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Disposition and excretion of 2,3,7,8-tetrachlorodibenzofuran in the rat
TOXICOLOGY
AND
Disposition
APPLIED
PHARMACOLOGY
and Excretion
55,
342-352
of 2,3,7,8-Tetrachlorodibenzofuran
L. S.BIRNBAUM,G. Environmental National
M. DECAD, AND H.B.
May
5, 1980; accepted
June
in the Rat1
MATTHEWS
Biology Branch. National Institute of Enrironmental Toxicology Program, Research Triangle Park, Norfh
Received Disposition
(1980)
Healrh Sciences Carolina 27709
and
13, 1980
Excretion of 2.3,7,8-Tetrachlorodibenzofuran in the Rat. BIRNBAUM, M., AND MATTHEWS, H. B. (1980). Toxicol. Appl. Pharmacol. 55,342-352. The absorption, distribution, and excretion of the highly toxic halogenated aromatic hydrocarbon, 2,3,7.8-tetrachlorodibenzofuran (TCDF) was studied in the male Fischer rat. [W]TCDF was completely absorbed after oral doses of 0.1 and 1.0 wmol/kg body wt. The distribution pattern was the same whether treatment was by oral or intravenous administration. The liver was the major depot of TCDF, with small amounts being redistributed to the skin and adipose tissue. TCDF was primarily excreted via the bile into the feces. Less than 6% was ever removed in the urine. More than half was excreted in the feces within 2 days. [“CITCDF-derived radioactivity in the tissues cochromatographed with the parent compound, while in the excreta, only metabolites were detected. Thus, TCDF is readily absorbed, metabolized, and excreted in the feces. This rapid detoxification may account for the relative resistance of the rat to the acute toxicity of TCDF. L. S., DECAD,
and
G.
2,3,7,8Tetrachlorodibenzofuran (TCDF) is one of the most toxic members of a class of polyhalogenated aromatics which have no known industrial use and yet are widespread in the environment. Polychlorinated dibenzofurans (PCDFs) are found as contaminants in commercial polychlorinated biphenyls (PCBs) (Vos et al., 1970) and in chlorinated phenol mixtures (Rappe et al., 1978). They have also been detected in fly ash and flue gases of incinerators, as well as in certain trichlorophenol-derived herbicides (Rappe et al., 1979) and in treated wood (Levin and Nilsson, 1977). In both structure and toxicity, TCDF is closely related to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The clinical symptoms of TCDD/TCDF poisoning are very similar to each other, varying mainly as to dose and severity (McConnell and Moore, ’ Portions of this paper were presented FASEB meeting, Anaheim, Calif. 0041-008X/80/1 Copyright All rights
10342-l
1$02.00/O
0 1980 by Acadermc Press. Inc. of reproduction in any form reserved.
at the
1980
342
1979). The single dose LD,,, for TCDF in guinea pigs, 5- 10 pLg/kg, is about three times as high as that reported for TCDD, while that for monkeys, 1 mg/kg, is about 20 times as high (Moore et al., 1979). The most consistent toxic symptom is progressive weight loss, while immunosuppression (Luster et al., 1979) and thymic atrophy have also been reported (McConnell and Moore, 1979). Epithelial cell disfunctions have been evidenced in some species by chloracne, skin lesions, hair loss, and edema. Although hepatomegaly is generally reported, pathological alterations of the liver are not found in all species, nor are disorders of the gastrointestinal tract, urinary bladder, etc. (Moore et a/., 1979). The only known large scale human exposure to PCDFs was in the “Yusho” incident in Japan. This was due to human ingestion of rich oil contaminated with polychlorinated biphenyls (PCBs) which were themselves contaminated with PCDFs,
TCDF
DISPOSITION
including TCDF (Nagayama ef al., 1976). It is estimated that some people may have consumed as much as 200 pg PCDFs/kg (Hayabuchi et al., 1979). The toxic symptoms-chloracne, gastrointestinal problems, fatigue, joint pain, etc.-are associated with exposure to a number of halogenated aromatics, but they have been unusually pronounced and persistent in the victims of the Yusho incident. It has been suggested that this is due to the PCDF contamination of the PCBs (Oishi et al., 1978). Although the acute toxicity of TCDF and several other polyhalogenated dibenzofurans has been examined in guinea pigs, mice, and monkeys (Moore et al., 1979), only preliminary studies or those involving a complex mixture of PCDFs have been carried out in rats (Moore et al., 1976; Oishi et al., 1978). No disposition studies using a purified PCDF have been reported. Such studies are useful in predicting the effects of chronic, low-dose exposure such as may occur with environmental contaminants. Mixtures of PCDFs have been shown to accumulate in the liver, adipose tissue, and spleen in mice with a whole-body halflife of approximately 2 weeks (Morita and Oishi, 1977). However, studies of other halogenated aromatics of defined composition (Goldstein et al., 1978) have demonstrated that the degree and position of chlorination dramatically effects the tissue distribution, excretion, and biological halflife of the molecule in question (Matthews and Anderson, 1975b). Since a similar situation may well exist for the PCDFs, we have examined the fate of purified, radiolabeled TCDF in the rat after a single oral or intravenous injection. We have followed its distribution in body tissues and excretion from the animals. METHODS Cl~emicals. thesized with
‘“C-Labeled difficulty
by
TCDF was custom synIIT Research Institute.
IN THE
RAT
343
Chicago. Illinois. The procedure used (Gray et ul., 1980) resulted in general labeling of the benzene rings with high specific activity (57 mCi/mmol) and purity (>99.9%). The purity was checked by using a Varian 3740 gas chromatograph equipped with a 25 m x 0.25 mm glass capillary column coated with OV-215 and electron capture detector. Helium flow through the column was 0.75 ml/min. The column temperature was maintained at 150°C. with the temperatures at the injector and detector being 230 and 28O”C, respectively. Makeup gas at the column outlet and at detector purge was 5% methane in argon. These procedures allow detection of impurities which vary both in number and position of chlorine atoms. The only impurity detected, trichlorodibenzofuran, was less than 0.1% of the total. Unlabeled TCDF had purity > 99%. Animnls. Male Fischer 344 rats, obtained from Charles River Breeding Laboratories (Portage, Mich.). were maintained in our colony at least I week prior to experimentation. The rats weighed 200-250 g and were 2-3 months old. For disposition studies. the rats were housed in individual metabolism cages allowing for separate collection of urine and feces. They were fed pelleted NIH 31 rat chow and water cld libidum. Fischer rats were used for these studies since many of the carcinogenesis testing programs and other bioassay experiments are being run with this strain which has the advantages of limited growth and low tumor incidence (Coleman et (II.. 1977). Trecrtment. For administration of TCDF. the desired amount of compound was first dissolved in a mixture of Emulphor:ethanol (1:l) (Emulphor EL-620 is a polyethoxylated vegetable oil preparation and was kindly supplied by GAF Corp., New York, N.Y.). No water was added to this mixture for oral administration by gavage of 0.1 or 1.0 pmol [14C]TCDF/kg because of solubility problems at the higher concentration of TCDF (equivalent to 306 &kg). The radioisotope was diluted as needed with unlabeled TCDF for the high-dose treatment. For intravenous administration, the low dose was used (0.1 wmol/kg = 30.6 &kg). The [“CITCDF was dissolved as above in Emulphor: ethanol ( 1: 1) and then diluted with 8 vol of distilled water. resulting in a final concentration of 0.1 pm01 ]“C]TCDF/ml Emulphor:ethanol:water (I: l:8). This solvent system has been shown to be suitable for other lipophilic compounds (Matthews and Anderson, 1975a). The iv dose was administered as 1.0 ml (30.6 pg)/kg into a tail vein for distribution studies or into an exposed femoral vein for studies of biliary excretion. Animals were treated between 9 and 10 AM and were sacrificed by cervical dislocation from 15 min to 3 weeks after TCDF administration. Three rats were used per time point. After sacrifice, the animals were immediately dissected. tissues removed and weighed,
344
BIRNBAUM.
DECAD,
and then stored at -20°C until sampling. The ‘C radioactivity in all tissues was analyzed by combustion to “CO, in a Harvey Biological Oxidizer (R. J. Harvey Instrument Corp., Hillsdale. N.J.). The ‘“CO, was counted in a Beckman Model LS-2SOliquid scintillation spectrometer (Beckman Co., Fullerton, Calif.) as previously described (Matthews and Anderson, 1975a). Samples of all tissues were oxidized directly, without homogenization. Body composition estimates for blood and muscle were 8 and 50% (Matthews and Anderson. 1975a). The values used for fat and skin, 11 and 16%. respectively, were determined by complete dissection of five 200 to 300-g male Fischer 344 rats in our laboratory. Rats sacrificed 10 and 21 days postTCDF treatment were also completely dissected in order to account for any effects of TCDF on body composition. Excretion of TCDF-derived radioactivity in the urine was determined by complete urine collections from each rat kept for 1 day or longer after treatment. Duplicate O.l-ml samples were counted in 20 ml Aquasol (New England Nuclear, Boston, Mass.), and any necessary quench corrections made. Fecal samples were air-dried, weighed, and ground to a fine powder with a mortar and pestle. Duplicate samples were oxidized and radioactivity determined as described above. Bilinry e.rcreriorl. Excretion of [“CITCDF-derived radioactivity in the bile was determined in animals anesthetized with pentobarbital, 50 mgikg, ip, and 50 mgikg, po (Matthews, 1978). After cannulating the common bile duct. 0.1 pmol/kg [“CITCDF (in Emulphor: ethanol:water (I: 1:8) at a concentration of 0.1 Fmol/ml) was injected into the femoral vein and serial bile samples collected for 6 hr. The volume of each sample was measured and the radioactivity of duplicate O.Ol-ml samples in 10 ml Aquasol was determined by liquid scintillation counting. At the end of the collection period, the animals were sacrificed and the tissue distribution of radioactivity determined. Metabolism oJ‘ [‘“C]TCDF. In order to ascertain whether or not the [“CITCDF was being metabolized and whether parent compound or derivative(s) was stored in the tissues or excreted, the tissues and excreta were extracted and analyzed as previously described (Matthews and Anderson, 1975a). The thinlayer system used in this study involved two solvent systems: ( I) chloroform:methanol (1: 1) or (2) methylene chloride:hexane (5: I). In these systems [“CITCDF migrated with an R, of 0.8 and metabolites had R, values less than 0.5. Following chromatography for IO cm on 250 p 20 x 20-cm silica gel G plates (Analtech, Inc., Newark, Del.), the plates were air-dried, scraped off in l-cm segments into 20 ml Aquasol, and radioactivity determined by liquid scintillation counting. Duru ana1ysi.s. The data for tissue distribution and excretion of [“C]TCDF (derived) radioactivity were
AND
MATTHEWS TABLE
TOTAL
TCDF-DERIVED
1
RADIOACTIVITY Percentage
I5 min
Tissue Blood Liver Fat Muscle Skin Kidneys Adrenals Thymus Spleen Testes Brain LLUgS Heart
1.84 34.63 4.43 25.34 5.18 1.20 0.10 0.15 0.16 0.19 0.25 1.36 0.28
” 0.1 Fmol/kg,
-+ k i ti* r f k ? 2 k f
total 180 min
0.57 0.75 0.74 1.31 0.20 0.05 0.08 0.03 0.01 0.02 0.04 0.19 0.08
0.70 41.36 10.02 5.87 6.57 0.25 0.06 0.13 0.03 0.22 0.17 0.20 0.05
k k * -c k ? t + t 2 -t k 2
0.14 3.61 1.00 0.37 0.28 0.04 0.02 0.02 0.01 0.02 0.02 0.02 0.01
IN TISSUES dose” I day 0.40 k 18.09 f 13.89 2 2.43 r 2.33 k 0.10 i co.01 0.02 f 0.01 t 0.14 f 0.03 k 0.04 f 0.01 k
0.11 2.03 3.40 1.97 0.31 0.02 0.01 0 0.09 0.01 0.02 0.01
iv.
analyzed by a nonlinear regression analyses computer program (Morales et al., 1979) based on the decay curves following an exponential pattern. The number of exponential terms was determined by the best fit (Matthews and Anderson, 1975a. b). In all cases the data is expressed as the mean t standard deviation or the estimate * standard error of the estimate.
N 2 3.
RESULTS Tissue Distribution
All of the major organs and tissues were sampled for their radioactive content at various time points after iv administration of 0.1 pmol/kg TCDF, three of which are shown in Table 1. The blood, liver, fat, skin, and muscle tissues accounted for more than 95% of the total unexcreted dose of [‘“ClTCDF derived radioactivity (between 85 and 90% of the injected dose was recovered). The nature of the radioactivity in these tissues was determined by extraction of the above tissues at 15 min, 3 hr, and 1 day after iv administration of 0.1 pmol/kg [‘“CITCDF. Extraction of muscle, fat, skin, and blood removed greater than 80% of the total radioactivity, while similar extractions recovered 98% of the radio-
TCDF
DISPOSITION
4/-
&
2
blood
I
ti
11
Id
3d Time
(hrs-days)
FIG. 1. Percentage of total TCDF dose in blood vs time. TCDF removal from blood between 15 min and 3 days after iv administration of 0.1 pmol/kg [“CITCDF. Each point represents the average value (*SD) obtained with three animals. The solid lines represent the computer drawn two-component exponential decay curve.
activity from liver. These values are nearly identical to what was determined using control tissues to which [‘“CITCDF was added during homogenization. In all cases, greater than 95% of the extractable radioactivity cochromatographed with the parent compound. No metabolites were detectable in the extracts from muscle and fat. By 1 day after treatment, approximately 2% of the radioactivity in skin was not TCDF, while less than 1% of the liver radioactivity was metabolite. In the blood, approximately 5% of the extracted radioactivity failed to cochromatograph with TCDF by Day 1. At the two earlier time points examined, all the extracted radioactivity cochromatographed with [‘“ClTCDF. Thus, the very great majority of the radioactivity present in rat tissues represents unmetabolized TCDF. After intravenous injection of [“‘CITCDF, the distribution of radioactivity as a function of time was examined more closely in the five major tissues depots: blood, liver, fat, skin, and muscle. In all cases, the loss of radioactivity from the tissue could be described by an exponential decay curve, consisting of one or more components. As can be seen in Fig. 1, TCDF is rapidly
IN THE
345
RAT
cleared from the blood, so that by the first time point, 15 min after iv administration, less than 2% of the injected dose remained in the blood. From this point on, a twocomponent exponential decay curve could be used to describe the clearance of TCDF from the blood from 15 min through 21 days (data for 10 and 21 days not shown). The half-lives and pool sizes of these two components are shown in Table 2. Since muscle accounts for a large percentage of the total body mass and has an intermediate rate of perfusion, it is not surprising that it contained approximately 25% of the TCDF dose after 15 min. However, as can be seen in Fig. 2, TCDF was rapidly cleared from muscle at a rate which could be best described by a two-component decay curve. The half-life for the first component was only 30 min, probably reflecting the blood flow through this tissue; the second component has a halflife of 17 hr (Table 2). Liver, fat, and skin all show an initial accumulation of radioactivity. In liver and skin, TCDF derived radioactivity reached a maximum at approximately 3 hr post-treatment, and then decreased at a biphasic rate (Figs. 3, 4, Table 2). The peak concentration in skin was significantly lower TABLE COMPONENTS
FOR THE FROM
2
DECAY
OF RADIOACTIVITY
TISSUES
Pool size (% total dose)
1
1.31 + 0.43 0.89 k 0.2
32.23 z 22.04 0.6 k 0.3
0.02 1.14
2
29.09 i- 6.40 31.39 k 3.30
7.02 2 2.71 0.55 t 0.08
0.10 1.25
Fat
I
17.85 t 1.18
0.19 2 0.04
3.75
MllSCk
I 2
24.85 2 2.36 6.73 t 1.52
29.42 i- 6.33 0.96 k 0.50
0.02 0.72
1
6.84 + 0.47 1.22 f 0.43
Tissue Blood
2 Liver
Skin
1
2
Decay rate (day-l )
Halflife (days)
Component
2
0.29
0.45
0.06 i;
0.41
11.09
1.55
346
BIRNBAUM,
DECAD,
muscle
.!
4t IlI
I
2 3h7h
Id
Tune
3d
(hrs-days)
FIG. 2. Percentage of total TCDF dose in muscle vs time. TCDF removal from muscle between 1.5 min and 3 days after iv administration of 0.1 pmol/kg [‘C]TCDF. Each point represents the average value (*SD) obtained with three animals. The solid lines represent the computer drawn two-component exponential decay curve.
AND
MATTHEWS
and the clearance of the radioactivity from the skin was slower than what was observed in the liver. The liver, however, was the major site of TCDF disposition, accounting for nearly 50% of the total dose by 3 hr after iv injection. The remaining major depot for TCDF in the rat was the adipose tissue (Fig. 5). The maximum accumulation of radioactivity in fat was reached at approximately 7 hr postadministration. The radioactivity was then cleared at a rate which could best be described by a one-component decay curve. The half-life for the decay of TCDF-derived radioactivity from the fat is approximately 3.7 days (Table 2). The tissues described above account for the major tissue volumes and the major depots for lipid-soluble compounds such as TCDF. However, TCDF was also distributed to all of the other tissues in the rat and in some cases, the concentrations were initially quite high. The concentration of TCDF per gram tissue is shown in Table 60,
50-
a I013
7
24 Time
(hrs)
Time
(days)
FIG. 3. Percentage of total TCDF dose in liver vs time. TCDF accumulation in the liver from 15 min to 3 hr is shown in (a). Removal of TCDF from the liver from 3 to 24 hr is shown in (a), and from 3 hr to 10 days in (b). The treatment was 0.1 PmoVkg [“CITCDF. iv. Each point represents the average value (f SD) obtained with three aminals. The solid lines for TCDF removal represent the computer drawn two-component exponential decay curve.
TCDF DlSPOSlTION
347
1N THE RAT
skin
a ‘13
7
24 Ttme
b 2l
(hrs)
3
IO Ttme (days)
21
FIG. 4. Percentage of total TCDF dose in skin vs time. TCDF accumulation in the skin from 15 to 3 hr is shown in (a). Removal of TCDF from the skin from 3 to 24 hr is shown in (a) and from 24 hr to 21 days in (b). The treatment was 0.1 PmoVkg [%]TCDF, iv. Each point represents the average value (*SD) obtained with three animals. The solid lines for TCDF removal represent the computer drawn two-component exponential decay curve.
3. Of the major sites of deposition described above the liver has the highest specific content, approximately equal to 1 nmol/g tissue. However, the adrenal gland had the
highest TCDF concentration per gram tissue, over 50% higher than the liver at initial time point. This radioactivity in adrenals is cleared quite rapidly, so that
b
20
5
FIG. 5. Percentage oftotal TCDF dose in fat vs time. TCDF accumulation is shown in (a). Removal of TCDF from the fat from 7 to 24 hr is shown 21 days in (b). The treatment was 0.1 pmol/kg, iv. Each point represents tained with three animals. The solid line for TCDF removal represents the decay curve.
IO Time
15
20
(days)
in the fat from 15 min to 7 hr in (a), and from 7 hr through the average value (?-SD) obcomputer drawn exponential
of the the by
-
348
BIRNBAUM,
DECAD,
1 day the adrenals contained only 5% of their initial specific activity. Other tissues which contained appreciable initial concentrations of TCDF were the lungs, kidney, heart, and thymus. In each of these cases, as observed for the adrenals, approximately 90% of this radioactivity was cleared within 24 hr. On the other hand, the specific activity of the liver decreased by only 50% during the same 24 hr. Oral Administration
TABLE
Blood Liver Fat Muscle Skin Kidneys Adrenals Thymus Spleen Testes Brain Lungs Heart
15 min 0.12 4.39 0.20 0.25 0.17 0.67 7.43 0.52 0.37 0.09 0.25 1.08 0.66
n 0.1 PmoVkg,
3
OF TCDF-RADIOACTIVITY CONTENT IN TISSUES
Percentage Tissue
2 k 2 + + + + + k t rf% + iv.
MATTHEWS TABLE
0.04 0.17 0.03 0.01 0.02 0.04 6.91 0.12 0.07 0.01 0.01 0.08 0.03
total
dose”/g
tissue
180 min 0.04 5.05 0.44 0.06 0.20 0.17 4.65 0.54 0.08 0.09 0.15 0.24 0.11
& ? r k + + t r 5 ? ? f k
0.01 0.35 0.07 0.00 0.01 0.03 1.26 0.13 0.02 0.01 0.03 0.02 0.00
4
DISTRIBUTION OF RADIOACTIVITY 3 DAYS ADMINISTRATION OF [“C]TCDF
AFTER
Route of administration OEil Tissue LlVU Fat Skin Total excreted In feces In urine
Environmental chemicals are rarely encountered by an intravenous route, however, by eliminating the absorption phase, such a treatment does greatly simplify a pharmacokinetic analysis. Therefore, it was considered necessary to demonstrate that the tissue distribution after an oral dose resulted in the same profile as that obtained after iv exposure. It was also important to demonstrate that the dose used was not saturating the system, thereby affecting the disposition. To answer these questions, a dose identical to the iv dose (0.1 pmollkg)
CONCENTRATION
AND
Dose:
1.0 &m&kg
I” 0 I pm&kg
0. I pm&kg
3.92 2 2.1x 9.79 k 3.55 1.10 c 0.59
5.02 2 0.42 4.64 z 0.75 1.07 z 0.19
5.87 t 0.27 Il.57 + 2.33 I.24 k 0.27
67.81 f 5.64 I .47 + 0.33
70.42 k 3 76 I 76 k 0.01
63.05 2 9.58 2.01 + 0.43
and one IO-fold higher (1 .O pmol/kg) was administered to the rats by oral gavage. A comparison of the major tissue depots 3 days after treatment with the high and low oral dose and the iv dose is shown in Table 4. Total recovery of administered radioactivity was approximately 85%. There was no significant difference in the liver or skin content between the different treatments. However, there may have been less TCDF in the fat of animals which received the low oral dose. There was no detectable difference in the TCDF content of muscle or blood which accounted for less than 1% of the total dose after the various treatments. The excretion data were also similar in both feces and urine for the high- and lowdose po and the low-dose iv route (Fig. 6).
1 day 0.03 2.22 0.64 0.03 0.07 0.08 0.34 0.07 0.02 0.06 0.02 0.07 0.02
2 t + 2 t 2 + k ? k k + f
0.01 0.37 0.11 0.02 0.01 0.01 0.14 0.03 0.00 0.02 0.00 0.02 0.02
Excretion
The excretion of [“‘CITCDF-derived radioactivity was analyzed by daily collection of urine and feces from individual animals held from 1 to 21 days post-treatment. Total cumulative excretion in urine and feces for 7 days is shown in Fig. 6. The data in Fig. 6 clearly demonstrate that the major route for excretion of this compound is via the feces. The majority of urinary excretion occurs within the first day, with no detectable radioactivity appearing in the
TCDF
DISPOSITION
IN THE
349
RAT
7-
bile
6-
. 0 I,uimol/kg, 0 Oi,umal/kg.PO A I O,umal/kg,PO
I
2
3
4 Time (days)
5
IV I
6
7
I
I
2
3 Time
4
5
6
Y 7
(days)
7. Daily excretion of TCDF-derived radioactivity in urine. The percentage of the total iv dose of 0. I pmol/kg [‘4C]TCDF excreted daily by rats for 7 days was plotted vs time. Each point represents the daily excretion of a single rat. Numbers in parentheses indicate the number of rats having the same excretion. The solid line represents the computer-drawn exponential decay curve. FIG.
3 Time
FIG. 6. Cumulative excretion of TCDF-derived material in urine and feces. Cumulative excretion of radioactivity, expressed as percentage of total dose, in the urine and feces for 7 days after iv administration of 0.1 pmol/kg [“C]TCDF (0). and for 3 days after po administration of 0.1 pmol/kg [“CITCDF (0) and 1.0 pmol/kg [‘QJTCDF (A). Each point represents the average cumulative percentage of the total dose excreted by three or more animals.
04'
2
4
5
6
Chrs)
FIG. 8. Cumulative excretion of TCDF-derived radioactivity in bile. Data obtained by bile duct cannulation followed by iv administration of 0.1 pmol [‘“C]TCDF/kg into the femoral vein. Results shown are the average value (*SD) for three animals.
urine after Day 6. The urinary excretion best fits a single exponential decay curve, with a decay rate of 0.5 day-’ (Fig. 7). Since the feces was the major route of excretion, the elimination via the bile was also studied to determine if the material in feces originated in bile and if enterohepatic circulation played a major role in the distribution of the [‘“CITCDF-derived radioactivity. As shown in Fig. 8, over 7% of an iv dose of TCDF was excreted in bile within 6 hr. This is in good agreement with the observed elimination of 24% of the total dose in feces within the first day and suggests that once the radioactivity appears in the bile, it enters the intestines and is excreted without recirculation. The compound is then removed via the feces following a single component exponential decay, having a half-life of slightly under 2 days (Fig. 9). The nature of the [14C]TCDF-derived radioactivity in the urine and bile was examined by thin-layer chromatography. In
350
BIRNBAUM,
DECAD,
the urine, no parent compound was detectable at any time point (Day 1 through Day 6) after treatment. However, the metabolite(s) exhibited increasing polarity as a function of time after dosing. In the bile, greater than 96% of the radioactivity was metabolite within 90 min after injection. By 3 hr, there was no detectable parent compound in bile. These results agree well with those obtained by extraction of feces followed by thin layer chromatography. During the first day, approximately 1% of the extracted radioactivity cochromatographs with [14C]TCDF. This value decreased to 0.5% during the second day, after which no parent compound was detectable in the feces. It should be pointed out that only 20-25% of the [14C]TCDF derived radioactivity was extractable from the excreted feces. This contrasts with essentially complete extraction of [14C]TCDF added to control feces just prior to extraction. This suggests that nonextractable radioactivity was not parent TCDF, but metabolites. The results presented here indicate that greater than 99% of the radioactivity which is excreted from the body was no longer TCDF but several metabolites of this compound. DISCUSSION The objectives of this investigation were to determine the absorption, distribution, and excretion of TCDF in male rats in order to better evaluate the toxicity of this compound. This study demonstrated that approximately 90% of an oral dose of TCDF was absorbed from the gut, and this absorption was not a function of the doses administered. Distribution of TCDF was initially to the liver and muscle, followed by some redistribution to skin and adipose tissue, in contrast to TCDD which accumulates in the liver and fat (Rose et al., 1976). This pattern was not affected by dose or route of exposure. Essentially all the radio-
AND
MATTHEWS
01’
5
IO Time ldoys)
15
20
FIG. 9. Daily excretion of TCDF-derived radioactivity in feces. The percentage of the total dose of 0.1 pmol of [‘“C]TCDF/kg excreted daily by rats for 2 1 days was plotted vs time. Each point represents the daily fecal excretion of one rat. Numbers in parentheses indicate the number of rats having the same excretion. The solid line represents the computer-drawn exponential decay curve.
activity present in the tissues cochromatographed with [‘“CITCDF, suggesting that greater than 95% of the tissue burden was unmetabolized compound. In the fat, essentially all the radioactivity appears to be TCDF. The primary route of excretion was in the feces. Less than 6% of the administered dose was ever excreted in the urine. Excretion was not affected by the dose, within the range studied or the route of administration. The rapid rate of excretion of TCDF was in contrast to that observed for TCDD, where a whole-body half-life of 3-5 weeks has been reported (Rose et al., 1976). All of the radioactivity excreted in the urine was metabolites of TCDF. The same was true for the radioactivity in bile and feces. Preliminary results suggested that intestinal flora may alter the biliary metabolites, since the chromatographic results for
TCDF
DISPOSJTION
extracts of bile and feces were quite distinct. The action of intestinal microorganisms on TCDF itself remains unknown although it has been shown here that microbial action does not alter the dose of TCDF which reaches the tissues following an oral vs an iv dose. Therefore, since virtually all of the TCDF-derived radioactivity excreted in the urine, bile and feces was in the form of several TCDF metabolites and storage of TCDF metabolites in the tissues was negligible, the more rapid clearance of TCDF vs TCDD (Rose et al., 1976) must be attributed to more rapid metabolism of TCDF by the rat. Some qualitative observations concerning the relationship of acute TCDF toxicity to its metabolism should be noted. By 2 days after treatment, regardless of dose or route of injection, the rats showed listlessness, excessive hair loss, and decreased weight gain. However, by Day 6 post-treatment, the hair loss had ceased and their weight gain returned to normal. Thus, 3 weeks after TCDF treatment, the rats were of normal weight and apparently healthy. There were no visible signs of thymic or splenic atrophy or liver hypertrophy. It seems that the metabolism of TCDF resulting in its excretion prevented the acute toxicity from progressing to lethality. Instead, the animals recovered. Of course, some permanent damage may have occurred which is not yet manifest. such as neoplastic transformation, shortened life span, etc. Thus, it appears that in the case of TCDF, the parent compound is the toxic agent. Metabolism results in detoxification. From these conclusions, we might predict that animals with higher rates of metabolism of TCDF will be more resistant to its toxic actions. Such a correlation, in fact, may exist in the rat itself in relation to the differential toxicity due to TCDD vs TCDF, the former of which is more slowly metabolized and is several times more toxic (Rose et al., 1976; Ramsey et al., 1979). The mechanism of toxicity of these compounds is still unclear (Neal et al., 1979).
IN THE
351
RAT
The effects are pleiotropic, affecting various organs and cell types. Several investigators have suggested that toxicity is mediated by an intracellular receptor which activates a battery of genes (Poland et al., 1979; Okey et al., 1979). In fact, there is a good correlation between the biological potency of various PCDD and PCDF congeners (McConnell et al., 1978) and their ability to induce aryl hydrocarbon hydroxylase activity by binding to the TCDD receptor (Poland et crl., 1979). The ability of the rat to metabolize TCDF and therefore clear it from the body may thus explain its relative resistance to the toxic effect of this compound. ACKNOWLEDGMENTS The authors cal assistance Oates, and the by Dr. Phillip
wish to acknowledge the expert techniof Ms. Minerva Fields and Mr. Tony purity determination made on [“CITCDF Albro.
REFERENCES COLEMAN,
G. L.,
G. W.,
FOSTER,
BARTHOLD, S. J.,
S. W.. AND
OSBALDISTON,
JONAS.
A.
M.
(1977).
Pathological changes during aging in barrier-reared Fischer 344 Male Rats. J. Gerorztol. 32, 258-277. GOLDSTEIN, J. A., VAN. D. (1978).
HAAS,
J. R.. LINDO.
P.. AND
HARA-
2.3.7,8-Tetrachlorodibenzofuran in a commercially available 99% pure polychlorinated biphenyl isomer identified as the inducer of hepatic cytochrome P-448 and aryl hydrocarbon hydroxylase in the rat. Dvlrg Mrrah. Di~pos. 6, 258-264.
GRAY,
A. P., MCCLELLAN.
W. J., AND
DIPINTO,
V. M.
(1980). Carbon-14-labeled 2.3,7,8-and 1.2,7.8-tetrachlorodibenzofuran. J. Label. Cumpo~~rds Radiopham.. 17, in press. HAYABUCHI,
H.,
YOSHIMURA.
T.,
AND
KURATSUNE,
M. (1979). Consumption of Toxic rice oil by ‘Yusho’ patients and its relation to the clinical response and latent period. Fond Comet. To.rid. 17, 455-461. LEVIN, J. 0.. AND NILSSON. C. A. (1977). Chromatographic determination of polychlorinated phenols. phenoxyphenols, dibenzofurans. and dibenzodioxins in wood-dust from worker environments. Che~zosphere 6, 443-448. LUSTER,
(1979). (TCDF Chern.
M.
I.,
FAITH,
R. E..
AND
LAWSON,
L.
D.
Effects of 2,3,7.8-tetrachlorodibenzofuran I on the immune system in guinea pigs. Drug Tmicol. 2, 49-60.
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B. (1978). A simple method for experiod of surgical anesthesia in rats. Sci. 28, 720-722. MATTHEWS, H. B., AND ANDERSON, M. W. (1975a). The distribution and excretion of 2,4,5,2’S’-pentachlorobiphenyl in the rat. Drug. Metab. Dispos. 3, 211-219. MATTHEWS. H. B., AND ANDERSON, M. W. (1975b). Effect of chlorination on the distribution and excretion of polychlorinated biphenyls. Drug. Merab. Dispos. 3, 371-380. MCCONNELL, E. E., AND MOORE, J. A. (1979). Toxicopathology characteristics of the halogenated aromatics. Ann. N.Y. Acad. Sci. 320, 138-150. MATTHEWS,
tending the Lab. Animal
MCCONNELL.
E..
MOORE,
J. A.,
HASEMAN,
J.,
AND
HARRIS, M. (1978). The chlorinated dibenzo-p-dioxin Toricol. Appl. Pharmacol.
comparative toxicity of in mice and guinea-pigs. 44, 335-356. MOORE, J. A.. &PTA, B. N.. AND Vos, J. G. (1976). Toxicity of 2,3,7.8-tetrachlorodibenzofuran-Preliminary results. Proc. NUI. Cot$. PC&, November 1976, pp. 77-80. MOORE, AND
J. A., HARRIS,
MCCONNELL.
E. E.,
M. W. (1979). of halogenated dibenzofurans and rhesus monkeys. Ann. 151-163.
MORALES, N. M.. AND MATTHEWS,
TUEY,
D.
DALGARD,
D. W..
Comparative toxicity in guinea pigs, mice, N.Y. Acad. Sri. 320. B.,
COLBURN,
W.
A..
H. B. (1979). Pharmacokinetics oral doses of selected polychlorinated in mice. Toxicol. Appl. Pharmacol.
of multiple biphenyls 48, 397-407. MORITA, M., AND OISHI. S. (1977). Clearance and tissue distribution of polychlorinated dibenzofurans in mice. Bu//. Euviron. Contam. Toxicol. 18, 61-66. NAGAYAMA, J.. DURATSUNE. M., AND MASUDA, Y. (1976). Determination of chlorinated dibenzofurans in kanechlors and “Yusho Oil.” Bull. Environ. Contarn. Toxicol. 15, 9- 13. NEAL,
R. A.,
BEATTY,
P. W.,
AND
GASIEWICZ,
T. A.
AND
MATTHEWS
(1979). Studies of the mechanisms of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Am. N.Y. Acad. Sci. 320, 204-213. OISHI, S., MORITA, M., AND FUKUDA, H. (1978). Comparative toxicity of polychlorinated biphenyls and dibenzofurans in rats. Toxicol. Appl. Pharmnco/. 43, 13-22. OKEY, A. B., BONDY, G. P., MASON, M. E., KAHL, G. F., EISEN, H. J., GUEN~HNER, T. M.. AND NEBERT, D. W. (1979). Regulatory gene product of the Ah locus. J. Bio/. Chem. 254. 11636- 11648. POLAND. A., GREENLEE. W. F., AND KENDE, A. S. (1979). Studies on the mechanism of action of the chlorinated dibenzo-p-dioxins and related compounds. Anrr. N.Y. Acad. Sci. 320, 214-230. RAMSEY. J. C., HEFNER, J. G.. KARBOWSKI. R. J.. BRAUN. W. H., AND GEHRINC, P. J. (1979). The in viva biotransformation of 2.3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat. To.ricol. Appl. Pharmucol. 48, A162. RAPPE, C., BUSER. H. R., AND BOSSHARDI. H. P. (1979). Dioxins, dibenzofurans, and other polyhalogenated aromatics: Production, use, formation. and destruction. Ann. N.Y. Acad. Sci. 320, 1-18. RAPPE,
C.,
MARKLUND,
S.. BUSER,
H. R..
AND
Boss-
HARDT, H. P. (1978). Formation of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) by burning or heating chlorophenates. Chermsphere 7, 209-281. ROSE, J. Q.. RAMSEY. J. C., WENTZLER, T. H., HUMMEL. R. A., AND GEHRING, P. J. (1976). The fate of 2.3,7.8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appi. Phurmaco!. 36, 209-226. Vos, J. G., KOEMAN, J. H.. VANDER MAAS, F. L.. TEH NOEVER DEBRAUW. M. C., AND Vos. R. H. (1970). Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated napthalene in two commercial polychlorinated biphenyls. Food Cosrnef. Toxic~~/. 8, 625-633.
AND
Disposition
APPLIED
PHARMACOLOGY
and Excretion
55,
342-352
of 2,3,7,8-Tetrachlorodibenzofuran
L. S.BIRNBAUM,G. Environmental National
M. DECAD, AND H.B.
May
5, 1980; accepted
June
in the Rat1
MATTHEWS
Biology Branch. National Institute of Enrironmental Toxicology Program, Research Triangle Park, Norfh
Received Disposition
(1980)
Healrh Sciences Carolina 27709
and
13, 1980
Excretion of 2.3,7,8-Tetrachlorodibenzofuran in the Rat. BIRNBAUM, M., AND MATTHEWS, H. B. (1980). Toxicol. Appl. Pharmacol. 55,342-352. The absorption, distribution, and excretion of the highly toxic halogenated aromatic hydrocarbon, 2,3,7.8-tetrachlorodibenzofuran (TCDF) was studied in the male Fischer rat. [W]TCDF was completely absorbed after oral doses of 0.1 and 1.0 wmol/kg body wt. The distribution pattern was the same whether treatment was by oral or intravenous administration. The liver was the major depot of TCDF, with small amounts being redistributed to the skin and adipose tissue. TCDF was primarily excreted via the bile into the feces. Less than 6% was ever removed in the urine. More than half was excreted in the feces within 2 days. [“CITCDF-derived radioactivity in the tissues cochromatographed with the parent compound, while in the excreta, only metabolites were detected. Thus, TCDF is readily absorbed, metabolized, and excreted in the feces. This rapid detoxification may account for the relative resistance of the rat to the acute toxicity of TCDF. L. S., DECAD,
and
G.
2,3,7,8Tetrachlorodibenzofuran (TCDF) is one of the most toxic members of a class of polyhalogenated aromatics which have no known industrial use and yet are widespread in the environment. Polychlorinated dibenzofurans (PCDFs) are found as contaminants in commercial polychlorinated biphenyls (PCBs) (Vos et al., 1970) and in chlorinated phenol mixtures (Rappe et al., 1978). They have also been detected in fly ash and flue gases of incinerators, as well as in certain trichlorophenol-derived herbicides (Rappe et al., 1979) and in treated wood (Levin and Nilsson, 1977). In both structure and toxicity, TCDF is closely related to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The clinical symptoms of TCDD/TCDF poisoning are very similar to each other, varying mainly as to dose and severity (McConnell and Moore, ’ Portions of this paper were presented FASEB meeting, Anaheim, Calif. 0041-008X/80/1 Copyright All rights
10342-l
1$02.00/O
0 1980 by Acadermc Press. Inc. of reproduction in any form reserved.
at the
1980
342
1979). The single dose LD,,, for TCDF in guinea pigs, 5- 10 pLg/kg, is about three times as high as that reported for TCDD, while that for monkeys, 1 mg/kg, is about 20 times as high (Moore et al., 1979). The most consistent toxic symptom is progressive weight loss, while immunosuppression (Luster et al., 1979) and thymic atrophy have also been reported (McConnell and Moore, 1979). Epithelial cell disfunctions have been evidenced in some species by chloracne, skin lesions, hair loss, and edema. Although hepatomegaly is generally reported, pathological alterations of the liver are not found in all species, nor are disorders of the gastrointestinal tract, urinary bladder, etc. (Moore et a/., 1979). The only known large scale human exposure to PCDFs was in the “Yusho” incident in Japan. This was due to human ingestion of rich oil contaminated with polychlorinated biphenyls (PCBs) which were themselves contaminated with PCDFs,
TCDF
DISPOSITION
including TCDF (Nagayama ef al., 1976). It is estimated that some people may have consumed as much as 200 pg PCDFs/kg (Hayabuchi et al., 1979). The toxic symptoms-chloracne, gastrointestinal problems, fatigue, joint pain, etc.-are associated with exposure to a number of halogenated aromatics, but they have been unusually pronounced and persistent in the victims of the Yusho incident. It has been suggested that this is due to the PCDF contamination of the PCBs (Oishi et al., 1978). Although the acute toxicity of TCDF and several other polyhalogenated dibenzofurans has been examined in guinea pigs, mice, and monkeys (Moore et al., 1979), only preliminary studies or those involving a complex mixture of PCDFs have been carried out in rats (Moore et al., 1976; Oishi et al., 1978). No disposition studies using a purified PCDF have been reported. Such studies are useful in predicting the effects of chronic, low-dose exposure such as may occur with environmental contaminants. Mixtures of PCDFs have been shown to accumulate in the liver, adipose tissue, and spleen in mice with a whole-body halflife of approximately 2 weeks (Morita and Oishi, 1977). However, studies of other halogenated aromatics of defined composition (Goldstein et al., 1978) have demonstrated that the degree and position of chlorination dramatically effects the tissue distribution, excretion, and biological halflife of the molecule in question (Matthews and Anderson, 1975b). Since a similar situation may well exist for the PCDFs, we have examined the fate of purified, radiolabeled TCDF in the rat after a single oral or intravenous injection. We have followed its distribution in body tissues and excretion from the animals. METHODS Cl~emicals. thesized with
‘“C-Labeled difficulty
by
TCDF was custom synIIT Research Institute.
IN THE
RAT
343
Chicago. Illinois. The procedure used (Gray et ul., 1980) resulted in general labeling of the benzene rings with high specific activity (57 mCi/mmol) and purity (>99.9%). The purity was checked by using a Varian 3740 gas chromatograph equipped with a 25 m x 0.25 mm glass capillary column coated with OV-215 and electron capture detector. Helium flow through the column was 0.75 ml/min. The column temperature was maintained at 150°C. with the temperatures at the injector and detector being 230 and 28O”C, respectively. Makeup gas at the column outlet and at detector purge was 5% methane in argon. These procedures allow detection of impurities which vary both in number and position of chlorine atoms. The only impurity detected, trichlorodibenzofuran, was less than 0.1% of the total. Unlabeled TCDF had purity > 99%. Animnls. Male Fischer 344 rats, obtained from Charles River Breeding Laboratories (Portage, Mich.). were maintained in our colony at least I week prior to experimentation. The rats weighed 200-250 g and were 2-3 months old. For disposition studies. the rats were housed in individual metabolism cages allowing for separate collection of urine and feces. They were fed pelleted NIH 31 rat chow and water cld libidum. Fischer rats were used for these studies since many of the carcinogenesis testing programs and other bioassay experiments are being run with this strain which has the advantages of limited growth and low tumor incidence (Coleman et (II.. 1977). Trecrtment. For administration of TCDF. the desired amount of compound was first dissolved in a mixture of Emulphor:ethanol (1:l) (Emulphor EL-620 is a polyethoxylated vegetable oil preparation and was kindly supplied by GAF Corp., New York, N.Y.). No water was added to this mixture for oral administration by gavage of 0.1 or 1.0 pmol [14C]TCDF/kg because of solubility problems at the higher concentration of TCDF (equivalent to 306 &kg). The radioisotope was diluted as needed with unlabeled TCDF for the high-dose treatment. For intravenous administration, the low dose was used (0.1 wmol/kg = 30.6 &kg). The [“CITCDF was dissolved as above in Emulphor: ethanol ( 1: 1) and then diluted with 8 vol of distilled water. resulting in a final concentration of 0.1 pm01 ]“C]TCDF/ml Emulphor:ethanol:water (I: l:8). This solvent system has been shown to be suitable for other lipophilic compounds (Matthews and Anderson, 1975a). The iv dose was administered as 1.0 ml (30.6 pg)/kg into a tail vein for distribution studies or into an exposed femoral vein for studies of biliary excretion. Animals were treated between 9 and 10 AM and were sacrificed by cervical dislocation from 15 min to 3 weeks after TCDF administration. Three rats were used per time point. After sacrifice, the animals were immediately dissected. tissues removed and weighed,
344
BIRNBAUM.
DECAD,
and then stored at -20°C until sampling. The ‘C radioactivity in all tissues was analyzed by combustion to “CO, in a Harvey Biological Oxidizer (R. J. Harvey Instrument Corp., Hillsdale. N.J.). The ‘“CO, was counted in a Beckman Model LS-2SOliquid scintillation spectrometer (Beckman Co., Fullerton, Calif.) as previously described (Matthews and Anderson, 1975a). Samples of all tissues were oxidized directly, without homogenization. Body composition estimates for blood and muscle were 8 and 50% (Matthews and Anderson. 1975a). The values used for fat and skin, 11 and 16%. respectively, were determined by complete dissection of five 200 to 300-g male Fischer 344 rats in our laboratory. Rats sacrificed 10 and 21 days postTCDF treatment were also completely dissected in order to account for any effects of TCDF on body composition. Excretion of TCDF-derived radioactivity in the urine was determined by complete urine collections from each rat kept for 1 day or longer after treatment. Duplicate O.l-ml samples were counted in 20 ml Aquasol (New England Nuclear, Boston, Mass.), and any necessary quench corrections made. Fecal samples were air-dried, weighed, and ground to a fine powder with a mortar and pestle. Duplicate samples were oxidized and radioactivity determined as described above. Bilinry e.rcreriorl. Excretion of [“CITCDF-derived radioactivity in the bile was determined in animals anesthetized with pentobarbital, 50 mgikg, ip, and 50 mgikg, po (Matthews, 1978). After cannulating the common bile duct. 0.1 pmol/kg [“CITCDF (in Emulphor: ethanol:water (I: 1:8) at a concentration of 0.1 Fmol/ml) was injected into the femoral vein and serial bile samples collected for 6 hr. The volume of each sample was measured and the radioactivity of duplicate O.Ol-ml samples in 10 ml Aquasol was determined by liquid scintillation counting. At the end of the collection period, the animals were sacrificed and the tissue distribution of radioactivity determined. Metabolism oJ‘ [‘“C]TCDF. In order to ascertain whether or not the [“CITCDF was being metabolized and whether parent compound or derivative(s) was stored in the tissues or excreted, the tissues and excreta were extracted and analyzed as previously described (Matthews and Anderson, 1975a). The thinlayer system used in this study involved two solvent systems: ( I) chloroform:methanol (1: 1) or (2) methylene chloride:hexane (5: I). In these systems [“CITCDF migrated with an R, of 0.8 and metabolites had R, values less than 0.5. Following chromatography for IO cm on 250 p 20 x 20-cm silica gel G plates (Analtech, Inc., Newark, Del.), the plates were air-dried, scraped off in l-cm segments into 20 ml Aquasol, and radioactivity determined by liquid scintillation counting. Duru ana1ysi.s. The data for tissue distribution and excretion of [“C]TCDF (derived) radioactivity were
AND
MATTHEWS TABLE
TOTAL
TCDF-DERIVED
1
RADIOACTIVITY Percentage
I5 min
Tissue Blood Liver Fat Muscle Skin Kidneys Adrenals Thymus Spleen Testes Brain LLUgS Heart
1.84 34.63 4.43 25.34 5.18 1.20 0.10 0.15 0.16 0.19 0.25 1.36 0.28
” 0.1 Fmol/kg,
-+ k i ti* r f k ? 2 k f
total 180 min
0.57 0.75 0.74 1.31 0.20 0.05 0.08 0.03 0.01 0.02 0.04 0.19 0.08
0.70 41.36 10.02 5.87 6.57 0.25 0.06 0.13 0.03 0.22 0.17 0.20 0.05
k k * -c k ? t + t 2 -t k 2
0.14 3.61 1.00 0.37 0.28 0.04 0.02 0.02 0.01 0.02 0.02 0.02 0.01
IN TISSUES dose” I day 0.40 k 18.09 f 13.89 2 2.43 r 2.33 k 0.10 i co.01 0.02 f 0.01 t 0.14 f 0.03 k 0.04 f 0.01 k
0.11 2.03 3.40 1.97 0.31 0.02 0.01 0 0.09 0.01 0.02 0.01
iv.
analyzed by a nonlinear regression analyses computer program (Morales et al., 1979) based on the decay curves following an exponential pattern. The number of exponential terms was determined by the best fit (Matthews and Anderson, 1975a. b). In all cases the data is expressed as the mean t standard deviation or the estimate * standard error of the estimate.
N 2 3.
RESULTS Tissue Distribution
All of the major organs and tissues were sampled for their radioactive content at various time points after iv administration of 0.1 pmol/kg TCDF, three of which are shown in Table 1. The blood, liver, fat, skin, and muscle tissues accounted for more than 95% of the total unexcreted dose of [‘“ClTCDF derived radioactivity (between 85 and 90% of the injected dose was recovered). The nature of the radioactivity in these tissues was determined by extraction of the above tissues at 15 min, 3 hr, and 1 day after iv administration of 0.1 pmol/kg [‘“CITCDF. Extraction of muscle, fat, skin, and blood removed greater than 80% of the total radioactivity, while similar extractions recovered 98% of the radio-
TCDF
DISPOSITION
4/-
&
2
blood
I
ti
11
Id
3d Time
(hrs-days)
FIG. 1. Percentage of total TCDF dose in blood vs time. TCDF removal from blood between 15 min and 3 days after iv administration of 0.1 pmol/kg [“CITCDF. Each point represents the average value (*SD) obtained with three animals. The solid lines represent the computer drawn two-component exponential decay curve.
activity from liver. These values are nearly identical to what was determined using control tissues to which [‘“CITCDF was added during homogenization. In all cases, greater than 95% of the extractable radioactivity cochromatographed with the parent compound. No metabolites were detectable in the extracts from muscle and fat. By 1 day after treatment, approximately 2% of the radioactivity in skin was not TCDF, while less than 1% of the liver radioactivity was metabolite. In the blood, approximately 5% of the extracted radioactivity failed to cochromatograph with TCDF by Day 1. At the two earlier time points examined, all the extracted radioactivity cochromatographed with [‘“ClTCDF. Thus, the very great majority of the radioactivity present in rat tissues represents unmetabolized TCDF. After intravenous injection of [“‘CITCDF, the distribution of radioactivity as a function of time was examined more closely in the five major tissues depots: blood, liver, fat, skin, and muscle. In all cases, the loss of radioactivity from the tissue could be described by an exponential decay curve, consisting of one or more components. As can be seen in Fig. 1, TCDF is rapidly
IN THE
345
RAT
cleared from the blood, so that by the first time point, 15 min after iv administration, less than 2% of the injected dose remained in the blood. From this point on, a twocomponent exponential decay curve could be used to describe the clearance of TCDF from the blood from 15 min through 21 days (data for 10 and 21 days not shown). The half-lives and pool sizes of these two components are shown in Table 2. Since muscle accounts for a large percentage of the total body mass and has an intermediate rate of perfusion, it is not surprising that it contained approximately 25% of the TCDF dose after 15 min. However, as can be seen in Fig. 2, TCDF was rapidly cleared from muscle at a rate which could be best described by a two-component decay curve. The half-life for the first component was only 30 min, probably reflecting the blood flow through this tissue; the second component has a halflife of 17 hr (Table 2). Liver, fat, and skin all show an initial accumulation of radioactivity. In liver and skin, TCDF derived radioactivity reached a maximum at approximately 3 hr post-treatment, and then decreased at a biphasic rate (Figs. 3, 4, Table 2). The peak concentration in skin was significantly lower TABLE COMPONENTS
FOR THE FROM
2
DECAY
OF RADIOACTIVITY
TISSUES
Pool size (% total dose)
1
1.31 + 0.43 0.89 k 0.2
32.23 z 22.04 0.6 k 0.3
0.02 1.14
2
29.09 i- 6.40 31.39 k 3.30
7.02 2 2.71 0.55 t 0.08
0.10 1.25
Fat
I
17.85 t 1.18
0.19 2 0.04
3.75
MllSCk
I 2
24.85 2 2.36 6.73 t 1.52
29.42 i- 6.33 0.96 k 0.50
0.02 0.72
1
6.84 + 0.47 1.22 f 0.43
Tissue Blood
2 Liver
Skin
1
2
Decay rate (day-l )
Halflife (days)
Component
2
0.29
0.45
0.06 i;
0.41
11.09
1.55
346
BIRNBAUM,
DECAD,
muscle
.!
4t IlI
I
2 3h7h
Id
Tune
3d
(hrs-days)
FIG. 2. Percentage of total TCDF dose in muscle vs time. TCDF removal from muscle between 1.5 min and 3 days after iv administration of 0.1 pmol/kg [‘C]TCDF. Each point represents the average value (*SD) obtained with three animals. The solid lines represent the computer drawn two-component exponential decay curve.
AND
MATTHEWS
and the clearance of the radioactivity from the skin was slower than what was observed in the liver. The liver, however, was the major site of TCDF disposition, accounting for nearly 50% of the total dose by 3 hr after iv injection. The remaining major depot for TCDF in the rat was the adipose tissue (Fig. 5). The maximum accumulation of radioactivity in fat was reached at approximately 7 hr postadministration. The radioactivity was then cleared at a rate which could best be described by a one-component decay curve. The half-life for the decay of TCDF-derived radioactivity from the fat is approximately 3.7 days (Table 2). The tissues described above account for the major tissue volumes and the major depots for lipid-soluble compounds such as TCDF. However, TCDF was also distributed to all of the other tissues in the rat and in some cases, the concentrations were initially quite high. The concentration of TCDF per gram tissue is shown in Table 60,
50-
a I013
7
24 Time
(hrs)
Time
(days)
FIG. 3. Percentage of total TCDF dose in liver vs time. TCDF accumulation in the liver from 15 min to 3 hr is shown in (a). Removal of TCDF from the liver from 3 to 24 hr is shown in (a), and from 3 hr to 10 days in (b). The treatment was 0.1 PmoVkg [“CITCDF. iv. Each point represents the average value (f SD) obtained with three aminals. The solid lines for TCDF removal represent the computer drawn two-component exponential decay curve.
TCDF DlSPOSlTION
347
1N THE RAT
skin
a ‘13
7
24 Ttme
b 2l
(hrs)
3
IO Ttme (days)
21
FIG. 4. Percentage of total TCDF dose in skin vs time. TCDF accumulation in the skin from 15 to 3 hr is shown in (a). Removal of TCDF from the skin from 3 to 24 hr is shown in (a) and from 24 hr to 21 days in (b). The treatment was 0.1 PmoVkg [%]TCDF, iv. Each point represents the average value (*SD) obtained with three animals. The solid lines for TCDF removal represent the computer drawn two-component exponential decay curve.
3. Of the major sites of deposition described above the liver has the highest specific content, approximately equal to 1 nmol/g tissue. However, the adrenal gland had the
highest TCDF concentration per gram tissue, over 50% higher than the liver at initial time point. This radioactivity in adrenals is cleared quite rapidly, so that
b
20
5
FIG. 5. Percentage oftotal TCDF dose in fat vs time. TCDF accumulation is shown in (a). Removal of TCDF from the fat from 7 to 24 hr is shown 21 days in (b). The treatment was 0.1 pmol/kg, iv. Each point represents tained with three animals. The solid line for TCDF removal represents the decay curve.
IO Time
15
20
(days)
in the fat from 15 min to 7 hr in (a), and from 7 hr through the average value (?-SD) obcomputer drawn exponential
of the the by
-
348
BIRNBAUM,
DECAD,
1 day the adrenals contained only 5% of their initial specific activity. Other tissues which contained appreciable initial concentrations of TCDF were the lungs, kidney, heart, and thymus. In each of these cases, as observed for the adrenals, approximately 90% of this radioactivity was cleared within 24 hr. On the other hand, the specific activity of the liver decreased by only 50% during the same 24 hr. Oral Administration
TABLE
Blood Liver Fat Muscle Skin Kidneys Adrenals Thymus Spleen Testes Brain Lungs Heart
15 min 0.12 4.39 0.20 0.25 0.17 0.67 7.43 0.52 0.37 0.09 0.25 1.08 0.66
n 0.1 PmoVkg,
3
OF TCDF-RADIOACTIVITY CONTENT IN TISSUES
Percentage Tissue
2 k 2 + + + + + k t rf% + iv.
MATTHEWS TABLE
0.04 0.17 0.03 0.01 0.02 0.04 6.91 0.12 0.07 0.01 0.01 0.08 0.03
total
dose”/g
tissue
180 min 0.04 5.05 0.44 0.06 0.20 0.17 4.65 0.54 0.08 0.09 0.15 0.24 0.11
& ? r k + + t r 5 ? ? f k
0.01 0.35 0.07 0.00 0.01 0.03 1.26 0.13 0.02 0.01 0.03 0.02 0.00
4
DISTRIBUTION OF RADIOACTIVITY 3 DAYS ADMINISTRATION OF [“C]TCDF
AFTER
Route of administration OEil Tissue LlVU Fat Skin Total excreted In feces In urine
Environmental chemicals are rarely encountered by an intravenous route, however, by eliminating the absorption phase, such a treatment does greatly simplify a pharmacokinetic analysis. Therefore, it was considered necessary to demonstrate that the tissue distribution after an oral dose resulted in the same profile as that obtained after iv exposure. It was also important to demonstrate that the dose used was not saturating the system, thereby affecting the disposition. To answer these questions, a dose identical to the iv dose (0.1 pmollkg)
CONCENTRATION
AND
Dose:
1.0 &m&kg
I” 0 I pm&kg
0. I pm&kg
3.92 2 2.1x 9.79 k 3.55 1.10 c 0.59
5.02 2 0.42 4.64 z 0.75 1.07 z 0.19
5.87 t 0.27 Il.57 + 2.33 I.24 k 0.27
67.81 f 5.64 I .47 + 0.33
70.42 k 3 76 I 76 k 0.01
63.05 2 9.58 2.01 + 0.43
and one IO-fold higher (1 .O pmol/kg) was administered to the rats by oral gavage. A comparison of the major tissue depots 3 days after treatment with the high and low oral dose and the iv dose is shown in Table 4. Total recovery of administered radioactivity was approximately 85%. There was no significant difference in the liver or skin content between the different treatments. However, there may have been less TCDF in the fat of animals which received the low oral dose. There was no detectable difference in the TCDF content of muscle or blood which accounted for less than 1% of the total dose after the various treatments. The excretion data were also similar in both feces and urine for the high- and lowdose po and the low-dose iv route (Fig. 6).
1 day 0.03 2.22 0.64 0.03 0.07 0.08 0.34 0.07 0.02 0.06 0.02 0.07 0.02
2 t + 2 t 2 + k ? k k + f
0.01 0.37 0.11 0.02 0.01 0.01 0.14 0.03 0.00 0.02 0.00 0.02 0.02
Excretion
The excretion of [“‘CITCDF-derived radioactivity was analyzed by daily collection of urine and feces from individual animals held from 1 to 21 days post-treatment. Total cumulative excretion in urine and feces for 7 days is shown in Fig. 6. The data in Fig. 6 clearly demonstrate that the major route for excretion of this compound is via the feces. The majority of urinary excretion occurs within the first day, with no detectable radioactivity appearing in the
TCDF
DISPOSITION
IN THE
349
RAT
7-
bile
6-
. 0 I,uimol/kg, 0 Oi,umal/kg.PO A I O,umal/kg,PO
I
2
3
4 Time (days)
5
IV I
6
7
I
I
2
3 Time
4
5
6
Y 7
(days)
7. Daily excretion of TCDF-derived radioactivity in urine. The percentage of the total iv dose of 0. I pmol/kg [‘4C]TCDF excreted daily by rats for 7 days was plotted vs time. Each point represents the daily excretion of a single rat. Numbers in parentheses indicate the number of rats having the same excretion. The solid line represents the computer-drawn exponential decay curve. FIG.
3 Time
FIG. 6. Cumulative excretion of TCDF-derived material in urine and feces. Cumulative excretion of radioactivity, expressed as percentage of total dose, in the urine and feces for 7 days after iv administration of 0.1 pmol/kg [“C]TCDF (0). and for 3 days after po administration of 0.1 pmol/kg [“CITCDF (0) and 1.0 pmol/kg [‘QJTCDF (A). Each point represents the average cumulative percentage of the total dose excreted by three or more animals.
04'
2
4
5
6
Chrs)
FIG. 8. Cumulative excretion of TCDF-derived radioactivity in bile. Data obtained by bile duct cannulation followed by iv administration of 0.1 pmol [‘“C]TCDF/kg into the femoral vein. Results shown are the average value (*SD) for three animals.
urine after Day 6. The urinary excretion best fits a single exponential decay curve, with a decay rate of 0.5 day-’ (Fig. 7). Since the feces was the major route of excretion, the elimination via the bile was also studied to determine if the material in feces originated in bile and if enterohepatic circulation played a major role in the distribution of the [‘“CITCDF-derived radioactivity. As shown in Fig. 8, over 7% of an iv dose of TCDF was excreted in bile within 6 hr. This is in good agreement with the observed elimination of 24% of the total dose in feces within the first day and suggests that once the radioactivity appears in the bile, it enters the intestines and is excreted without recirculation. The compound is then removed via the feces following a single component exponential decay, having a half-life of slightly under 2 days (Fig. 9). The nature of the [14C]TCDF-derived radioactivity in the urine and bile was examined by thin-layer chromatography. In
350
BIRNBAUM,
DECAD,
the urine, no parent compound was detectable at any time point (Day 1 through Day 6) after treatment. However, the metabolite(s) exhibited increasing polarity as a function of time after dosing. In the bile, greater than 96% of the radioactivity was metabolite within 90 min after injection. By 3 hr, there was no detectable parent compound in bile. These results agree well with those obtained by extraction of feces followed by thin layer chromatography. During the first day, approximately 1% of the extracted radioactivity cochromatographs with [14C]TCDF. This value decreased to 0.5% during the second day, after which no parent compound was detectable in the feces. It should be pointed out that only 20-25% of the [14C]TCDF derived radioactivity was extractable from the excreted feces. This contrasts with essentially complete extraction of [14C]TCDF added to control feces just prior to extraction. This suggests that nonextractable radioactivity was not parent TCDF, but metabolites. The results presented here indicate that greater than 99% of the radioactivity which is excreted from the body was no longer TCDF but several metabolites of this compound. DISCUSSION The objectives of this investigation were to determine the absorption, distribution, and excretion of TCDF in male rats in order to better evaluate the toxicity of this compound. This study demonstrated that approximately 90% of an oral dose of TCDF was absorbed from the gut, and this absorption was not a function of the doses administered. Distribution of TCDF was initially to the liver and muscle, followed by some redistribution to skin and adipose tissue, in contrast to TCDD which accumulates in the liver and fat (Rose et al., 1976). This pattern was not affected by dose or route of exposure. Essentially all the radio-
AND
MATTHEWS
01’
5
IO Time ldoys)
15
20
FIG. 9. Daily excretion of TCDF-derived radioactivity in feces. The percentage of the total dose of 0.1 pmol of [‘“C]TCDF/kg excreted daily by rats for 2 1 days was plotted vs time. Each point represents the daily fecal excretion of one rat. Numbers in parentheses indicate the number of rats having the same excretion. The solid line represents the computer-drawn exponential decay curve.
activity present in the tissues cochromatographed with [‘“CITCDF, suggesting that greater than 95% of the tissue burden was unmetabolized compound. In the fat, essentially all the radioactivity appears to be TCDF. The primary route of excretion was in the feces. Less than 6% of the administered dose was ever excreted in the urine. Excretion was not affected by the dose, within the range studied or the route of administration. The rapid rate of excretion of TCDF was in contrast to that observed for TCDD, where a whole-body half-life of 3-5 weeks has been reported (Rose et al., 1976). All of the radioactivity excreted in the urine was metabolites of TCDF. The same was true for the radioactivity in bile and feces. Preliminary results suggested that intestinal flora may alter the biliary metabolites, since the chromatographic results for
TCDF
DISPOSJTION
extracts of bile and feces were quite distinct. The action of intestinal microorganisms on TCDF itself remains unknown although it has been shown here that microbial action does not alter the dose of TCDF which reaches the tissues following an oral vs an iv dose. Therefore, since virtually all of the TCDF-derived radioactivity excreted in the urine, bile and feces was in the form of several TCDF metabolites and storage of TCDF metabolites in the tissues was negligible, the more rapid clearance of TCDF vs TCDD (Rose et al., 1976) must be attributed to more rapid metabolism of TCDF by the rat. Some qualitative observations concerning the relationship of acute TCDF toxicity to its metabolism should be noted. By 2 days after treatment, regardless of dose or route of injection, the rats showed listlessness, excessive hair loss, and decreased weight gain. However, by Day 6 post-treatment, the hair loss had ceased and their weight gain returned to normal. Thus, 3 weeks after TCDF treatment, the rats were of normal weight and apparently healthy. There were no visible signs of thymic or splenic atrophy or liver hypertrophy. It seems that the metabolism of TCDF resulting in its excretion prevented the acute toxicity from progressing to lethality. Instead, the animals recovered. Of course, some permanent damage may have occurred which is not yet manifest. such as neoplastic transformation, shortened life span, etc. Thus, it appears that in the case of TCDF, the parent compound is the toxic agent. Metabolism results in detoxification. From these conclusions, we might predict that animals with higher rates of metabolism of TCDF will be more resistant to its toxic actions. Such a correlation, in fact, may exist in the rat itself in relation to the differential toxicity due to TCDD vs TCDF, the former of which is more slowly metabolized and is several times more toxic (Rose et al., 1976; Ramsey et al., 1979). The mechanism of toxicity of these compounds is still unclear (Neal et al., 1979).
IN THE
351
RAT
The effects are pleiotropic, affecting various organs and cell types. Several investigators have suggested that toxicity is mediated by an intracellular receptor which activates a battery of genes (Poland et al., 1979; Okey et al., 1979). In fact, there is a good correlation between the biological potency of various PCDD and PCDF congeners (McConnell et al., 1978) and their ability to induce aryl hydrocarbon hydroxylase activity by binding to the TCDD receptor (Poland et crl., 1979). The ability of the rat to metabolize TCDF and therefore clear it from the body may thus explain its relative resistance to the toxic effect of this compound. ACKNOWLEDGMENTS The authors cal assistance Oates, and the by Dr. Phillip
wish to acknowledge the expert techniof Ms. Minerva Fields and Mr. Tony purity determination made on [“CITCDF Albro.
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