* Department of Pathology and
Department of Environmental Health, College of Veterinary Medicine and Biomedical Sciences, Center for Environmental Toxicology and Technology, Colorado State University, Fort Collins, Colorado 80523
Received June 20, 2001; accepted November 9, 2001
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ABSTRACT |
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Key Words: carcinogenesis; polychlorinated biphenyls; liver; rat; promotion; altered hepatic foci; GST-P.
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INTRODUCTION |
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Polychlorinated biphenyls (PCBs) are halogenated aromatic hydrocarbons that have been used widely in industry and are now recognized as worldwide environmental pollutants (Safe, 1994). The biological effects induced by PCBs are dependent on the molecular structure of individual congeners (McFarland and Clarke, 1989
; Safe, 1994
). Congeners lacking or having one chlorine substitution in the ortho position with an additional chlorine in the para position of the biphenyl ring have a more co-planar conformation. PCB congeners substituted in both para positions and at least two meta positions are structurally related to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and they elicit comparable biological effects including binding with the aryl hydrocarbon receptor (AhR), induction of the monooxygenase enzymes cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2), thymic involution, the induction of a wasting syndrome, increased mortality, and a variety of toxic effects in the liver including carcinogenesis (Safe, 1990
). PCB congeners, which bind the AhR and elicit the previously described effects, will be referred to as AhR agonist congeners in this text. Introduction of two or more chlorines in an ortho position results in decreased co-planarity between the two phenyl rings due to stearic interactions. Such PCB congeners exhibit liver tumor promoting activity that is "phenobarbital-like." These compounds are not as potent as the AhR agonist congeners but, like phenobarbital (PB), can induce hepatic tumor promotion. PCBs of this less planar group induce CYP2B1 and CYP2B2 or both CYP1A1/2 and CYP2B1/2 (Safe, 1994
; Safe et al., 1985
). The induction of CYP2B1 by less planar PCBs congeners is mediated by the nuclear receptor constitutive androstane (CAR) (Muangmoonchai et al., 2001
) and will be referred to as CAR agonists in this text.
Human epidemiologic data on the carcinogenicity of PCBs have been equivocal. While no overall increases in cancer-related mortality could be correlated with occupational PCB exposure, some studies reported nonstatistically significant increases of specific cancers, including the grouping of liver, gall bladder, and biliary tract cancers (Kimbrough, 1995). Although the carcinogenicity of PCBs in humans has not been established, the carcinogenic effects of individual PCBs and mixtures of PCBs in laboratory animals have been amply demonstrated (Mayes et al., 1998
).
PCBs in the environment are mixtures of congeners with differing physical and biological characteristics and their impact on biological systems may be due to interactions between different congeners. Despite this, there are relatively few relevant data on such interactive biological effects and risk estimation remains based on toxicologic data for individual PCB congeners (Safe, 1994). Understanding the interactions between PCB 126 and PCB 153 is important due to the potent hepatotoxicity of PCB 126 (Kimbrough, 1995
) and the ubiquitous nature of PCB 153 (Cogliano, 1998
; Hansen, 1998
). This study was designed to evaluate and quantify the interactive hepatic tumor promoting effects of these two environmentally relevant PCBs.
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MATERIALS AND METHODS |
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Animals and care.
Thirty-day-old, female F344 rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and allowed to acclimate to altitude (Fort Collins, CO; 5000 feet) for 4 weeks. Following acclimation, rats were randomized by weight and divided into treatment groups. All animals were housed (3 per cage) in polycarbonate cages with corncob bedding and stainless steel wire tops and were maintained at 25°C with 55% humidity with lighting maintained on a 12-h light/12-h dark cycle. Control and exposed animals were given food (Harland Tedkad NIH-07 diet; Madison, WI) and deionized water ad libitum. Rats were observed for clinical state twice daily, and food and water consumption and body weight were evaluated 3 times weekly. This study was conducted in accordance with the National Institutes of Health guidelines for the care of laboratory animals. Animals were housed in facilities fully accredited by the Association for Accreditation of Laboratory Animal Care.
Experimental design.
To evaluate promotional potential we used a medium-term bioassay for promoters of hepatocarcinogenesis (Ito et al., 1989). This assay employs placental glutathione-S-transferase positive (GST-P+) liver cell foci as markers of preneoplasia in the rat after treatment with the known initiator DEN followed by partial hepatectomy and exposure to a test chemical or chemical mixture. In contrast to some other known rat hepatic preneoplastic markers, such as glucose-6-phosphatase, adenosine triphosphatase,
-glutamyltransferase and glucose-6-phosphatase dehydrogenase, GST-P appears to be more stable after withdrawal of carcinogens from the diet. Ease of visualization has also established GST-P as one of the best markers for detection of early liver lesions for analysis of hepatocarcinogenesis and in rapid bioassay methods for carcinogens. Although not all the liver foci detected may necessarily develop into tumors, good correlation (93% concordance) is obtained between the induction of GST-P positive foci and the incidence of hepatocellular carcinomas in parallel long-term assays. (Hasegawa and Ito, 1994
). At the start of the study, rats received either a single ip injection of DEN (200 mg/kg) dissolved in 0.9% saline or an injection of saline only. Fourteen days after initial DEN/saline treatment, oral gavage administration of PCBs dissolved in 1 ml of corn oil vehicle or corn oil alone was begun. Several dose levels of the two PCBs in corn oil, either alone or in combination were used (Fig. 1
). The low PCB doses were aimed at achieving levels roughly comparable to what has been found in human tissues. The high PCB doses (PCB 126 at 1.0 and 10 µg/kg and PCB 153 at 100010,000 µg/kg) were based on reported studies with PCB promotion of liver foci (Bager et al., 1995
; Hemming et al., 1993
, 1995
). Groups 1 and 2 consisted of DEN-initiated single PCB treated animals. Group 3 contained DEN-initiated PCB combination treated animals. Group 4 contained non-DEN-initiated high dose individual and combined PCB treated animals. This group served to assay the potential of high dose PCBs alone and in combination to induce GST-P foci without prior DEN exposure. Groups 5 and 6 contained the DEN control and vehicle control animals respectively. On Day 21 after initial DEN/saline treatment, a 2/3 partial hepatectomy was performed on all animals. Gavages were administered 3 times weekly through the remainder of the 8-week study. On Day 56 after initial treatment, rats were sacrificed by exsanguination via the abdominal aorta while under isoflorane anesthesia.
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Number and area of GST-P+ foci greater than 0.2 mm diameter per liver sections examined were quantified. GST-P+ foci were measured using a Leitz light microscope coupled with the Bioquant® IV computerized histomorphometry program (B and M Biometrics, Nashville, TN). These measurements consisted of manually outlining the GST-P+ foci on the liver sections and allowing software to compute the enclosed area. Reference areas of liver sections were measured on a separate system using a Dage CCD72 MTI camera (Dage Corporation, Michigan City, IN) connected to a Bioquant® for Windows system (Version 2.6). In this system, images of the liver sections were projected on a computer terminal and automatically outlined and measured by the computer software. Results for the treatment and control groups were expressed as foci area in mm2 of GST-P+ foci more than 0.2 mm in diameter per area of liver sections examined in cm2. The use of foci > 0.2 mm has been shown to provide the best correlation with the development of hepatocellular carcinoma (Tatematsu et al., 1988).
Analysis of PCBs in the liver and adipose tissue.
Samples of liver and fat from animals were analyzed for their PCB content. Tissues were extracted by a modification of published procedures (de Faubert Maunder et al., 1964; Mills et al., 1963
). PCB isomer determinations were by gas chromatography and 63Ni pulsed electron-capture detector operated at 32°C. Analysis was performed with dual column confirmation using a 0.25 mm x 60 m DB-5 capillary column and a 0.32 mm x 30 m DB-608 capillary column. Each set of 10 samples included one reagent blank and a control spike of calibration standard. Evaluation of data was based on residues found in the reagent blank, recovery of a matrix spike of all analytes, and recovery of a PCB (BZ#183) spike from all samples.
Statistical analysis.
Numbers and areas of GST-P foci greater than 0.2 mm in diameter were analyzed using two-way ANOVA for unequal group size, with PCB 126 dose and PCB 153 dose as the 2 main effects including their interaction. This analysis was followed by post hoc comparisons of the PCB treatment group means compared to DEN-corn oil control via Dunnett's multiple comparison procedure. PCB tissue concentrations and changes in liver and body weights were analyzed by one-way ANOVA using Tukey's pairwise comparisons procedure where DEN-initiated, PCB-treated animals were compared to DEN-corn oil controls. All statistical tests were performed using the Minitab statistical software package, release 10.5 (Minitab, State College, PA).
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RESULTS |
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Adipose tissue levels increased in a dose dependent manner in rats treated with PCB 126 or PCB 153 alone (Tables 4 and 5). PCB 126 fat concentration was significantly increased (p < 0.05) compared with PCB 126 exposure alone only after one mixture exposure (PCB 126 1.0 µg/kg + PCB 153 100 µg/kg; Table 4
). PCB 153 fat concentrations after combined PCB 126/153 exposure were not significantly different at any dose compared with PCB 153 exposure alone (Table 5
).
Quantitative Morphometric Evaluation of GST-P Positive Foci
Treatment of non-DEN initiated rats with PCBs alone or in combination did not result in formation of GST+ foci (Group 4, data not shown), thus indicating that the PCBs alone did not have initiating potential in our study. Treatment with PCB 126 or PCB 153 alone resulted in a significant (p < 0.01) dose dependent increase in GST-P+ foci area and number compared with DEN controls (Figs. 2 and 3) at the highest doses. Treatment with the mixture of PCB 126 and 153 resulted in significantly increased foci area and number (p < 0.01) in the 3 highest dose groups (Fig. 4
). When the two PCBs were combined, PCB 153 had an antagonistic effect on PCB 126 GST-P formation. The interaction between the two PCBs after subtraction of the DEN control responses from the PCB-induced responses is shown in Figure 5
. This response was less than additive and analysis (two-way ANOVA using a general linear model) showed this negative interaction to be significant (p < 0.001). PCB mixture responses were less than the predicted additive values of the individual PCBs for all dose groups.
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DISCUSSION |
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Mixtures of AhR agonist and CAR agonist PCB congeners have been reported to influence the occurrence of foci in different ways. A mixture of PCB 77 and PCB 47 was reported by Sargent et al. (1991) to give a seemingly synergistic effect in one dose combination. In a study by Bager et al. (1995), 3 different doses of PCB 126 in combination with one dose of PCB 153 also responded in a synergistic manner. Berberian et al. (1995), however, did not observe any synergistic effects on foci following PCB 77 and PCB 153 exposure, but did report the number and volume of altered hepatic foci were increased by both congeners individually. Our findings that both AhR agonist and CAR agonist PCB congeners can act as promoters of carcinogenesis individually are consistent with the findings of Berberian et al. (1995) and other investigators (Buchmann et al., 1986, 1991
; Cogliano, 1998
; Haag-Grönlund et al., 1997
, 1998
; Safe, 1994
; Silberhorn et al., 1990
). Our finding of antagonistic foci formation differed from reported studies of synergism between these two PCB congeners and other mixtures studies with more planar and less planar congeners (Bager et al., 1995
; Sargent et al., 1991
). This finding was surprising because our studies were based on similar reported dosing schemes (Bager et al., 1995
). A more recent publication, however, is in accordance with our findings of antagonism (Haag-Grönlund et al., 1998
). Haag-Grönlund et al. (1998) reported a combination of PCB 126 and PCB 153 resulted in a weak antagonistic effect for volume fraction of altered hepatic foci. Interestingly, van der Plas et al. (1999) report that addition of PCB 153 to a mixture of AhR agonist polyhalogenated aromatic hydrocarbons, including TCDD and PCB 126, resulted in a higher mean foci volume and volume fraction when compared to the mixture without PCB 153, thus suggesting additivity.
While there has been extensive research on carcinogenicity of dioxins and dioxin-like AhR agonist PCBs, the actions of the CAR agonist PCB congeners are less well studied, as are potential interactions between the 2. Although it is accepted that dioxin-like PCBs mediate their toxic effects via the Ah receptor, little is known about the mechanisms of action of CAR agonist congeners. The varied results obtained from mixtures of AhR agonist and CAR agonist PCBs challenge the hypothesis that the AhR route is the only mechanism involved in tumor promotion by PCB and suggest that other mechanisms may play an important role in the interactive tumor promoting effects of PCBs.
It has been stressed that nonadditive interactions between AhR agonist PCBs or TCDD with CAR agonist PCBs appear to be affected by toxicokinetic interactions (van den Berg et al., 1994) and that knowledge of actual tissue retention is critical both for understanding mechanisms and for providing realistic risk assessments (Giesy and Kannan, 1998
). TCDD binding in liver is related to induction of a specific hepatic binding protein, CYP1A2, and by binding the AhR (van Birgelen et al., 1996
). Selective binding of the AhR by PCB 126 also occurs (Chu et al., 1996
). High doses of PCB 153 are reported to alter the distribution of TCDD, increasing the concentration in liver and decreasing the concentration in adipose tissues via induction of hepatic CYP1A2, which subsequently binds TCDD (De Jongh et al., 1995
). PCB 153 concentration can be increased in liver by a high dose of TCDD (van Birgelen et al., 1996
). PCB 153 is lipophilic and, since TCDD appears to increase PCB 153 levels in the liver, it has been suggested that this is related to increased fat in liver cells due to toxicity of the TCDD (van Birgelen et al., 1996
).
Our data indicate an increase in PCB 153 concentrations in liver when administered in combination with PCB 126 compared with PCB 153 alone. Increased levels of PCB 153 in the liver would be expected to increase promotional effects based on the effect of the chemical alone, rather than to decrease them, as we have seen. Thus, distribution changes alone could not account for the antagonistic promotional effects. While the mechanism of this distribution change is not clear, the rats in the highest dose groups (PCB 126 at 10 µg/kg and PCB 153 at 5000 and 10,000 µg/kg) did have significant weight loss compared to controls. Loss of adipose tissue could have led to redistribution of the PCB 153 from the fat to the liver. As previously mentioned, one study suggested that TCDD toxicity caused hepatic lipidosis and this increased PCB 153 levels in liver because of its lipophilicity (van Birgelen et al., 1996). This is unlikely in our study, since we saw no histologic evidence of hepatic toxicity or visible fat increase after exposure to individual congeners or mixtures. PCB 126 fat concentration after combined PCB 126/153 exposure increased only at one dose combination (PCB 126 1.0 µg/kg + PCB 153 100 µg/kg) and this was a minimal change. The specific congeners in the mixture, the dose levels of the individual congeners, route of administration, duration of exposure, and species and strain of animal no doubt affect such toxicokinetic interactions. Regardless, our results suggest toxicokinetic interactions alone cannot explain the antagonistic promotional response seen with the PCB mixture.
Understanding the mechanism of interaction between PCB 126 and PCB 153 is important because they are among the most environmentally important congeners (Kimbrough, 1995; McFarland and Clarke, 1989
) and are found in significant quantities in human tissues (Cogliano, 1998
; Hansen, 1998
). Currently risk estimation is based on toxicologic data on individual PCBs, mainly the AhR agonist congeners (Giesy and Kannan 1998
; Safe, 1994
; van den Berg et al., 1998
; Whysner and Williams, 1996
). The toxic equivalency factor (TEF) concept is used to facilitate the risk assessment of PCBs and related substances, the potency of the most toxic dioxin, TCDD, is used as a standard and set to 1, and other dioxin-like chemicals are assigned TEFs that are fractions thereof. The total toxic equivalency (TEQ) of a mixture is the sum of the individual potencies. Several assumptions are made when utilizing the TEF scheme. First, all interactions are presumed to be additive in nature even though nonadditive PCB interactions are reported. TEFs are assigned based on a chemical's affinity to bind to the AhR, which CAR agonist PCB congers are unable to do. Therefore, the current system does not include the potential impact of CAR agonist PCB congeners even though they are known promoters. It has been stressed that without consideration of the toxicity of the CAR agonist PCBs, current risk assessment for the PCBs is incomplete (Fischer et al., 1998
; Hansen, 1998
; van den Berg et al., 1998
). With respect to this, our lowest dose of PCB 153 (10 µg/kg body weight) resulted in adipose tissue levels of 872 ± 107 ng/g, only 23 times those reported in human tissues (Hansen, 1998
; Stellman et al., 1998
). Our low dose of PCB 126 (0.1 µg/kg body weight) resulted in tissue levels that were at the lower limit of detection by our analysis system, which is highly sensitive (capable of detecting 8 ng/g and above), again emphasizing the environmental relevance of these studies. Our data demonstrate antagonistic hepatic tumor promoting activity in a mixture of AhR agonist and CAR agonist PCBs, which results in a less than additive GST-P foci formation. The less than additive response was present in all 5 PCB 126/PCB 153 combinations, including low doses of PCB 153 (10 and 100 µg/kg) that alone did not show any evidence of promotional activity (Fig. 5
).
In summary, results from this study confirm that individual treatment with either the CAR agonist PCB 153 or AhR agonist PCB 126 lead to the formation of GST-P+ altered enzymatic hepatic foci and that both congeners can act as promoters of hepatocarcinogenesis. Additionally, treatment with a mixture of PCB 126 and 153 resulted in less than additive promotional interactions. These results suggest that risk estimation for mixtures containing multiple PCBs cannot be based on a model of simple additivity as has been proposed.
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ACKNOWLEDGMENTS |
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NOTES |
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