* Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS-1021, Indianapolis, Indiana 46202; and
ExxonMobil Biomedical Sciences Inc., Toxicology and Environmental Sciences Division, Annandale, New Jersey
Received June 1, 2001; accepted August 16, 2001
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ABSTRACT |
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Key Words: DEHP; phenobarbital; liver; peroxisome proliferation; DNA synthesis; gap-junctional intercellular communication.
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INTRODUCTION |
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Chronic dietary administration of diethylhexyl phthalate (DEHP), a commercially used plasticizer, has been shown to produce liver tumors in rats and mice (Kluwe et al., 1982). Neither DEHP nor its primary metabolite, mono-2-ethylhexyl phthalate (MEHP), showed direct evidence of mutagenic activity in bacterial and mammalian mutagenicity assays (Budroe and Williams, 1993
; Dybing et al., 1995
), suggesting that the induction of the observed rodent liver tumors may be the result of epigenetic processes. DEHP has been shown to function as a tumor promoter in the development of liver tumors in rats, initiated by diethylnitrosamine (Ward et al., 1986
). Previous studies have indicated that the hepatic effects of DEHP, which are the consequence of peroxisomal proliferation, appear to require a functioning peroxisomal proliferator-activated receptor (PPAR) alpha (Ward et al., 1998
). These hepatic effects display threshold dose-response characteristics, (Cattley, 1987; David et al., 1999
; Kluwe, et al., 1982
) and are reversible (David et al., 1999
; Kluwe et al., 1982
), characteristics of tumor promoters. Furthermore, the incidence of hepatocellular neoplasms in rats (12,500 mg/kg) and mice (6000 mg/kg) fed DEHP for 78 weeks, followed by a 26-week recovery period, was reduced compared to animals treated for 104 weeks (David et al., 1999
). The exact mechanism(s) by which peroxisomal proliferating agents such as DEHP induce hepatic cancer in rodents are not precisely known, but may be related to the modulation of peroxisomal ß oxidation, the PPAR
receptor, gap-junctional intercellular communication, and replicative DNA synthesis (Isenberg et al., 2000
).
Treatment with DEHP produced a pleiotropic response including hyperplasia and hypertrophy (Wada et al., 1992), induction of several metabolic enzymes (Reddy et al., 1980
), and inhibition of gap-junctional communication (Isenberg et al., 2000
; Krutovskikh et al., 1995
) in rodent liver. Furthermore, the observation of enhanced peroxisomal ß oxidation increased replicative DNA synthesis and suppression of gap-junctional communication (Isenberg et al., 2000
) and demonstrated a species-specific response to peroxisome proliferators (Thomas and Thomas, 1992
). Increased cell replication produced by peroxisomal proliferators may enhance the growth of hepatic lesions from spontaneously initiated cells into hepatic focal lesions (Cattley et al., 1991
, 1998
; Huber et al., 1991
). Nongenotoxic carcinogens, including peroxisomal proliferators and phenobarbital, produce a strong, transient increase in replicative DNA synthesis during the first few weeks of exposure. This increase in replicative DNA synthesis returned to baseline levels within 24 weeks of treatment (Reddy et al., 1979
; Schulte-Hermann et al., 1983
; Yeldandi et al., 1989
). Peroxisome proliferators have been shown to selectively enhance the growth of preneoplastic lesions through enhanced cell replication (Cattley et al., 1991
; Cattley and Popp, 1989
; Isenberg et al., 1997
; ). A transient increase in hepatic replicative DNA synthesis followed administration of DEHP in rats and mice, but not in other species (Isenberg et al., 2000
; Pugh et al., 2000
).
Gap junctions are transmembrane channels formed at the area of contact between cells that permit the transfer of small molecules (< 1000 Da) and appear to be involved in the maintenance of homeostasis in multicellular organisms (Klaunig and Ruch, 1990; Yamaski et al., 1993
). Gap-junctional intercellular communication may mediate cell proliferation through regulating the passage of either growth stimulatory or inhibitory molecules between adjacent cells (Trosko et al., 1990
). Dysfunctional gap-junctional communication may result in the disruption of regulated cell division and may enhance preneoplastic cell growth (Klaunig and Ruch, 1990
; Trosko et al., 1990
; Yamaski et al., 1993
). Many hepatic tumor promoters and nongenotoxic carcinogens inhibit gap-junctional intercellular communication in rodent hepatocytes in vitro (Klaunig and Ruch, 1990
; Trosko et al., 1990
; Yamaski et al., 1993
). Furthermore, several studies have shown that phenobarbital-induced suppression of gap junction intercellular communication in hepatocytes is reversible upon withdrawal of phenobarbital treatment (Neveau et al., 1990
; Ruch and Klaunig, 1988
). Inhibition of gap-junctional intercellular communication has been observed following treatment with MEHP and another peroxisome proliferator, nafenopin, in primary cultured rat, but not in hamster or guinea pig hepatocytes (Elcock et al., 1998
). Finally, recent studies by our group demonstrated dietary administration of tumorigenic concentrations of DEHP suppressed gap-junctional intercellular communication in rat and mouse liver, but not in hamster liver, within one week of treatment (Isenberg et al., 2000
). This inhibition continued throughout the duration of the study (6 weeks) (Isenberg et al., 2000
). These studies indicate that inhibition of GJIC in rodent liver correlated with the reported species- and concentration-specific carcinogenicity of DEHP.
The present investigation evaluated the persistence and reversibility of the effect of DEHP administration on rodent liver following treatment with and subsequent withdrawal of DEHP from the diet. In these mechanistic studies, concentrations of DEHP and phenobarbital (a well-studied hepatic tumor promoter) were selected to correspond to tumorigenic doses in chronic studies. Removal of DEHP from the diet has been shown to reduce the carcinogenic response observed in chronically treated rodents (David et al., 1996, 1997
, 1999
); however, the effects of suspension of treatment on GJIC inhibition have not been previously documented.
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MATERIALS AND METHODS |
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Animals and experimental design.
Six- to eight-week-old male B6C3F1 mice and F344 rats were purchased from Harlan Sprague-Dawley Co. (Indianapolis, IN). Animals were housed in polycarbonate cages in an AALAC-accredited animal facility at the Indiana University School of Medicine. Animal handling procedures followed NIH recommendations. The animals received NIH-07 diet in pelletized form and deionized water ad libitum during a 1-week acclimation period. Male F344 rats and B6C3F1 mice were used in Study 1 and male F344 rats in Study 2 (reversal study). In the first study, animals were randomly allocated to treatment and control groups and fed either control diet, DEHP diet (6000 mg /kg NIH-07), or phenobarbital diet of (500 mg/kg NIH-07) for 18 months. Liver weight, peroxisomal activity, inhibition of gap-junctional intercellular communication, replicative DNA synthesis, and the hepatic concentration of DEHP and its metabolites were determined in 5 animals from each group, as described below.
In a second study, the ability of F344 male rats to recover from the short-term effects of DEHP and phenobarbital was examined. Rats were randomly placed into one of the following treatment groups (15 animals/group) and fed diets that contained, NIH-07 diet (Control), 500 mg PB/kg NIH-07 diet, or 20,000 mg DEHP/kg NIH-07 diet. Following treatment, the test compounds were removed from the diet and the rats were fed NIH-07 diet for an additional 1, 2, or 4 weeks. Five rats from each group were selected after 2, 4, and 6 weeks, sacrificed, and we evaluated indicators of peroxisomal proliferation and inhibition of gap-junctional intercellular communication.
Osmotic mini-pumps (model 2ML1, Alzet Co., Palo Alto, CA) containing 5-bromo-2-deoxyuridine (BrdU; 16 mg/ml of PBS) were surgically implanted subcutaneously on the dorsal sides of rats from each treatment group 7 days prior to sacrifice. At termination, animals were humanely killed, weighed, and necropsied. The livers were removed in toto, weighed, separated by lobe, and sectioned into 12 mm sections. Fresh liver sections were used to assess inhibition of gap-junctional intercellular communication or frozen for evaluation of peroxisomal ß oxidation. The remaining liver sections from each lobe were placed in cassettes, fixed in formalin for 48 h, and embedded in paraffin. Serial sections (5 µm) from each block were used to measure replicative DNA synthesis.
Replicative DNA synthesis.
Measurement of replicative DNA synthesis was by immunohistochemical detection of BrdU, which had incorporated into the DNA of cells that had undergone replicative DNA synthesis. Incorporation of BrdU into nuclei (labeled cells) was visualized by comparison to hemotoxylin-counterstained nuclei of unlabeled cells. A section of duodenum was included on every slide as a positive control to ensure proper staining and incorporation of BrdU. The hepatic-labeling index was calculated by dividing the number of labeled hepatocytes by the total number of hepatocytes examined and multiplying by 100. At least 5000 hepatocytes from each animal were examined. The labeling index was calculated for each treatment group.
Peroxisomal beta oxidation.
Hepatic peroxisomal ß oxidation activity was measured as described by Lazarow and DeDuve, 1976. Briefly, liver samples (0.250.50 grams) from each animal were homogenized in a 0.25-M sucrose buffer on ice. The samples were centrifuged for 10 min at 3500 RPM at 4°C and the supernatants retained. Tritonx (2%) was added to the supernatants and peroxisomal ß oxidation activity was measured spectrophotometrically at 340 nm as the cyanide-insensitive reduction of NAD+, using palmitoyl-CoA as a substrate (Lazarow and DeDuve, 1976).
Gap-junctional intercellular communication.
Gap-junctional intercellular communication in intact rat liver was determined by the direct measurement of lucifer yellow dye transfer in liver slices, utilizing in situ dye transfer (ISDT) as previously described (Isenberg et al., 2000). Immediately after sacrifice, a representative 25-mm strip of liver was rinsed in sterile ISDT buffer, placed in a 5-ml lucifer-yellow dye solution (1 mg Ly-CH/1 mL DTA buffer) and 2 to 3 straight incisions were made in the tissue with a double-edged razor blade. Following transfer of the dye (37°C for 5 min), the dye solution was removed and the liver slice was rinsed in ISDT buffer. The slice was then fixed in 10% phosphate-buffered formalin for 2448 h. Following the formalin fixation, the slices were embedded in JB4 monomer plastic (Polysciences, Inc., Warrington, PA) and cured for at least 48 h. Serial sections (5 µm) were cut and a computer-based imaging system (BDS Image, Oncor, Inc., Gaithersberg, MD) was used to evaluate the length of dye transfer through the incisions in each liver slice. A standard curve based on the size and amount of dye present in 100 individual control, phenobarbital- and DEHP-treated cells was generated. This standard curve was used to convert the length of dye transfer generated in arbitrary units by BDS Image to a distance of dye transfer (in mm) and the number of cells communicating.
Tissue analysis for DEHP and metabolites.
Extraction and high-pressure liquid chromatography (HPLC) analysis of DEHP, MEHP, and phthalic acid from liver were carried out as previously described by Isenberg et al. (2000). Approximately 100 to 300 mg of liver was homogenized in 1 ml of methanol, rinsed twice with 1 ml of acetonitrile, and the rinses added to the methanol homogenate. The compounds of interest were extracted by continuous mixing at 04°C for 12 h, followed by centrifugation (15 min, 12,000 x g at 4°C). The resulting supernatants were analyzed by HPLC (Hewlett-Packard 1050 solvent delivery pump, autoinjector, variable wavelength detector, and Hewlett Packard 3396A integrator, Palo Alto, CA). The analyte concentrations were determined by injection of 10 µl of liver supernatant onto a 5 µm hypersil ODS C18 reverse phase column (4.6 x 150 mm). The mobile phase consisted of 40% 50 mM KH2PO4 (pH 3.0) and 60% acetonitrile with a flow rate of 1.5 ml/min. Ultraviolet absorbance was monitored at 254 nm. Calibration curves were prepared from analytical standards on each day of analysis and were linear between 1 and 100 µM. Analyte concentrations were determined on calibration curves (plot of the analyte peak area versus concentration). The results, corrected for the extraction recovery (95%), were expressed as mean ± standard deviation.
Statistics.
Statistical differences (p < 0.05) from control values for all data were determined by 2-way ANOVA followed by a Dunnett's test. The least-squares-means, post hoc test was used for analysis of the tissue data, which are expressed as the mean ± standard deviation (SD).
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RESULTS |
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DISCUSSION |
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Although the role of gap-junctional intercellular communication in the carcinogenic process has not been precisely defined, a correlation between inhibition of gap-junctional intercellular communication and tumor promotion has been demonstrated (Elcock et al., 1998; Klaunig and Ruch, 1990
; Trosko et al., 1990
; Yamaski et al., 1993
). This implies that the loss of control over cellular growth activity associated with inhibition of gap-junctional intercellular communication may permit the proliferation of transformed cells.
In the present study, induction of peroxisomal proliferation and inhibition of gap-junctional intercellular communication were observed in rats and mice treated for 18 months with 6000 mg/kg DEHP. These data indicate that peroxisome proliferation and inhibition of gap-junctional intercellular communication persist upon continuous treatment with DEHP. Similar data for these parameters have been obtained in studies of shorter duration (Isenberg et al., 2000). In contrast to the peroxisomal proliferation and gap-junctional intercellular communication data, replicative DNA synthesis was elevated only during the first 2 weeks of treatment and then returned to levels similar to controls.
These results suggest that induction of peroxisome proliferation and/or inhibition of gap-junctional intercellular communication, along with modification of DNA synthesis, may be important for the development of hepatocellular cancer in sensitive rodent species. While the induction of peroxisome proliferation has been suggested as accounting for the carcinogenic response observed upon administration of peroxisome proliferators, increased cell proliferation appears to be responsible for the growth of hepatic lesions from spontaneously and chemically initiated cells into hepatic focal lesions (Cattley et al., 1991; Cattley and Popp, 1989
; Huber et al., 1991
; Isenberg et al., 1997
).
Gap-junctional intercellular communication appears to be essential to the regulation of hepatocyte proliferation and may mediate cell proliferation through regulating the passage of either growth stimulatory or inhibitory molecules between adjacent cells. Therefore, dysfunctional gap-junctional intercellular communication may result in the disruption of regulated cell division and be an important contributor to the enhancement of preneoplastic cell growth by tumor promoters such as phenobarbital and DEHP (Klaunig and Ruch, 1990; Trosko et al., 1990
; Yamaski et al., 1993
). Furthermore, many hepatic tumor promoters and nongenotoxic carcinogens inhibit gap-junctional intercellular communication in rodent hepatocytes in vitro (Elcock et al., 1998
; Klaunig and Ruch, 1990
; Trosko et al., 1990
; Yamaski et al., 1993
). Recent studies in our group demonstrated dietary administration of tumorigenic concentrations of DEHP suppressed gap-junctional intercellular communication in intact rat and mouse liver within one week of treatment, and this inhibition continued throughout the duration of the study (6 weeks; Isenberg et al., 2000
). Those studies indicate that inhibition of gap-junctional intercellular communication in rodent liver correlated with the reported species- and concentration-specific carcinogenicity of DEHP. Given the present observation of persistent inhibition of gap-junctional intercellular communication following 18 months of treatment and previous observations of subchronic inhibition of gap-junctional intercellular communication in rodent liver following treatment with carcinogenic concentrations of DEHP, it seems probable that inhibition of gap-junctional intercellular communication may be involved in the carcinogenicity of DEHP.
These studies also examined the reversibility of peroxisomal proliferation, inhibition of gap-junctional intercellular communication, and replicative DNA synthesis in rats. In the present study, dietary exposure to DEHP for 2 weeks increased the relative liver weight. Since the induction of peroxisomal ß oxidation correlates with proliferation of peroxisomal organelles (Sharma et al., 1988), the increase in relative liver weight appears to be due to a hypertrophic and hyperplastic response to DEHP. Upon removal of DEHP from the diet, the relative liver weight was reduced. The reduction in the relative liver weight corresponded to the reduction in peroxisomal activity and replicative DNA synthesis. While peroxisomal activity and replicative DNA synthesis returned to control values, the relative liver weight remained slightly elevated. This increase appears to be due to a persistent elevation in the liver weight and may be due to liver cell increases related to the dynamics of cell proliferation and apoptosis. Phenobarbital produced similar responses.
Enhanced cell proliferation and suppression of apoptosis have been reported to be important characteristics of nongenotoxic tumor promoter treatment (Schulte-Hermann, 1983). Several investigators have shown that treatment with the nongenotoxic carcinogens phenobarbital and DEHP enhance cell proliferation above basal levels in normal and putative preneoplastic cells (Schulte-Hermann et al., 1983). However, the duration and extent of proliferation varies. Similar to previous studies, the present study demonstrated that the nongenotoxic hepatic tumor promoters, phenobarbital and DEHP, produce a transient increase in proliferation followed by a return to the basal rate of cell proliferation (Schulte-Hermann et al., 1983
). Furthermore, upon withdrawal of DEHP from the diet, replicative DNA synthesis returned to control levels. In previous studies by our group, treatment with the same concentration of DEHP (20,000 mg/kg) for 4 weeks produced a 2-fold increase in replicative DNA synthesis (Isenberg et al., 2000
). Therefore, the reduction in replicative DNA synthesis following removal of DEHP from the diet for 2 weeks may not be completely related to the transient nature of DEHP-induced hyperplasia.
In addition to enhancing cell proliferation, treatment with nongentoxic hepatocarcinogens such as nafenopin and phenobarbital appears to confer resistance to apoptosis in a manner that contributes to the hepatic tumor-promoting effect (Bursch et al., 1984, 1985
, 1990
; Cattley and Popp, 1989
; Kolaja et al., 1996a
). The observation that some nongenotoxic carcinogens suppress apoptosis in vitro and in vivo suggests a role for the dysregulation of apoptosis in the cancer process. Several studies have demonstrated the regression of hepatic focal lesions following promoter withdrawal (Bursch et al., 1984
; Kolaja et al., 1996b
) involves enhanced apoptosis. Therefore, the reduction in liver weight observed following withdrawal of the tumor promoters, DEHP and phenobarbital, in the present study may be related to downregulation in the number of peroxisomes or peroxisomal enzymes and/or a reduction in the number of hepatocytes through apoptosis.
The present study demonstrated inhibition of gap-junctional intercellular communication is persistent with treatment and reversible when treatment is removed. In addition, studies in primate liver (Pugh et al., 2000) and human cells in culture (Baker et al., 1996
) demonstrate that inhibition of gap-junctional intercellular communication shows the same species specificity as the tumor response data. Therefore, it is possible that enhanced cell replication provides the initial stimulus allowing transformed cells to be expressed, and that inhibition of gap-junctional intercellular communication then facilitates the development of these cells into tumors.
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NOTES |
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