoxyuridine (BrdU, 16 mg/ml in phosphate-buffered saline) were surgically implanted subcutaneously on the dorsal side in anesthetized animals 7 days prior to sacrifice. After the indicated treatment duration, animals were sacrificed by diethyl ether asphyxiation, weighed, and necropsied, including the withdrawal of blood samples from the vena cava. Livers were removed, weighed, separated by lobes, and sectioned into samples for determination of gap junctional intercellular communication (GJIC), replicative DNA synthesis, and peroxisomal beta-oxidation activity (PBOX), as described previously (Isenberg et al., 2000).
Tissue analysis for parent phthalate and metabolites.
Extraction and subsequent analysis by high-pressure liquid chromatography (HPLC) of DINP-1, MINP-1, and PA from liver and serum were performed in a manner as previously described for DEHP and metabolites (Isenberg et al., 2000). Extraction recoveries were 99%, 97%, and 93% for PA, MINP-1, and DINP-1, respectively, and the reported values were corrected accordingly.
Data evaluation.
All data are expressed as the mean ± standard deviation (SD). A total of three to five animals were evaluated for each experimental group unless otherwise indicated. Statistical differences (p < 0.05) from control values were determined by two-way ANOVA followed by a Dunnett's test (Gad and Weil, 1988). The two-way ANOVA followed by a Least Squares Means post-hoc test was used to evaluate the tissue and serum analysis data for DINP-1, MINP-1, and PA.
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RESULTS |
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Relative Liver Weight
Overall, high dietary doses of the six phthalates studied produced statistically significant increases of relative liver weights in male F344 rats after both 2 and 4 weeks of treatment (Figs. 1A and 1B
). Exceptions were DINP-1 at 2 weeks (Fig. 1A) and DNOP at 4 weeks (Fig. 1B), where no increase was observed. The low dietary dose of 1000 ppm was without effect on relative liver weight except for a small, statistically significant increase with DIHP at 2 weeks (Fig. 1A) that was not observed after 4 weeks.
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In male B63F1 mice, the high dietary doses of 6000 ppm DINP-1, DINP-A, DIDP, and D711P produced statistically significant increases of relative liver weights after 2 weeks (Fig. 2A
). Relative liver weight remained increased at 4 weeks for high dose groups treated with DINP-1, DINP-A, and D711P (Fig. 2B). Relative liver weights were elevated at 4 weeks for high dose groups treated with DIDP, but were not statistically different from the control group (Fig. 2B). Neither DIHP nor DNOP increased relative liver weights of mice in the high dose groups at either the 2- or 4-week observation (Figs. 2A and 2B). The low dietary doses of 500 ppm did not increase relative liver weights in mice at either 2 or 4 weeks (Figs. 2A and 2B), but a slight decrease of relative liver weight was observed for DINP-1 at 2 weeks only (Fig. 2A).
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Peroxisomal Beta-Oxidation (PBOX)
In rats, PBOX activities were elevated at the 12,000 ppm high dose of the branched phthalates DINP-1, DINP-A, DIDP, and DIHP at both 2 and 4 weeks (Figs. 3A and 3B
). With D711P and DNOP treatment in rats, statistically significant elevations of PBOX were seen at the high doses only at the 2-week observation (Fig. 3A) and not at 4 weeks (Fig. 3B). In rats, dietary phthalates at the low dose of 1000 ppm did not increase PBOX activity at either 2 or 4 weeks (Figs. 3A and 3B).
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In mice, elevations of PBOX were observed at the high dose of all six phthalates after both 2 and 4 weeks (Figs. 4A and 4B
). DNOP also produced a statistically significant elevation of PBOX at the low dose of DNOP after 4 weeks only (Fig. 4B). In mice, the low doses of 500 ppm of the other phthalates did not affect PBOX activity after 2 or 4 weeks (Figs. 4A and 4B).
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Gap Junctional Intercellular Communication (GJIC)
In rats, a reduction of dye transfer distance indicated an inhibition of GJIC at 2 weeks for groups treated with 12,000 ppm of DINP-1 and DINP-A (Fig. 5A
) and at 4 weeks for groups treated with 12,000 ppm DINP-A and D711P (Fig. 5B). Statistically significant effects on dye transfer distance (and GJIC) were not observed in rats treated with the high doses of DIDP, DIHP, or DNOP at either 2 or 4 weeks (Figs. 5A and 5B). In rats, the low doses of 1000 ppm dietary phthalates produced no evidence for inhibition of GJIC at either 2 or 4 weeks (Figs. 5A and 5B).
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In mice, a significant inhibition of GJIC was seen at 2 weeks for the group treated with 6000 ppm DINP-A, but not for high-dose groups treated with the other phthalates (Fig. 6A
). GJIC was inhibited at 4 weeks for the groups of mice treated with 6000 ppm DINP-1 and DINP-A, but not for the groups treated with high doses of DIDP, DIHP, D711P, nor DNOP (Fig. 6B). In mice, GJIC was not inhibited at 2 or 4 weeks by the low doses (500 ppm) of any of the tested phthalates (Figs. 6A and 6B).
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Replicative DNA Synthesis
In rats, elevations of periportal DNA synthesis were seen for the high-dose treatment groups treated with all six phthalates at the 2-week observation (Fig. 7A
). Periportal DNA synthesis remained elevated at the 4-week observation in groups of rats treated with the high doses of DINP-A, DIDP, DIHP, and DNOP, but neither DINP-1 nor D711P (Fig. 7B). At the low doses, statistically significant increases of periportal DNA synthesis were observed with DIDP at the 4-week observation and with DIHP at both 2 and 4 weeks. Low doses of 1000 ppm DINP-1, DINP-A, D711P, and DNOP showed no statistically significant effects on periportal DNA synthesis. Centrilobular DNA synthesis, determined only for the DINP-1 and DINP-A groups, showed similar effects as the observations for periportal DNA synthesis, but the labeling index was slightly less than the periportal DNA synthesis labeling index (data not shown). Thus, statistically significant elevations of centrilobular DNA synthesis were seen at the 2-week observations for rats treated with 12,000 ppm DINP-1 and DINP-A and at the 4-week observation for rats treated with 12,000 ppm DINP-A only.
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In mice, significant elevations of periportal DNA synthesis were seen at 2 weeks for the high-dose treatment groups treated with DINP-1, DIDP, and DIHP, and also for the low-dose groups treated with DIDP, DIHP, and D711P (Fig. 8A
). At the 4-week observation, periportal DNA synthesis remained elevated for the groups that received both the low and high dose of DIDP and for the high dose of DIHP, but returned to control levels for the high-dose groups that received DINP-1 and D711P (Fig. 8B). No effects on periportal DNA synthesis were observed for either the high or low dose of DINP-A or DNOP at either 2 or 4 weeks. Low doses of DINP-1, DINP-A, and DNOP were without effect on periportal DNA synthesis in mice at both 2 and 4 weeks. Centrilobular DNA synthesis, determined only for the DINP-1 and DINP-A groups, showed similar effects as the observations for periportal DNA synthesis, but the labeling index was slightly less than the periportal DNA synthesis labeling index (data not shown). Thus, only the high dose of DINP-1 showed an elevation of centrilobular DNA synthesis at 2 weeks only.
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Analysis of DINP and Primary Metabolites in Liver and Serum
Limited evaluations were conducted in rats and mice treated with DINP-1 for the hepatic and serum content of the parent metabolite (DINP-1), the monoester metabolite (MINP-1), and the phthalic acid metabolite (PA) (Table 1
). DINP-1, MINP-1, and PA were essentially nondetectable in the control groups, or the concentrations were very low and highly variable.
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DINP-1 was detected in the liver of rats and mice at 2 and 4 weeks, but was essentially nondetectable in the serum. At 2 weeks, the concentration of DINP-1 was slightly greater in the livers of the high group than in the low-dose group for both rats and mice. However DINP-1 concentrations did not show this dose response in the liver at 4 weeks or in the serum at either 2 or 4 weeks. The concentrations of MINP-1 in liver and serum increased with both dose and with time in rats and mice. Thus, the concentrations of MINP-1 in liver and serum were greater in rats treated with 12,000 ppm than in those treated with 1000 ppm, and greater in mice treated with 6000 ppm than with 500 ppm DINP-1. The concentrations of MINP-1 in liver and serum were greater after 4 weeks than after 2 weeks of treatment. Further, on a µmole basis, MINP-1 was more abundant than DINP-1 and PA in the liver and serum of both rats and mice. Concentrations of PA were low relative to MINP-1 in the liver and serum of both species. In rats, PA increased in a time-dependent manner in both liver and serum. In mice, there was a time-dependent increase of PA in the liver and serum at the low dose of 500 ppm, but not at the high dose of 6000 ppm. Further, the concentrations of PA did not show a dose-related increase in the liver or serum of rats or mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in relative liver weight (Fig. 1
), PBOX activity (Fig. 3), GJIC (Fig. 5), and DNA synthesis (Fig. 7) in rats and mice treated with the six phthalate esters are summarized in Table 2. In rats, hepatic effects seen with the two isomers of DINP-1 and DINP-A at 2 and 4 weeks correlated with the occurrence of liver tumors in previous chronic feeding studies (Table 2). At the lower nontumorigenic doses of DINP-1 and DINP-A in male rats, there were no statistically significant alterations of any of the hepatic end points (Figs. 1, 3, 5, and 7). Collectively, these observations were consistent with previous chronic feeding studies, which showed liver tumors at 12,000 ppm DINP-1 but not at 6000 ppm DINP-1 (Butala et al., 1996; Lington et al., 1997), and at 5000 ppm DINP-A but not at 500 ppm DINP-A (EU, 1997; Monsanto, 1986a). It should be noted that the differences in the no observed effect level (NOEL) for liver tumors produced by DINP-1 and DINP-A largely reflect the limited doses examined. Between the two separate chronic feeding studies on DINP-1 in rats, dietary doses of 0, 300, 500, 1500, 3000, 6000, and 12,000 ppm were used (Butala et al., 1996; Lington et al., 1997). DINP-A, a noncommercial product, was evaluated in rats in a single study at dietary doses of 0, 500, 5000, and 10,000 ppm (EU, 1997; Monsanto, 1986a).
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In mice, DINP-1 was tested in a 2-year chronic feeding study where doses included 0, 500, 1500, 4000, and 8000 ppm (Butala et al., 1997
). No chronic data are available at this time for DINP-A in mice. Liver tumors were observed in male and female mice at DINP-1 dietary doses of 1500 ppm and greater (Butala et al., 1997). Thus, the high dose of 6000 ppm used to treat male mice in the present study falls within the range of tumorigenic doses reported by Butala et al. (1997). In the present study, at the 2 and 4-week treatment with 6000 ppm DINP-1, an increase in relative liver weight (Fig. 2), PBOX activity (Fig. 4), and DNA synthesis (Fig. 8), and a decrease in GJIC (Fig. 6) were seen in mice. At the lower nontumorigenic 500-ppm dose of DINP-1 in male mice, there were no significant changes in PBOX (Fig. 4), GJIC (Fig. 6), or DNA synthesis (Fig. 8) relative to control. A slight decrease in relative liver weight was seen at this dose (Fig. 2). Thus, the high-dose and low-dose effects of DINP-1 on mouse liver also showed a good correlation with the potential to produce liver tumors. In a similarly designed study addressing the effects of DEHP in rats and mice (Isenberg et al., 2000), we showed that DEHP produced increased relative liver weight, increased PBOX activity, inhibition of GJIC, and increased DNA synthesis at DEHP doses that were consistent with the tumorigenic activity of this compound.
The other phthalates evaluated in the present study (DIHP, DIDP, D711P, and DNOP) were all observed to produce some elevation of PBOX activity at doses comparable to DINP-1, DINP-A, and DEHP (Table 2). These observations were consistent with previously reported effects of these phthalates. Barber et al. (1987) reported that DIDP was the most potent inducer of PBOX in rats when compared with structurally related phthalates as in this study (Fig. 3). In general, the branched alkyl chain phthalates appeared to be more potent inducers of PBOX activity than the linear (DNOP) or mixed (D711P) phthalates in both rats (Fig. 3) and mice (Fig. 4). Similar observations have been reported by other investigators (Barber et al., 1987; Gray et al., 1983; Lake et al., 1984, 1986, 1987; Mann et al., 1985; Moody and Reddy, 1978). Chronic data are limited or not existent for DIHP, DIDP, D711P, and DNOP, therefore the potential use of the above biologic end points (DNA synthesis, GJIC, PBOX, and liver weight) in predicting the carcinogenic effects of these phthalates remains unresolved. However, the mechanistic linkage between blockage of cell-to-cell communication (GJIC), induction of DNA synthesis, and induction of PBOX with nongenotoxic carcinogenesis indicates that these end points may be valuable in understanding the relative risk of these agents in humans. Peroxisomal proliferators, including the phthalates DEHP and DINP, have been consistently observed to lack hepatic effects in human hepatocytes and in nonhuman primate in vitro and in vivo test models (Ashby et al., 1994; IARC, 1995; Kurata et al., 1998; Pugh et al., 1999; Rhodes et al., 1986).
In conclusion, similar to DEHP (Isenberg et al., 2000), these data indicate that DINP and other C7–C11 phthalates produced effects after 2–4 weeks of treatment on important mechanistic end points (GJIC, DNA synthesis, PBOX, liver weight) in the liver of rats and mice. The hepatic effects of DINP-1 and DEHP occurred at doses that were consistent with the hepatic tumorigenic responses of these phthalates in chronic feeding studies in these species. Furthermore, these phthalates exhibited a threshold for inducing cellular events in liver, as doses that lacked tumorigenic activity had little to no effect on these hepatic end points.
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ACKNOWLEDGMENTS |
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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.
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NOTES |
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Presented in part at the February 1999 Winter Toxicology Forum in Washington, DC and at the 38th Annual Meeting of the Society of Toxicology in New Orleans, LA, March 1999.
1 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. 
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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