* Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, 46202; and
Exxon Biomedical Sciences, Inc., East Millstone, New Jersey 08875
Received June 8, 1999; accepted January 11, 2000
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ABSTRACT |
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Key Words: di-2-ethylhexyl phthalate (DEHP); gap-junctional intercellular communication (GJIC); rodent liver; peroxisome proliferator; in situ dye transfer (ISDT).
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INTRODUCTION |
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Chronic feeding of DEHP to rodents resulted in an increased incidence of liver tumors in male Fischer 344 rats (hepatocellular carcinoma or neoplastic nodules) at dietary doses of 12,000 ppm (~670 mg/kg/day) but not at 6000 ppm (~320 mg/kg/d). In male B6C3F1 mice, there was an increase of hepatocellular carcinoma or adenoma at dietary doses of 3000 ppm (~390 mg/kg/d) and 6000 ppm (~780 mg/kg/day) (Kluwe et al., 1982). At a dietary dose of 20,000 ppm (estimated daily dose of ~1000 mg/kg/day), the hepatic tumor incidence in F344 rats was 78% (Rao et al., 1987
; 1990
). More recent studies utilizing lower doses of DEHP noted an increase in hepatocellular tumors in male F344 rats following treatment with 2500 ppm (estimated daily dose of ~140 mg/kg/day), but not at 500 ppm (estimated as 30 mg/kg/day) (David et al., 1996
). Evaluation of effects in male mice at lower doses of DEHP also demonstrated an increased incidence of hepatocellular carcinomas and adenomas at 1500 ppm, but not at 500 ppm (David et al., 1997
). An approximate daily dose of greater than 200 mg/kg/day in susceptible species appears to induce hepatocellular cancer.
In the absence of chronic carcinogenicity data for dietary administration of DEHP in Syrian Golden hamsters, continuous lifetime inhalation exposure to DEHP (15 ± 5 µg/m3 maximum total dose of 710 mg DEHP/kg body weight [bw]) or weekly intraperitoneal injections of DEHP (3 g/kg/week, maximum total dose of 54 g DEHP/kg bw) revealed no increase in hepatic tumor formation (Schmezer et al., 1988). Studies utilizing peroxisome proliferators with greater potency than DEHP, such as the potent ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid, Wy-14,643) and the moderately potent hepatocarcinogens (clofibrate, nafenopin), indicate hamsters are less responsive than rats and mice to the other hepatic effects of peroxisome proliferators (Lake et al., 1986
; Marsman et al., 1992
). In hamsters, the peroxisome proliferators, Wy-14,643, nafenopin, and clofibric acid, produced a modest increase in the relative liver weight and peroxisomal enzyme activity (PBOX) (Lake et al., 1993
; 1989
; 1986
). Furthermore, Wy-14,643 and nafenopin treatment produced no increase in hepatic replicative DNA synthesis in hamster liver (Lake et al., 1993
) or primary cultured hepatocytes (James and Roberts, 1996
).
Following dietary administration of DEHP, the hydrolysis products of DEHP (notably 2-ethylhexanol and mono-2-ethylhexyl phthalate [MEHP]) rather than the intact diester (Albro and Lavenhar, 1989) are absorbed across the intestine of mammalian species. DEHP hydrolase activity in pancreatic juices, intestinal contents, and/or intestinal tissue of mice is greater than that in rats, which is greater than that observed in hamsters (Albro and Lavenhar, 1989
; Albro and Thomas, 1973
). Other studies have shown that MEHP appears to be the active metabolite of DEHP responsible for the induction peroxisomes and inhibition GJIC in rat and mouse hepatocytes (Cornu et al., 1992
; Klaunig et al., 1988
; Lington et al., 1994
; Mitchell et al., 1985
). Hepatic tissue and plasma concentration differences and different metabolic profiles for DEHP amongst species with differing susceptibilities to DEHP-induced tumorigenicity may offer an explanation for the mechanism of DEHP-induced tumorigenicity.
Gap junctions are transmembrane channels formed at the area of contact between adjacent cells. These channels permit the transfer of small molecules (<1 kDa) between the cytoplasm of adjacent cells (Klaunig and Ruch, 1990; Yamasaki, 1990
) and appears to be involved in growth, development, and differentiation of tissues, as well as in the maintenance of homeostasis in multicellular organisms (Loewenstein, 1988
). GJIC appears to be involved in the regulation of cell proliferation through the passage of either growth stimulatory or inhibitory molecules between adjacent cells (Loewenstein and Kanno, 1966
). Many hepatic tumor promoters and nongenotoxic carcinogens inhibit GJIC in rodent hepatocytes in vitro (Klaunig et al., 1990
; Trosko et al., 1990
; Yamasaki, 1990
, 1993). GJIC appears to be an essential feature in the regulation of hepatocyte proliferation. This dysfunctional GJIC may result in the disruption of regulated cell division and be a potential mechanism by which tumor promoters, such as DEHP, enhance preneoplastic cell growth (Klaunig et al., 1990
; Trosko et al., 1990
; Yamasaki, 1990
; Yamasaki et al., 1993
). The inhibition of GJIC in vitro has demonstrated a good correlation with tumor promoting activity and has been used as a short-term assay for the detection of tumor promoters. However, concerns, such as the physiological relevance of the concentration examined and limitations of cultured cell viability and duration, complicate in vitro analysis of GJIC and the relevance to its effect in vivo.
Inhibition of GJIC in tissues appears to correlate to the increased replicative DNA synthesis observed following treatment with tumor promoters (Bager et al., 1994; Fitzgerald et al., 1989
; Krutovskikh et al., 1995
; Neveu et al., 1990
; 1994
). Reduced immunohistochemical detection of the gap junction proteins, connexins, or reduced levels of connexin mRNA have been observed in preneoplastic hepatic focal lesions and hepatocellular adenomas and carcinomas (Fitzgerald et al., 1989
; Neveu et al., 1990
; Tsuda et al., 1995
). Therefore, while immunohistochemical detection and measurement of mRNA levels of gap junction proteins may permit an estimation of the functionality of gap junctions, it does not measure the functional capacity of gap junctions.
The present study utilized a novel technique, in situ dye transfer (ISDT), to examine the effect of DEHP on GJIC in intact rodent liver and we compared it to other endpoints associated with the peroxisome proliferator-induced hepatocarcinogenicity (increased relative liver weight, peroxisome proliferation, and hepatic replicative DNA synthesis). The purpose of this study was to obtain dose-response information for DEHP on selected liver endpoints in rodents in subacute (1- to 6-week) dietary studies. Male B6C6F1 mice, Fischer 344 rats, and Syrian Golden hamsters were selected for these studies because this combination of species reflects differences in susceptibility to the hepatic effects and tumorigenicity of DEHP.
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MATERIALS AND METHODS |
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Animals and treatment.
Male Fischer 344 rats, B6C3F1 mice, and Syrian Golden hamsters (6 to 8 weeks of age) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and housed under standard conditions in an AALAC-accredited laboratory animal research center (LARC) at the Indiana University School of Medicine (Indianapolis, IN). All animals were maintained in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (US DHEW, 1978). Animals were housed in polycarbonate cages with microbarrier isolation tops, bedding, deionized water, and maintained on a 12-h light/dark cycle. During a 1-week acclimation period, all animals received pelletized NIH-07 diet and deionized water ad libitum. At the conclusion of the acclimation period, the animals were randomly placed into treatment groups (5 animals per group). Treatment groups are as indicated:
Gap-Junctional Intercellular Communication (GJIC).
GJIC was determined by direct measurement of dye flow in liver slices using in situ dye transfer (ISDT) as previously described by Landis et al. (1997). Immediately upon euthanasia, a representative 25-mm strip of liver was removed, rinsed in sterile ISDT buffer containing 5.6 mM dextrose, 0.34 mM anhydrous potassium phosphate, 5.36 mM potassium chloride, 136.9 mM sodium chloride and 0.34 mM anhydrous sodium phosphate (pH 7.2) and placed in 5 ml of lucifer yellow CH dye (1 mg/ml ISDT buffer). Two to 3 straight incisions perpendicular to the cut edge were made in the tissue with a double-edged razor blade. The tissue was then placed in the dark at 37°C for 5 min to permit dye transfer. The liver slice was then removed from the dye solution, rinsed in ISDT buffer, and fixed in 10% phosphate buffered formalin at room temperature for 2448 h, in the dark. Following the formalin fix, the slices were progressively dehydrated in alcohol for one h, infiltrated with catalyzed JB4 monomer embedding solution (Polysciences, Inc., Warrington, PA) for 2 h, embedded in plastic molding cups (Polysciences, Inc., Warrington, PA), covered with EBH-2 plastic block holders (Polysciences, Inc., Warrington, PA), and cured in the dark for at least 48 h. Serial sections (5 µM) were cut and computer-based imaging (BDS Image, Oncor, Inc., Gaithersburg, MD) was used to evaluate the length of dye transfer from the incision into the surrounding hepatocytes in each liver slice, as a measurement of GJIC. A standard curve was generated based on the size and amount of dye present in 100 individual, randomly selected control cells. This standard curve was used to convert the length of dye transfer in arbitrary units assigned by BDS Image to the distance of dye transfer (in mm) and the number of cells communicating. The calculation is made using the equations:
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The length of transfer for each sample was determined by averaging the length of dye transfer in 5 to 10 individual measurements from each animal. The average length of dye transfer for each treatment group was obtained by determining the mean ± standard deviation from each animal in that treatment group.
Replicative DNA synthesis.
Measurement of replicative DNA synthesis was performed by immunohistochemical demonstration of BrdU formalin fixed, paraffin embedded sections of liver as previously described (Eldridge et al., 1990; Isenberg et al., 1997
). Zonal examination of labeling in hepatocytes was performed as described previously (Barrass et al., 1993
). The hepatic labeling index represents the number of labeled hepatocytes divided by the total number of hepatocytes and multiplied by 100. A total of at least 1000 hepatocytes were examined for each animal in each treatment. The average labeling index for each treatment group was obtained by determining the mean±standard deviation from each animal in that treatment group.
Peroxisomal Beta-Oxidation (PBOX).
The induction of PBOX activity correlates with an increased number of peroxisomes (Foster et al., 1986; Lin, 1987
; Sharma et al., 1988
), therefore measurement of PBOX activity provides an assessment of the induction of enzyme activity and organelle proliferation. Hepatic PBOX activity in liver samples (0.250.50 gs) from each animal was measured as previously described (Lazarow and DeDuve, 1976; Isenberg et al., 1997
).
Tissue analysis for DEHP and metabolites.
Extraction and high pressure liquid chromatography (HPLC) analysis of DEHP, MEHP, and PA from liver was performed as previously described by Dine et al. (1991). Briefly, approximately 100 to 300 mg of liver was homogenized in 1 ml of methanol, rinsed twice with 1 ml acetonitrile, and added to the methanol homogenate. Compounds of interest were extracted by continuous mixing at 04°C for 12 h, centrifuged (15 min, 12,000 x g at 4°C) and the supernatants removed for HPLC analysis (Hewlett-Packard 1050 solvent delivery pump, autoinjector, variable wavelength detector, and Hewlett Packard 3396A integrator (Palo Alto, CA). DEHP, MEHP, and PA were measured by injection of 10 µL of liver homogenate supernatant onto a 5 µm hypersil ODS C18 reversed phase column (4.6 x 150 mm). The mobile phase consisted of 85% 50 mM KH2PO4 (pH 3.0) and 15% acetonitrile with a flow rate of 1.0 ml/min for PA analysis, 40% 50 mM KH2PO4 (pH 3.0) and 60% acetonitrile with a flow rate of 1.5 ml/min for MEHP analysis, and 100% acetonitrile with a flow rate of 1.5 ml/min for DEHP analysis. Ultraviolet absorbance was monitored at 254 nm. Calibration curves were prepared from analytical standards on each day of analysis and were linear between 1100 µM. Analyte concentrations were determined on calibration curves (plot of the analyte peak area versus concentration). Extraction recovery was 99% for PA, 95% for MEHP, and 97% for DEHP using these methods, and results were corrected for the extraction recovery. A total of 3 to 5 animals were evaluated from each treatment group and the results are expressed as mean ± standard error (SE). Concentrations of 2 other primary DEHP metabolites (2-ethylhexanoic acid and 2-ethylhexanol, the cleaved alkyl side chains) were not determined, because previous studies have demonstrated that these metabolites do not induce tumors (Astill et al., 1996) or peroxisomal-mediated changes in hepatocytes (Cornu et al., 1992
; Lington et al., 1994
; Mitchell et al., 1985
).
Statistics.
Statistical differences (p < 0.05) from control values for all data was determined by 2-way ANOVA followed by a Dunnett's test (Gad and Weil, 1988). The least-squares means post hoc test was used for analysis of the determination of the hepatic concentration of DEHP and metabolites. A total of 3 to 5 animals were evaluated for each experimental group. Data are expressed as the mean ± standard deviation (SD).
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RESULTS |
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In male B6C3F1 mice, 2 and 4 weeks of treatment with 500 ppm DEHP increased the body weight gain over control. 6000 ppm DEHP had no effect on body weight gain following either of the time points examined in the present study (data not shown).
An increase in body weight gain over control in male Syrian Golden hamsters was observed following dietary administration of 1000 ppm DEHP for 2 weeks. We observed no effect on body weight gain following dietary treatment with 1000 ppm for 4 weeks or 6000 ppm for 2 and 4 weeks (data not shown).
Relative Liver Weight
Concentration-related effects on the relative liver weight (RLW) (ratio of liver weight to terminal body weight x 100) were observed in rats following dietary administration of DEHP (Table 1). The RLW increased over control following 1 (5.7 versus 4.4%), 2 (6.4 versus 4.6%), 4 (5.8 versus 4.1%), and 6 (5.6 versus 3.7%) weeks of treatment with 6000 ppm DEHP. Furthermore, dietary administration of 20,000 ppm DEHP increased the RLW when compared to control following 1 (6.4 versus 4.4%), 2 (7.2 versus 4.6%), 4 (7.3 versus 4.1%), and 6 (7.2 versus 3.7%) weeks of treatment (Table 1
). The largest increase in RLW was observed following treatment with 20,000 ppm DEHP for 6 weeks (94% increase over control). Additionally, the non-tumorigenic concentration of DEHP (1000 ppm) had no effect on the RLW following 2 weeks of treatment (Table 1
). However, dietary administration of 12,000 ppm DEHP for 2 weeks increased the RLW over control (7.2 versus 4.6%) (Table 1
).
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In hamsters, dietary administrations of 1000 ppm DEHP for 2 weeks had no effect on the RLW (Table 1). However, following 4 weeks of treatment, an increase in the RLW was observed over control (6.2 versus 5.0%). Dietary administration of 6000 ppm increased the RLW over control following 2 (7.1 versus 5.8%), and 4 (6.2 versus 5.0%) weeks of treatment (Table 1
).
Peroxisomal Beta-Oxidation (PBOX)
Previous studies suggested the hepatocarcinogenic effect of phthalate esters such as DEHP may be related to the induction of peroxisomal enzymes (Reddy and Rao, 1989; Lake et al., 1990
). The present study examined the effect of DEHP on PBOX activity as a measure of the induction of peroxisomal enzymes and peroxisomal proliferation. In rats, 6000 ppm DEHP increased PBOX activity over control following 1, two2, 4, and 6 weeks of dietary treatment (Table 1
). Furthermore, treatment with 20,000 ppm DEHP increased PBOX activity following 1, 2, 4, and 6 weeks of treatment. Additionally, 2 weeks of treatment with 1000 ppm had no effect on PBOX activity (Table 1
). However, treatments with 12,000 ppm DEHP for 2 weeks increased PBOX activity (Table 1
).
In mice, the non-tumorigenic concentration of DEHP, 500 ppm, had no effect on PBOX activity following 2 and 4 weeks of dietary treatment (Table 1). An increase over control in PBOX activity was observed following 2 and 4 weeks of treatment with the tumorigenic concentration of DEHP, 6000 ppm (Table 1
).
Dietary administration of 1000 and 6000 ppm DEHP to hamsters had no effect and increased PBOX activity over control, respectively, following 2 weeks and 4 weeks of treatment (Table 1).
These data indicate DEHP induced a time- and concentration-dependent increase in PBOX activity in rats and mice. Hamsters were less responsive to the induction of PBOX activity than rats and mice.
Gap-Junctional Intercellular Communication (GJIC)
Previous studies in primary cultured hepatocytes demonstrated a relationship between the inhibition of GJIC and tumor promoting activity (Klaunig et al., 1990; Trosko et al., 1990
; Yamaski, 1990; Yamaski et al, 1993). The present study utilized a novel technique, in situ dye transfer (ISDT), to examine GJIC in intact rodent liver treated with DEHP. In rats, dietary administration of 6000 ppm DEHP reduced GJIC following 1, 2, 4, and 6 weeks of treatment (Fig. 1
). Treatment with 20,000 ppm DEHP reduced GJIC following one, two, four and six weeks of dietary administration (Fig. 1
). Additionally, the non-tumorigenic concentration of DEHP, 1000 ppm, had no effect on GJIC, however 12,000 ppm DEHP reduced GJIC following two weeks of treatment (Fig. 1
).
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Replicative DNA Synthesis
In rats, dietary administrations of 6000 ppm DEHP increased replicative DNA synthesis in periportal and centrilobular hepatocytes following 1 and 2 weeks of treatment. As seen in Figure 3, 6000 ppm had no effect on replicative DNA synthesis at any of the other time points examined in the present study. Following 1, 2, and 4 weeks of treatment with 20,000 ppm DEHP, an increase over control in replicative DNA synthesis was observed in periportal hepatocytes (Fig. 3
). No increase in periportal replicative DNA synthesis was evident following 6 weeks of treatment with 20,000 ppm DEHP. Furthermore, replicative DNA synthesis in centrilobular hepatocytes was increased over control following 1 and 2 weeks of dietary administration of 20,000 ppm DEHP (Fig. 3
). The non-tumorigenic concentration of DEHP, 1000 ppm, had no effect on periportal or centrilobular replicative DNA synthesis following 2 weeks of dietary treatment (Fig. 3
). However, the tumorigenic concentration of DEHP, 12,000 ppm, increased periportal and centrilobular hepatocellular replicative DNA synthesis following 2 weeks of treatment (Fig. 3
).
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These studies indicate DEHP induces a rapid, transient increase in replicative DNA synthesis in periportal and centrilobular hepatocytes in a concentration-dependent manner in rats and mice. The increase in replicative DNA synthesis observed in the present study was larger in periportal hepatocytes than centrilobular hepatocytes.
Analysis of DEHP and Primary Metabolites in Liver
Concentration of DEHP in rodent liver.
Evaluation of the hepatic concentration of DEHP and the 2 primary metabolites, MEHP and PA, showed species differences (Figs. 611). Importantly, the hepatic concentration of DEHP, MEHP, and PA in untreated animals was minimal or below the analytical detection limit. MEHP was the most prominent metabolite observed in all species. Hepatic PA concentrations were determined to be slightly greater than DEHP concentrations.
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The concentrations of DEHP in the livers of mice treated with 500 and 6000 ppm DEHP were 4.0 and 9.5 µmol DEHP/g tissue and 3.2 and 7.3 µmol DEHP/g tissue, respectively, following 2 and 4 weeks of treatment (Fig. 7).
The concentration of DEHP in the livers of hamsters treated with 1000 and 6000 ppm DEHP for 2 and 4 weeks are shown in Figure 7: 2.2 and 3.4 µmol DEHP/g tissue were observed following treatment with 1000 ppm DEHP for 2 and 4 weeks (Fig. 7
). The concentration of DEHP in the livers of hamsters fed 6000 ppm DEHP for 2 and 4 weeks were 5.2 and 5.0 µmol DEHP/g tissue (Fig. 7
).
Concentration of MEHP in rodent liver.
The hepatic MEHP concentration increased in a concentration-dependent manner through a 6-week DEHP treatment in rats and through 4 weeks of treatment in mice and hamsters. Following treatment with 6000 ppm DEHP for 1, 2, 4, and 6 weeks, the hepatic MEHP concentrations were 11.9, 22.3, 39.7, and 74.3 µmol MEHP/g tissue (Fig. 8). The concentration of MEHP in the liver of rats fed 20,000 ppm DEHP for 1, 2, 4, and 6 weeks was 50.9, 60.1, 86.3, and 151.9 µmol MEHP/g tissue, respectively (Fig. 8
). Following a 2-week treatment with 1000 and 12,000 ppm DEHP, 8.7 and 42.8 µmol MEHP/g tissue was observed.
In mice, a concentration- and time-dependent increase in the hepatic concentration of MEHP was observed following treatment with DEHP. Hepatic MEHP concentration in mice fed 500 and 6000 ppm DEHP was 28.1 and 62.1 µmol MEHP/g tissue and 40.2 and 71.6 µmol MEHP/g tissue following 2 and 4 weeks of treatment (Fig. 9).
The concentration of MEHP in the liver of hamsters fed 1000 and 6000 ppm DEHP for 2 and 4 weeks is shown in Figure 9: 10.4 and 21.0 µmol MEHP/g tissue was observed following treatment with 1000 ppm DEHP for 2 and 4 weeks (Fig. 9
). The concentration of MEHP in the liver of hamsters fed 6000 ppm DEHP for 2 and 4 weeks was 8.7 and 8.1 µmol MEHP/g tissue (Fig. 9
).
Concentration of PA in rodent liver.
Following treatment with 6000 ppm DEHP for 1, 2, 4, and 6 weeks, the concentrations of PA in rat liver were 1.8, 7.1, 10.7, and 4.6 µmol PA/g tissue (Fig. 10). The hepatic PA concentrations following treatment with 20,000 ppm DEHP for 1, 2, 4, and 6 weeks were 5.0, 9.9, 18.4, and 7.1 µmol PA/g tissue (Fig. 10
). Following a 2-week treatment with 1000 and 12,000 ppm DEHP, 4.9 and 9.0 µmol PA/g tissue were observed in rat liver.
The concentration of PA in the liver of mice fed 500 and 6000 ppm DEHP for was 1.7 and 9.6 µmol PA/g tissue and 2.8 and 10.1 µmol PA/g tissue following two and four weeks of treatment (Fig. 11).
The concentrations of PA in the livers of hamsters treated with 1000 and 6000 ppm DEHP for 2 and 4 weeks are shown in Figure 11: 5.0 µmol PA/g tissue was observed following treatment with 1000 ppm DEHP for both 2 and 4 weeks (Fig. 11
). The concentration of PA in the livers of hamsters fed 6000 ppm DEHP following 2 and 4 weeks was 8.7 and 8.1 µmol PA/g tissue (Fig. 11
).
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DISCUSSION |
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The hypertrophic response is characterized by an increase in peroxisome number, volume, and enzymatic activity. Following treatment with peroxisome proliferators, the enzymatic profile of the peroxisome is altered. The expression of the enzymes responsible for peroxisomal beta oxidation of very long-chain fatty acids, acyl CoA oxidases, are induced, whereas the expression of other enzymes in the peroxisome are unaltered or reduced (Goel et al., 1986; Lake et al., 1987
; 1990
; Reddy and Rao, 1989
). The enzymes involved in the oxidation of fatty acids produce H2O2 as a byproduct of beta-oxidation. As a result of an increased production of H2O2 and the unaltered expression of the H2O2 detoxifying enzymes within the peroxisome, Reddy and coworkers proposed that a state of oxidative stress is induced (Reddy et al., 1980
). These reactive oxygen species (ROS) may then leak into the cytoplasm and bind to or react with cellular macromolecules and/or DNA and may be responsible for the observed hepatocarcinogenicity of these compounds in rodents (Reddy et al., 1980
). However, the present study and others (Lake et al., 1993
) demonstrated that species non-responsive to peroxisome proliferator-induced hepatocarcinogenicity, such as Syrian Golden hamsters, are susceptible to the induction of peroxisomes, albeit not as large a response as observed in the sensitive species. Additionally, treatment with an anti-carcinogen, rotenone, prevents the Wy-14,643-induced increase in replicative DNA synthesis and mitosis without inhibiting the induction of PBOX activity (Cunningham et al., 1995
; Isenberg et al., 1997
). These studies demonstrated the anticancer activity of rotenone against hepatic focal lesion growth in mice suggests the alteration of hepatocellular proliferation is more important in the carcinogenicity of Wy-14,643 than the induction of peroxisomal activity (Isenberg et al., 1997
). Although some evidence for the oxidative stress hypothesis secondary to peroxisomal proliferation exists, measurement of oxidative damage reveals only a weak correlation between the induction of PBOX and the observed carcinogenicity. The present study and others demonstrate a weak correlation between the induction of PBOX and the reported carcinogenicity of DEHP (Eacho et al., 1991
; Elcombe et al., 1986; Marsman et al., 1988
). Furthermore, an increase in the RLW in rats, mice, and hamsters was observed in the present study. As the susceptibility to the hepatocarcinogenic effect of DEHP appears to be species-specific (Huber et al., 1996
; Kluwe et al., 1982
; Thomas and Thomas, 1984
), evaluation of PBOX and RLW may not be a predictive indicator of the susceptibility of different species to DEHP-induced hepatocarcinogenesis.
Alternatively, the hyperplastic response attributed to peroxisome proliferators is characterized by a transient increase in replicative DNA synthesis. Most peroxisome proliferators produce a transient increase in replicative DNA synthesis, primarily in periportal hepatocytes, that subsides by the second or third week of treatment (Barrass et al., 1993; Lake et al., 1993
; Marsman et al., 1988
). The effect of peroxisome proliferators on centrilobular hepatocytes appears to be primarily the induction of peroxisomal enzymes and, to a lesser extent, replicative DNA synthesis (Barrass et al., 1993
; Bell et al., 1991
; Eacho et al., 1991
; Eldridge et al., 1990
; Marsman et al., 1988
;). The induction of replicative DNA synthesis may promote the development of spontaneously initiated hepatocytes to neoplasms. Examination of oxidative stress in young and old Wistar rats revealed no difference in oxidative damage between the young and aged rats; however, the old rats were more sensitive to the induction of hepatocellular carcinomas by nafenopin (Huber et al., 1991
). The induction of PBOX and the subsequent oxidative damage by the strongly carcinogenic peroxisome proliferator, Wy-14,643, is roughly equivalent to that of the weakly carcinogenic peroxisome proliferator, DEHP (Lake et al., 1986
; Marsman et al., 1992
). Unlike the induction of PBOX, treatment with the strongly carcinogenic peroxisome proliferator, Wy-14,643, produces a large and sustained increase in replicative DNA synthesis in rats but not in hamsters (Lake et al., 1993
), whereas the weakly carcinogenic peroxisome proliferator, DEHP, produces a weaker and transient increase in replicative DNA synthesis (Marsman et al., 1988
; Price et al., 1991
). The present study demonstrated high concentrations of DEHP (20,000 ppm in rats and 6000 ppm in mice) produced a large increase in replicative DNA synthesis that was sustained through 4 weeks of treatment. However, the effect of DEHP on replicative DNA synthesis in hamsters was minimal. These results correlate with the reported species-specific hepatocarcinogenicity of DEHP. The observed increase in centrilobular replicative DNA synthesis may be related to the concentration utilized in the present study.
Gap-junctional intercellular communication (GJIC) involves the passage of ions and/or small molecules involved in the control of cellular growth, differentiation, and death. Reduced GJIC has been associated with rapidly proliferating cells and tissue (Kren et al., 1993; Neveu et al., 1995
). Examination of GJIC in neoplastic cells also reveals a lack of intercellular communication between cancer and normal cells (Esinduy et al., 1995
; Fentiman et al., 1979
; Loewenstein and Kanno, 1966
; Yamasaki et al., 1987
). Furthermore, hepatocytes within hepatic lesions have a reduced amount or loss of gap junction proteins and mRNA when compared to the normal surrounding non-lesion hepatocytes (Fitzgerald et al., 1989
; Janssen-Timmen et al., 1986
; Krutovskikh et al., 1991
; Neveu et al., 1990
; Oyamada et al., 1990
; Sakamoto et al, 1992
; Wilgenbus et al., 1992
). However, transfection of the cDNA encoding the protein(s) that come together to form the functional gap junction or co-culture with subpopulations of communicating cells (Esinduy et al., 1995
) results in the recovery of communication and loss of tumorigenicity (Eghbali et al., 1991
; Mehta et al., 1991
; Naus et al., 1992
; Rose et al., 1993
; Zhu et al., 1991
). Furthermore, in the present study with DEHP and following treatment with phenobarbital and following partial hepatectomy (unpublished data), the reduction of GJIC appears to correspond to enhanced replicative DNA synthesis following acute treatment (12 weeks). However, as the treatment duration is extended, reduced GJIC does not necessarily correlate with enhanced replicative DNA synthesis. Therefore, reduced GJIC is consistently observed in liver undergoing replicative DNA synthesis. These findings indicate that the loss of the control of cell growth through GJIC may play an important role in the development of neoplasms.
Several studies have examined the effect of treatment with tumor promoters including peroxisome proliferators on GJIC in vitro (Ruch et al., 1987; Klaunig et al., 1988
, 1990
; Ruch and Klaunig, 1988
; Leibold et al., 1994
) and in vivo (Krutovskikh et al., 1995
). These studies demonstrated that most tumor-promoting compounds inhibit GJIC in vitro and in vivo. In the present study utilizing ISDT to evaluate GJIC in intact rat, mouse and hamster liver, DEHP produced a concentration-dependent suppression of GJIC in rat and mouse liver. These results are similar to in vitro studies that demonstrated the primary metabolite of DEHP, MEHP, inhibits GJIC in primary cultured rat and mouse hepatocytes (Klaunig et al., 1988
; Lington et al., 1994
). Studies from our lab indicate that MEHP does not suppress GJIC in primary cultured hamster hepatocytes (Baker et al., 1996
). The present study revealed DEHP had no effect on GJIC in Syrian Golden hamster liver. Since, in vitro analysis of GJIC demonstrates a strong correlation with tumor-promoting activity and DEHP is a tumor promoter in rats and mice, but not hamsters, the analysis of GJIC in rodent liver may provide a useful endpoint for the evaluation of hepatic tumor-promoting potential.
The present study demonstrates a modest reduction (<30%) in GJIC that appears to correlate with the carcinogenic potential of DEHP in susceptible species. It is difficult to determine the level of inhibition of GJIC necessary to promote the formation of hepatocellular neoplasms, however, a more detailed concentration and time-response study with DEHP and further evaluation of other nongenotoxic hepatocarcinogens may help further illustrate the level of inhibition of GJIC necessary for the promotion of hepatocellular neoplasms. However, a reduction of GJIC by 2030% was demonstrated in male F344 rats and B6C3F1 mice fed concentrations of DEHP (6000 ppm) that produced hepatocellular neoplasms following chronic treatment (18 and 24 months) (unpublished data).
Analysis of hepatic DEHP, MEHP, and PA concentrations in the present study indicated accumulation of the active metabolite of DEHP, MEHP, in liver is slightly greater in mice than rats and much greater than in hamsters. Comparison of published values from lifetime studies show average daily intake at a 6000-ppm dietary dose of approximately 300 mg/kg/day male rats, with male mice consuming at least 2-fold more at 600 to 780 mg/kg/day (from NTP bioassay, Kluwe et al., 1982; and as calculated by Huber et al., 1996). Yet, the hepatic concentrations of MEHP are greater than 2-fold those observed in rats at this dose. The hepatic concentrations of DEHP and PA were equivalent amongst the 3 species examined in this study. Therefore, the low concentrations of DEHP and PA detected in the livers of all 3 species examined indicate that there is minimal direct absorption of DEHP and PA. This suggests that the majority of DEHP was absorbed as the MEHP metabolite and MEHP appears to be the active metabolite responsible for the hepatic effects of DEHP. Additionally, the concentration of MEHP in the livers of rats and mice was much greater than the hepatic concentration in hamsters. Therefore, the present study and others indicate species differences in esterase activity and GI absorption may be involved in the species-specific response following exposure to DEHP (Albro and Lavenhar, 1989; Albro and Thomas, 1973
; Lhugeunot et al., 1986). This metabolic difference may account for the species sensitivity to the hepatic effects of the phthalate esters. The observation of metabolic profiles and hepatic responses to phthalate esters, similar to those observed in non-responsive species, may permit the extrapolation of short-term markers of carcinogenicity to humans.
The present study demonstrated that the reported species-specificity for tumorigenicity observed in response to treatment with DEHP may be related to reduced GJIC and enhanced cell replication. The species-specific response may be attributed to differing metabolic profiles in rats, mice, and hamsters. In the present study, the hepatic MEHP concentration was elevated in rats and mice. While hepatic MEHP concentrations in hamsters were also elevated, this elevation was approximately 10-fold less than in rats and mice, and may be insufficient to produce a sustained reduction of GJIC and increase in replicative DNA synthesis associated with the carcinogenicity of DEHP. Therefore, examination of replicative DNA synthesis and GJIC in the livers of peroxisome proliferator-treated animals may permit the assessment of the carcinogenic potential of the phthalate esters or other peroxisome proliferators.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at the Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS-1021, Indianapolis, IN 46202. Fax: (317) 274-7787. E-mail: jklauni{at}iupui.edu.
Presented in part at the 38th annual meeting of the Society of Toxicology in New Orleans, LA, March 1999.
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