* 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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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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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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Animals and treatment.
Male B6C3F1 mice and male Fischer 344 (F344) rats (68 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:
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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DISCUSSION |
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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 C7C11 phthalates produced effects after 24 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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NOTES |
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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 462025120. Fax: (317) 274-7787. E-mail: jklauni{at}iupui.edu.
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