Center for Environmental Toxicology and Technology,
* Department of Environmental Health and
Department of Pathology, Colorado State University, Fort Collins, Colorado 805231680
Received July 20, 1999; accepted October 6, 1999
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ABSTRACT |
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Key Words: hexachlorobenzene; pentachlorobenzene; 1,2,4,5-tetrachlorobenzene; 1,4-dichlorobenzene; medium-term bioassays; carcinogenicity.
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INTRODUCTION |
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Although 2-year cancer bioassays in rodents are the most reliable method of determining the carcinogenic potential of xenobiotics, short- and medium-term bioassays have been developed in an attempt to lessen the burden of potential carcinogen screening. The use of glutathione S-transferase (GSTP1-1) expression as a marker for preneoplastic lesions in liver is a well established method, and has been used extensively in medium-term bioassays to identify potential hepatocarcinogens (Ogiso et al., 1990
). GSTP1-1 focus formation used in conjunction with initiation/promotion medium-term study designs, has been shown to correctly identify 97% of known genotoxic hepatocarcinogens and 86% of known nongenotoxic hepatocarcinogens (Hasegawa and Ito, 1994
). The sensitivity and specificity of GSTP1-1 focus formation in liver sections as an endpoint in medium-term study designs has made this combination a reliable tool for screening agents for hepatocarcinogenicity (Hasegawa and Ito, 1990
).
Earlier studies from our laboratory, using GSTP1-1 focus formation in a medium-term initiation/promotion study design, have shown that pentachlorobenzene (PeCB) (Thomas et al., 1998b) and 1,2,4,5-tetrachlorobenzene (TeCB) cause promotion of DEN initiated GSTP1-1 foci, whereas 1,4-dichlorobenzene does not (Gustafson et al., 1998
). Earlier studies have also shown that hexachlorobenzene (HCB) is positive for GSTP1-1 focus promotion using the same study design (Cabral et al., 1996
). Results from studies with PeCB and HCB, within this medium-term study design, have shown that PeCB and HCB treatment causes the induction of non-focal GSTP1-1, and that this induction co-localizes with the induction of c-fos, c-jun, and CYP1A2 in the liver (Thomas et al., 1998c
). Other studies with PeCB showed that liver glutathione concentrations and glutathione-associated enzymes were altered (Thomas et al., 1998b
). Alterations in glutathione-associated enzymes included increases in glutathione reductase (GR) and
-glutamylcysteine synthetase (
-GCS) in the centrilobular region. These changes in GR and
-GCS in the centrilobular region were accompanied by a decrease in GSTP1-1 foci in the centrilobular region, which suggests that these biochemical changes may play some role in the negative selection of foci in the centrilobular region.
The studies presented here compare the carcinogenic potential of 4 chlorobenzene isomers: 1,4-dichlorobenzene (DCB), TeCB, PeCB, and HCB. The potential was determined using a medium-term initiation/promotion protocol, as well as observed changes in glutathione status, non-focal GSTP1-1 induction, and the induction of c-fos, CYP1A2, CYP2B1/2, and CYP2E1 in response to exposure to equimolar levels of these 4 chlorobenzene isomers. These studies provide a unique opportunity to use medium-term carcinogenicity assays, in conjunction with molecular and biochemical measurements, to attempt to identify specific changes in liver associated with hepatocarcinogenicity in this series of isomers.
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MATERIALS AND METHODS |
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Animals and Treatment
Male F344 rats (30 days of age) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and allowed to acclimate for an additional 4 weeks. Following acclimation, animals were randomized by weight and assigned to treatment groups as shown in Figure 1. Groups 2 through 5 consisted of 18 rats each, while 12 rats were allocated to Group 1 (vehicle control), for each study. At Week 0, Groups 3, 4, and 5 received a single intra peritoneal (ip) injection of DEN (200 mg/kg) dissolved in 0.9% saline. Groups 1 and 2 received an injection of saline only. After a 2-week recovery period, chemical treatment was started with daily gavage administration of the appropriate chlorobenzene isomer in a corn oil vehicle through the remainder of the 8-week study. Groups 2 and 5 received chlorobenzene isomer at a dose of 0.4 mmol/kg/day, while Group 4 received 0.1 mmol/kg/day. Groups 1 and 3 received the corn oil vehicle only, by gavage At Week 3, a partial hepatectomy was performed on all animals. Control and treated animals were given food (Harlan Tekad NIH-07 Diet, Madison, WI) and water ad libitum and lighting was set on a 12-h light/dark cycle. These studies were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Animals were housed in a fully accredited American Association for Accreditation of Laboratory Animal Care (AAALAC) facility.
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For studies measuring the induction of CYP1A2, CYP2B1/2, and CYP2E1 in liver, animals were dosed with 0.1 mmol/kg/day of the appropriate chlorobenzene by gavage in corn oil for 1 week. Animals were killed as described above following the 7 days of daily treatment, and livers were removed and stored at 80°C until microsomal fractions were isolated.
Immunohistochemical Staining and Liver Foci Analysis
Acetone-fixed liver sections were deparaffinized in xylene and rehydrated by passage through an alcohol series. Endogenous peroxidase was quenched in 3% hydrogen peroxide for 10 min. The slides were rinsed with deionized water and placed in phosphate buffered saline (PBS) (pH 7.4; 2.7 mM KCl, 0.14 M NaCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4). A standard avidin/biotin (ABC) protocol was followed using an immunoperoxidase kit specific for the primary antibody. GSTP1-1 primary-antibody incubations were done at 37°C for 15 min. A final incubation with 3-amino-9-ethyl carbazole (AEC) was then carried out. Slides were then counterstained with hematoxylin for histologic evaluation.
GSTP1-1 foci were measured using a Leitz light microscope coupled with the Bioquant image analysis system (Version IV). Measurements consisted of manually outlining the GSTP1-1 foci on liver sections and allowing software to compute the area within the outline. Reference areas of liver sections were measured on a separate system using a Dage CCD72 MTI camera (Dage Corporation, Michigan City IN) connected with a Bioquant system (Version 2.60). In this system, images of the liver sections were projected on a computer terminal and automatically outlined and measured by the computer software. Comparisons between treatment groups were made using numbers and areas of foci of more than 0.2 mm in diameter normalized to the reference area.
GSH and GSSG Measurements
GSH and GSSG concentrations were measured in rat livers using a microtiter-plate assay as previously described (Vandeputte et al., 1994). Briefly, approximately 0.5 g of 80°C frozen liver was homogenized in 2 ml of ice-cold 10 mM HCl, an aliquot was taken for total protein determination, and a 480-µl aliquot of tissue homogenate was added to 120 µl of 6.5% (w/v) 5-sulfosalicylic acid. Samples were incubated on ice for 10 min, followed by centrifugation for 10 min. The supernatant was collected and stored on ice until used. Twenty µl of blank, standard, or unknown were added to a 96-well plate, followed by twenty µl of sodium phosphate buffer (143 mM, pH 7.4) with 6.3 mM EDTA to neutralize the samples. Two hundred µl of assay mix, consisting of 143 mM sodium phosphate (pH 7.4), 6.3 mM EDTA (ethylenediaminetetraacetic acid), 1 mM DTNB, and 0.34 mM NADPH, was then added to each well, followed by a 5-min incubation at room temperature. The reaction was begun by the addition of 40 µl of glutathione reductase (8.5 IU/ml in 143 mM sodium phosphate buffer, pH 7.4), and the rate determined by measuring the change in absorbance at 405 nm using a Bio-Tek 9000 plate reader.
GSSG content was determined by following the same procedure, except samples were incubated for one h with 2-vinylpyridine (5 % (v/v) final concentration) prior to the assay. GSH (glutathione), and GSSG (glutathione disulfide) concentrations were calculated from standard curves generated using known concentrations of GSH and GSSG.
Western Blotting
For Western-blot analysis of c-fos and GSTP1-1 samples were prepared by homogenizing approximately 0.5 g of frozen liver tissue in 1.5 ml of 10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1 % (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (w/v) NP-40, and 100 µg/ml phenylmethylsulfonyl fluoride. Homogenized samples were incubated on ice for 30 min , followed by centrifugation at 15,000 x g for 20 min. The resulting supernatant was collected, and total protein concentration determined in the sample. For Western-blot analysis of CYP1A2, CYP2B1/2, and CYP2E1, microsomal fractions were isolated using a standard differential centrifugation procedure and total protein concentration determined. The amount of total protein and the percent SDSPAGE gel used was determined for GSTP1-1, CYP1A2, CYP2B1/2, CYP2E1, and c-fos Western blots, independently based on empirical data and the molecular weight of the protein. Filters were then blocked overnight at 4°C with TBST (10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 0.1 % (v/v) Tween 20) containing 15% nonfat dried milk (NFDM). Filters were then incubated with the appropriate primary antibody in TBST with 15% NFDM for 1 h at room temperature. Following the addition of primary antibody, the filter was washed 3 times with TBST. Filters were then incubated with the appropriate secondary antibody, a species-specific IgG horseradish-peroxidase conjugate, in TBST with 15% NFDM for 1 h at room temperature. Following incubation with the secondary antibody, filters were washed 3 times in TBST. The blots were then developed using ECL detection. Quantitation of blots was done using an imaging densitometer (BioRad).
Total Protein Determination
Total protein in samples was determined using the BCA Protein Assay Reagent with bovine serum albumin as a standard.
Statistical Analysis
Numbers and areas of GSTP1-1 foci greater than 0.2 mm in diameter were analyzed using a standard unbalanced analysis of variance. For the high dose group, between-group post tests were performed using Bonferroni`s adjustment for multiple comparisons (DeVore and Peck, 1996). Comparisons between the low-dose treatment and its respective controls were made using a Student`s t-test. All statistical tests were performed using the Minitab statistical software package, release 10.5 (Minitab, Inc., State College, PA).
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RESULTS |
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DISCUSSION |
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Although the NTP cancer bioassay is considered the definitive method for assessing carcinogenicity, this mode of assessment is too resource-intensive and time-consuming. That is probably the main reason why only about 500 chemicals have been assessed with the NTP/NCI cancer bioassay protocol since its inception 37 years ago. To efficiently obtain information on the approximately 70,000 chemicals in commerce as well as the huge numbers of chemical mixtures relevant to public health, an alternative and predictive toxicology approach must be developed. In this paper, we attempt to strengthen Ito's medium-term, liver-focus bioassay by correlating molecular and biochemical indices with carcinogenic potential as expressed by the increase in GSTP1-1 focus formation.
The present study was undertaken to compare the ability of 4 chlorobenzene isomers, DCB, 1,2,4,5-tetrachlorobenzene (TeCB), pentachlorobenzene (PeCB), and HCB to induce the formation of GSTP1-1 positive foci in an initiation/promotion medium-term assay, as well as to quantify molecular and biochemical changes that seem to be associated with foci formation. Previous studies from our laboratory, and others, have shown that HCB is positive for carcinogenicity in the context of this medium-term assay study design (Benjamin et al., 1996; Cabral et al., 1996
), and that PeCB (Thomas et al., 1998b
) and TeCB are positive, while DCB is negative (Gustafson et al., 1998
). The results from these studies are compiled here as normalized to the DEN control, to account for subjective differences in foci scoring. These data show that although TeCB, PeCB, and HCB all test positive in this assay at both 0.1 and 0.4 mmol/kg/day, HCB induces significantly more foci. This phenomena is not just a factor of dose, however, as none of the compounds that tested positive showed any significant dose-responsiveness between 0.1 and 0.4 mmol/kg/day. Previous studies with HCB, using this medium-term assay design, have shown a clear dose-responsiveness when HCB was given in the diet in a range of 0.6150 ppm; however the dose-responsiveness followed a trend that suggested a plateau level for foci induction (Cabral et al., 1996
). Results measuring the promoting effect of phenobarbital in a similar inititation/promotion protocol have shown similar results at high doses (Kitano et al., 1998
).
Gross pathology of treated versus control livers showed a marked increase in liver weight in TeCB-, PeCB-, and HCB-treated groups. Histologic examination of liver sections showed centrilobular hepatocellular hypertrophy, karyomegaly, and anisocytosis in these same groups. DCB-treated animals showed none of these gross pathologic or histologic changes, showing that induction of these changes is consistent with foci-promoting ability with these 4 isomers. Previous studies from our laboratory have also shown no increase in cell proliferation, as measured by proliferating-cell nuclear-antigen (PCNA) labeling, in response to PeCB exposure as used in these studies (Thomas et al., 1998b). These studies actually showed a decrease in cell proliferation in response to PeCB exposure at 0.4 mmol/kg/day, suggesting that induced cell proliferation is not responsible for the measured promotional effect.
Studies to address changes in molecular or biochemical indicators, that may play a role in chlorobenzene-induced GSTP1-1 foci formation, were done both in animals that received the same treatment regimen as those in focus formation studies, with the exception that they were not DEN-initiated, as well as in animals that received only 7 consecutive days of low-dose chlorobenzene treatment. The molecular and biochemical indicators measured were CYP1A2, CYP2B1/2, CYP2E1, c-fos, non-focal GSTP1-1, and glutathione. These markers were chosen based on previous studies from our laboratory that suggested they might be important indicators that play a role in foci development.
Analysis of glutathione levels in control and chlorobenzene isomer-treated animals showed that reduced glutathione (GSH) levels were unchanged in response to treatment with DCB, TeCB, or PeCB and were slightly elevated in response to HCB. However, oxidized glutathione (GSSG) levels were significantly decreased in response to PeCB and TeCB treatment. These decreases in GSSG in response to PeCB and TeCB were accompanied by a significant increase in the GSH:GSSG ratio. Previous studies from our laboratory have localized the biochemical changes thought to be responsible for the decrease in GSSG to an induction in glutathione reductase (GR) and -glutamylcysteine synthetase (
-GCS), specifically in the centrilobular region (Thomas et al., 1998b
). It has been proposed that these changes in the GSH:GSSG ratio in response to PeCB treatment may play a role in the negative selection of GSTP1-1 foci in the centrilobular region, as this induction is accompanied by a decrease in the relative number of foci in the centrilobular versus non-centrilobular regions. This may account for the relative increase in GSTP1-1 foci with HCB as compared to TeCB and PeCB, as HCB did not induce significant decreases in GSSG or increases in the GSH:GSSG ratio.
The induction of non-focal GSTP1-1 expression is localized to the centrilobular region, and co-localized with CYP1A2, c-fos, c-jun, and porphyrin accumulation in PeCB- and HCB-treated animals (Thomas et al., 1998c). The fact that induction of non-focal GSTP1-1 was seen in response to treatment with PeCB and HCB, but not in TeCB- and DCB-treated animals, suggests that this induction may be linked to porphyrin accumulation, since TeCB and DCB do not cause porphyrin accumulation in the liver (Carlson, 1977
; Chu et al., 1983
).
Induction of CYP1A2 and CYP2B1/2 correlated to the presence or absence of promoting ability in this series of chlorobenzene isomers, and the fold induction of CYP1A2 was consistent with the relative focus-promoting abilities of HCB, PeCB, and TeCB. Previous studies have shown a correlation between induction of CYP2B isozymes and tumor-promoting ability with phenobarbital and barbiturate-like compounds (Diwan et al., 1988; Nims et al., 1987
; Wolff et al., 1991
). Induction of CYP1A1 and CYP1A2 has also been used as an indicator of TCDD-induced changes in growth kinetics in a 2-cell clonal growth model that effectively describes TCDD-induced promotion (Conolly and Andersen, 1997
). However, quantitative correlations between the induction of CYP1A2 and preneoplastic foci formation are tenuous due to the non-linearity of CYP1A1 and CYP1A2 induction by TCDD (Vanden Heuval et al., 1994
). Other studies have shown that levels of CYP1A1 induction do not predict the promoting potency of dioxins (Schrenk et al., 1994
). Taken together, these other studies suggest that the increased induction of CYP2B1/2 by HCB, when compared to PeCB and TeCB, is an effective indicator for the increased GSTP1-1 foci area and numbers seen in this study with HCB.
The goal of these studies was to measure the hepatocarcinogenic potential of these 4 chlorobenzene isomers, along with molecular and cellular changes in the liver that occurred due to chlorobenzene exposure. We are currently developing physiologically based pharmacokinetic (PBPK) models for chlorobenzene isomers in normal (Thomas et al., 1998a), and partially hepatectomized animals (Thomas et al., 1999
), that will allow for prediction of target tissue dosimetry. Our long-term goal is to use the results from short- and medium-term assays, along with target tissue dosimetry from PBPK models, to predict target tissue effects. The next step in the implementation of this approach is to test other chlorobenzene isomers in short-term assays, and to measure the molecular and cellular endpoints presented here. Those results can then be used to predict the effect of the tested isomers in the medium-term, liver-focus bioassay. This type of alternative and predictive approach to toxicology has been proposed to be an effective way to undertake the arduous task of screening the enormous number of chemicals and chemical mixtures that need to be defined in terms of their potential toxicities (Yang et al., 1998
). Although this approach may seem far-fetched, the use of predictive models for tissue dosimetry, along with mechanistic data that may predict disease outcome, is already being implemented into risk-assessment guidelines (Page et al., 1997
).
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: School of Pharmacy, Campus Box C-238, UCHSC, 4200 E. Ninth Ave., Denver, CO 80262.
3 Present address: McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706.
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