Tissue Distribution and Half-Lives of Individual Polychlorinated Biphenyls and Serum Levels of 4-Hydroxy-2,3,3`,4`,5-pentachlorobiphenyl in the Rat

Mattias Öberg*, Andreas Sjödin{dagger},1, Helena Casabona*, Ingrid Nordgren*, Eva Klasson-Wehler{dagger} and Helen Håkansson*,2

* Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden; and {dagger} Department of Environmental Chemistry, Stockholm University, Stockholm, Sweden

Received June 4, 2002; accepted September 10, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was done to generate kinetic data on individual congeners of chlorinated biphenyls in the low dose range, which could be of value in the risk assessment procedure. Male Sprague-Dawley rats were given a single oral dose of a mixture of polychlorinated biphenyls (CBs) containing either CBs 105, 118, 138, 153, 156, 157, 170, and 180 (A-mix) or CBs 28, 52, 77, 87, and 101 (B-mix). Liver, serum, and adipose tissue were collected after 6 h up to 135 days, from rats given the A-mix, and after 6 h up to 4 days from rats given the B-mix. CB concentrations were measured in liver, serum, and adipose tissue. In addition, this study provides kinetic data of one of the major CB metabolites, 4-hydroxy-2,3,3`,4`,5-pentachlorobiphenyl (4-OH-CB107). The low doses used resulted in serum CB concentrations similar to human background serum concentrations. In the A-mix experiment all CBs show high initial liver and serum concentrations followed by redistribution into adipose tissue. Differences between congeners were correlated to molecular weight. High molecular weight correlated to lower uptake and slower redistribution. During dynamic steady-state the tissue concentrations decreased with a calculated first order rate between 54–129 days for halving the concentrations (half-life). Most of the decrease in concentration was explained by the growth-related increase of tissue masses in general and adipose tissue in particular. In the B-mix experiment, the concentrations of CBs in adipose tissue decreased with between 25 and 59% from day 1 to day 4. These results show that the B-mix congeners, given at low dose, have longer half-lives than previously reported in high dose studies. Partition coefficients between body compartments are reported and for the first time a high and congener specific liver-to-serum ratio of CB 77 is observed.

Key Words: distribution; partition coefficient; half-life; polychlorinated biphenyls; PCB; liver; adipose tissue; serum; rat; 4-hydroxy-2,3,3`,4`,5-pentachlorobiphenyl.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polychlorinated biphenyls (PCB) are commercial products that are prepared by the chlorination of biphenyls. PCB oil has been widely used in industry, e.g., as heat transfer fluid, dielectric fluid, and flame retardant. The high lipophilicity and stability of PCB results in biomagnification and bioconcentration (reviewed by Geyer et al., 2000Go). Today PCB are found almost everywhere in the biosphere, including human tissues, in spite of the fact that they have been banned in most industrialized countries. Acute PCB exposure has been seen in accidents, such as in 1968 in Yoshu, Japan, where rice oil, by mistake, was contaminated with PCB (Taki et al., 1969Go). Accidental contamination of food is still occurring, as seen in Belgium during the spring of 1999, when old PCB was mixed into cattle feed, resulting in exposure to humans via animal products (van Larebeke et al., 2001Go). In normal situations humans are exposed to low chronic doses. In 1996 the average daily PCB intake in Sweden was reported to be about 0.05 µg/kg body weight (Darnerud et al., 1995Go), although in some local areas the intake of persistent organohalogen pollutants can be considerably higher than the average.

Since the use of PCB was banned in many countries, the levels in human milk have declined (Norén and Meironyte, 2000Go; Solomon and Weiss, 2002Go). Nevertheless, present human background levels are suspected to adversely impact health (van Leeuwen and Younes, 1998Go). Individual congeners of chlorinated biphenyls (CBs) show a broad spectrum of toxic effects. Effects of high concern for the general population are reproductive and developmental toxicity (Ahlborg et al., 1992Go). In particular, children exposed in utero to background levels have shown subtle cognitive and motor developmental delays, which persist into school age (Jacobson and Jacobson, 1997Go; Vreugdenhil et al., 2002Go). More optimal intellectual stimulation provided by a more advantageous parental and home environment may counteract these effects (Vreugdenhil et al., 2002Go). Non- and mono-ortho CB congeners with chlorine substituents in both para and at least two meta positions are approximate isostereomers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These CBs, which elicit similar toxic effects, probably by binding to the aryl hydrocarbon receptor (AhR; Safe, 1990Go, 1994Go), are hereby referred to as dioxin-like. Other CB congeners, herein referred to as nondioxin-like, cause adverse effects by other mechanisms of action, not always well investigated (Fischer et al., 1998Go; Giesy and Kannan, 1998Go; Seegal et al., 1990Go). Lack of toxicity data for individual CBs in the low dose range still hamper a proper human health risk assessment of CBs. Even less information is available for the toxicokinetic behavior of individual CBs in the low dose range (Ahlborg et al., 1992Go). Kinetic data based on previous studies with high doses of technical PCB mixtures and short followup times are of limited value for risk assessment procedure. It is known, for example, that high doses of organohalogen pollutants can cause altered kinetic behavior as a result of toxic effects, protein induction, or interactions (de Jongh et al., 1993aGo; Santostefano et al., 1997Go). There are few half-lives reported on CBs. In one of the most extensive studies the absorption and elimination rates of individual CB congeners in a commercial mixture (3 mg Kanechlor/day during 5 days) were examined during 90 days and the 79 analyzed congeners were classified into three classes (Tanabe et al., 1981Go). Most high-chlorinated CBs were reported to have half-lives longer than 90 days. The lower-chlorinated CBs were classified into two other classes with shorter initial half-lives of about 1 to 2 days.

The present study was done to generate kinetic data for individual CB congeners in the low dose range. Such information is of value in the risk assessment procedure. The 13 selected congeners were chosen to include CBs of relevance to human exposure via common food items and with variable chlorine substitution, but without high dioxin-like toxic equivalency factors. This study reports CB concentrations in liver, serum, and adipose tissue, the rates by which CB concentrations decrease during dynamic steady-state, and tissue partition coefficients. In addition, it provides kinetic data for 4-hydroxy-2,3,3`,4`,5-pentachlorobiphenyl (4-OH-CB107), which is a major metabolite of CBs 105 and 118 (Sjödin et al., 1998Go). This metabolite is also important for its blood specific binding and endocrine disrupting potential. In addition, P450 activities (EROD and PROD) as well as vitamin A levels are reported to demonstrate that the study was performed at a dose level, which is unlikely to interfere with the kinetics of the given compounds. EROD have together with vitamin A been demonstrated to be among the most sensitive biochemical responses to the exposure of dioxin-like compounds (Fattore et al., 2000Go; Fletcher et al., 2001Go; van Birgelen et al., 1995Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male Sprague-Dawley rats obtained from B & K Universal AB (Sollentuna, Sweden), weighing approximately 225 g at the beginning of the study, were used. The animals were housed in wire-bottomed plastic cages, with six animals in each cage. The temperature was maintained at 21 ± 1°C and the illumination cycle (12 h light, 12 h dark) was automatically controlled. Before treatment, the animals were kept for a 7 to 10 day acclimation period. Throughout the experiment, the animals were provided with tap water ad libitum and they had free access to a standard pellet diet (R36, Lactamin AB, Vadstena, Sweden). The experimental protocol, including animal housing and care during the study was approved by the Stockholm Northern Animal Ethic Committee (Stockholm, Sweden).

Chemicals.
The CBs used in this study were synthesized by Professor Bergman's group at the Department of Environmental Chemistry, Stockholm University, Sweden. All CBs were prepared as previously described and purified on a charcoal column (Bergman et al., 1990Go; Sundström, 1973Go). The following CBs, as numbered by IUPAC, were used: 28, 52, 77, 87, 101, 105, 118, 138, 153, 156, 157, 170, and 180 (Table 1Go). PCBs 40, 97, 137, 189, and 4-OH-CB193 were used as internal quantification standards. All other chemicals were of analytical grade and commercially obtained.


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TABLE 1 Characteristics of the Chlorinated Biphenyls (CBs) Used in the Two Mixtures and the Analyzed Hydroxy Metabolite
 
Experimental design.
Rats were given a single oral dose of a CB mixture in corn oil containing either CBs 105, 118, 138, 153, 156, 157, 170, and 180 (A-mix) or CBs 28, 52, 77, 87, and 101 (B-mix). All A-mix congeners were reported to have half-lives longer than 90 days (Tanabe et al., 1981Go). The lower-chlorinated CBs in the B-mix were all classified into classes with short initial half-lives of about 1 to 2 days by Tanabe et al.(1981)Go.

The given amount per rat, was 0.03 µmol/CB (= 47 to 56 µg/kg) of the A-mix and 0.1 µmol/CB (= 119 to 151 µg/kg) of the B-mix. Each mixture was given in a total volume of 0.5 ml. The time points for termination of rats given the A-mix were set to 6 h, and 1, 5, 15, 45, and 135 days, whereas rats given the B-mix were terminated at 6 h, and 1, 2, and 4 days. At each time point, blood was withdrawn from the abdominal aorta under mebumal anesthesia (90 mg/kg). Every group contained six animals. Total body weights were recorded, and liver, kidneys, thymus, and peritesticular fat were collected and weighed. The collected tissues were frozen and stored at –70°C until further examination. Blood was centrifuged at 3000 rpm for 5 min and the serum was stored at –70°C. Control animals were given pure corn oil and were followed for 6 h, and 4, 15, and 135 days, respectively.

Biochemical analyses.
Vitamin A was extracted from frozen liver, kidney, and serum samples and was quantified as retinol by high pressure liquid chromatography (HPLC) as previously described (Håkansson et al., 1987Go). The column used was a Nucleosil 5µm C18, eluted with methanol/water (95:5). Detection of retinol was made with a JASCO 821-FP fluorescence detector.

Hepatic activities of the monooxygenase enzymes cytochrome P450 1A1 and 2B1/2 were measured, by O-dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), respectively. The formation of resorufin was measured fluorimetrically ({lambda}ex = 522 nm, {lambda}em = 586 nm) in liver S9 fractions containing 10 µM dicumarol at 37°C using a Shimadzu RF-5000 spectrofluorometer (Burke et al., 1985Go; Lubet et al., 1985bGo).

Chemical analyses.
The extraction procedure for tissue samples was slightly modified from a previously published method (Hovander et al., 2000Go; Jensen et al., 1983Go). Briefly, tissues were homogenized in acetone:hexane (7:2). The solvent was transferred to 0.1 M phosphoric acid in a 1% sodium chloride solution in water. Additional solvent, hexane:methyl tert-butyl ether (9:1) was added. After rocking the organic phase against the acidified water phase and subsequent centrifugation, the organic phase was transferred to a preweighted sample tube. Lipid content was thereafter determined gravimetrically. The extracts were treated with concentrated sulphuric acid in order to remove coextracted lipids.

Serum and adipose tissue were analyzed for CBs by gas chromatography with electron capture detector (GC-ECD) using a Varian 3400 equipped with a DB5 column (30 m, 0.25 µm stationary phase, 0.25 mm i.d.; J&W Scientific, Folsom, CA). Quantification was made using single point calibration after verification of a linear response. The injector and detector temperatures were 250 and 360°C, respectively. The GC oven was programmed from 80°C (2 min), to 300°C (10°C/min) and isothermally for 5 min. Hydrogen was used as the carrier gas and nitrogen as the makeup gas. For separation of congeners in liver tissues from rats exposed to the A-mix, a slightly different temperature program was used with a start at 150°C followed by a 4°C/min rise up to 280°C. For detection of congeners in the liver tissues a mass spectrometer (Hewlett-Packard 5970) was used. CB 28 was analyzed at m/z 256 and 258 with CB 21 as internal standard (IS). CBs 52 and 77 were analyzed at m/z 290 and 292 with CB 40 as IS. CBs 87, 101, 105, and 118 were analyzed at m/z 324 and 326 with CB 97 as IS. CBs 138, 153, 156, and 157 were analyzed at m/z 358 and 360 with CB 137 as IS. PCBs 170 and 180 were analyzed at m/z 392 and 394 with CB 189 as IS. Quantification was based on peak-height measurements using a linear standard curve.

Serum was analyzed with respect to 4-OH-CB107 by GC using the same equipment and program as for CB analysis with 4-OH-CB193 as IS. The phenolic compounds were derivatized to their corresponding methylethers by the addition of ethereal diazomethane prior to analysis as described earlier (Hovander et al., 2000Go).

Kinetic calculations.
Calculations of rates of decreasing CB concentrations were made using only data from observed dynamic steady-state, an assumption defined as a condition with constant partition during concentration decline in all examined compartments (Segel, 1988Go; Segel and Slemrod, 1989Go). The data were fitted to a first order elimination curve; , where Ct is the concentration of the CB congener in different tissues at time t1, C0 is the concentration at the beginning of the decrease, ke is the elimination rate in days-1. The half-lives (t1/2) in different tissues were calculated from the ke values by the equation t1/2 = ln2/ke. The calculated half-lives of total amount in liver and adipose tissue is based on measured liver weights and a model where adipose tissue weight is related to total body weight (BW) as 0.0199 x BW + 1.664 (Bailey et al., 1980Go). The TableCurve 2D from Jandel Scientific, USA, was used. Partition coefficients between body compartments were calculated as the average distribution ratios during steady-state.

Statistical analysis.
The statistical analyses were carried out with the SPSS for Windows statistical package and data from exposed rats were compared with control rats using Wilcoxon's Rank Correlation Test. Comparisons were considered significant at the p < 0.05 level. Principal Component Analysis (PCA) was used to identify systematic differences between the congeners in the A-mix experiment using normalized concentration data from all tissues and individual animals. The PCA was made using Simca-P 8.0 (Umetrics AB, Sweden).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Weights, Clinical and Biochemical Effects
No animals died during the study and no abnormal clinical signs were observed. No treatment related effect on body weight, organ weights, or organ lipid fractions were seen for any of the two mixtures given (data not shown). The average body weight increased from 216 at the beginning of experiments to 530 g after 135 days and liver weights increased from 9 to 17 g during the same period.

Transient increases in hepatic EROD activity were observed in both experiments (Figs. 1A and 1BGo). The maximum induction was 6- and 22-fold, respectively, in the two experiments, 24 h after exposure. For both exposures (A-mix and B-mix) during the whole time range, PROD activity showed an increase in the range 1.5–3.5 times the control activity. There were no treatment-related changes in either the total hepatic vitamin A content or in serum or kidney concentrations (data not shown).



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FIG. 1. Hepatic ethoxy- and pentoxyresorufin-O-dealkylation activities (EROD and PROD), for male Sprague-Dawley rats given a single oral dose of two different mixtures of polychlorinated biphenyls (CBs). (A) and (C) show results from the A-mix experiment including CBs 105, 118, 138, 153, 156, 157, 170, and 180. (B) and (D) show results from the B-mix experiment including CBs 28, 52, 77, 87, and 101. *Significant difference (p < 0.05) as compared to the corresponding control or the mean control value.

 
Concentrations of CBs in Liver, Serum, and Adipose Tissue
A-mix CB congeners.
Liver concentration profiles of all A-mix CB congeners showed a biphasic course of concentration decrease (Table 2Go) as visualized for CB 105 in Figure 2Go. At 6 h, individual CB concentrations were in the range of 280–626 pmol/g. There was a decrease of about 80% in hepatic CB concentrations between 6 and 24 h after exposure. Thereafter, the concentration decreased at a slower rate during dynamic steady-state (between days 15 and 135). The liver concentration half-lives calculated during this period were found to be in the range of 54–120 days (Table 3Go). At the end of the experiment, liver concentrations of individual congeners were in the range of 3–6 pmol/g for all congeners (Table 2Go).


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TABLE 2 Concentrations of Chlorinated Biphenyls (CBs) and 4-Hydroxy-2,3,3`,4`,5-pentachlorobiphenyl (4-OH-CB107) in Male Sprague-Dawley Rats
 


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FIG. 2. Mean concentration over time of CB 105 in adipose tissue, liver and serum and the hydroxy metabolite (4-OH-CB107) concentration in serum of male Sprague-Dawley rats given a single oral dose of a mixture consisting of CBs 105, 118, 138, 153, 156, 157, 170, and 180 (0.03 µmol/CB).

 

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TABLE 3 Half-Lives (Days) Based on Decreasing Concentrations and Total Amount of Chlorinated Biphenyls (CBs) in Liver, Serum, and Adipose Tissue of Sprague-Dawley Rats
 
Serum concentrations of all A-mix CBs were close to one-tenth of the liver concentrations and showed the same biphasic decreasing pattern (Table 2Go) as visualized for CB 105 in Figure 2Go. At 6 h, individual CB concentrations were in the range of 16–55 pmol/g. Between 6 and 24 h after exposure the concentrations decreased by more than 80%. The serum concentration half-lives calculated during dynamic steady-state were in the range of 81–117 days (Table 3Go). After 135 days, concentrations between 0.2 and 0.5 pmol/g were found for the A-mix CBs (Table 2Go).

The adipose tissue concentrations of all A-mix CBs increased during the first five days followed by a slow decreasing phase from day 15 (Table 2Go) as visualized for CB 105 in Figure 2Go. Adipose tissue concentrations were in general higher than the liver levels, with the exception for the first time point. The observed maxima of adipose tissue CB concentrations were in the range of 360–709 pmol/g. The adipose tissue concentration half-lives calculated during dynamic steady-state ranged from 77 to 129 days (Table 3Go). After 135 days the adipose tissue levels were in the range of 115–151 pmol/g (Table 2Go).

B-mix CB congeners.
Hepatic CB-concentrations, 6 h after administration to the B-mix, were in the range of 107–560 pmol/g (Table 4Go). The highest concentration was found for CB 77 and the lowest for CB 52. The decrease of CB concentration in the liver was most rapid during the first day. Between 6 and 24 h after exposure there was a decrease of between 57 and 85%. A rapid decrease in hepatic CB concentration was observed for all five congeners, but was most prominent for CB 77. Between one and four days after exposure the concentration of CB 77 dropped by 95%, while the other showed a decrease of concentration between 57 and 71%. The slowest decrease was found for CB 28. At the end of the experiment, four days after exposure, CB 28 was found at the highest concentration (41 pmol/g), whereas CBs 77 and 52 were found at the lowest concentrations (8 pmol/g).


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TABLE 4 Concentrations of Chlorinated Biphenyls (CBs) in Male Sprague-Dawley Rats following a Single Oral Dose of CBs 28, 52, 77, 87, and 101
 
Serum concentrations varied between 11 and 49 pmol/g 6 h after treatment (Table 4Go). The highest initial concentration was found for CB 101. The B-mix CB concentrations in serum showed a rapid decrease during the first day. Between 6 and 24 h after exposure, the CB concentrations decreased by 62–82%. The slowest rate of decrease was found for CB 28. Between one and four days after exposure the concentration of CB 28 decreased with 60%, while the concentrations of other CBs were diminished by 67–80%. Four days after exposure, CB 101 was still the most abundant congener (3 pmol/g). The lowest serum concentration was found for CB 77 throughout the experiment.

The concentrations of B-mix CBs in adipose tissue increased until 24 h after exposure and decreased thereafter. The maximum concentration in adipose tissue was observed 24 h after exposure and ranged between 550 and 1727 pmol/g, with the highest concentration of CB 28 and the lowest of CB 77 (Table 4Go). From that time point to the end of the experiment a decrease of 20 to 59% was observed. At the end, CB concentrations in adipose tissue ranged between 227 and 1357 pmol/g.

Hydroxy metabolite.
The hydroxy metabolite 4-OH-CB107 was present in serum at all time points (Table 2Go). The concentration increased during the first five days, reached a plateau of about 6.5 pmol/g between days 5 and 15, and decreased thereafter (Fig. 2Go). After 135 days, the concentration of this metabolite was 0.2 pmol/g. After 15 days, the ratios between serum metabolite and the parental CBs 105 and 118 concentrations were 9 and 6, respectively. At the end of the experiment the ratios were 0.7 and 0.4, respectively.

Distribution Ratios
A-mix CB congeners.
Six h after administration of the A-mix, the liver-to-adipose tissue distribution ratios were about 1 for CBs 105 and 118 (Table 2Go). The corresponding ratios were 2 for CBs 138, 153, 156, and 157, while for CBs 170 and 180 the ratios were 5 and 5.5, respectively. During the period of dynamic steady-state (defined as constant distribution ratios between all body compartments) distribution ratios between liver and adipose tissue were in the range of 0.02 to 0.05 (Table 5Go). Liver-to-serum ratios varied between 7 and 22 for the different congeners and remained stable over the full time course of the study (Table 5Go). The adipose tissue-to-serum ratios were in the range of 2 to 14 after 6 h. During the dynamic steady-state period, ratios between adipose tissue and serum ranged between 316 and 517 for the different congeners (Table 5Go).


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TABLE 5 Tissue Partition Coefficients during Dynamic Steady-State between Liver (L), Adipose Tissue (A), and Serum (S) for Chlorinated Biphenyls (CBs) in Rat
 
B-mix CB congeners.
No period of dynamic steady-state was observed for any of the CBs in the B-mix. Six h after exposure, the distribution ratios between liver and adipose tissue varied between 0.2 and 1.2 for the different congeners (based on data in Table 4Go). The only congener with a ratio above one was CB 77. This liver retention of CB 77 declined at the end of the experiment. Liver-to-serum ratios were stable from 6 h and throughout the experiment, with the exception of CB 77 that showed an initial liver retention. The other congeners showed average liver-to-serum distribution ratios between 3 and 18 (Table 5Go). The liver-to-adipose tissue ratios were in the range of 0.2 to 2.5 after 6 h. Between days 1 and 4 the distribution was more and more shifted to the adipose tissue resulting in liver-to-adipose tissue ratios between 0.01 and 0.05 (based on data in Table 4Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose-Related Effects
The present study was performed with exposure doses that caused minimal biochemical effects and gave no signs of clinical or toxic effects. The transient hepatic EROD induction, which measures cytochrome P450 1A (CYP1A) enzyme activity (Bandiera et al., 1982Go), in the A-mix experiment was significant only before 15 days after exposure (Figs. 1A and 1BGo). The period of significant induction correlates with high concentrations in the liver. No altered EROD activity was seen during the time period when half-lives were measured. High activities of phase one metabolic enzymes (e.g., CYP1A) may explain the reported prolongation of half-lives with decreasing doses that has been observed for dioxins by others (Carrier et al., 1995bGo). The hepatic EROD induction seen after exposure to the B-mix was about 15 times the control activity after 24 h and decreased to about two times the control activity after four days. This transient induction, which could be a result of the non-ortho substituted CB 77, may have influenced the elimination rate. In both the A-mix and the B-mix experiments, up to a three-fold induction of hepatic PROD activity was seen throughout (Figs. 1C and 1DGo), independent of internal hepatic CB concentrations. It has been shown that PROD activity is related to CYP2B and can be induced by certain CBs (Connor et al., 1995Go; Lubet et al., 1985aGo). Among the congeners in the present study CBs 87, 101, 153, and 180 are all known to induce CYP2B, whereas CBs 105, 118, 156, 157, 138, and 170 are known to induce both CYP1A and 2B (McFarland and Clarke, 1989Go). The PROD induction is not likely to depend on the treatment nor likely to influence the kinetics.

In addition, the exposure was similar to present human exposure. Serum concentrations of {Sigma}CB during the dynamic steady-state of the A-mix experiment were found to be between 550 to 1100 ng/g lipid (Table 2Go). The median plasma levels of {Sigma}CB among people with low fish intake, is reported to be 780 ng/g lipid, of which 660 ng/g are from the eight CBs in the A-mix (Sjödin et al., 2000Go). The doses were lower than the dose (3 µg TEQ/kg) reported to cause reciprocal kinetic influence between dioxin-like substances (Carrier et al., 1995aGo). On a dioxin toxic equivalency (TEQ) basis, as defined by the World Health Organization (WHO; van Leeuwen and Younes, 1998Go), the rats were exposed to a TEQ dose of 0.05 µg/kg (A-mix), or 0.01 µg TEQ/kg (B-mix). The present study was also performed at doses (0.35 and 0.60 mg {Sigma}CB/kg, respectively) lower than the doses of nondioxin-like CBs that have been reported to cause shifted liver retention and potentiation of EROD activity (de Jongh et al., 1993bGo). In the study performed by de Jongh et al.(1993b)Go interactions were observed between CB 153 and 156 when given at doses of 99 and 15 mg/kg, respectively.

A-Mix CB Congeners
Tissue distribution and decreasing concentrations.
All the examined A-mix CBs were found to have a high initial liver and serum concentration after uptake through the gastrointestinal tract (Table 2Go). The following concentration decrease in liver and increase in adipose tissue demonstrates the redistribution to the adipose tissue (Fig. 2Go). This redistribution occurred during the first week after administration until dynamic steady-state was established. There may have been a significant amount of metabolism and redistribution of CBs prior to the initial sampling time. The distribution results in this study generally confirm previous observations for CB 153 in the rat (Matthews and Anderson, 1975Go).

A comparison of the eight individual A-mix CBs, by making a PCA for all normalized concentration data for individual rats, showed that the results were clustered in three groups in the second principal component (Fig. 3AGo). These groups correspond to the number of substituted chlorine atoms, which seems to be the most important quality for the differences in kinetic behavior among the CBs in the A-mix. Lipophilicity, measured as log Kow (Table 1Go), does not coincide with the clusters, showing that the impact of log Kow is not as large as reported elsewhere (van de Waterbeemd et al., 2001Go), even if molecular weight partially corresponds with log Kow. The loading plot of the second principal component (Fig. 3BGo) summarize the differences over time in all body compartments and shows that the CB congeners differ with respect to liver, adipose tissue, and serum concentrations at early time points, and in serum concentrations at the end of the experiment. The heptachlorinated CBs 170 and 180 showed a pattern characterized by higher concentration in the liver and serum at early time points, lower concentration in adipose tissue towards the beginning of the experiment, and lower serum concentrations towards the end. The pentachlorinated CBs 105 and 118 showed the opposite pattern with lower initial liver and serum concentrations, higher initial concentration in adipose tissue, and higher serum concentrations towards the end of the experiment. The lower initial concentration of the penta-CBs in the liver and serum could be explained by a more rapid flow through the liver and into the adipose tissue. After 6 h, most of the penta-CBs may already have passed the liver. Passive diffusion has been shown to depend on permeability rather than distribution coefficients (Andersen, 1991Go). Since adipose tissue is known to be the main deposit of lipophilic persistent contaminants in the body, the adipose concentrations reflect differences in the total body burden and, if the metabolism is slow, also the uptake. During the whole experimental time, most pronounced at early time points, the penta-CBs had the highest concentrations in adipose tissue and our conclusion was therefore that the lower chlorinated CBs had a higher uptake, a statement that is in accordance with previous results by Tanabe et al.(1981)Go who report an absorption of almost 85% for the penta-CBs and an absorption of about 75% for the hepta-CBs.



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FIG. 3. Principal Component Analysis (PCA) generated from individual concentration data normalized to total CB concentration. The model has two significant components (r2 = 0.987 and Q2 = 0.969). (A) Scores in two components with three clusters corresponding to number of chlorine atoms. (B) Loadings of the second component for all analyzed tissues.

 
When dynamic steady-state was established in the A-mix experiment, a slow and similar decrease of concentrations took place in all examined compartments as visualized for CB 105 in Figure 2Go. The constant tissue distribution ratios during this time period indicate that the diffusion between compartments in the body is much faster than the elimination from the body (Segel and Slemrod, 1989Go). In the tissue samples of rats exposed to the A-mix, concentration half-lives were found in the range of 54–129 days (Table 3Go). Relatively high variation between individuals and a slow decreasing rate made it difficult to determine exact and comparable values. In order to get more accurate t1/2 values, experimental time periods of at least three times the half-lives are needed, in this case about one year (Phillips et al., 1989Go).

Concentration in a tissue is a unit inversely proportional to the mass of the tissue and decreasing concentrations of persistent pollutants has been shown to partly be an effect of dilution (Lutz et al., 1977Go). This dilution-effect has also been seen in children occasionally exposed to PCB (Yakushiji et al., 1984Go). The Sprague-Dawley rats in the present study grew from about 350 to 500 g during the period of steady-state, i.e., 43% increase in body weight, while liver weights increased with 19% during the same time. A recalculation of the half-lives, based on total amount in the liver, showed prolonged times for all congeners by about 40% (Table 4Go). A change in body composition may also alter the distribution and can cause redistribution between body compartments when a dynamic steady-state is established. While the proportion of adipose tissue increases the lipophilic pollutants are "drawn" from other parts of the body into the adipose tissue. The proportion of adipose tissue as percent per body weight (BW) in Sprague-Dawley rats has been estimated to be 0.0199 x BW + 1.664 (Bailey et al., 1980Go). This means that in the present study the adipose tissue amount of rats increased from 30 to 58 g. In fact, the relative liver weight in this study decreased from 4 to 3% of total body weight. Some of the decrease of hepatic CB concentrations may be an effect of a second redistribution to the adipose tissue. Our conclusion from this study is that the decrease of CB concentrations in the tissues is mainly due to growth of the rat and specifically due to the increase of adipose tissue masses. A recalculation of half-lives based on an estimation of total amount in adipose tissue show that for several congeners almost no elimination at all could be detected during the study period (Table 3Go). In rats with constant mass of adipose tissue, the excretion terminal half-life of CB 153 has been calculated to be 478 days (Wyss et al., 1986Go), compared to a concentration half-life of about 100 days in this study. However, these t1/2 values are not directly comparable. Nevertheless, the long half-live values in different tissues of the Sprague-Dawley rat calculated in this study provide good evidence for the extreme stability of these substances. Very few half-lives of the examined CBs, measured under low dose conditions, are reported in the literature. Of the congeners in the present study the previously reported half-lives for total body burden was given as > 90 days for CBs 105, 118, 153, 138, 156, 170, and 180 (Tanabe et al., 1981Go). In Rhesus monkeys, half-lives for the CBs in mix-A are reported to vary between 0.5 and 1.5 years (Mes et al., 1995Go) and in humans about 3.7 years (Ryan et al., 1993Go). The results in the present study show that low dose elimination rates in the rat do not differ much from other species.

Partition between body compartments.
Lipophilic compounds are normally distributed in the fat depots within the body, as confirmed in the concentration order in the present study as visualized for CB 105 in Figure 2Go: adipose tissue > liver > serum. The same order has been reported by Lutz et al.(1977)Go. The examined CBs of the A-mix all have liver-to-adipose tissue ratios of about 0.03 (Table 5Go). This is in good accordance with Lutz et al.(1977)Go who reported a ratio of 0.03 in rats for CB 153. The liver-to-adipose tissue ratio is often used to describe and compare partition among persistent organohalogen compounds (Haag-Grönlund et al., 1997Go; van den Berg et al., 1994Go). In the body, the distribution between liver and adipose tissue is dependent on an intermediary compartment, the serum. In the present study, CB 153 was found to have an adipose tissue-to-serum ratio of 316 (Table 5Go). In comparison, others have reported an adipose tissue-to-serum ratio of 270 for this congener (Wolff et al., 1982Go). For all examined congeners Wolff et al.(1982)Go found somewhat lower human adipose tissue/serum ratios than observed in this study. The partition between liver and serum seem to be stable from the first time point, at 6 h after treatment, for all thirteen congeners, with the exception of CB 77 (Table 5Go). If a compound does not follow the same distribution as similar substances, this could be a sign of specific protein binding in a special compartment. This has been reported for dioxin and the dioxin-like CBs 126 and 169, which all bind to the hepatic enzyme CYP1A2 and where a shift towards the liver is observed in liver-to-adipose tissue ratios (de Jongh et al., 1993bGo; Diliberto et al., 1997aGo; Santostefano et al., 1997Go; Yoshimura et al., 1985Go). Experiments with mice deficient for CYP1A2 do not show this shifted ratio (Diliberto et al., 1997bGo). In the B-mix experiment, a liver specific initial retention for the non-ortho CB77 was observed. This retention could be explained by binding to the AhR-dependent CYP1A2 induced by the Ah-receptor. The liver retention also correlates to the AhR-dependent EROD activity.

B-Mix PCB Congeners
Tissue distribution and decreasing concentrations.
Previous data suggested that the CB congeners in the B-mix have initial half-lives in the range of 1–2 days (Tanabe et al., 1981Go). For CBs 28, 52, and 101 Tanabe et al.(1981)Go reported a biphasic elimination with a second half-life (whole body) of 6, 3, and 35 days, respectively. In the same study CB 87 was reported to have a half-life of about 2 days whereas CB 77 was not included. Our results suggest that the half-lives of the B-mix CBs are significantly longer than reported by Tanabe et al.(1981)Go. The previously reported short half-lives may be an effect of the relatively high dose given. The maximum accumulation in the adipose tissue occurred 24 h after administration as compared with 5 days for the congeners in the A-mix experiment. The trichlorinated CB 28 seemed to have an effective uptake, detected as a high adipose tissue concentration at early time points, and a slow elimination rate. It is known from environmental samples that CB 28 often occurs at high concentrations, but the tissue retention of this low chlorinated congener still has to be explained (Glynn et al., 2000Go). A structure-related model of CB metabolism has been proposed (Parham and Portier, 1998Go). This model depends mostly on the degree of chlorination, coplanarity, and chlorine free meta-para and ortho-meta sites (double positions). As in the results of this study, the proposed model gives a lower elimination rate if the number of chlorine increases, but no correlation with free double positions could be detected in the present study. For example, CB 28, which has three free double positions, shows a high tissue retention in the present study (Table 4Go). Lowest concentrations were seen of the tetrachlorinated CB 52. This congener is the only congener among the ones studied with two free meta-para sites, and a clear relationship between the number of free meta-para sites and a high degree of metabolism has been shown (Tanabe et al., 1981Go). This site is easily epoxidized and thereafter the congener can be conjugated and excreted. The tetrachlorinated CB 77 is characterized by a high initial relative liver concentration and a fast elimination. The high liver retention may potentiate the metabolism, since the half-lives of total body burden have been shown to depend on liver concentration (Carrier et al., 1995bGo). The two pentachlorinated CBs 87 and 101 seem to have similar kinetic behavior. CB 87 is found in slightly lower concentrations and this may reflect lower uptake and/or higher metabolism. Tanabe et al.(1981)Go reported that CB 87 has a much faster whole-body elimination rate compared to CB 101. Such a big difference could not be detected in this study. Due to the short study period (4 days) dynamic steady-state was not achieved for the B-mix congeners. To better describe the kinetics of these B-mix CBs the study period would need to be 40 days or more.

Hydroxy Metabolite
In this study the main hydroxy metabolite from CBs 105 and 118 was analyzed in serum (Table 2Go; Sjödin et al., 1998Go). The possible metabolic routes are shown in Figure 4Go. In humans, this serum metabolite has been shown to be one of the major hydroxylated CB metabolites, constituting 12–62% of all analyzed OH-CBs (Sandau et al., 2000Go; Sjödin et al., 2000Go). In the present study, 4-OH-CB107 reached a concentration about 10 times higher than the parental compounds after five days. From day 15, when the parental compounds are in a dynamic steady-state condition, there was a fast, nonlinear decrease on the metabolite concentration. It is worth noting that the metabolite shows serum concentrations similar to the parental compounds at both 45 and 135 days after exposure. This is similar to previous results, where 20 days after iv exposure to 3 µmol/kg of CBs 105 and 118, the plasma concentration ratio between parent CBs and 4-OH-CB107 was found to be 0.7 and 1.4, for CBs 105 and 118, respectively (Sjödin et al., 1998Go). During the last 90 days in the present study, the concentration of the OH metabolite decreased by 59%, which corresponds to a first order decrease with a half-life of about 77 days. These data confirm previous findings, which demonstrate that hydroxylated CB metabolites have a selective retention in the blood (Bergman et al., 1994Go). In mouse plasma, the 4-OH-CB107 concentration was 15 times higher than the parent CB 105 concentration five days after exposure (Klasson Wehler et al., 1993Go). The selective retention in blood of OH metabolites may be due to their ability to bind to transthyretin (TTR), a thyroxin and retinol transporting protein. The meta positions of the 4-OH-CB107 are halogenated in the same ring as the para-substituted hydroxy group, which allows the metabolite to compete with thyroxin for the TTR binding site (Lans et al., 1993Go). By interfering with TTR the OH metabolite can disturb not only thyroxin, but also retinoid transport (Brouwer et al., 1988Go).



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FIG. 4. Suggested metabolic routes for formation of 4-OH-CB 107 from CBs 105 and 118 according to Sjödin et al. (1998)Go.

 
Conclusions
During the dynamic steady-state CBs 105, 118, 138, 153, 156, 157, 170, and 180 showed decreased concentrations with half-lives in the range 54 to 124 days. Most of the decrease in concentration was explained by the growth-related increase of tissue masses in general and adipose tissue in particular. A PCA showed a correlation between molecular weight and bioavailability and distribution between these congeners. The CBs 28, 52, 77, 87, and 101 were eliminated at slower rates than previously reported and for the first time a high and congener-specific liver-to-serum ratio of CB 77 was observed. This study also provides serum concentration data on the hydroxy metabolite 4-OH-CB107 that was present at concentrations similar to the parental CBs 105 and 118. The low doses used in this study are comparable with the human background situation. The dynamic steady-state reached makes the results valuable in the human health risk assessment procedure.


    ACKNOWLEDGMENTS
 
The authors sincerely thank Ellu Manzoor for chemical analysis and Joost de Jongh for sharing toxicokinetic knowledge. This study was supported by funds from the Swedish Environmental Protection Agency.


    NOTES
 
1 Present address: National Center for Environmental Health, 4770 Buford Highway, MS F-17, Atlanta, GA 30341. Back

2 To whom correspondence should be sent at Institute of Environmental Medicine (IMM), Karolinska Institutet, P.O. Box 210, SE-171 77 Stockholm, Sweden. Fax: 46-8-34-38-49. E-mail: helen.hakansson{at}imm.ki.se. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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