Initiating Activity of 4-Chlorobiphenyl Metabolites in the Resistant Hepatocyte Model

Parvaneh Espandiari*, Howard P. Glauert*,{dagger}, Hans-Joachim Lehmler*, Eun Y. Lee*,{ddagger}, Cidambi Srinivasan§ and Larry W. Robertson*,{dagger},1

* Graduate Center for Toxicology, {dagger} Graduate Center for Nutritional Sciences, {ddagger} Department of Pathology and Laboratory Medicine, and § Department of Statistics, University of Kentucky Medical Center, Lexington, Kentucky 40536

Received December 14, 2003; accepted January 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently reported that several mono- to tetrachlorinated biphenyls have initiating activity in the livers of Fischer 344 rats. In the present study, we investigated the metabolic activation of one of those compounds, 4-chlorobiphenyl (PCB 3). Monohydroxy (400 µmol/kg), dihydroxy (200 µmol/kg), and quinone (100 µmol/kg) metabolites of PCB 3 were evaluated for their initiating activity. Fischer 344 male rats were fasted for 4 days; 24 h after feeding again, they were injected (ip) with metabolites, vehicle, or diethylnitrosamine (DEN, 20 or 40 mg/kg). All animals were treated with selection agents as follows: three daily p.o. doses of 2-acetylaminofluorene (2-AAF, 30 mg/kg), followed by a single p.o. dose of carbon tetrachloride (2 ml/kg) and three additional daily treatments of 2-AAF. Rats were killed 2 weeks after the last 2-AAF intubation. Livers were evaluated for changes in morphology, and the number and volume of {gamma}-glutamyl transpeptidase-positive foci were measured. Of the metabolites tested, only one monohydroxy and one quinoid metabolite showed initiating activity. The metabolic activation of PCB 3, therefore, proceeds via parahydroxylation and oxidation to the ortho 3,4-quinone, the ultimate carcinogen. This is the first report to demonstrate that specific PCB metabolites possess initiating activity in the rodent liver in vivo. The results support the conclusion that 4-OH PCB 3 and 3,4-BQ PCB 3 act as proximate and ultimate carcinogenic metabolites resulting from the bioactivation of PCB 3 in rat liver.

Key Words: polychlorinated biphenyls (PCBs); PCB 3; hepatocarcinogenesis; initiation; Solt-Farber; altered hepatic foci.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During most of the last century, the commercial use of polychlorinated biphenyls (PCBs) was widespread. Mixtures of these compounds were used in transformers and capacitors as dielectrics, in hydraulic systems, in the formulation of lubricating and cutting oils, in pesticides, and as additives in paints, copying paper, adhesives, sealants, and plastics (ATSDR, 2000Go; Hansen, 1999Go; Robertson and Hansen, 2001Go). Although the production of PCBs has been restricted in many countries since the 1970s, they are still used in closed systems. Also, PCBs remain widely dispersed in the environment, due in part to their resistance to physical and biological degradation and their ability to be transported over longer distances (Lang, 1992Go; Miller et al., 2001Go; Safe, 1994Go). The biologic and toxic effects of the 209 congeneric PCBs (plus many atropisomers) and their mixtures are varied due to differences in structure, which determine their relative rates of metabolism, the spectra of metabolites produced (James, 2001Go), and the numerous receptors with which PCBs and their progeny interact (Bandiera, 2001Go; Pessah and Wong, 2001Go). In animal models, PCB commercial mixtures are complete carcinogens (Mayes et al., 1998Go; Silberhorn et al., 1990Go). In two-stage hepatocarcinogenesis, commercial PCB mixtures as well as individual congeners have activity as promoters (Glauert et al., 2001Go; Silberhorn et al., 1990Go). PCBs that are agonists of the aryl hydrocarbon receptor (PCB 77, 3,3',4,4'-tetrachlorobiphenyl and PCB 126, 3,3',4,4',5-pentachlorobiphenyl) and PCBs that are agonists of the constitutively active receptor (PCB 153, 2,2',4,4',5,5'-hexachlorobiphenyl) are active (Glauert et al., 2001Go).

Several studies have focused on the metabolism of lower chlorinated PCBs, since they are substrates of the hepatic enzymes cytochrome P450 1A and 2B and may be metabolically activated to electrophiles, namely arene oxides and (semi)quinones (Amaro et al., 1996Go; McLean et al., 1996aGo,bGo; Safe, 1984Go; Sundstrom et al., 1976Go). These reactive metabolites may bind to cellular nucleophiles like glutathione and to macromolecules like DNA, RNA, protein, including nuclear protein, and hemoglobin (Ludewig, 2001Go; McLean et al., 1996bGo; Narbonne and Daubeze, 1980Go; Oakley et al., 1996bGo; Pereg et al., 2001Go; Tampal et al., 2003Go). The generation of reactive oxygen species (ROS) during oxidative metabolism of PCBs has been demonstrated (McLean et al., 2000Go; Srinivasan et al., 2001Go), as has the resulting DNA damage like 8-oxo-deoxyguanosine formation and DNA strand breaks (Oakley et al., 1996aGo; Srinivasan et al., 2001Go).

Recently, using the resistant hepatocyte model (also known as the Solt-Farber protocol) (Semple-Roberts et al., 1987Go; Solt et al., 1977Go; Tsuda et al., 1980Go), we tested the initiating activities of certain lower chlorinated PCBs in our laboratory. We reported that PCBs 3, 15 (4,4'-dichlorobiphenyl), 77, and 52 (2,2',5,5' tetrachlorobiphenyl) were active as initiators in male Fisher 344 rats (Espandiari et al., 2003Go). In the present study, our goal was to investigate and clarify the mechanism of activation of PCB 3 by preparing and testing all known PCB 3 metabolites as initiators of hepatocarcinogenesis. In an earlier study, we identified three monohydroxy metabolites of PCB 3 (2-OH PCB 3, 3-OH PCB 3, and 4-OH PCB 3) and three dihydroxy metabolites of PCB 3 (2,3-diOH PCB 3, 2,5-diOH PCB 3, and 3,4-diOH PCB), the three one-ring precursors of ortho- and paraquinones (Fig. 1; McLean et al., 1996aGo). Based on these data, our working hypothesis was that metabolic activation was achieved through sequential hydroxylation reactions, followed by oxidation to a quinone structure, the ultimate mutagen/carcinogen. To test this progression, we employed decreasing doses of metabolites. The activity of the parent PCB, PCB 3, was demonstrated at a dose of 600 µmol/kg in a previous study (Espandiari et al., 2003Go). Therefore, we evaluated each metabolite at a dose based on where it was found within the sequence: 400 µmol/kg for monohydroxy metabolites, 200 µmol/kg for dihydroxy metabolites, and 100 µmol/kg for para- and orthoquinones. Similar approaches have been employed in unraveling the metabolic activation sequence of polycyclic aromatic hydrocarbons (Thakker et al., 1985Go).



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FIG. 1. The metabolic activation of the PCB 3 4-chlorobiphenyl.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


Chemicals.
2-Acetylaminofluorene (2-AAF), carboxymethy-cellulose, carbon tetrachloride (CCl4), and DEN were purchased from Sigma Chemical Co. (St. Louis, MO).

Study compounds.
The six hydroxy derivatives of PCB 3, 2-OH PCB 3, 3-OH PCB 3, 4-OH PCB 3, 2,3-diOH PCB 3, 2,5-diOH PCB 3, and 3,4-diOH PCB 3, and 1,4-BQ PCB 3 were synthesized and characterized as described previously (Amaro et al., 1996Go; McLean et al., 1996aGo). 2,3- and 3,4-Dihydroxy PCB 3 were oxidized to the respective orthoquinones, 2,3-BQ and 3,4-BQ PCB 3, 24 h before administration of the test compounds to the animals, using the following methodology: the dihydroxy biphenyl was dissolved in deuterated chloroform and twice the amount (by weight) of silver(I)oxide was added. Then, the mixture was stirred for 2 to 3 h in a nitrogen atmosphere in the dark to minimize side reactions. Silver(I)oxide was filtered off to yield a clear, dark brown solution. The purity of the respective orthoquinone was determined by 1H NMR spectroscopy. The solvent was removed under a gentle stream of nitrogen and the brown residue was dried under vacuum to remove solvent residues. The orthoquinones were stored in a nitrogen atmosphere at –20°C for 24 h, suspended in corn oil, and immediately injected into the animals. Control experiments showed some decomposition of 2,3-BQ PCB 3 after 24 h, whereas the decomposition of the 3,4-BQ PCB 3 appeared to be minimal during this time frame.

Analytical data for 2,3-BQ from 2,3-diOH PCB3 are as follows: 1H NMR (400 MHz, CDCl3); and {delta} = 6.41 (ddd, J = 10.0, 1.2, 0.9 Hz, 6H), 7.06 (dd, J = 6.4, 1.2 Hz, 5H), 7.15 (ddd, J = 10.0, 6.4, 1.2 Hz, 4H), and 7.35–7.43 (m, 4H) ppm. The 1H NMR spectrum of 3,4-BQ PCB 3 was in agreement with previously published data (Amaro et al., 1996Go).

Experimental design.
All animal studies were carried out in accordance with University guidelines. Male Fischer 344 rats (150–175 g) were maintained in plastic cages in a controlled environment at 22°C with a 12-h light-dark cycle. Rats were fed an unrefined diet (Purina rodent laboratory chow, Purina Mills, St. Louis, MO) and provided water ad libitum; 1 week later, rats were divided into different groups. In each study, animals were first subjected to 96 h of fasting. Then, 24 h after feeding again, rats were injected (ip) with a single bolus dose of the suspected PCB initiating agent (2-OH PCB 3, 3-OH PCB 3, or 4-OH PCB 3 at 400 µmol/kg; 2,3-diOH PCB 3, 2,5-diOH PCB 3, or 3,4-diOH PCB 3 at 200 µmol/kg; or 2,3-BQ PCB 3, 1,4-BQ PCB 3, or 3,4-BQ PCB 3 at 100 µmol/kg) or of vehicle (corn oil, 5 or 10 ml/kg) or diethylnitrosamine (DEN, 20 or 40 mg/kg, via gavage) as a positive control. Then, 2 weeks later, all rats received three daily doses of 2-AAF (30 mg/kg) via gavage, followed by a single dose of CCl4 (2 ml/kg) via gavage (diluted 1:1 with corn oil) and three additional daily treatments of 2-AAF (Semple-Roberts et al., 1987Go; Solt et al., 1977Go; Tsuda et al., 1980Go). The animals were euthanized 2 weeks after the last 2-AAF injection by CO2 asphyxiation. The livers were surgically removed, weighed, and carefully examined for the presence of clearly visible nodules. Liver tissue samples from each lobe were removed, fixed in neutral buffered formalin, and embedded in paraffin for histologic analysis. These pieces were sectioned into 5 µm slices, stained with hematoxylin and eosin, and examined by light microscopy. Hepatic lesions were classified as follows: (1) foci of cellular alteration (clear cell foci, eosinophilic foci, basophilic foci, and mixed foci); (2) hepatocellular adenoma; and (3) hepatocellular carcinoma, using the National Toxicology Program Nomenclature for Hepatoproliferative Lesions of Rats (Maronpot et al., 1986Go).

Altered hepatic foci (AHF) analysis.
Random pieces from each lobe were placed into cryomolds (Miles Laboratories, Naperville, IL) filled with Tissue-Tek OCT compound, placed on dry ice until frozen, and stored at –80°C. Cryostat sections (10 µm) were prepared from each frozen tissue and stained for {gamma}-glutamyltranspeptidase (GGT). The number and volume of GGT-positive foci were quantified using a computer digitizing system. The analyses of the number of foci/cm3 (Saltykov method), foci/liver (Saltykov method), and the volume fraction (Delesse method) were carried out as described previously (Campbell et al., 1982Go, 1986Go; Xu et al., 1998Go).

Statistical analysis.
The data for the primary variables, focal volume, foci/liver, and foci/cm3, are summarized as means ± SE for each treatment. The statistical analysis focused on the comparison of the effects, as measured by the primary variables, of the PCB treatment groups with the control (corn oil) for each experiment. The statistical methodology for the analysis of foci/liver and foci/cm3 was based on an overdispersed generalized linear model with logarithm as link function and negative binomial distribution. The parameters of the model were estimated by the method of maximum likelihood and the goodness of fit of the model was assessed by the Pearson chi-square value adjusted for overdispersion. The p values for the significance of the differences between the PCB treatment groups and the control were determined by Wald's procedure. The above statistical analyses were performed with the aid of the statistical software PROC GENMOD of SAS, version 8 (SAS Institute, Inc., Cary, NC). The use of negative binomial regression model in this analysis was necessitated by the discrete nature of the two primary variables foci/liver and foci/cm3 involving counts of foci. It is well established that Poisson or negative binomial regression models are best suited (Espandiari et al., 2002Go, 2003Go; Wiencke et al., 1999Go) for analyzing discrete data. In this study, the negative binomial model was chosen because it gave a better model fit. The analysis of focal volume for the comparison of treatment groups with the control was based on ANOVA for the general linear regression model, and the computations were carried out using the statistical software GLM of SAS version 8 (SAS Institute, Inc.).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have reported that several lower chlorinated PCBs have initiating activity in the resistant hepatocyte model (Espandiari et al., 2003Go). In the present study, we examined which metabolite(s) of PCB 3 could lead to tumor initiation. Initially, animals were fasted for 4 days, followed by feeding for 24 h before administration of the putative initiating agents. The use of fasting instead of partial hepatectomy was chosen as a less stressful procedure for the animals, and fasting is effective for detecting initiating agents (Espandiari et al., 2002Go; Laconi et al., 1995Go). The metabolites selected for testing (Fig. 1) were those primary and secondary metabolites of PCB 3 identified in a detailed metabolism study conducted in our laboratory (McLean et al., 1996aGo).

In all studies, rats treated with PCB 3 metabolites did not differ significantly from control rats either in their body weights or liver weights (data not shown). However, in each study where the selection groups received DEN as a positive control (20 or 40 mg/kg), body weights were significantly decreased and liver weights were significantly increased compared with other treatment groups (data not shown).

In the first experiment, the initiating activities of two primary metabolites of PCB 3, the 3- and the 4-OH PCB 3 metabolites (400 µmol/kg), were investigated. The 4-OH PCB 3-treated rats showed significant increases in the number of GGT-positive foci per cm3 of liver and per liver and in the volume fraction occupied by GGT-positive foci. The number and the volume of the GGT-positive foci were also increased to about the same level by 3-OH PCB 3, but this apparent increase was not statistically significant because of the large biological variability of the group (Table 1). The incidence of microscopic adenomas was not significantly increased by either PCB metabolite or by DEN.


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TABLE 1 Effects of PCB 3 Metabolites, 3-OH and 4-OH PCB 3, on the Production of {gamma}-Glutamyltranspeptidase-Positive Foci and on the Induction of Hepatocellular Adenomas

 
In the second experiment, metabolites possibly derived from 3-OH PCB 3 or 4-OH PCB 3, two dihydroxy and one quinone metabolite, were tested. The dihydroxy compounds, 3,4- and 2,5-diOH PCB 3, were administered at a dose of 200 µmol/kg and 1,4-BQ PCB 3 was given at a dose of 100 µmol/kg. None of the PCB 3 metabolites in this experiment led to significant increases in the number or volume of GGT-positive foci compared with the vehicle group (Table 2). The 1,4-BQ-treated animals showed structural liver damage. The histopathologic analysis showed microscopically recognizable adenomas in some animals in different groups, but there were no statistically significant differences (Table 2).


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TABLE 2 Effects of PCB 3 Metabolites, 3,4-diOH PCB 3, 2,5-diOH PCB 3, and 1,4-BQ PCB 3, on the Production of {gamma}-Glutamyltranspeptidase-Positive Foci and on the Induction of Hepatocellular Adenomas

 
In the third experiment, 2-OH PCB 3, 2,3-diOH PCB 3, and the two orthoquinones 2,3- and 3,4-BQ PCB 3 were investigated. Only 3,4-BQ caused a statistically significant increase in the number and volume of GGT-positive foci (Table 3).


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TABLE 3 Effects of PCB 3 Metabolites, 2-OH PCB 3, 2,3-diOH PCB 3, 2,3-BQ PCB 3, and 3,4-BQ PCB 3, on the Production of {gamma}-Glutamyltranspeptidase-Positive Foci and on the Induction of Hepatocellular Adenomas

 
The hepatocellular adenomas appeared in some rats treated with the lower dose of DEN (20 mg/kg) and in all rats receiving the higher dose (40 mg/kg, Tables 13). Also, DEN significantly increased the number of GGT-positive foci per cm3 and per liver as well as the focal volume. All the rat livers with significantly increased GGT-positive foci or adenomas also had cellular alterations, mainly basophilic, eosinophilic, or mixed.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PCBs as a class are relatively persistent environmental pollutants that are generally resistant to metabolic attack. Routine analysis for PCBs in environmental matrices ignores all but extractable, parent PCBs, and risk analysis and assignment of toxicity equivalency values assumes only binding of parent PCBs to a single receptor. Although metabolism of congeneric PCBs may be slow and generally slower with increasing chlorination, metabolic progeny of PCBs may play important roles in toxification (James, 2001Go; Letcher et al., 2000Go; Mills et al., 1985Go).

Many PCBs undergo hydroxylation reactions catalyzed by cytochromes P450. Monohydroxy metabolites are formed either as a result of direct insertion of a hydroxyl group or via isomerization of an arene oxide intermediate (Crawford and Safe, 1979Go; Shimada et al., 1981Go). A second hydroxylation, also catalyzed by cytochromes P450, leads to the dihydroxy metabolites catechols and hydroquinones (McLean et al., 1996aGo). When the two hydroxyl groups are ortho (catechols) or para (hydroquinones) to each other, a subsequent oxidation may form a new electrophile class, the ortho- or paraquinones. During an investigation of the microsomal metabolism of PCB 3, McLean and coworkers (McLean et al., 1996aGo) identified the mono- and dihydroxy metabolites of this PCB (Fig. 1). These metabolites included catechols and hydroquinones, the three one-ring precursors of ortho- and paraquinones. In subsequent in vitro studies, these authors showed that numerous enzymes, such as peroxidases and prostaglandin synthase, were capable of catalyzing the oxidation of these catechols and hydroquinones to quinones (McLean et al., 1996bGo; Oakley et al., 1996bGo). They also showed that these electrophiles bind to both S- and N-nucleophiles, including protein and DNA (Amaro et al., 1996Go; McLean et al., 1996bGo; Oakley et al., 1996bGo; Pereg et al., 2001Go, 2002Go; Srinivasan et al., 2002Go; Tampal et al., 2003Go). During this oxidation, ROS were produced that are themselves capable of inducing DNA damage (McLean et al., 2000Go; Oakley et al., 1996aGo; Srinivasan et al., 2001Go).

In the present study, the parahydroxylation metabolite 4-OH PCB 3, a major microsomal metabolite in PCB 3 metabolism (McLean et al., 1996aGo), was active. 4-OH PCB 3 can be hydroxylated/oxidized to 3,4-BQ PCB 3, also active in this study. Although we expected the intermediate metabolite in this scheme, 3,4-diOH PCB 3, to be positive, it was not. There are several possible reasons for the lack of initiating activity by 3,4-diOH PCB 3. First, the catechol may be very short-lived once it is injected into the animal, which may be due to its rapid conjugation and excretion or its inherent instability in that it degrades quickly. Second, in the metabolic activation of PCB 3, the 4-OH PCB 3 is directly oxidized to 3,4-BQ in one step, bypassing the catechol. Third, ROS generated in the oxidation of the monohydroxy metabolites, and not the catechol, are responsible for the genotoxicity seen. ROS generated with polycyclic aromatic hydrocarbon orthoquinones have recently been shown to cause mutations in p53 (Yu et al., 2002Go). Finally, the sensitivity of the assay is diminished due to the weak initiating activity of the selection agents (see Tables 13). By comparison, vehicle-treated rats that did not receive the selection agents had no altered hepatic foci (data not shown). Therefore, the effects of treatments may not be statistically significant, although they may have biologic activity. The initiating activity found after 3-OH PCB 3, although not statistically significant, was of the same general magnitude as that seen following 4-OH PCB 3. We cannot exclude the possibility that some activation is occurring via other metabolites.

This is the first report to demonstrate that specific PCB metabolites possess initiating activity in the rodent liver in vivo. Although we cannot exclude the contributions of other metabolites such as 3-OH PCB 3, our results support the conclusion that 4-OH PCB 3 and 3,4-BQ PCB 3 act as proximate and ultimate carcinogenic metabolites resulting from the bioactivation of PCB 3 in rat liver.


    ACKNOWLEDGMENTS
 
We thank Ms. Cynthia Long, Divinia Stemm, Izabela Korwel, and Drs. Gabriele Ludewig, Nilufer Tampal, and Job Tharappel for their assistance during this research. This study was supported by grant number P42 ES 07380 from the National Institute of Environmental Health Sciences and by the Kentucky Agricultural Experiment Station. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Sciences, National Institutes of Health.


    NOTES
 

1 To whom correspondence should be addressed at the University of Iowa, Department of Occupational and Environmental Health, College of Public Health, 100 Oakdale Campus #219 IREH, Iowa City, IA 52242-5000. Fax: (319) 335-4290. E-mail: larry-robertson{at}uiowa.edu


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 DISCUSSION
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