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Liver accumulation of 2,3,7,8-tetrachloro-[3H]dibenzofuran in mice: modulation by treatments with polychlorinated biphenyls
Choa~-ak~x~:al ~tottx~aw FI SEVILR SK'IFNTIFI( PI,'BLISItl RS IRFLA~.D
Chemico-Biological Interactions 89 (1993) 89-102
Liver accumulation of 2,3,7,8-tetrachloro-[3H]dibenzofuran in mice: modulation by treatments with polychlorinated biphenyls P.O. Darnerud *a, U. T6rnwall b, ,~,. Bergman c, I. Brandt b aDepartment of Toxicology. Uppsala University, BMC, Box 594, S-75124 Uppsala, Sweden bDepartments of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, BMC, Box 573, S-751 23 Uppsala, Sweden CEnvironmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
(Received 9 September 1992; revision received 7 April 1993; accepted 21 April 1993)
Abstract
The distribution of 2,3,7,8-tetrachloro-[3H]dibenzofuran ([3H]TCDF; 40 ttg/kg) resembled that earlier reported for 2,3,7,8-tetrachlorodibenzo-p-dioxin, with a strong accumulation in the liver and a selective uptake in the nasal olfactory mucosa of adult and fetal mice. Pretreatments with a series of selected congeners of polychlorinated biphenyls (PCBs), i.e.. I (IUPAC)-77, 1-105, I-118, 1-126, 1-153, 1-156, 1-169, and a commercial preparation, Aroclor 1254 (25-100 mg/kg body wt. i.p.), were found to modulate the hepatic uptake of [3H]TCDF (24 h post-3H-injection). At a short pretreatment time (4 h), non-ortho-chlorinated congeners decreased the uptake of [3H]TCDF equivalents in the liver (e.g., 1-126 = 3,3',4,4',5-pentachlorobiphenyl: 340 of control), while several mono- and di-ortho PCB congeners and Aroclor 1254 increased the hepatic uptake of [3HITCDF (e.g., 1-156= 2,3,3',4,4',5-hexachlorobiphenyl: 183% of control). At a longer pretreatment time (48 h), both a non-ortho (I-169=3,3',4,4',5,5'-hexachiorobiphenyl) and mono-ortho PCB congener(s) (e.g. 1-156) markedly increased the hepatic 3H-uptake (190%), a probable effect of an induction of hepatic binding sites for TCDF. Ethoxyresorufin-O-deethylase activities, regarded to mirror the metabolic activity of cytochrome P-450 IA 1 (CYP IA 1), were strongly and time-dependently induced after 1-169, but not after 1-156, pretreatment (25 mg/kg). The initial liver concentrations of the two PCB congeners were similar and increased for 1-169 but not for 1-156 at later time points. In conclusion, the results show a selective uptake of |3H]TCDF in the mouse liver and nasal olfactory mucosa of both dam and fetus. The uptake of [3H]TCDF in the liver is influenced both by dose and pre-exposure with PCBs. The presence of a PCB-sensitive, * Corresponding author, National Food Administration, Toxicology Unit, Box 622, S-75126 Uppsala, Sweden. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03200-E
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but CYP IA l-independent, hepatic binding site for TCDF is suggested• Consequently, pharmacokinetic interactions with PCBs complicate the toxicity assessment of TCDF in complex mixtures• Key words: Polychlorinated dibenzofuran; PCB; Co-exposure; Interactions; 3H-labelled;
Mouse; Distribution; Liver; Olfactory mucosa
1. Introduction
An important step in understanding the mechanism of action of halogenated dibenzo-p-dioxins/furans (PCDDs/Fs) was the discovery of a cytosolic receptor for these chemicals, the Ah receptor [1,2]• More recently, other binding sites for PCDDs/Fs and related compounds have been suggested in the rodent liver [3], one of these identified as the cytochrome P-450 species IA2 (form d; CYP IA2). This enzyme, induced by Ah receptor ligands, would likely explain the strong hepatic binding of a 2,3,7,8-dibenzo-p-dioxin (TCDD) analog [4,5], 2,3,4,7,8-pentachlorodibenzofuran [6,7], 3,3',4,4',5,5'-hexabromobiphenyl [8] and related compounds. In the environment mixtures of these and other pollutants are present, and the study of combinations of compounds regarding body accumulation and distribution is of relevance for predicting their toxic potential• Thus, the resulting accumulation of dibenzodioxin-like compounds in the body may be the consequence both of the presence of inducible binding sites in the body and the persistence of the compounds. The aim of the present paper was to define target tissues for TCDF accumulation in the mouse, and to describe the effects on organ TCDF uptake after exposure to a series of individual PCB congeners• The results of the distribution study of [3H]TCDF resemble those obtained earlier for TCDD in rats and mice [9,101, where a strong hepatic accumulation of radioactivity and a specific labelling of the nasal olfactory mucosa were observed• The present study revealed different effects on the hepatic accumulation of [3H]TCDF due to the PCB congener selected and pretreatment time. These congener-dependent effects are suggested to be related to the molecular structure (e.g., degree of planarity and number of chlorinated orthopositions of the biphenyl molecule) and metabolic inertness of different PCB congeners. Changes in concentration of the large hepatic pool of TCDF may be of significance for toxicity in non-hepatic tissues such as the lymphatic system• However, other types of (non-additive) interactions may also result from co-exposure to TCDD and PCB compounds, and antagonistic effects on immunotoxicity [11] and teratogenicity [12] has been reported• 2. Materials and methods 2.1• Chemicals
2,3,7,8-Tetrachloro-[4,6-3H]dibenzofuran (3H-TCDF) (18.9 Ci/mmol, purity > 98%) and unlabelled TCDF was purchased from Chemsyn Science Laboratories (Lenexa, Kansas, USA). The numbering system of PCB congeners as suggested by
P.O. Darnerudet al./ Chem.-Biol. Interact. 89 (1993) 89-102
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Table i PCB congeners used in the study: full chemicalname and their corresponding IUPAC nomenclature Name
Abbreviation
3,3',4,4'-tetrachlorobiphenyl 2,3,3 ' ,4,4'-pentachlorobiphenyl 2,3' ,4,4' ,5-pentachlorobiphenyl 3',3 ',4,4 ' ,5-pentachlorobiphenyl 2,2' ,4,4',5,5'-hexachlorobiphenyl 2,3,3',4,4 ' ,5-hexachlorobiphenyl 3,3 ',4,4 ',5,5 '-hexachlorobiphenyl
1-77 1-105 1-118 1-126 1-153 1-156 1-169
IUPAC is used in the present study [13]. Thus, the PCB congeners 1-77, 1-105, I-118, 1-126, 1-153, 1-156 and 1-169 (see Table 1 for full chemical names) were synthesized according to Sundstr6m [14]. Aroclor 1254 was produced by Monsanto (USA) and B-naphthoflavone (BNF) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals
NMRI Mice 20-23 g (Alab, Sollentuna, Sweden) were kept at 22°C under controlled light conditions (12 h light/12 h dark) and were given pelleted food (R3, Ewos, S6dert~lje, Sweden) and tap water ad libitum. Females were mated overnight and the presence of vaginal plugs the following morning confirmed mating and was denoted day 0 of pregnancy. 2.3. Whole-body autoradiography
Non-pregnant mice (n = 2) were given [3H]TCDF (40 /~g/kg body wt.; - 50 tLCi/animal) intravenously (iv.; dissolved in 20 t~l dimethylsulfoxide) and sacrificed in carbon dioxide atmosphere 7 and 28 days later. Pregnant mice (n = 5) were given the same [3H]TCDF dose on day 10, 14, 16 or 17 of gestation and were sacrificed on day 17, i.e., 7, 3, 1 day(s) or 8 h after injection, respectively. On day 16, one mouse was given a high dose (800 ~,g/kg body wt.) of TCDF and sacrificed after 24 h. In a separate study, two mice were given either BNF (80 mg/kg body wt.) or corn oil (vehicle) intraperitoneally (i.p.) 44 h before TCDF injection on day 16 of gestation; the animals were sacrificed 24 h thereafter. The animals were embedded in carboxymethyicellulose, frozen to -70°C and dry sectioned for autoradiography according to Ullberg [15]. The sections were exposed to X-ray film (Hyperfilm 3H, Amersham Sweden AB, Soina) which was developed after appropriate exposure time. 2.4. Quantitative pretreatment study
Female non-pregnant mice (n = 4) were given the different PCB congeners (see Table 1) or Aroclor 1254 i.p. (doses, see Table 3). Control animals (n = 4-6) were given the vehicle only (corn oil; 0.2 ml/20 g). Four hours later the mice were injected iv. with [3H]TCDF (2.6 nmol(0.83 ug)/kg body wt.; 1 ~Ci/animal), and 24 h after the [3H]TCDF administration the mice were sacrificed by cervical dislocation. Sep-
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arate groups of mice, pretreated with 1-105, 1-156 or 1-169 48 h before [3H]TCDF but otherwise handled similarly, were included in the study. Selected tissues, i.e., liver, fat tissue, right kidney, lung tissue and thymus (for 1-156 and Aroclor 1254, and for all compounds at the 48-h pretreatment study, only liver, fat and kidney), were excised. The tissue specimens were weighed, dissolved in Soluene 100 (PackardCanberra Sweden, Uppsala) over night, and subjected to liquid scintillation after addition of toluene-based scintillation cocktail (Toluene-Scint, Packard). The selection of two PCB pretreatment time points, 4 and 48 h, were based on the assumption that the 4 h pretreatment may lead to interactive effects at existing binding sites, whereas 48 h would be sufficient time to induce synthesis of new cytochrome P-450, or other binding proteins. The three PCB congeners in the 48 h study were chosen as representatives for congeners which increase (I-156), decrease (I-169) and have no effect (I-105) on hepatic [3H]TCDF uptake at short pretreatment.
2.5. Ethoxyresorufin-O-deetylase (EROD) measurements The effects of the PCB congeners I-156 and 1-169 (25 mg/kg body wt. i.p.) on hepatic microsomal enzyme activities were studied in vitro using ethoxyresorufin as substrate [161. The animals (females; n = 3-4/group) were sacrificed 4 h, 28 h and 72 h after PCB injection. The incubation time was 4-10 min and about 20 #g microsomal protein was added. EROD data was determined and analytical data collected for 1-156 and 1-169 because these congeners had been examined regarding hepatic [3H]TCDF uptake at two time points, and the fact that they gave opposite effects after short pretreatment time. 2.6. Analysis of 1-156 and 1-169 liver residues The hepatic tissue concentration of animals treated with I- 156 and I- 169 (the same mice as in the EROD study described above) was analysed by GC using a Varian 3400 gas chromatograph equipped with an electron capture detector (ECD) and a DB-5 fused capillary column (30 m, I.D. 0.25 mm) from J&W. Injections were made in the splitless mode and the column temperature was programmed: 80°C (2 min) 10°C/min to 300°C that was kept for 10 min. Liver specimens from animals in each group were pooled and the total fresh weight (f.w.) was determined for each sample. The samples were homogenized in aceton: hexane (3;5 ml, 25:1) and centrifuged. The solvent was removed and hexane: methyl tert-buthyl ether (MTBE) (2.5 ml (9:1)) was added before the sample was shaken and centrifuged. The solvent was removed and the procedure was repeated twice. The pooled organic phases were partitioned with 5 ml of a sodium chloride (0.9%)/phosphoric acid (0. i M) aqueous solution [17]. The organic phase was removed and the aqueous phase was extracted twice with hexane (1 ml). The hexane phase was pooled with the organic solvent and the solvent mixture was evaporated. The weight of the extracted lipids (l.w.) was determined. Hexane (2.0 ml) was added to the lipid residue and the lipids were finally destructed by use of conc. sulphuric acid (1.0 ml). The hexane phase was used for GC (ECD) analysis of 1-156 and 1-169, respectively. Livers from a group of control mice were also analyzed. 2,3,3 ',4,4',5,5 '-Heptachlorobiphenyi (I-189) was used as internal standard (IS).
mucosa
liver
fetuses
Fig. 1. Whole-body autoradiogram of a cryosectioned pregnant mouse sacrificed 24 h after [3H]TCDF injection (40 .g/kg body wt.). Note the strong hepatic accumulation and the specific labelling of the nasal olfactory mucosa.
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3. Results 3.1. Whole-body autoradiography
The dominant uptake of [3H]TCDF (40 ttg/kg) occurred in the liver (Fig. 1). The intrahepatic distribution was irregular, with a higher uptake around the central vein (Fig. 2a). The gallbladder contained high levels of [3H]TCDF-derived radioactivity, possibly metabolites. Other less dominant sites of labelling were the fat and fecal contents. Radioactivity was visible up to 3 days post-injection in the kidney (concentrated to the cortico-medullary region), and specifically in the nasal olfactory mucosa (Fig. 1). However, practically no labelling was observed in the endocrine system, including the thyroid gland, adrenals, thymus, bone marrow, lymph nodes and spleen. The radioactivity at all accumulation sites gradually disappeared; after 7 days only the liver was labelled and 28 days after[3H]TCDF administration the body was void of radioactivity. In pregnant mice (40 #g/kg [3H]TCDF) there was a faint and transient accumulation of radioactivity in the fetal liver and intestines, most accentuated 24 h postinjection. A thin slit of radioactivity surrounding the fetal membranes was also observed, probably representing labelling of the uterine fluid and/or the yolk sac placenta (Figs. 1 and 3a). At a higher dose of [3HITCDF (800 ttg/kg, 24 h survival time), the maternal liver accumulation became homogeneously distributed (Fig. 2b). In the fetuses a stronger
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Fig. 2. Detailof the liversfrom autoradiogramsof pregnant mice24 h after administrationof (a) 40 v.g/kg body wt. and (b) 800 #g/kg of [3HITCDF, respectively. Observe the mottled, unhomogeneous distribution in (a).
fetuses
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elf.
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Fig. 3. Detail of the uterine regions from autoradiograms, from Fig. 2. Note the specific uptake in the olfactory mucosa and the hepatic and intestinal accumulation in fetuses exposed to the higher [3H]TCDF dose (b).
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P.O. Darnerud et al. ~Chem.-Biol. Interact. 89 (1993) 89-102
Table 2 Uptake of [3H]TCDF-derived radioactivity in various organs of the mouse 24 h after [3HITCDF injection a Organ
Tissue T C D F conc. pmol/g wet wt.
Liver Fat tissue Kidney (right) Lung Thymus
5.8 3.9 0.5 0.6 0.5
± ± ± ± ±
0.7 b 0.3 0.07 0.04 0.05
~[3HITCDF dose: 2.6 nmol(0.83 ~g)/kg body wt. (i.v.). bMean ± S.D. (n = 6).
uptake was generally seen and the nasal passages were specifically labelled (Fig. 3b). The BNF pretreatment did not appreciably change the autoradiographic distribution of [3HITCDF.
3.2. Quantitative pretreatment study The uptake of [3H]TCDF-derived radioactivity in different tissues from mice in the control group is given in table 2. As seen in the Table, the liver showed the highest concentration of radioactivity followed by the fat tissue, whereas the uptake in lung, kidney and thymus was low. Some of the compounds used in the short-time pretreatment study did significantly change the retention of radioactivity in the liver 24 h after [3HITCDF injection (Table 3): 1-156, 1-153 and Aroclor 1254 increased the hepatic retention of [3H]TCDF-derived radioactivity (183, 138 and 166% of control, respectively),
Table 3 Effect of short-time (4 h) pretreatment with selected compounds on hepatic uptake of [3H]TCDF in mice Substance
n
PCB dose (mg/kg)
Hepatic T C D F concl (pmol/g wet wt.)
% of control
Vehicle (control) 1-77 a 1-105 1-118 1-126 1-153 1-156 1-169 Aroclor 1254
6 4 4 4 4 4 4 4 3
-25 b 25 25 25 100 25 25 100
5.8 4.3 6.2 6.1 1.9 8.0 10.6 4.9 9.6
-75 107 106 34 138 183 85 166
+ 0.8 c .4- 0.4 **d :t: 1.6 .4- 1.0 -4- 0.2*** ± 1.0* -4- 2.1"* ± 0.7 (9.0-10.6)**
aAbbreviations, see Table 1. bMice were pretreated 4 h before administration of [3H]TCDF (2.6 nmol(0.83 ttg)/kg body wt.) and sacrificed 24 h after injection of radioactivity. ¢-4- S.D. {Aroclor 1254: range; n = 3). dSignificantly different from control values; Student's t-test (* P < 0.05, ** P <0.01, ***P < 0.001).
P.O. Darnerud et al. / Chem.-Biol. Interact. 89 (1993) 89-102
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1-126 dose (mg/kg) Fig. 4. Dose-related decrease in hepatic [3H]TCDF concentration after pretreatment with the PCB congener 1-126 (2.5,5,10 and 25 mg/kg body wt.). The relative values represent means ± S.D. (n = 4). Significant differences from controls in Student's t-test (P < 0.001) were observed at 10 and 25 mg/kg.
whereas 1-126 and 1-77 decreased the retention (34 and 75%). To conclude, the strongest decrease in hepatic retention of [3H]TCDF was observed for the coplanar 1-126, while the mono-ortho substituted H 5 6 gave rise to the strongest increase in retention of 3H-TCDF, at short-time pretreatment and a dose level of 25 mg/kg of the PCB congener. In the case of 1-126, three additional doses were tested. The result showed that the decrease in hepatic retention of [3H]TCDF was dose dependent (Fig. 4). Lower Table 4 Effects of long-term (48 h) pretreatment with selected PCB congeners on hepatic retention of [~H]TCDF in mice Substance
n
Vehicle (control) 1-105 1-156 1-169
4 4 4 4
PCB dose (mg/kg)
Hepatic T C D F conc. (pmol/g wet wt.)
% of control
25 25 25
5.0 ~9.7 ~ 9.5:1: 9.8:1:
-194 190 196
Footnotes in Table 3 valid (except pretreatment time).
0.9 1.0"** 2.0** 1.0***
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doses of 1-156 and Aroclor 1254 (12.5 and 25 mg/kg, respectively) resulted in nonsignificant increases in hepatic 3H-levels. At the long pretreatment time (48 h), all the PCB congeners studied (I-105, 1-156 and 1-169) markedly (190%) increased the retention of [3H]TCDF in the liver (Table 4). The effect of PCB treatments on the uptake of [3H]TCDF in extrahepatic tissues was in most cases not profound. However, at the short-time pretreatments several PCB congeners (I-77, 1-105, I-118, 1-153, 1-169) resulted in a slight (not less than 70% of control) but significant decrease of [3H]TCDF retention in the lung. In fat, significantly decreased 3H-levels were observed after pretreatments with 1-153 (69%), and in thymus with 1-153 (91%). In contrast, an increase in the uptake of [3H]TCDF (139%) was observed in the kidney after 1-126 treatment. At long-time pretreatments, all the three PCB congeners studied gave rise to a decreased uptake of [3H]TCDF in fat (46-69%). The BNF pretreatment increased the hepatic uptake of radioactivity (162% of control), whereas the retention in fat was significantly decreased (43%). 3.3. E R O D m e a s u r e m e n t s
Results from the E R O D measurements on microsomes from 1-156- and 1-169treated mice, are given in Table 5. A time-dependent increase in E R O D induction is seen after 1-169 pretreatment, whereas 1-156 values are similar to controls. 3.4. G C congener analysis
The concentration of the pretreatment substances 1-156 and 1-169 in liver tissue is given in Table 5. On a fat weight basis, the values after 4 h are identical for the two congeners (120 ng/mg fat), and increases with time for 1-169 (up to 230 ng/mg fat) but not for 1-156.
Table 5 Hepatic congener concentration and microsomal EROD induction at three time points after treatment with the PCB congeners !-156 and 1-169 (dose 25 mg/kg) Substance
Treatment time (h)
n
Liver conc.~
EROD activity (range)b
Vehicle 1-156 1-156 1-156 1-169 1-169 1-169
72 4 28 72 4 28 72
3 4 3 3 4 2 3
-120 130 80 120 150 230
0.048 (0.022-0.096) 0.083 (0.031-0.165) 0.108 (0.058-0.182) 0.117 (0.060-0.228) 0.0.50 (0.028-0.064) 0.675 (0.541-0.809) 2.32 (0.994-3.214)
a Liver concentration of congener in ng/mg extracted fat (pooled samples). b Ethoxyresorufin-O--deethylase(EROD) activity in nmol formed resorufin/min/mg protein.
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4. Discussion
The distribution of [3H]TCDF in the mother and fetus was similar to that earlier found for TCDD [9,101 with pronounced uptake in the liver and nasal olfactory mucosa. The retention of [3H]TCDF seemed to be less pronounced than that of TCDD - - a finding that is supported by earlier results by Weber and Birnbaum [18]. A similar retention difference between chlorinated dioxins and furans in the liver of rats has also been observed [19]. Among the coplanar PCBs, similarities to TCDF regarding tissue distribution are observed, especially for 1-126 [20]; for example, both TCDF and 1-126 give rise to strong hepatic accumulation. However, the coplanar 1-77 results in a different tissue distribution in mice as compared to 1-126 and 1-169, characterized by a limited accumulation in liver and fat, a marked labelling of the blood and excreta, and a pronounced uptake in fetal blood and tissues [21,22]. The fast metabolism of 1-77 to hydroxylated metabolites, some of which have high binding affinity to transthyretin in plasma [23], could be one explanation to the specific distribution pattern of this congener. According to our autoradiographical results, administration of a higher molar dose of TCDF (132 vs. 5296 nmol/kg body wt.; but with an equal dose of [3H]TCDF) seemed to increase the hepatic pool of [3H]TCDF. A similar observation was made by Poland and coworkers [4,5] who reported TCDD (100 nmol/kg) to strongly increase the hepatic binding of [125I]2-iodo-3,7,8-trichlorodibenzo-pdioxin (0.1 nmol/kg). The TCDD binding species was reported to be an induced form of cytochrome P-450, i.e.: CYP IA2 (P-450d) [24]. In the present quantitative study the liver retention of [3H]TCDF was approximatively 15% of the total 3H dose for non-pretreated animals, as compared to 1-2% of 125Idose in studies on the TCDD analog by Poland and coworkers. Besides being two different compounds, one additional explanation to the discrepancy may be the fact that the tracer dose used in our study (2.6 nmol/kg body wt. of [3H]TCDF) was 26 times higher (on molar basis) than that of [125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin (0.1 nmol/kg) used by Poland and coworkers. This means that our [3H]TCDF dose seems to be high enough to induce de novo synthesis of a cytochrome P-450 species with [3H]TCDF binding properties. Altogether, these results indicate that the hepatic fraction of a TCDF (TCDD) dose will increase with increasing doses. However, at a high enough dose, induction will be maximal and saturation of hepatic binding sites will most likely occur. The presence of a large hepatic pool of [3H]TCDF, similar to that reported for TCDD, made it of interest to study how other xenobiotics with structures similar to TCDF could modify the hepatic binding and retention of [3HITCDF, and thereby also the concentration of [3H]TCDF in other tissues. Presumably, a decrease in the hepatic pool of TCDF would result in an increase of TCDF in extrahepatic target tissues. In our study, only the three coplanar congeners 1-77, 1126 and 1-169 decreased the liver accumulation at the short (4 h) pretreatment time. Reasons for the ability of coplanar congeners to displace [3H]TCDF would be their persistency and relatively high affinity for the hepatic binding sites, including the Ah
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receptor and cytochrome P-450 species. In contrast some PCB congeners, i.e., 1-156, increased the hepatic pool of [3H]TCDF after the 4 h pretreatment, suggesting an increase in binding sites for this compound in analogy with the situation after increasing the dose of [3H]TCDF (see above). On one hand this would leave less for extrahepatic target sites (inducing e.g., teratogenicity), but on the other hand an increased liver binding would also most probably lead to an increase in the total retention time of T C D F in the body, which may increase the toxicity. At longer pretreatment time all PCB congeners studied, including 1-156 and the non-ortho 1169, increased hepatic binding of [3H]TCDF. If a decrease in peripheral target organ concentrations of T C D F is the consequence, these results could be supported by several teratogenicity studies on TCDD, which conclude that teratogenic effects are antagonized within certain dose limits by co-treatment with Aroclor 1254 [25] and 1-153 [12,26]. Also T cell-mediated immunotoxic effects of T C D D are antagonized by treatment with commercial PCB mixtures [11,27]. However, other studies have suggested that e.g., teratogenic effects of 2,3,4,7,8-pentachlorodibenzofuran and 1-156 are additive [28], and that the antagonistic properties of pure PCB congeners seem to be restricted to certain structural forms [29]. The latter study also suggested 1-156 to have additive effects on TCDD-mediated immunotoxicity. Another factor to take into account is that all the compounds that induced hepatic [3H]TCDF binding and retention in the present short-time pretreatment study (I153, 1-156 and Aroclor 1254), are also known to induce Ah receptor levels [30,31]. The increase in the levels of Ah receptors would result in de novo synthesis of cytochrome P-450 binding sites for TCDF. The fact that the hepatic [3H]TCDF concentration is increased both after short and long 1-156 treatment time whereas EROD activities are not induced, suggests that CYP IAI is not directly involved in the hepatic accumulation of [3H]TCDF. Moreover, CYP IA1 does not seem to mirror the induction status of CYP IA2, the proposed main binding site of T C D F and related compounds in the liver. The small effects of I-156 on EROD induction in our study is somewhat in contrast with earlier results of Davis and Safe [29], who observed a quite substantial induction (ca. 3 nmol resorufin/min/mg protein) using a similar PCB dose and treatment time (3 days). This difference may be due to the use of different strain and sex of mice. Regarding hepatic congener concentrations, our results show similar initial levels of 1-156 and 1-169; this rules out the possibility of a concentration difference as explanation to the differential effects of the tested PCB congeners. To summarize, a specific uptake of radioactivity in the liver and nasal olfactory mucosa of both dam and fetus was observed in mice exposed to [3HITCDF. The body distribution was dependent on the T C D F dose, probably determined by induction of T C D F binding sites in the liver. Regarding co-administration of T C D F and PCB congeners, the observed differential effects on [3H]TCDF distribution, depending on congener and pretreatment time, could be due to induction of [3HlTCDF-binding sites, competition for binding sites and rate of elimination. It is notable that at the longer pretreatment time all PCB congeners studied showed a potential to increase the hepatic [3H]TCDF pool; this may reflect an 'environmental' effect of a continuous exposure to Ah receptor binding PCB congeners.
P.O. Darnerud et al. ~Chem.-Biol. Interact. 89 (1993) 89-102
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5. Acknowledgements Economic support was given by the Research Committee of the Swedish Environmental Protection Agency.
6. References I
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14 15 16
17
A. Poland and J.C. Knutson, 2,3,7,8-Tetrachlorodibenzo-p-di0xin and related halogenated hydrocarbons: examination of the mechanism of toxicity, Ann. Rev. Pharmacol. Toxicoi., 22 (1982) 517-554. J.P. Whitlock, The regulation of gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin, Pharmacol. Rev., 39 (1987) 147-161. L.R. Curtis, N.I. Kerkvliet, L. Baecher-Steppan and H.M. Carpenter, 2,3,7,8-Tetrachlorodibenzo-pdioxin petreatment of female mice altered tissue distribution but not hepatic metabolism of a subsequent dose, Fund. Appl. Toxicol. 14 0990) 523-531. A. Poland, P. Teitelbaum and E. Glover, [a251] 2-1odo-3,7,8-trichlorodibenzo-p-dioxin-binding species in mouse liver induced by agonists for the Ah receptor: characterization and identification, Mol. Pharmacol., 36 (1989) 113-120. A. Poland, P. Teitelbaum, E. Glover and A. Kende, Stimulation of in vivo and in vitro hepatic binding of [125I]2-iodo-3,7,8-dibenzo-p-dioxin by the administration of agonists for the Ah receptor, Mol. Pharmacol., 36 (1989) 121-127. J. Kuroki, N. Koga and H. Yoshimura, High affinity of 2,3,4,7,8-pentachlorodibenzofuran to cytochrome P-450 in the hepatic microsomes of rats, Chemosphere, 15 (1986) 731-738. H. Yoshimura, J. Kuroki, N. Koga, H. Kuroki, Y. Masuda, N. Fukasaku and M. Hasegawa, High accumulation of 2,3,4,7, 8-pentachlorodibcnzofuran to hepatic microsomes of rats, J. Pharm. Dyn., 7 (1984) 414-419. R. Voorman and S.D. Aust, Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome P-450d to the cytochrome and inhibition of its estradiol 2-hydroxylase activity, Toxicol. Appl. Pharmacol., 90 (1987) 69-78. L.-E. Appelgren, I. Brandt, E.B. Brittebo, M. Gillner and J.-~. Gustaafsson, Autoradiography of 2,3,7,8-tetrachloro[ 14C]dibenzo-p-dioxin (TCDD): accumulation in the nasal mucosa, Chemosphere, 12 (1983) 545-548. M. Gillner, E.B. Brittebo, I. Brandt, P. S6derkvist, L.-E. Appelgren and J.-~. Gustafsson, Uptake and binding of 2,3,7, 8-tetrachlorodibenzo-p-dioxin in the olfactory mucosa of mice and rats, Cancer Res., 47 (1987) 4150-4159. D. Davis and S. Safe, Dose-response immunotoxicities of commercial polychlorinated biphenyls (PCBs) and their interaction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, Toxicol. Lett., 48 (1989) 35-43. R.E. Morrissey, M.W. Harris, J.J. Diliberto and L.S. Birnbaum, Limited P C B antagonism of TCDD-induced malformations in mice, Toxicol. Lett.,60 (1992) 19-25. R. Fischerand K. Ballschmiter,Congener-specificidentificationof technicalP C B mixtures by capillary gas chromatography on a n-octylmethyl siliconephase (SB-Octyl 50) with an electroncaptureand mass-selectivedetection,Anal. Chem., 335 (1989) 457-463. G. Sundstr6m, Polychlorinated biphenyls. II. Synthesis of some tetra-and penta-chlorobiphenyls, Acta Chem. Scand., 22 0973) 600-604. S. Ullberg, Studies on the distributionand fate of 3sS-labelledbenzylpenicillinin the body, Acta Radiol., Suppl. 118 (1954) I-II0. T. Sinjari,U. T6rnwall and P.O. Darnerud, Induction of 7-ethoxyresorufin-O-deethylase(EROD) activity in mice foetuses by the PCB--congener 3,3'4,4"-tetrachlorobiphenyl, Xenobiotica, Xenobiotica, 23 (1993) I07--I 14. S. Jensen, L. Reuterg~rdh and B. Janson, Analytical methods for measuring organochlorines and methyl mercury by gas chromatography. AOS/SIDAS Manual of methods in aquatic environment
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research. Part 9. Analysis of metals and organochlorines in fish, FAO Fish. Tech. Pap., 212 (1983) 21-33. 18 H. Weber and L.S. Birnbaum, 2,3,7,8-Tetrachlorodihenzo-p-dioxin (TCDD) and 2,3,7,8tetrachlorodihenzofuran (TCDF) in pregnant C57BL/6N mice: Distribution to the embryo and excretion, Arch. Toxicol., 57 (1985) 159-162. 19 M. Berg van den. J. Wijnen van, H. Wever and W. Seinen` Selective retention of toxic polychlorinated dihenzo-p-dioxins and dibenzofurans in the liver of the rat after intravenous administration o( a mixture, Toxicolo.gy, 55 (1989) 173-182. 20 E. Klasson Wehler, J. J6nsson, A. Bergman, I. Brandt and P.O. Darnerud, 3,3 ', 4,4'-Tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl: tissue-localization and metabolic fate in the mouse, Chemosphere, 19 (1989) 809-812. 21 P.O. Darnerud, I. Brandt, E. Klasson-Wehler, A. Bergman` R. d'Argy, L. Dcncker and G.O. Sperber, 3,3',4,4'-Tetrachioro[ '4C]biphenyl in pregnant mice: enrichment of phenol and methyl sulphone metabolit~ in late gestational fetuses, Xenobiotica, 16 (1986) 295-306. 22 E. Klasson Wehk't, A. Bergman, I. Brandt, P.O. Darnerud and C.A. Wachtmeister, 3,3',4,4'T e t r a c M o r o ~ e n y l : Excretion and tissue retention of hydroxylated metabolites in the mouse, Drug Metab. I)isptm., 17 (1989) 441-448. 23 A. Brouwet and K.J. van den Berg, Binding of a metabolite of 3,4,3',4'-tetrachlorobiphenyl to transthyrefin reduces vitamin A transport by inhibiting the formation of the protein complex carrying both ~ ami thyroxine, Toxicol. Appl. Pharmacol., 85 (1986) 301-312. 24 D.W. Nehert, D.R. Nelson, M.J. Coon, R.W. Estabrook, R. Fcyeteisen, Y. Fujii-Kuriyama, F.J. Gonzalez, F.P. Gtamgerich, I.C. Gunsalus, E.F. Johnson, J.C. Loper, R. Sato, M.R. Waterman and D.J. WaxnttL The P450 superfamily: Update on new sequences, gen¢ mapping, and recommended nomenclature, DNA Cell Biol., l0 (1991) 1-14. 25 J.M. Haake, S. Safe, K. Mayura and T.D. Phillips, Aroclor 12.54 as an antagonist of the teratogcnicity ol" 2,3,7,g-tetrachlorodibenzo-p-dioxin, Toxicol. Lett., 39 (1987) 299-306. 26 L. Bieget, M. Harris, D. Davis, R. Rosengren` L. Safe and S. Safe, 2,2',4,4',5,5'Hexacblorobi~l'ml~ as a 2,3,7,8-tetrachlorodibcnzo-p-dioxin antagonist in C57BL/6J mice, Toxicol. Appl. Pharnmcxfl., 97 (1989) 561-571. 27 R. BamniV,~,, D. Davis, T. Zacharewski, I. Tizard and S. Safe, Aroclor 1254 as 2,3,7,8-tetrachlorodihenzo-p-dioxin aatagonist: effects on enzyme induction and immunotoxicity, Toxicology, 46 (1987) 2942. 28 L.S. Birnlmum, M.W. Harris, D.D. Crawford and R.E. Morrissey, Tetatogenic effects of polychlorinated dibenzofurans in combination in C57BL/6N mice, Toxicol. Alapl. Pharmacol., 91 (1987) 246-255. 29 D. Davis and S. Safe, lmmunosuppressive activities of polychlorinated biphenyls in C57BL/6N mice: Structure-*Krtivity relationships as Ah receptor agonists and partial antagonists, Toxicology 63 (1990) 97-11 I. 30 R. Bannister and S. Safe, Synergistic interactions of 2,3,7,8-TCDD and 2,2',4,4',5,5'-hexachlorobiphenyl in C57BL/6J and DBA/2J mice: Role of the Ah receptor, Toxicology, 44 (1987) 159-169. 31 M.A. Denomme, B. Leece, A. Li, R. Towner and S. Safe, Elevation of 2,3,7,8-tetrachlorodihenzo-pdioxin (TCDO) rat hepatic receptor by polychlorinatcd biphenyls. Structure-activity relationships, Biochem. Phammcol., 35 (1986) 277-282.
Chemico-Biological Interactions 89 (1993) 89-102
Liver accumulation of 2,3,7,8-tetrachloro-[3H]dibenzofuran in mice: modulation by treatments with polychlorinated biphenyls P.O. Darnerud *a, U. T6rnwall b, ,~,. Bergman c, I. Brandt b aDepartment of Toxicology. Uppsala University, BMC, Box 594, S-75124 Uppsala, Sweden bDepartments of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, BMC, Box 573, S-751 23 Uppsala, Sweden CEnvironmental Chemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
(Received 9 September 1992; revision received 7 April 1993; accepted 21 April 1993)
Abstract
The distribution of 2,3,7,8-tetrachloro-[3H]dibenzofuran ([3H]TCDF; 40 ttg/kg) resembled that earlier reported for 2,3,7,8-tetrachlorodibenzo-p-dioxin, with a strong accumulation in the liver and a selective uptake in the nasal olfactory mucosa of adult and fetal mice. Pretreatments with a series of selected congeners of polychlorinated biphenyls (PCBs), i.e.. I (IUPAC)-77, 1-105, I-118, 1-126, 1-153, 1-156, 1-169, and a commercial preparation, Aroclor 1254 (25-100 mg/kg body wt. i.p.), were found to modulate the hepatic uptake of [3H]TCDF (24 h post-3H-injection). At a short pretreatment time (4 h), non-ortho-chlorinated congeners decreased the uptake of [3H]TCDF equivalents in the liver (e.g., 1-126 = 3,3',4,4',5-pentachlorobiphenyl: 340 of control), while several mono- and di-ortho PCB congeners and Aroclor 1254 increased the hepatic uptake of [3HITCDF (e.g., 1-156= 2,3,3',4,4',5-hexachlorobiphenyl: 183% of control). At a longer pretreatment time (48 h), both a non-ortho (I-169=3,3',4,4',5,5'-hexachiorobiphenyl) and mono-ortho PCB congener(s) (e.g. 1-156) markedly increased the hepatic 3H-uptake (190%), a probable effect of an induction of hepatic binding sites for TCDF. Ethoxyresorufin-O-deethylase activities, regarded to mirror the metabolic activity of cytochrome P-450 IA 1 (CYP IA 1), were strongly and time-dependently induced after 1-169, but not after 1-156, pretreatment (25 mg/kg). The initial liver concentrations of the two PCB congeners were similar and increased for 1-169 but not for 1-156 at later time points. In conclusion, the results show a selective uptake of |3H]TCDF in the mouse liver and nasal olfactory mucosa of both dam and fetus. The uptake of [3H]TCDF in the liver is influenced both by dose and pre-exposure with PCBs. The presence of a PCB-sensitive, * Corresponding author, National Food Administration, Toxicology Unit, Box 622, S-75126 Uppsala, Sweden. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03200-E
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but CYP IA l-independent, hepatic binding site for TCDF is suggested• Consequently, pharmacokinetic interactions with PCBs complicate the toxicity assessment of TCDF in complex mixtures• Key words: Polychlorinated dibenzofuran; PCB; Co-exposure; Interactions; 3H-labelled;
Mouse; Distribution; Liver; Olfactory mucosa
1. Introduction
An important step in understanding the mechanism of action of halogenated dibenzo-p-dioxins/furans (PCDDs/Fs) was the discovery of a cytosolic receptor for these chemicals, the Ah receptor [1,2]• More recently, other binding sites for PCDDs/Fs and related compounds have been suggested in the rodent liver [3], one of these identified as the cytochrome P-450 species IA2 (form d; CYP IA2). This enzyme, induced by Ah receptor ligands, would likely explain the strong hepatic binding of a 2,3,7,8-dibenzo-p-dioxin (TCDD) analog [4,5], 2,3,4,7,8-pentachlorodibenzofuran [6,7], 3,3',4,4',5,5'-hexabromobiphenyl [8] and related compounds. In the environment mixtures of these and other pollutants are present, and the study of combinations of compounds regarding body accumulation and distribution is of relevance for predicting their toxic potential• Thus, the resulting accumulation of dibenzodioxin-like compounds in the body may be the consequence both of the presence of inducible binding sites in the body and the persistence of the compounds. The aim of the present paper was to define target tissues for TCDF accumulation in the mouse, and to describe the effects on organ TCDF uptake after exposure to a series of individual PCB congeners• The results of the distribution study of [3H]TCDF resemble those obtained earlier for TCDD in rats and mice [9,101, where a strong hepatic accumulation of radioactivity and a specific labelling of the nasal olfactory mucosa were observed• The present study revealed different effects on the hepatic accumulation of [3H]TCDF due to the PCB congener selected and pretreatment time. These congener-dependent effects are suggested to be related to the molecular structure (e.g., degree of planarity and number of chlorinated orthopositions of the biphenyl molecule) and metabolic inertness of different PCB congeners. Changes in concentration of the large hepatic pool of TCDF may be of significance for toxicity in non-hepatic tissues such as the lymphatic system• However, other types of (non-additive) interactions may also result from co-exposure to TCDD and PCB compounds, and antagonistic effects on immunotoxicity [11] and teratogenicity [12] has been reported• 2. Materials and methods 2.1• Chemicals
2,3,7,8-Tetrachloro-[4,6-3H]dibenzofuran (3H-TCDF) (18.9 Ci/mmol, purity > 98%) and unlabelled TCDF was purchased from Chemsyn Science Laboratories (Lenexa, Kansas, USA). The numbering system of PCB congeners as suggested by
P.O. Darnerudet al./ Chem.-Biol. Interact. 89 (1993) 89-102
91
Table i PCB congeners used in the study: full chemicalname and their corresponding IUPAC nomenclature Name
Abbreviation
3,3',4,4'-tetrachlorobiphenyl 2,3,3 ' ,4,4'-pentachlorobiphenyl 2,3' ,4,4' ,5-pentachlorobiphenyl 3',3 ',4,4 ' ,5-pentachlorobiphenyl 2,2' ,4,4',5,5'-hexachlorobiphenyl 2,3,3',4,4 ' ,5-hexachlorobiphenyl 3,3 ',4,4 ',5,5 '-hexachlorobiphenyl
1-77 1-105 1-118 1-126 1-153 1-156 1-169
IUPAC is used in the present study [13]. Thus, the PCB congeners 1-77, 1-105, I-118, 1-126, 1-153, 1-156 and 1-169 (see Table 1 for full chemical names) were synthesized according to Sundstr6m [14]. Aroclor 1254 was produced by Monsanto (USA) and B-naphthoflavone (BNF) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Animals
NMRI Mice 20-23 g (Alab, Sollentuna, Sweden) were kept at 22°C under controlled light conditions (12 h light/12 h dark) and were given pelleted food (R3, Ewos, S6dert~lje, Sweden) and tap water ad libitum. Females were mated overnight and the presence of vaginal plugs the following morning confirmed mating and was denoted day 0 of pregnancy. 2.3. Whole-body autoradiography
Non-pregnant mice (n = 2) were given [3H]TCDF (40 /~g/kg body wt.; - 50 tLCi/animal) intravenously (iv.; dissolved in 20 t~l dimethylsulfoxide) and sacrificed in carbon dioxide atmosphere 7 and 28 days later. Pregnant mice (n = 5) were given the same [3H]TCDF dose on day 10, 14, 16 or 17 of gestation and were sacrificed on day 17, i.e., 7, 3, 1 day(s) or 8 h after injection, respectively. On day 16, one mouse was given a high dose (800 ~,g/kg body wt.) of TCDF and sacrificed after 24 h. In a separate study, two mice were given either BNF (80 mg/kg body wt.) or corn oil (vehicle) intraperitoneally (i.p.) 44 h before TCDF injection on day 16 of gestation; the animals were sacrificed 24 h thereafter. The animals were embedded in carboxymethyicellulose, frozen to -70°C and dry sectioned for autoradiography according to Ullberg [15]. The sections were exposed to X-ray film (Hyperfilm 3H, Amersham Sweden AB, Soina) which was developed after appropriate exposure time. 2.4. Quantitative pretreatment study
Female non-pregnant mice (n = 4) were given the different PCB congeners (see Table 1) or Aroclor 1254 i.p. (doses, see Table 3). Control animals (n = 4-6) were given the vehicle only (corn oil; 0.2 ml/20 g). Four hours later the mice were injected iv. with [3H]TCDF (2.6 nmol(0.83 ug)/kg body wt.; 1 ~Ci/animal), and 24 h after the [3H]TCDF administration the mice were sacrificed by cervical dislocation. Sep-
92
P.O. Darnerud et al. ~Chem.-Biol. Interact. 89 (1993) 89-102
arate groups of mice, pretreated with 1-105, 1-156 or 1-169 48 h before [3H]TCDF but otherwise handled similarly, were included in the study. Selected tissues, i.e., liver, fat tissue, right kidney, lung tissue and thymus (for 1-156 and Aroclor 1254, and for all compounds at the 48-h pretreatment study, only liver, fat and kidney), were excised. The tissue specimens were weighed, dissolved in Soluene 100 (PackardCanberra Sweden, Uppsala) over night, and subjected to liquid scintillation after addition of toluene-based scintillation cocktail (Toluene-Scint, Packard). The selection of two PCB pretreatment time points, 4 and 48 h, were based on the assumption that the 4 h pretreatment may lead to interactive effects at existing binding sites, whereas 48 h would be sufficient time to induce synthesis of new cytochrome P-450, or other binding proteins. The three PCB congeners in the 48 h study were chosen as representatives for congeners which increase (I-156), decrease (I-169) and have no effect (I-105) on hepatic [3H]TCDF uptake at short pretreatment.
2.5. Ethoxyresorufin-O-deetylase (EROD) measurements The effects of the PCB congeners I-156 and 1-169 (25 mg/kg body wt. i.p.) on hepatic microsomal enzyme activities were studied in vitro using ethoxyresorufin as substrate [161. The animals (females; n = 3-4/group) were sacrificed 4 h, 28 h and 72 h after PCB injection. The incubation time was 4-10 min and about 20 #g microsomal protein was added. EROD data was determined and analytical data collected for 1-156 and 1-169 because these congeners had been examined regarding hepatic [3H]TCDF uptake at two time points, and the fact that they gave opposite effects after short pretreatment time. 2.6. Analysis of 1-156 and 1-169 liver residues The hepatic tissue concentration of animals treated with I- 156 and I- 169 (the same mice as in the EROD study described above) was analysed by GC using a Varian 3400 gas chromatograph equipped with an electron capture detector (ECD) and a DB-5 fused capillary column (30 m, I.D. 0.25 mm) from J&W. Injections were made in the splitless mode and the column temperature was programmed: 80°C (2 min) 10°C/min to 300°C that was kept for 10 min. Liver specimens from animals in each group were pooled and the total fresh weight (f.w.) was determined for each sample. The samples were homogenized in aceton: hexane (3;5 ml, 25:1) and centrifuged. The solvent was removed and hexane: methyl tert-buthyl ether (MTBE) (2.5 ml (9:1)) was added before the sample was shaken and centrifuged. The solvent was removed and the procedure was repeated twice. The pooled organic phases were partitioned with 5 ml of a sodium chloride (0.9%)/phosphoric acid (0. i M) aqueous solution [17]. The organic phase was removed and the aqueous phase was extracted twice with hexane (1 ml). The hexane phase was pooled with the organic solvent and the solvent mixture was evaporated. The weight of the extracted lipids (l.w.) was determined. Hexane (2.0 ml) was added to the lipid residue and the lipids were finally destructed by use of conc. sulphuric acid (1.0 ml). The hexane phase was used for GC (ECD) analysis of 1-156 and 1-169, respectively. Livers from a group of control mice were also analyzed. 2,3,3 ',4,4',5,5 '-Heptachlorobiphenyi (I-189) was used as internal standard (IS).
mucosa
liver
fetuses
Fig. 1. Whole-body autoradiogram of a cryosectioned pregnant mouse sacrificed 24 h after [3H]TCDF injection (40 .g/kg body wt.). Note the strong hepatic accumulation and the specific labelling of the nasal olfactory mucosa.
olf.
Int. contents
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3. Results 3.1. Whole-body autoradiography
The dominant uptake of [3H]TCDF (40 ttg/kg) occurred in the liver (Fig. 1). The intrahepatic distribution was irregular, with a higher uptake around the central vein (Fig. 2a). The gallbladder contained high levels of [3H]TCDF-derived radioactivity, possibly metabolites. Other less dominant sites of labelling were the fat and fecal contents. Radioactivity was visible up to 3 days post-injection in the kidney (concentrated to the cortico-medullary region), and specifically in the nasal olfactory mucosa (Fig. 1). However, practically no labelling was observed in the endocrine system, including the thyroid gland, adrenals, thymus, bone marrow, lymph nodes and spleen. The radioactivity at all accumulation sites gradually disappeared; after 7 days only the liver was labelled and 28 days after[3H]TCDF administration the body was void of radioactivity. In pregnant mice (40 #g/kg [3H]TCDF) there was a faint and transient accumulation of radioactivity in the fetal liver and intestines, most accentuated 24 h postinjection. A thin slit of radioactivity surrounding the fetal membranes was also observed, probably representing labelling of the uterine fluid and/or the yolk sac placenta (Figs. 1 and 3a). At a higher dose of [3HITCDF (800 ttg/kg, 24 h survival time), the maternal liver accumulation became homogeneously distributed (Fig. 2b). In the fetuses a stronger
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Fig. 2. Detailof the liversfrom autoradiogramsof pregnant mice24 h after administrationof (a) 40 v.g/kg body wt. and (b) 800 #g/kg of [3HITCDF, respectively. Observe the mottled, unhomogeneous distribution in (a).
fetuses
w.~,r
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elf.
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Fig. 3. Detail of the uterine regions from autoradiograms, from Fig. 2. Note the specific uptake in the olfactory mucosa and the hepatic and intestinal accumulation in fetuses exposed to the higher [3H]TCDF dose (b).
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P.O. Darnerud et al. ~Chem.-Biol. Interact. 89 (1993) 89-102
Table 2 Uptake of [3H]TCDF-derived radioactivity in various organs of the mouse 24 h after [3HITCDF injection a Organ
Tissue T C D F conc. pmol/g wet wt.
Liver Fat tissue Kidney (right) Lung Thymus
5.8 3.9 0.5 0.6 0.5
± ± ± ± ±
0.7 b 0.3 0.07 0.04 0.05
~[3HITCDF dose: 2.6 nmol(0.83 ~g)/kg body wt. (i.v.). bMean ± S.D. (n = 6).
uptake was generally seen and the nasal passages were specifically labelled (Fig. 3b). The BNF pretreatment did not appreciably change the autoradiographic distribution of [3HITCDF.
3.2. Quantitative pretreatment study The uptake of [3H]TCDF-derived radioactivity in different tissues from mice in the control group is given in table 2. As seen in the Table, the liver showed the highest concentration of radioactivity followed by the fat tissue, whereas the uptake in lung, kidney and thymus was low. Some of the compounds used in the short-time pretreatment study did significantly change the retention of radioactivity in the liver 24 h after [3HITCDF injection (Table 3): 1-156, 1-153 and Aroclor 1254 increased the hepatic retention of [3H]TCDF-derived radioactivity (183, 138 and 166% of control, respectively),
Table 3 Effect of short-time (4 h) pretreatment with selected compounds on hepatic uptake of [3H]TCDF in mice Substance
n
PCB dose (mg/kg)
Hepatic T C D F concl (pmol/g wet wt.)
% of control
Vehicle (control) 1-77 a 1-105 1-118 1-126 1-153 1-156 1-169 Aroclor 1254
6 4 4 4 4 4 4 4 3
-25 b 25 25 25 100 25 25 100
5.8 4.3 6.2 6.1 1.9 8.0 10.6 4.9 9.6
-75 107 106 34 138 183 85 166
+ 0.8 c .4- 0.4 **d :t: 1.6 .4- 1.0 -4- 0.2*** ± 1.0* -4- 2.1"* ± 0.7 (9.0-10.6)**
aAbbreviations, see Table 1. bMice were pretreated 4 h before administration of [3H]TCDF (2.6 nmol(0.83 ttg)/kg body wt.) and sacrificed 24 h after injection of radioactivity. ¢-4- S.D. {Aroclor 1254: range; n = 3). dSignificantly different from control values; Student's t-test (* P < 0.05, ** P <0.01, ***P < 0.001).
P.O. Darnerud et al. / Chem.-Biol. Interact. 89 (1993) 89-102
100
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1-126 dose (mg/kg) Fig. 4. Dose-related decrease in hepatic [3H]TCDF concentration after pretreatment with the PCB congener 1-126 (2.5,5,10 and 25 mg/kg body wt.). The relative values represent means ± S.D. (n = 4). Significant differences from controls in Student's t-test (P < 0.001) were observed at 10 and 25 mg/kg.
whereas 1-126 and 1-77 decreased the retention (34 and 75%). To conclude, the strongest decrease in hepatic retention of [3H]TCDF was observed for the coplanar 1-126, while the mono-ortho substituted H 5 6 gave rise to the strongest increase in retention of 3H-TCDF, at short-time pretreatment and a dose level of 25 mg/kg of the PCB congener. In the case of 1-126, three additional doses were tested. The result showed that the decrease in hepatic retention of [3H]TCDF was dose dependent (Fig. 4). Lower Table 4 Effects of long-term (48 h) pretreatment with selected PCB congeners on hepatic retention of [~H]TCDF in mice Substance
n
Vehicle (control) 1-105 1-156 1-169
4 4 4 4
PCB dose (mg/kg)
Hepatic T C D F conc. (pmol/g wet wt.)
% of control
25 25 25
5.0 ~9.7 ~ 9.5:1: 9.8:1:
-194 190 196
Footnotes in Table 3 valid (except pretreatment time).
0.9 1.0"** 2.0** 1.0***
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doses of 1-156 and Aroclor 1254 (12.5 and 25 mg/kg, respectively) resulted in nonsignificant increases in hepatic 3H-levels. At the long pretreatment time (48 h), all the PCB congeners studied (I-105, 1-156 and 1-169) markedly (190%) increased the retention of [3H]TCDF in the liver (Table 4). The effect of PCB treatments on the uptake of [3H]TCDF in extrahepatic tissues was in most cases not profound. However, at the short-time pretreatments several PCB congeners (I-77, 1-105, I-118, 1-153, 1-169) resulted in a slight (not less than 70% of control) but significant decrease of [3H]TCDF retention in the lung. In fat, significantly decreased 3H-levels were observed after pretreatments with 1-153 (69%), and in thymus with 1-153 (91%). In contrast, an increase in the uptake of [3H]TCDF (139%) was observed in the kidney after 1-126 treatment. At long-time pretreatments, all the three PCB congeners studied gave rise to a decreased uptake of [3H]TCDF in fat (46-69%). The BNF pretreatment increased the hepatic uptake of radioactivity (162% of control), whereas the retention in fat was significantly decreased (43%). 3.3. E R O D m e a s u r e m e n t s
Results from the E R O D measurements on microsomes from 1-156- and 1-169treated mice, are given in Table 5. A time-dependent increase in E R O D induction is seen after 1-169 pretreatment, whereas 1-156 values are similar to controls. 3.4. G C congener analysis
The concentration of the pretreatment substances 1-156 and 1-169 in liver tissue is given in Table 5. On a fat weight basis, the values after 4 h are identical for the two congeners (120 ng/mg fat), and increases with time for 1-169 (up to 230 ng/mg fat) but not for 1-156.
Table 5 Hepatic congener concentration and microsomal EROD induction at three time points after treatment with the PCB congeners !-156 and 1-169 (dose 25 mg/kg) Substance
Treatment time (h)
n
Liver conc.~
EROD activity (range)b
Vehicle 1-156 1-156 1-156 1-169 1-169 1-169
72 4 28 72 4 28 72
3 4 3 3 4 2 3
-120 130 80 120 150 230
0.048 (0.022-0.096) 0.083 (0.031-0.165) 0.108 (0.058-0.182) 0.117 (0.060-0.228) 0.0.50 (0.028-0.064) 0.675 (0.541-0.809) 2.32 (0.994-3.214)
a Liver concentration of congener in ng/mg extracted fat (pooled samples). b Ethoxyresorufin-O--deethylase(EROD) activity in nmol formed resorufin/min/mg protein.
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4. Discussion
The distribution of [3H]TCDF in the mother and fetus was similar to that earlier found for TCDD [9,101 with pronounced uptake in the liver and nasal olfactory mucosa. The retention of [3H]TCDF seemed to be less pronounced than that of TCDD - - a finding that is supported by earlier results by Weber and Birnbaum [18]. A similar retention difference between chlorinated dioxins and furans in the liver of rats has also been observed [19]. Among the coplanar PCBs, similarities to TCDF regarding tissue distribution are observed, especially for 1-126 [20]; for example, both TCDF and 1-126 give rise to strong hepatic accumulation. However, the coplanar 1-77 results in a different tissue distribution in mice as compared to 1-126 and 1-169, characterized by a limited accumulation in liver and fat, a marked labelling of the blood and excreta, and a pronounced uptake in fetal blood and tissues [21,22]. The fast metabolism of 1-77 to hydroxylated metabolites, some of which have high binding affinity to transthyretin in plasma [23], could be one explanation to the specific distribution pattern of this congener. According to our autoradiographical results, administration of a higher molar dose of TCDF (132 vs. 5296 nmol/kg body wt.; but with an equal dose of [3H]TCDF) seemed to increase the hepatic pool of [3H]TCDF. A similar observation was made by Poland and coworkers [4,5] who reported TCDD (100 nmol/kg) to strongly increase the hepatic binding of [125I]2-iodo-3,7,8-trichlorodibenzo-pdioxin (0.1 nmol/kg). The TCDD binding species was reported to be an induced form of cytochrome P-450, i.e.: CYP IA2 (P-450d) [24]. In the present quantitative study the liver retention of [3H]TCDF was approximatively 15% of the total 3H dose for non-pretreated animals, as compared to 1-2% of 125Idose in studies on the TCDD analog by Poland and coworkers. Besides being two different compounds, one additional explanation to the discrepancy may be the fact that the tracer dose used in our study (2.6 nmol/kg body wt. of [3H]TCDF) was 26 times higher (on molar basis) than that of [125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin (0.1 nmol/kg) used by Poland and coworkers. This means that our [3H]TCDF dose seems to be high enough to induce de novo synthesis of a cytochrome P-450 species with [3H]TCDF binding properties. Altogether, these results indicate that the hepatic fraction of a TCDF (TCDD) dose will increase with increasing doses. However, at a high enough dose, induction will be maximal and saturation of hepatic binding sites will most likely occur. The presence of a large hepatic pool of [3H]TCDF, similar to that reported for TCDD, made it of interest to study how other xenobiotics with structures similar to TCDF could modify the hepatic binding and retention of [3HITCDF, and thereby also the concentration of [3H]TCDF in other tissues. Presumably, a decrease in the hepatic pool of TCDF would result in an increase of TCDF in extrahepatic target tissues. In our study, only the three coplanar congeners 1-77, 1126 and 1-169 decreased the liver accumulation at the short (4 h) pretreatment time. Reasons for the ability of coplanar congeners to displace [3H]TCDF would be their persistency and relatively high affinity for the hepatic binding sites, including the Ah
I O0
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receptor and cytochrome P-450 species. In contrast some PCB congeners, i.e., 1-156, increased the hepatic pool of [3H]TCDF after the 4 h pretreatment, suggesting an increase in binding sites for this compound in analogy with the situation after increasing the dose of [3H]TCDF (see above). On one hand this would leave less for extrahepatic target sites (inducing e.g., teratogenicity), but on the other hand an increased liver binding would also most probably lead to an increase in the total retention time of T C D F in the body, which may increase the toxicity. At longer pretreatment time all PCB congeners studied, including 1-156 and the non-ortho 1169, increased hepatic binding of [3H]TCDF. If a decrease in peripheral target organ concentrations of T C D F is the consequence, these results could be supported by several teratogenicity studies on TCDD, which conclude that teratogenic effects are antagonized within certain dose limits by co-treatment with Aroclor 1254 [25] and 1-153 [12,26]. Also T cell-mediated immunotoxic effects of T C D D are antagonized by treatment with commercial PCB mixtures [11,27]. However, other studies have suggested that e.g., teratogenic effects of 2,3,4,7,8-pentachlorodibenzofuran and 1-156 are additive [28], and that the antagonistic properties of pure PCB congeners seem to be restricted to certain structural forms [29]. The latter study also suggested 1-156 to have additive effects on TCDD-mediated immunotoxicity. Another factor to take into account is that all the compounds that induced hepatic [3H]TCDF binding and retention in the present short-time pretreatment study (I153, 1-156 and Aroclor 1254), are also known to induce Ah receptor levels [30,31]. The increase in the levels of Ah receptors would result in de novo synthesis of cytochrome P-450 binding sites for TCDF. The fact that the hepatic [3H]TCDF concentration is increased both after short and long 1-156 treatment time whereas EROD activities are not induced, suggests that CYP IAI is not directly involved in the hepatic accumulation of [3H]TCDF. Moreover, CYP IA1 does not seem to mirror the induction status of CYP IA2, the proposed main binding site of T C D F and related compounds in the liver. The small effects of I-156 on EROD induction in our study is somewhat in contrast with earlier results of Davis and Safe [29], who observed a quite substantial induction (ca. 3 nmol resorufin/min/mg protein) using a similar PCB dose and treatment time (3 days). This difference may be due to the use of different strain and sex of mice. Regarding hepatic congener concentrations, our results show similar initial levels of 1-156 and 1-169; this rules out the possibility of a concentration difference as explanation to the differential effects of the tested PCB congeners. To summarize, a specific uptake of radioactivity in the liver and nasal olfactory mucosa of both dam and fetus was observed in mice exposed to [3HITCDF. The body distribution was dependent on the T C D F dose, probably determined by induction of T C D F binding sites in the liver. Regarding co-administration of T C D F and PCB congeners, the observed differential effects on [3H]TCDF distribution, depending on congener and pretreatment time, could be due to induction of [3HlTCDF-binding sites, competition for binding sites and rate of elimination. It is notable that at the longer pretreatment time all PCB congeners studied showed a potential to increase the hepatic [3H]TCDF pool; this may reflect an 'environmental' effect of a continuous exposure to Ah receptor binding PCB congeners.
P.O. Darnerud et al. ~Chem.-Biol. Interact. 89 (1993) 89-102
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5. Acknowledgements Economic support was given by the Research Committee of the Swedish Environmental Protection Agency.
6. References I
2 3
4
5
6
7
8
9
10
11
12 13
14 15 16
17
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