Environ. Sci. Technol. 2005, 39, 3513-3520
Congener-Specific Tissue Distribution of Aroclor 1254 and a Highly Chlorinated Environmental PCB Mixture in Rats I Z A B E L A K A N I A - K O R W E L , †,‡ KERI C. HORNBUCKLE,§ AARON PECK,§ GABRIELE LUDEWIG,† LARRY W. ROBERTSON,† WIESLAW W. SULKOWSKI,‡ PARVANEH ESPANDIARI,⊥ C. GARY GAIROLA,⊥ AND H A N S - J O A C H I M L E H M L E R * ,† Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, Iowa 52242, Department of Environmental Chemistry and Technology, University of Silesia, Szkolna 9, 40-006 Katowice, Poland, Department of Civil and Environmental Engineering, University of Iowa, Iowa City, Iowa 52242, and Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40546
Polychlorinated biphenyl (PCB) mixtures were synthesized and marketed in Eastern European countries, but little is known about their composition, distribution, or toxicity. PCBcontaminated soil from the former production site of the Polish PCB mixture Chlorofen was collected, and the PCBs were extracted. An in vivo study was performed to investigate the PCB tissue distribution and biochemical effects of this soil extract in immature male SpragueDawley rats. Rats were administered 0.05 mmol/kg soilextracted PCBs or Aroclor 1254 and sacrificed 7 days later, and congener-specific PCB profiles in selected tissues were determined. Distribution of ΣPCBs (sum of 120 congeners) in tissues was primarily a function of lipid content of the tissues, except for the spleen, which retained more PCB than other tissues. Multivariate analysis of the PCB congener data showed that (a) congener profiles in tissues had changed, as compared to the parent mixture; (b) disposition and redistribution of individual congeners in vivo differed between both mixtures; and (c) more highly chlorinated congeners were retained in the spleens of both treatment groups. Differences in the induction of cytochrome P-450 1A and 2B subfamilies reflected the homologue composition of the respective mixtures and predict a different toxicity profile for Chlorofen than for Aroclor 1254.
Introduction Polychlorinated biphenyls (PCBs) are an important class of environmental contaminants (1, 2). They are resistant to * Corresponding author phone: (319)335-4414; fax: (319)335-4290; e-mail:
[email protected]. † Department of Occupational and Environmental Health, University of Iowa. ‡ University of Silesia. § Department of Civil and Environmental Engineering, University of Iowa. ⊥ University of Kentucky. 10.1021/es047987f CCC: $30.25 Published on Web 04/16/2005
2005 American Chemical Society
chemical and thermal degradation, properties that make them useful for a number of technical applications, such as use as dielectric and hydraulic fluids, in transformers and capacitors, and in sealants. The same properties also result in their lipophilicity and provide biological stability. As a consequence, PCBs can not only be found in almost all environmental samples, but they also bioaccumulate and biomagnify in animals and humans. Their manufacture was banned in the U.S. in the 1970s because of serious concerns about their adverse effects in the environment and on human health. The total world production of PCBs was estimated to be 1 million tons (3). This number does not include mixtures manufactured in Eastern and Central European countries, where lack of protective measures resulted in both local and widespread environmental contamination. Only limited information about the PCB mixtures produced in Eastern Europe is currently available. The environmental and human health risks resulting from exposure to these mixtures are largely unknown. One example of an Eastern European PCB mixture is Chlorofen. Approximately 1000 tons of Chlorofen were manufactured from 1966 to 1970 in Dabrowa Gornicza Zabkowice, Poland (4). The mixture is highly chlorinated, with an average of 7.3 chlorines per molecule, and has a homologue profile with octa- and nona-chlorinated PCBs as main groups and a chlorine content of 64% (4). Chlorofen has a unique congener profile differing from other technical PCB mixtures, such as Aroclors, Clophens or Kanechlors (4). The PCB congener distribution of Chlorofen is somewhere between Aroclor 1262 and Aroclor 1268. Several congeners, such as PCB 180, 194, and 201, are particularly enriched in Poland’s Chlorofen but less so in the technical mixture from other countries. There are few reports of the toxicity of Chlorofen. The effects of Chlorofen on the mortality of fish, daphnia, and snails showed that daphnia was the most sensitive of these three species, with an LD50 of 1 mg L-1 after 24-h exposure (5). Chlorofen caused a decrease in body weight in fish, but it had no effect on snails. In an in vitro study, the Ah receptor response to Chlorofen along with other technical mixtures was reported (6). The relative potencies compared to TCDD were in the range of 3.8 × 10-8 to 7.6 × 10-7, almost an order of magnitude less compared to all Aroclors studied. To our knowledge, no in vivo mammalian toxicity studies with Chlorofen or environmental mixtures from Chlorofencontaminated sites have been performed. Some inferences regarding Chlorofen’s toxicity can be made on the basis of previous publications with other PCB mixtures. Chlorofen has a congener profile that is to some extent comparable to Aroclor 1260, another highly chlorinated PCB mixture. It is, therefore, likely that Chlorofen, like Aroclor 1260, will increase liver weights and induce several hepatic cytochrome P-450s (CYP), with CYPB2 being the most sensitive biomarker of exposure (7). To understand the toxicity of Chlorofen and to assess the health risk of the population living near the Chlorofen production site, it is important to correlate in vivo toxicity studies with the PCB congener profiles in the target tissues (8). Surprisingly few studies have investigated the PCB congener profiles in tissues and attempted to correlate total PCB and congener levels with biological effects in typical laboratory animals, such as the rat. A recent publication by Imsilp and co-workers shows that exposures of female Sprague-Dawley rats either by direct contact or airborne exposure to a PCB-contaminated soil results in different PCB profiles and compositions in target tissues, which is linked VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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to different biological effects (9). A similar observation was made in a study that compared an air extract and a soil extract from a PCB-contaminated Superfund site (10). The extracts, which also contained polychlorinated dibenzofurans and polychlorinated dibenzodioxins, not only had different compositions but also caused different biological effects. Even less information is available with regard to the biological effect caused by individual congeners present in a PCB mixture. The persistence of enzyme induction observed even after termination of PCB exposure is thought to be due to persistent congeners, such as PCBs 138 and 153 (11). PCB 153 has also been shown to distribute regionally in the brain (12). Both chlorination level and substitution pattern are important determinants of the PCB congener profiles in animal tissues. Less highly chlorinated PCBs (1-4 chlorines) do not accumulate in tissues, because they are typically readily metabolized and disappear from tissue profiles (13, 14), except from adipose tissue (15). These congeners are sometimes referred to as episodic congeners (9, 16). 2,4- and 3,4-Substituted congeners are more persistent, especially if there is 4-substitution on the second ring (9, 14, 15). As a result of the disappearance of episodic PCB congeners, PCB profiles in tissues are different from the profile of the parent mixture profile (17). The present study investigates the differences in tissue distribution and the potential effects of PCB congeners in rats dosed with an environmental mixture obtained from soil contaminated with Chlorofen. For comparison with Chlorofen, a second group of rats was similarly exposed to an equimolar dose of Aroclor 1254, a well-investigated PCB mixture. In both cases, the PCB congener distribution of 120 congeners was measured in nine tissues. Principle component analysis (PCA) was employed as a data reduction tool to identify congener subsets showing significant changes between (a) parent mixtures and animal tissues and (b) different tissues within one treatment group. The results from this study allow us to further understand how different chlorination levels and, to some extent, the structure of PCB congeners affect their distribution and total concentration in selected tissues and their biological effects.
Experimental Section Chemicals. The PCB congener nomenclature used in this manuscript is based on the nomenclature proposed by Ballschmiter and Zell (18). The Aroclor 1254 mixture selected for this study has been previously employed in several toxicological studies in our laboratory and, on the basis of a comparison with published profiles (19), resembles batch G4 (R2 ) 0.935). The Chlorofen standard was provided by Dr. K. Kannan of the Wadsworth Center, New York State Department of Health, Albany, New York. The soil sample was collected in 2001 at the Chlorofen manufacturing site in Dabrowa Gornicza Zabkowice in Southern Poland. The PCB extraction from the soil sample followed standard U.S. EPA methods (20, 21) and has been described in detail elsewhere (22). In short, 5 g of the dried and sieved soil sample was mixed with sodium sulfate and Soxhlet-extracted twice for 7 h with a hexane/acetone mixture (1:1, 100 mL). The extract was shaken in a separatory funnel with concentrated sulfuric acid to obtain a colorless extract. After evaporation of the solvent, ∼10 mg of the PCB soil extract mixture was obtained. The total PCB content was determined as the weight of the dried extract. The average molecular weight of the soil extract was estimated to be 395 g mol-1 (average chlorine per molecule, 6.94). Animal Treatment. Eleven 1-month-old male SpragueDawley rats were obtained from Harlan (Indianapolis, IN) and allowed to acclimatize for 1 week. Animals were randomly divided into three groups and injected intraperitoneally with a single dose of the soil extract mixture (20 mg/kg b.w., 0.05 3514
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mmol/kg b.w., 5 mL/kg, n ) 3) or Aroclor 1254 (16 mg/kg b.w., 0.05 mmol/kg b.w., 5 mL/kg, n ) 4). Control animals received the vehicle alone (corn oil, 5 mL/kg, n ) 4). Rats were euthanized on day 7. Blood was collected by cardiac puncture, and serum was prepared by centrifugation. Whole blood and serum were stored at -20 °C. Tissues (adipose tissue, brain, heart, liver, lung, kidney, spleen, and whole skin) were collected, and their net weights were determined. All tissue samples were stored in aluminum foil at -20 °C. Tissue Extraction Procedure. PCBs were extracted from all tissues using standard U.S. EPA methods (20, 21). Approximately 0.5 g of a randomly selected tissue sample was homogenized with 11 g of sodium sulfate. The recovery standard solution consisting of PCBs 14, 65, and 166 (500 µL 504 µg/L in hexane) was added, and the sample was Soxhletextracted for 8 h using hexane/acetone (1:1 v/v). The extract was concentrated using a rotary evaporator. Lipid cleanup was performed with an aliquot of the concentrated PCB extract by eluting it through a column containing 1 g of sodium sulfate, 6 g of Florisil (100-200 mesh, activated at 260 °C for ∼16 h), and 1 g of sodium sulfate. An internal standard solution consisting of PCBs 30 and 204 (100 µL; 100.8 µg/L in hexane) was added. Sulfur cleanup was performed by shaking a 2-mL aliquot of the PCB extract with 2 mL of 2-propanol and 2 mL of tetrabutylammonium sulfite, adding 8 mL of nanopure water, and reshaking the sample. The organic extract was removed, mixed with 2 mL of concentrated sulfuric acid, and allowed to stand for at least 24 h before the PCB analysis. Blood/Serum Extraction Procedure. Whole blood and serum samples were extracted according to the procedure reported by Gill and co-workers (23). After protein precipitation with acetic acid, recovery standards solution was added (50 µL; PCBs 14, 65, 166; 504 µg/L in hexane). The samples were extracted three times with hexane/dichloromethane (9:1 v/v). The extracts were concentrated and cleaned using a Florisil column as described above and with sulfuric acid. The internal standard solution (PCB 30 and 204; 100 µL; 100.8 mg/L in hexane) was added before the PCB analysis. Lipid Content Determination. The lipid content in all tissues was determined gravimetrically (24) from an aliquot of the hexane/acetone extract. Total cholesterol and triglycerides in aliquots of the serum samples were determined using a commercial test kit (Chol and Trig/GB tests for Roche/ Hitachi 917 system; Roche Diagnostics, Indianapolis, IN). Blood lipids were calculated using the formula TL ) 2.27 × TC + TG + 0.623, where TL ) total lipids, TC ) total cholesterol, TG ) total triglicerides (25). PCB Analysis. The PCB analysis was performed using a HP 6890 gas chromatograph with a 60-m DB5 capillary column and a 63Ni µ-ECD detector. The detector temperature was 300 °C. The oven temperature program was as follows: 100 °C held for 1 min, then increased by 1 °/min from 100 to 240 °C, 10 °/min from 240 to 280 °/min, hold for 20 min. Using this temperature program, 90 chromatographic peaks representing 120 PCB congeners were qualified and quantified using the internal standard method and the LMMB Mullin mixture as the PCB standard mixture (24). The detector response was linear over the entire concentration range encountered in this study. A detailed description of the quality assurance measures, and the recovery rates are given in the Supporting Information. Biochemical Assays. Liver microsomes were prepared by differential centrifugation as described previously (26). The total cytochrome P-450 content of the microsomes was quantified using the method of Omura and Sato (27). The resorufin assay developed by Lagueux for a microplate reader was employed to determine EROD (ethoxyresorufin-Odealkylase) and PROD (pentoxyresorufin-O-dealkylase) activities in the liver microsomes (28). The concentrations were
TABLE 1. Biological Effect of PCB Mixtures on Treated Rats biological effect
control
Aroclor 1254
soil extract
body weight change (%) liver weight change (%) spleen weight change (%) thymus weight change (%) total cytochrome P-450 (nmol/mL) PROD (nmol/mg protein/min) EROD (nmol/mg protein/min)
33.3 ( 1.5 4.98 ( 0.10 0.40 ( 0.03 0.37 ( 0.06 0.57 ( 0.16 n.d.b n.d.b
32.7 ( 1.2 4.70 ( 0.47 0.40 ( 0.02 0.38 ( 0.04 0.60 ( 0.03 0.62 ( 0.55 4.03 ( 1.23d
33.77 ( 1.8 5.35 ( 0.10a 0.42 ( 0.02 0.31 ( 0.04 0.68 ( 0.13 2.83 ( 0.72c,d 0.14 ( 0.10
a Statistically different from Aroclor 1254, P ) 0.01. from control, P ) 0.01.
b
Not detectable. c Statistically different from Aroclor 1254, P ) 0.05.
d
Statistically different
FIGURE 1. Tissue distribution of total PCBs in nanograms of PCB per gram wet weight of tissue in (a) Aroclor 1254-treated rats and (b) soil extract treated rats and in nanograms of PCB per gram of lipid in (c) Aroclor 1254-treated rats and (d) soil extract treated rats (please see the Supporting Information for the absolute values of the total PCB concentrations and the results from the statistical analysis). calculated from measured absorbance against the standard curve in the concentration range 0 to 125 pmol/well and with an excitation filter at 530 nm and emission at 590 nm. The microsomal samples (∼50 µg protein in each well) were incubated with 100 µL of sodium phosphate and 25 µL of 7 mM NADPH for 2 min at 37 °C. The respective alkoxyresorufin (10 µL) was added to initiate the enzymatic reaction, which proceeded for 30 min at 37 °C before acetonitrile containing 300 µg/mL of protein-binding fluorochrome fluorescamine was added. Statistical Analysis. The Tukey Multiple Comparison Procedure modified by Games and Howell (29) was applied to compare the data from the biochemical assays shown in Table 1. The significance level for these comparisons was set at 0.05. The analysis of statistical differences between total PCB concentrations in tissues from both treatment groups was performed using two-way Anova with a post-hoc Tukey test. The significance level required was P < 0.05. The statistical software Systat Version 8.0 (Point Richmond, CA) was used for the above-mentioned statistical analyses. The PCB data set of single congener concentrations, normalized by total PCB, was preprocessed with autoscaling and used as input for the PCA (30). Both analyses were carried
out with Matlab (Mathworks, Natick, MA, version 6.5.1). Significant differences between the single PCB congener content in parent mixtures, tissues, or groups of PCB data obtained from the PCA were shown by plotting the differences of the respective means. The following formula was used to calculate the mean of the PCB concentrations of the congener under investigation,
∆levels(groupA-groupB) ) levelPCBxgroupA - levelPCBxgroupB (1) where x is the Ballschmitter and Zell number of the PCB congener (18), groups A and B are the data sets that are compared (e.g., parent mixture versus tissues, tissues from animals treated with Aroclor and soil extract, different tissues within one treatment group etc.), ∆levelPCBxgroup are the means of the levels of PCBx of the respective group, and ∆levels(groupA-groupB) is the difference of the means from group A and group B. Please see the Supporting Information for a graphical presentation of the results of our statistical analysis using eq 1. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Comparison of congener profiles (content as percent of total PCB) of parent mixtures and selected tissues: (A) Aroclor 1254, (B) soil extract, (C) liver from Aroclor 1254-treated rat, (D) liver from soil-extract-treated rat, (E) spleen from Aroclor 1254-treated rat, (F) spleen from soil-extract-treated rat. The congeners are presented in elution order.
Results and Discussion Analysis of Total PCB Concentration. Total and lipid adjusted PCB concentrations in all tissues are presented in Figure 1. As expected for the highly lipophilic PCBs (31), the highest PCB concentrations were found in the adipose tissue on whole weight basis in both treatment groups. On a molar as well as a weight-adjusted basis, the total PCB content was significantly higher in the spleen (P < 0.005) but significantly lower in the skin (P < 0.005) in the soil extract treatment group, as compared to the Aroclor group (data not shown), although in both cases, the differences were not large. Lipidadjusted PCB levels were similar in most tissues, with the exception of the spleen, where the lipid-adjusted PCB levels were much higher when compared to the other tissues investigated. 3516
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To our knowledge, this is the first report of retention of PCBs in the spleen, as compared to other organs on a lipidadjusted basis. Considering the short exposure time of 7 days, our findings most likely reflect different redistribution of PCBs in the two treatment groups rather than metabolic processes. PCBs are initially distributed into highly perfused organs, such as the liver, lung, kidney, and spleen (32). Subsequently, PCBs are redistributed into poorly perfused storage organs, such as the adipose tissue and the fat depots under the skin (33). We hypothesize that the significantly higher PCB levels in the spleen of soil-extract-treated animals are due to a slower redistribution of more highly chlorinated PCB congeners from this highly perfused organ into storage tissues. On the basis of our gravimetrical lipid determination and the respective wet organ weight, the spleen has a lipid
FIGURE 3. Approximate percentage of homologue groups in parent mixtures and selected rat tissues of (A) the Aroclor and (B) soil extract treatment group. The data presented here do not include coeluting congeners for which the coeluting congener belonged to different groups, because the fraction of each single congener is not known. As a result of this approximation, 24% of the total PCB content in the Aroclor 1254 and 15% of the total PCB content in the soil extract treatment group is not taken into consideration. content of 1.5 ( 0.6%, which is much smaller when compared to other tissues, such as the liver (3.2 ( 0.9%) and the kidney (3.2 ( 0.2%). A smaller fat content in the spleen has also been reported for other mammals (31). It seems counterintuitive that this lower fat content causes selective retention of more highly chlorinated and, thus, highly lipophilic PCBs in the spleen, but with the present data, we cannot discount this possibility. PCB Profiles and Prevalence of Homologue Groups. The PCB profiles of the liver and spleen from rats belonging to both treatment groups and of both parent mixtures are shown in Figure 2. Additional profiles of tissues can be found in the Supporting Information. The relative contribution of PCBs by homologues is illustrated in Figure 3. As can be seen in both Figures, the composition of both parent mixtures undergoes changes in the biological matrix. For example, Aroclor 1254, which consists of mostly moderately chlorinated PCB congeners, displays an overall increase in more highly chlorinated homologues (g6 Cl atoms) and a decrease in less highly chlorinated homologues (e5 Cl atoms) in the spleen relative to the original mixture. All other tissues show a similar trend in the homologue composition (data not shown). In contrast, the soil extract, which consists predominantly of hepta- and octachlorobiphenyls, shows an overall increase in less highly chlorinated homologues (e4 Cl atoms) and a decrease in more highly chlorinated homologues (g7 Cl atoms) in most tissues. It is noteworthy that the PCB homologue profile in the spleen and the soil extract are similar (R2 ) 0.988). This similarity was also noted
FIGURE 4. Projections of PC1all versus PC2all (A) and PC1all versus PC3all (B) for all tissues and parent mixtures data set (PCAall). in a previously published hierarchical cluster analysis of the PCB data from this study (34). Congener-Specific Analysis of the PCB Data. One goal of this study was to investigate differences in the tissue distribution and the potential effects of the Polish PCB mixture Chlorofen using congener-specific PCB data. Typically, PCB congener profiles from similar animal studies are analyzed visually to identify PCB congeners that show an increase or decrease in the tissues relative to the respective parent mixture; however, such an approach may be biased, and important congener-specific changes may be overlooked (9). Principle component analysis (PCA) is frequently employed to analyze PCB profiles of environmental samples (35, 36). We used PCA to reduce the data and to identify congeners for further analysis. Equation 1 was used to confirm that the congeners identified by PCA, indeed, are significantly different among the respective groups of data (see Supporting Information). Principal Component Analysis of All Tissue Data (PCAall). Three principle components (PCs) accounting for 52.0% of the data variance were analyzed. As shown in Figure 4, PC1all separates Aroclor from soil extract samples, with highly chlorinated/persistent congeners versus moderately chlorinated congeners contributing to its construction. Specifically, PCBs 180, 187 + 182 + 175, 194, 201, and 208 + 195 are significantly higher in the soil extract, whereas PCBs 118, 128, and 163 + 138 are significantly higher in Aroclor. The congeners characteristic of the soil extract mixture, especially PCB 180, need to be taken into consideration when studying the health effects or assessing the risk of exposure to PCB VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Projection of PC1Aroclor and PC2Aroclor for Aroclor 1254 and tissues of rat treated with the mixture alone. Different symbols were used for each tissue and Aroclor 1254.
FIGURE 6. Projection of PC1soil and PC2soil for Chlorofen, soil extract, and tissues of rat treated with the mixture alone. Each tissue, Chlorofen, and soil extract contaminated with Chlorofen are assigned with a different symbol. mixtures originating from Chlorofen. PCB 180 is relevant in this context because this congener has been recently associated with a higher risk of prostate cancer (37). This raises the question of whether men exposed to Chlorofen, either as a result of occupational or environmental exposure, are at a higher risk of prostate cancer due to an increased exposure to/body burden of PCB 180. PC2all separates the PCB samples from the analysis blanks and, therefore, was not further analyzed. PC3all explains 9.7% of the total data variance and separates the parent mixtures from tissues (Figure 4B), providing further evidence for changes in the composition of both PCB mixtures in the biological matrix. 3518
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Two additional PCAs, PCAAroclor and PCAsoil, were performed using the data from the respective treatment groups to investigate which congeners exhibit the most significant changes in the tissues. Principal Component Analysis of the Aroclor 1254 Group (PCAAroclor). For the Aroclor treatment group, we analyzed two PCs which describe 78.6% of data variance (Figure 5). PC1 separates Aroclor 1254- from Aroclor 1254-treated tissues. PCBs 118, 153 + 105 + 132, 163 + 138, and 146, which have high loadings on PC1, are higher in the animal tissues than in the parent mixture. It is not surprising that PCBs 138 and 153, which are typically considered to be highly
persistent, are part of the group of congeners with increased levels in the animal tissues. PCBs 44, 52, 70 + 76, 84 + 92, 95, 97, and 110 + 77, all of which have a 2,2′,3-substitution pattern, have low loadings on PC1 and are significantly reduced in the animal tissues. This suggests that less highly chlorinated congeners are metabolized and eliminated from the animals. The rapid elimination of these and related congeners has previously been observed for mammals (9, 38, 39). PC2Aroclor separates tissues, that is, spleen and heart from adipose, skin, and lung, thus allowing the identification of congeners with different levels in these tissues. All PCB congeners with low loadings on PC2Aroclor have a 2,4,4′substitution pattern and are, therefore, persistent. These congeners tend to have higher levels in “storage tissues”, such as adipose tissue and skin. Congeners with high loadings on PC2Aroclor, that is, PCBs 174, 180, 183, 194, and 203 + 196, have higher levels in the spleen. These congeners, with few exceptions, are highly chlorinated, suggesting retention of some nonmetabolizable congeners in the spleen. This is similar to our finding that the soil extract, which is characterized by highly chlorinated PCB congeners, such as PCB 180, is preferentially retained in the spleen and indicates that the same phenomenon is also happening in Aroclor 1254-treated rats. Principal Component Analysis of the Soil Extract Group (PCAsoil). PCA of the soil extract treatment group data was performed analogously to the Aroclor 1254 treatment group. Only PC1soil, which separates Chlorofen, soil extract, and spleen from the other tissues, was further analyzed (Figure 6). The congeners with low loadings on PC1soil are persistent congeners, that is, PCBs 151 + 185, 157 + 200, 199, 174, and 203 + 196, which are present at higher levels in the parent mixture. Among the more highly chlorinated PCB congeners, only the persistent PCB congeners 138 and 153 have higher levels, that is, accumulate, in tissues. This finding is surprising because it suggests that there are differences in the disposition, and possibly the elimination, of some highly chlorinated and persistent PCB congeners. In addition to PCBs 138 and 153, PCBs 28, 47, 66, 74, and 118 have increased levels in tissues or begin to appear in the PCB tissue profiles (except of the spleen) when compared to the parent mixture. Biochemical Effects. As little as 2 mg of Aroclor 1254 or Phenoclor DP6 per kg of body weight, a dose that resembles typical environmental exposures and was used in this study, is sufficient to cause a positive response in some enzymatic assays of activities of hepatic cytochrome P-450 (40, 41). Major effects of PCB exposure, such as changes in liver weights and total cytochrome P-450, as well as ethoxyresorufin O-dealkylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activities in the liver were investigated and are summarized in Table 1. Liver weights were significantly elevated in the soil-extract-treated animals, as compared to the controls and the Aroclor 1254-treated rats. No differences were observed for body, thymus, and spleen weights, and no increase of total cytochrome P-450 content in the liver was noted. The lack of these effects, which are characteristic for PCB exposure, is most likely the result of the low dose, the selected time point or the lack of statistical power. However, both PCB mixtures increased EROD activity (CYP1A), and the soil extract increased PROD activity (CYP2B) in the livers, as compared to controls. In the soil-extract-treated animals, PROD activity in liver microsomes was 4.5 times higher than in Aroclor 1254-treated rats. In the Aroclor 1254-treated rats, EROD activity was 28.7 times higher, as compared to soilextract-treated animals. The observation that the highly chlorinated Chlorofen is a good inducer of CYP2B is in agreement with two earlier studies. Ngui and Bandiera have shown that CYP2B is a sensitive biomarker of exposure to Aroclor 1260, which has
a congener profile comparable to that of the Chlorofen soil extract (7). Similarly, Li and Hansen demonstrated that a soil extract altered BROD (benzylresorufin-O-dealkylase) and PROD activities before and after removal of arylhydrocarbon receptor (AhR) agonists by charcoal treatment (42). After removal of the AhR agonists, the purified PCB soil extract contained mostly PCB congeners with multiple ortho substitutions and resulted in greatly enhanced BROD and PROD activities. This is comparable to our study in which Aroclor 1254, which is rich in Ah receptor agonists, induces CYP1A, whereas the soil extract, which is rich in PCB congeners with multiple ortho substituents, predominantly increases CYP2B activity. This suggests that the Chlorofen mixture may pose human health risks that are comparable to more highly chlorinated Aroclors, such as Aroclor 1260.
Acknowledgments The authors gratefully acknowledge Dr. M. P. Jones (University of Iowa) for help with the statistical analysis. I. KaniaKorwel gratefully acknowledges the support of a Fulbright Junior Research Grant and a Kosciuszko Foundation Grant. This publication was made possible by Grant nos. ES05605, ES07380, and ES12475 from the National Institute of Environmental Health Sciences, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.
Supporting Information Available A short description of quality assurance and recovery rates, tables with total PCB concentration in tissues, and the PCB composition in Aroclor 1254 and the soil extract, additional PCB tissue profiles, and difference plots for PCB congeners with high absolute loading values from the PCA analyses are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Robertson, L. W.; Hansen, L. G. PCBs Recent Advances in Environmental Toxicology and Health Effect; University Press of Kentucky: Lexington, 2001. (2) Hansen, L. G. The ortho side of PCBs; Kluwer Academic Publishers: 1998. (3) Erickson, M. D. Introduction: PCB Properties, Uses, Occurrence and Regulatory History In PCBs Recent Advances in Environmental Toxicology and Health Effects; Robertson, L. W., Hansen, L. G., Ed.; The University Press of Kentucky: Lexington, KY, 2001. (4) Falandysz, J.; Yamashita, N.; Tanabe, S.; Tatsukawa, R. Composition of PCB isomers and congeners in technical chlorofen formulation produced in Poland. Int. J. Environ. Anal. Chem. 1992, 47, 129-136. (5) Luczak, J.; Rybak, M.; Zycinski, D. Wpływ Chlorofenu na organizmy wodne oraz charakterystyka chemiczna zmian w s´rodowisku wodnym pod wpływem tego preparatu. Rocz. Panstw. Zak. Hig. 1976, 27, 555-561. (6) Villeneuve, D. L.; Khim, J. S.; Kannan, K.; Giesy, J. P. In vitro response of fish and mammalian cells to complex mixtures of polychlorinated naphthalenes, polychlorinated biphenyls and polycyclic aromatic hydrocarbons. Aquat. Toxicol. 2001, 54, 125-141. (7) Ngui, J. S.; Bandiera, S. M. Induction of hepatic CYP2B is a more sensitive indicatior of exposure to Aroclor 1260 than CYP1A in male rats. Toxicol. Appl. Pharmacol. 1999, 161, 160-170. (8) Birnbaum, L. S. Overview of 3rd biannual International PCB Workshop. In PCBs: More Recent Advances; Hansen, L. G., Robertson, L. W., Ed.; University of Illinois Press: Champaign, IL, 2005. (9) Imsilp, K.; Hansen, L. PCB profiles in mouse skin biopsies and fat from an environmental mixture. Environ. Toxicol. Pharmacol. 2005, 19, 71-84. (10) Li, M.-H.; Hansen, L. G. Enzyme induction and acute endocrine effects in prepubertal female rats receiving environmental PCB/ PCDF/PCDD mixtures. Environ. Health Perspect. 1996, 104, 712722. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Received for review December 20, 2004. Revised manuscript received March 21, 2005. Accepted March 22, 2005. ES047987F
