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Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection
FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.
31, 184–191 (1996)
0090
Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection MATTI VILUKSELA,*,† THANG V. DUONG,* BERNHARD U. STAHL,*,‡ XUELIN LI,* JOUKO TUOMISTO,† AND KARL K. ROZMAN*,‡ *Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417; †Department of Toxicology, National Public Health Institute, Kuopio, Finland; and ‡Section of Environmental Toxicology, GSF-Institut fu¨r Toxikologie, 85758 Neuherberg, Federal Republic of Germany Received July 27, 1995; accepted January 29, 1996
Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection. VILUKSELA, M., DUONG, T. V., STAHL, B. U., LI, X., TUOMISTO, J., AND ROZMAN, K. K. (1996). Fundam. Appl. Toxicol. 31, 184–191. Toxicokinetics of a nontoxic intravenous dose of 14C-labeled TCDD were studied in two substrains of Long–Evans (L-E) rats with a fivefold difference in sensitivity in terms of TCDD-induced mortality. The Turku/AB Long–Evans rat (T L-E) is the most sensitive rat strain with an oral LD50 of 17.7 mg/kg, whereas the Charles River Long–Evans rat (CR L-E) is a more resistant strain (oral LD50 95.2 mg/kg). Samples of 18 tissues were collected 1, 2, 4, 8, 16, and 32 days after dosing and analyzed for radioactivity. Body weight and fecal and urinary excretion of radioactivity were monitored daily during the 32-day study period. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% in 32 days compared with only 16% in T L-E rats. This difference was not caused by toxicity, because the weight gain was identical in control and TCDD-treated rats of both substrains. Tissue concentrations of [14C]TCDD-associated radioactivity and area under the curve (AUC) values were lower in CR L-E than in T L-E rats. The most pronounced differences were found in thymus, white adipose tissue, brown adipose tissue, and adrenals. The decrease of TCDD concentration in tissues was faster in CR L-E than in T L-E rats, whereas fecal and urinary excretion was faster in T L-E than in C L-E rats. Elimination half-life was 20.0 days in T L-E rats and 28.9 days in CR L-E rats. Differential toxicokinetics of TCDD in the two L-E substrains provide a likely explanation for the greater sensitivity of the T L-E strain, since observed differences in tissue concentrations and AUC values are in good agreement with the difference in susceptibility. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics seems to be a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the difference in susceptibility. More rapid excretion of TCDD in T L-E rats than in CR L-E rats is clearly a result of higher tissue concentrations in T L-E rats. However, this faster excretion rate is not sufficient to counterbalance the much slower dilution by growth in T L-E rats than in CR L-E rats. Thus, dilution by growth can be a more 0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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important factor in determining the toxicokinetics and toxicity of TCDD in rodents than is excretion. q 1996 Society of Toxicology
TCDD is extremely toxic to some animal species, with huge inter- and intraspecies variations in sensitivity regarding acute toxicity (reviewed by Pohjanvirta and Tuomisto, 1994). For example, a more than a 1000-fold sensitivity difference has been reported between two rat strains (Pohjanvirta et al., 1993). The Han/Wistar (Kuopio), a substrain of the Wistar rat, is probably the most TCDD-resistant laboratory animal. It has been impossible to determine the LD50 for this strain in an acute study because of solubility limitations of TCDD (Pohjanvirta et al., 1993). The most sensitive rat strain is the Turku/AB Long–Evans substrain, being only slightly less sensitive than the guinea pig, which is the most TCDD-sensitive species. Extreme sensitivity to TCDD, however, is not a specific feature of the Long–Evans strain, since the Charles River substrain seems to be about five times more resistant to TCDD than the Turku/AB substrain (oral LD50 in male CR L-E rats is 95.2 mg/kg vs 17.7 mg/ kg in male T L-E rats) (Fan and Rozman, 1994; Pohjanvirta et al., 1993). Toxicokinetic factors clearly can explain only partially the differential sensitivity of various species to TCDD (Van den Berg et al., 1994). In fact, kinetic factors were reported to have at most a contributory impact on the intraspecies difference in TCDD-induced lethality between the most sensitive (T L-E) and the most resistant (Han/Wistar, Kuopio) rat strains (Pohjanvirta et al., 1990). On the other hand, recent studies from this laboratory suggested that a relatively small sensitivity difference between male and female Sprague–Dawley rats (cf. Beatty et al., 1978; Kociba et al., 1976, 1978) is likely to be due to toxicokinetics alone rather than to gender-specific physiological differences (Weber et al., 1993; Li et al., 1995). To reduce the confounding effects of physiological dissimilarities between species or even strains, we have utilized in the present study the fivefold intrastrain sensitivity differ-
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ence to examine the role of toxicokinetics in the toxicity of TCDD. Intravenous route of administration was chosen to allow an accurate evaluation of the toxicokinetic profile as well as to permit comparisons with two previous kinetic studies from this laboratory with the same route of administration and a comparable study design (Weber et al., 1993; Li et al., 1995). MATERIALS AND METHODS Animals. Male outbred CR L-E were obtained from Charles River (Boston, MA). Male inbred L-E (Turku/AB) rats (T L-E), originally obtained from the National Laboratory Animal Center of the University of Kuopio (Kuopio, Finland), were bred at the Laboratory Animal Resources of the University of Kansas Medical Center (Kansas City, KS). Rats were about 8 weeks old at the beginning of the experiments. They were kept individually in stainless steel wire-bottom cages or polycarbonate metabolism cages for urine and feces collection (32-day time point). Their maintenance consisted of Purina 5001 rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum. Their room was artificially illuminated from 6 AM to 6 PM and air-conditioned with a Honeywell DeltaNet W1044 computer controller (Minneapolis, MN) programmed to maintain the ambient temperature at 21–227C and relative humidity at 40–60%. Rats were acclimated to experimental conditions for 1 week before dosing. Emulsions. 14C-labeled TCDD (ED-902H, sp act 122 mCi/mmol) was obtained from Cambridge Isotope Laboratories Inc. (Woburn, MA). The solvent (toluene) was evaporated and [14C]TCDD redissolved in corn oil (Sigma) by stirring for 5 days. Thirty milligrams of cholic acid was dissolved in 9 ml of Ringer’s solution with the aid of 1 N NaOH, after which 30 mg of lecithin (Sigma) was added. The pH was adjusted to 8, and 1 ml of corn oil containing [14C]TCDD was added and shaken and the emulsion sonicated (Sonifier 185, micro-tip, Branson Sonic Power, Danbury, CT). The final emulsion was stored in a refrigerator and found to be stable for at least 3 months. Treatment. Animals were randomly allocated into groups of four or six (groups sampled on Day 32) and given a slow (1 min) intravenous injection of 14C-labeled TCDD emulsion into the lateral tail vein using Quick-Cath over-the-needle Teflon catheters (22 gauge, Baxter, Deerfield, IL). CR L-E rats received a single dose of 5.6 mg/kg (2.12 mCi/kg) and T L-E rats 2.0 mg/kg (0.76 mCi/kg) of [14C]TCDD in a volume of 4 ml/kg. The doses were selected to be as low as possible (to avoid toxicity), but still sufficient to obtain a reasonable level of detection. Body weights, urine volumes and dry feces weights were recorded daily for the 32-day groups. Rats were killed by decapitation 1, 2, 4, 8, 16, and 32 days after dosing. Tissue collection. Serum (from trunk blood), liver, kidney, thymus, heart, pancreas, lung, spleen, brain, thyroids, adrenals, and testes were weighed and duplicate samples (except for thyroids and adrenals) of about 100–200 mg prepared for analysis. Duplicate samples of the same size were also taken from epididymal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), proximal large intestine, small intestine (jejunum), muscle, and dorsal skin. The intestines were flushed with saline and the fat was scraped off from the skin with a scalpel. Tissue samples were digested overnight by 1 ml of Soluene-350 tissue solubilizer (Packard Instrument Co., Downers Grove, IL) at 507C. Radioactivity was determined, after the addition of 10 ml scintillation cocktail (Hionic Fluor, Packard Instrument Co.), in a TriCarb-1900AC liquid scintillation counter (Packard Instrument Co.). Duplicate aliquots of 1 ml were procured from serum and urine, and radioactivity was measured after addition of 10 ml scintillation cocktail. Feces were dried and ground, and radioactivity was determined after combustion of about 100 mg duplicate samples in a sample oxidizer (Packard 306B biological oxidizer).
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FIG. 1. Body weight development of the [14C]TCDD-treated Charles River (open circles) and Turku/AB (solid circles) Long–Evans rats (32-day groups). The dotted lines represent untreated control groups of both strains. (CR L-E control rats were slightly older and thus heavier than the TCDD-treated rats.) Group means { SE, n Å 6, except for controls, n Å 4.
Calculations. Radioactivity found in various tissues was converted to percentage of total dose recovered per gram of tissue (or total tissue) or percentage of total dose excreted per day (urine, feces). Uptake rate constants were calculated by linear regression. Rate constants of elimination (one-compartment model), areas under the curve (AUC), and half-lives were calculated on a mainframe computer using the SAS software package. Sigma-minus plots (log percentage of administered dose remaining to be excreted vs day) were constructed for fecal, urinary, and combined excretions.
RESULTS
Body weight development of the 32-day groups and their controls is shown in Fig. 1. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% (146 g) compared to 16% (41 g) in T L-E rats. The difference in growth rate was not caused by toxicity, since untreated control rats gained weight similar to those treated with TCDD of both substrains. Percentages of dose (radioactivity) recovered per gram of tissue are depicted in Tables 1A and 1B, absolute organ weights in Tables 2A and 2B, and percentages of dose per total tissue in Tables 3A and 3B. The only remarkable changes in absolute organ weights (Tables 2A and 2B) were the same reported also by others, i.e., increased liver weight (more prominent in CR L-E rats) and decreased thymus weights (more pronounced in T L-E rats). The time course of [14C]TCDD-associated radioactivity in selected tissues is illustrated in Fig. 2. The tissue distribution profile of TCDD was distinctly and consistently different in the two substrains. All tissue concentrations were lower in CR L-E than in T L-E rats with peak concentrations being typically reached between 1 and 2 days (CR L-E) versus 4 to 8 days (T L-E) after dosing (Fig. 2; Tables 1A, 1B, 3A, and 3B). It appears that this pattern of tissue distribution is at least partly due to slow release of TCDD from the emulsion (vehicle) trapped in the lymphatic system and/or to redistribution of TCDD from highly perfused to poorly perfused tissues.
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TABLE 1A Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [11000]:a Charles River Long–Evans Rats Time after injection Tissue Adrenals BAT Brain Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT Liver/WAT a
1 day 101.5 { 231.1 { 8.4 { 35.9 { 31.3 { 17.9 { 1507.1 { 43.3 { 6.4 { 28.7 { 6.8 { 32.3 { 28.1 { 38.8 { 7.0 { 46.0 { 60.5 { 183.2 { 8.2
2 days 10.4 21.3 0.4 12.4 2.6 1.0 80.8 6.2 0.6 2.8 0.3 1.3 2.0 9.3 0.6 3.2 12.1 15.2
73.9 { 257.5 { 6.9 { 29.6 { 31.0 { 22.5 { 1777.5 { 52.0 { 5.8 { 21.9 { 8.7 { 40.6 { 25.4 { 59.9 { 5.8 { 43.0 { 43.5 { 290.6 { 6.1
4 days
21.6 13.7 0.5 7.5 3.8 4.1 211.0 10.9 0.3 2.9 1.8 3.4 3.9 23.2 0.6 6.1 5.2 63.2
72.9 { 211.4 { 7.3 { 25.4 { 27.2 { 14.0 { 1759.8 { 37.2 { 5.0 { 19.2 { 7.4 { 41.0 { 23.8 { 33.3 { 5.1 { 43.6 { 21.1 { 299.2 { 5.9
8 days
5.0 12.5 1.4 9.1 2.7 1.0 241.0 5.9 0.2 1.7 0.8 2.7 1.9 6.1 0.2 2.7 3.5 18.4
53.0 { 128.6 { 5.2 { 22.8 { 25.0 { 11.0 { 1635.0 { 34.3 { 3.6 { 13.3 { 5.3 { 23.8 { 15.0 { 52.6 { 4.7 { 25.6 { 19.6 { 262.5 { 6.2
9.6 11.7 1.4 7.3 3.1 0.6 152.0 8.1 0.2 1.5 0.7 1.6 1.3 22.4 0.8 0.7 3.3 9.9
16 days 32.5 92.2 4.1 21.9 16.9 8.6 841.2 28.9 3.0 9.1 3.2 14.1 11.0 32.5 2.8 24.1 19.7 171.0
{ 2.4 { 4.1 { 1.2 { 10.0 { 4.9 { 0.8 { 106.2 { 8.6 { 0.4 { 0.8 { 0.5 { 1.0 { 1.7 { 13.8 { 0.1 { 2.7 { 4.1 { 14.0 4.9
32 days 22.2 { 47.2 { 2.7 { 19.3 { 13.2 { 4.8 { 234.2 { 26.1 { 2.1 { 5.3 { 0.9 { 4.5 { 6.5 { 41.1 { 1.7 { 12.5 { 12.3 { 77.4 { 3.0
2.4 2.3 0.4 7.5 4.5 0.4 31.2 9.0 0.3 0.3 0.1 0.6 0.5 18.3 0.1 0.9 2.7 2.2
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined.
AUCs were also lower in CR L-E rats than in T L-E rats (Table 4). In both substrains the highest tissue concentrations and AUCs were measured in liver, followed by WAT, BAT,
and adrenals. The most remarkable differences in peak concentrations between the two substrains were found in thymus (8-fold) and WAT (4.9-fold). Skin, adrenals, testes, BAT,
TABLE 1B Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [11000]:a Turku/AB Long–Evans Rats Time after injection Tissue Adrenals BAT Brain Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT Liver/WAT a
1 day 369.4 { 699.4 { 22.2 { 70.2 { 99.2 { 48.0 { 1227.5 { 114.3 { 17.4 { 69.0 { 10.7 { 66.2 { 64.6 { 95.9 { 15.9 { 313.8 { 113.8 { 424.1 { 2.9
33.3 17.6 3.1 16.2 17.7 4.2 164.1 24.7 1.9 3.6 1.2 4.6 5.1 20.3 1.3 32.2 11.4 43.8
2 days 372.0 { 731.5 { 17.1 { 45.6 { 85.9 { 45.2 { 1438.2 { 93.4 { 14.8 { 60.2 { 12.0 { 123.5 { 58.8 { 65.8 { 15.6 { 239.7 { 143.6 { 712.6 { 2.0
36.7 52.7 1.7 3.9 6.8 3.4 103.1 7.9 1.8 2.9 1.4 8.9 6.5 4.7 1.0 13.5 22.4 52.7
4 days 419.4 { 889.4 { 19.1 { 53.4 { 78.2 { 51.3 { 1691.1 { 109.4 { 20.5 { 75.6 { 13.6 { 172.5 { 66.2 { 69.7 { 19.0 { 368.5 { 195.4 { 1158.1 { 1.5
8 days
26.6 63.5 1.7 5.1 4.0 4.0 107.0 10.4 0.9 10.9 1.4 18.3 5.3 11.4 1.0 31.1 8.2 92.7
366.6 { 702.1 { 15.9 { 35.6 { 71.7 { 48.9 { 2149.4 { 95.8 { 14.2 { 61.8 { 16.5 { 168.6 { 63.4 { 52.8 { 19.0 { 281.3 { 86.1 { 1451.2 { 1.5
11.5 26.2 0.6 1.3 1.7 3.3 195.9 3.5 0.3 4.3 1.1 23.4 2.0 2.8 1.5 25.4 23.2 59.7
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined.
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16 days 242.4 { 538.1 { 14.4 { 62.0 { 63.1 { 35.3 { 1513.6 { 96.6 { 14.7 { 46.3 { 8.1 { 99.1 { 50.1 { 95.4 { 13.6 { 242.9 { 68.1 { 1167.9 { 1.3
18.7 45.5 2.8 25.1 12.7 3.2 22.4 22.3 0.8 6.6 0.2 16.0 3.6 27.2 1.2 32.1 19.3 29.8
32 days 175.8 { 314.6 { 21.3 { 54.9 { 52.2 { 39.0 { 819.1 { 79.3 { 20.6 { 65.5 { 5.5 { 45.4 { 42.9 { 102.8 { 24.9 { 169.6 { 272.4 { 667.1 { 1.2
22.6 9.3 3.4 18.9 14.7 2.4 58.7 22.3 2.5 10.1 0.5 3.8 3.7 32.9 2.9 20.0 28.0 20.1
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TABLE 2A Absolute Organ Weights (g):a Charles River Long–Evans Rats Time after injection Tissue
1 day
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids a
0.052 0.240 1.875 1.024 2.685 13.477 1.153 0.884 0.647 3.036 0.590 0.018
{ { { { { { { { { { { {
2 days
0.006 0.014 0.066 0.032 0.097 0.617 0.048 0.059 0.028 0.155 0.055 0.001
0.047 0.262 1.792 0.984 2.477 15.008 1.113 0.774 0.630 3.221 0.423 0.015
{ { { { { { { { { { { {
4 days
0.005 0.025 0.048 0.052 0.045 0.866 0.077 0.069 0.034 0.083 0.050 0.001
0.057 0.139 1.854 1.034 2.669 16.807 1.132 0.962 0.705 3.353 0.459 0.018
{ { { { { { { { { { { {
8 days
0.003 0.013 0.054 0.075 0.210 1.240 0.030 0.057 0.068 0.164 0.034 0.001
0.053 0.106 1.880 1.148 2.270 17.427 1.510 0.879 0.643 3.332 0.372 0.017
{ { { { { { { { { { { {
16 days
0.001 0.009 0.053 0.156 0.206 0.868 0.112 0.066 0.038 0.083 0.021 0.000
0.052 0.158 1.811 1.352 2.558 17.174 1.859 0.775 0.637 3.391 0.403 0.024
{ { { { { { { { { { { {
32 days
0.003 0.016 0.051 0.215 0.180 1.139 0.580 0.100 0.041 0.116 0.032 0.002
0.054 0.135 1.632 1.158 2.558 15.561 1.328 0.815 0.626 3.448 0.469 0.019
{ { { { { { { { { { { {
0.002 0.007 0.296 0.069 0.182 1.125 0.111 0.087 0.035 0.120 0.048 0.002
Mean { SE, n Å 4 (6 for the 32-day group).
thyroids, muscle, and kidney showed 3.2- to 4.3-fold differences. The most pronounced differences in terms of AUC values were found in thymus (10.2-fold), adrenals (6.9-fold), skin (6.2-fold), WAT (5.8-fold), and BAT (5-fold). Most notably, the substrain differences in liver peak concentrations and AUCs were only 1.2- and 1.5-fold, respectively. The decrease in TCDD tissue concentrations was faster in CR L-E than in T L-E rats (Table 5). Tissue half-lives of [14C]TCDD-associated radioactivity in CR L-E and T L-E rats were 9.4 and 13.3 days for serum, 10.5 and 16.9 days for liver, and 14.5 and 22.4 days for WAT, respectively. In contrast to the disappearance of TCDD from tissues, its fecal and urinary excretion occurred at a lower rate in CR L-E rats than in T L-E rats (Fig. 3). In T L-E rats the excretion followed a strictly
linear pattern (r Å 0.987) on a semilogarithmic plot during 32 days, whereas in CR L-E rats fecal and total (fecal / urinary) excretion curves flattened out, indicating retarded elimination during the second half of the observation period. The elimination half-life of TCDD, calculated from total excretion using linear regression, was 20.0 days in T L-E rats. Because of retarded excretion of TCDD in CR L-E rats a curve fit was obtained according to the equation 1.2
y Å a(1 0 b 1 e0c1t ), where a Å 41.658, b Å 01.3715, and c Å 0.035237, which described accurately (r Å 0.999) the total elimination curve
TABLE 2B Absolute Organ Weights (g):a Turku/AB Long–Evans Rats Time after injection Tissue
1 day
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids a
0.049 0.095 1.568 0.795 2.194 9.488 1.005 0.707 0.422 2.612 0.232 0.014
{ { { { { { { { { { { {
2 days
0.003 0.007 0.030 0.028 0.151 0.279 0.030 0.074 0.028 0.089 0.014 0.001
0.052 0.095 1.602 0.749 2.040 10.045 1.001 0.523 0.449 2.542 0.223 0.012
{ { { { { { { { { { { {
4 days
0.001 0.004 0.004 0.015 0.030 0.249 0.029 0.038 0.018 0.023 0.012 0.001
0.053 0.115 1.571 0.770 2.181 10.852 1.030 0.657 0.404 2.578 0.225 0.013
{ { { { { { { { { { { {
0.001 0.007 0.017 0.019 0.090 0.371 0.033 0.048 0.064 0.037 0.015 0.002
8 days 0.051 0.092 1.605 0.777 2.058 10.949 1.012 0.702 0.487 2.701 0.185 0.013
{ { { { { { { { { { { {
0.0005 0.008 0.041 0.026 0.053 0.268 0.021 0.066 0.021 0.071 0.007 0.001
16 days 0.056 0.089 1.561 0.856 2.198 11.451 1.170 0.598 0.481 2.618 0.135 0.014
Mean { SE, n Å 4 (6 for the 32-day group).
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{ { { { { { { { { { { {
0.001 0.008 0.054 0.028 0.071 0.639 0.114 0.051 0.025 0.190 0.006 0.001
32 days 0.056 0.084 1.679 0.870 2.431 11.353 1.181 0.619 0.502 2.902 0.110 0.014
{ { { { { { { { { { { {
0.002 0.007 0.017 0.037 0.035 0.319 0.050 0.037 0.013 0.036 0.005 0.001
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TABLE 3A Percentage of Dose (Radioactivity) Recovered per Total Tissue [11000]:a Charles River Long–Evans Rats Time after injection Tissue
1 day
2 days
4 days
8 days
16 days
32 days
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids WATb
0.053 { 0.008 0.555 { 0.069 0.157 { 0.009 0.368 { 0.124 0.843 { 0.079 204 { 19 0.506 { 0.086 0.258 { 0.040 0.254 { 0.064 0.211 { 0.022 0.274 { 0.035 0.011 { 0.003 4218
0.032 { 0.009 0.671 { 0.065 0.123 { 0.009 0.286 { 0.062 0.764 { 0.084 264 { 30 0.587 { 0.151 0.166 { 0.017 0.394 { 0.175 0.186 { 0.019 0.179 { 0.024 0.006 { 0.000 6910
0.042 { 0.003 0.293 { 0.032 0.135 { 0.026 0.276 { 0.119 0.717 { 0.066 287 { 20 0.422 { 0.071 0.183 { 0.015 0.231 { 0.045 0.171 { 0.015 0.201 { 0.023 0.004 { 0.001 7474
0.028 { 0.005 0.137 { 0.017 0.097 { 0.024 0.295 { 0.139 0.583 { 0.111 281 { 11 0.536 { 0.165 0.115 { 0.010 0.359 { 0.176 0.157 { 0.025 0.096 { 0.007 0.003 { 0.0005 7088
0.017 { 0.002 0.147 { 0.020 0.073 { 0.020 0.268 { 0.109 0.416 { 0.102 141 { 10 0.491 { 0.137 0.069 { 0.008 0.199 { 0.076 0.095 { 0.004 0.098 { 0.015 0.005 { 0.001 5118
0.012 { 0.001 0.063 { 0.003 0.046 { 0.011 0.232 { 0.100 0.321 { 0.097 35.7 { 4.4 0.340 { 0.126 0.044 { 0.006 0.248 { 0.105 0.058 { 0.005 0.059 { 0.007 0.002 { 0.001 2717
a b
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined. Values for WAT calculated by assuming that the carcass fat content is 9% of body weight (Pohjanvirta et al., 1990).
in CR L-E rats. According to this the disposition half-life of TCDD was 28.9 days in CR L-E rats. The excreted radioactivity (94.1% CR L-E and 90.2% T L-E) was found in feces, whereas urine contributed only 5.9 and 9.8% to the total excretion, respectively. In 32 days CR L-E and T L-E rats excreted 52.4 and 69.7% of the total radioactivity, respectively.
tivity to TCDD. This approach was undertaken to clarify the role of toxicokinetics in the sensitivity difference between genetically closely related substrains. The doses of TCDD administered were nontoxic for both substrains as shown by their body weight development. The doses were 9 and 17 times lower than the corresponding oral LD50 in T L-E and CR L-E rats, respectively (Pohjanvirta et al., 1993; Fan and Rozman, 1994). T L-E rats received the lowest possible dose that would allow accurate detection of radioactivity in tissues with the lowest concentrations. The most distinct feature in the body weight development was a
DISCUSSION
This study was performed to compare the toxicokinetics of TCDD in two substrains of L-E rats with different sensi-
TABLE 3B Percentage of Dose (Radioactivity) Recovered per Total Tissue [11000]:a Turku/AB Long–Evans Rats Time after injection Tissue
1 day
2 days
4 days
8 days
16 days
32 days
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids WATb
0.181 { 0.018 0.662 { 0.049 0.347 { 0.048 0.564 { 0.139 2.155 { 0.390 117 { 19 1.149 { 0.250 0.490 { 0.066 0.396 { 0.073 0.416 { 0.043 0.727 { 0.082 0.016 { 0.003 9703
0.192 { 0.021 0.694 { 0.054 0.274 { 0.027 0.343 { 0.035 1.748 { 0.117 144 { 9 0.930 { 0.062 0.311 { 0.007 0.295 { 0.023 0.397 { 0.023 0.532 { 0.026 0.016 { 0.003 16,569
0.220 { 0.012 1.015 { 0.080 0.299 { 0.026 0.409 { 0.032 1.708 { 0.127 183 { 10 1.126 { 0.105 0.496 { 0.075 0.298 { 0.086 0.488 { 0.024 0.825 { 0.080 0.025 { 0.003 26,844
0.188 { 0.006 0.643 { 0.065 0.254 { 0.005 0.277 { 0.018 1.475 { 0.045 235 { 19 0.970 { 0.041 0.431 { 0.040 0.256 { 0.009 0.513 { 0.035 0.525 { 0.063 0.012 { 0.003 33,667
0.136 { 0.011 0.481 { 0.073 0.223 { 0.040 0.543 { 0.228 1.393 { 0.302 173 { 8 1.200 { 0.406 0.282 { 0.056 0.465 { 0.141 0.359 { 0.050 0.333 { 0.055 0.010 { 0.003 28,252
0.097 { 0.011 0.264 { 0.028 0.357 { 0.056 0.476 { 0.166 1.268 { 0.355 92.8 { 6.5 0.931 { 0.256 0.409 { 0.077 0.510 { 0.154 0.720 { 0.079 0.189 { 0.028 0.038 { 0.004 18,111
a b
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined. Values for WAT calculated by assuming that the carcass fat content is 9% of body weight (Pohjanvirta et al., 1990).
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TABLE 4 Area under the Curves (AUCs)a for [14C]TCDD in Tissues of Male Turku/AB Long–Evans (T L-E) and Charles River Long– Evans (CR L-E) Rats AUC Tissueb
T L-E
CR L-E
TL-E/CRL-E ratio
Adrenals BAT Kidney Liver Lung Serum Skin Small intestine Thymus WAT
8.57 17.04 1.82 44.88 2.54 0.31 3.35 1.70 7.66 32.58
1.24 3.42 0.52 29.49 0.77 0.11 0.54 0.39 0.75 5.67
6.91 4.98 3.50 1.52 3.30 2.82 6.20 4.36 10.21 5.75
a Unit is ‘‘percentage of dose per gram tissue 1 days’’ over the 32-day observation period. b Only tissues suitable for analysis in both substrains were included.
were higher in T L-E rats than in CR L-E rats. Peak concentrations of most tissues were also reached at later time points in T L-E rats, facilitating the detection of an uptake phase for TABLE 5 Kinetic Rate Constants for [14C]TCDD in Tissues of Male Turku/AB Long–Evans (T L-E) and Charles River Long–Evans (CR L-E) Rats FIG. 2. Time courses of [14C]TCDD-associated radioactivity in some tissues of Charles River (open symbols) and Turku/AB (solid symbols) Long–Evans rats. (A) Liver, kidney, adrenals, and serum. (B) White adipose tissue (WAT), large intestine, thymus, and brain. Group means { SE, n Å 4 or 6 (32-day groups).
considerably slower growth rate of T L-E rats compared to CR L-E rats, although their body weights were very similar at the time of dosing (Fig. 1). From the toxicokinetic point of view this is a crucial difference, because distribution, tissue concentrations and elimination are all critically influenced by growth rate which leads to dilution by growth. Tissue distribution of TCDD in the L-E substrains followed the general pattern observed in rodents (cf. Neal et al., 1982; Van den Berg et al., 1994) in that the highest concentrations were found in liver and WAT. Similar to previous studies (Neal et al., 1982; Pohjanvirta et al., 1990; Weber et al., 1993; Li et al., 1995) tissues with lowest concentrations were brain, serum, testis, and muscle. Also, elimination half-lives (calculated from combined urinary and fecal excretion) and the relative proportions of fecal and urinary excretion were within the range previously reported in other rat strains. In general, tissue concentrations and related AUCs of TCDD
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T L-E
CR L-E
Tissue
Uptakea
Eliminationb
Adrenals Brain BAT Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT
0.0002 —c 0.0008 —c
0.0362 —c 0.0424 —c 0.0293 —c 0.0410 0.0139 —c —c 0.0522 0.0490 0.0165 —c —c 0.0281 —c 0.0309
—c 0.0013 —c —c 0.0004 —c —c 0.0003 —c 0.0024
Uptake
0.0019
0.0001
0.0004
Elimination 0.0701 0.0522 0.0662 0.0330 0.0471 0.0532 0.0658 0.0404 0.0450 0.0830 0.0735 0.1009 0.0546 —c 0.0500 0.0470 0.1007 0.0479
a Uptake rate constant calculated by linear regression. The unit is ‘‘days01.’’ Open space means no uptake phase was detected. b Elimination rate constant calculated by SAS one-compartment model. The unit is days01. Negative sign omitted for clarity. c No rate constant could be calculated.
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many tissues in this substrain. In most tissues, however, TCDD concentrations of T L-E rats were clearly higher already on Day 1 after dosing, when body weights of the substrains were equal and no dilution by growth had occurred yet. Therefore, it is concluded that tissue uptake of TCDD is more efficient in T L-E than in CR L-E rats. The faster growth rate of CR L-E rats resulting in faster dilution of TCDD tissue concentrations is also likely to have partially contributed to the earlier appearance of peak tissue concentrations in this substrain compared with T L-E rats. This is also the most likely explanation for the faster tissue elimination of TCDD in CR L-E than in T L-E rats. Sprague–Dawley rats seem to have a faster tissue uptake of TCDD than the L-E substrains. Intravenous administration of TCDD to Sprague–Dawley rats resulted in most cases in peak tissue concentrations within 1 day (Weber et al., 1993; Li et al., 1995), whereas a tissue uptake very similar to the present data was observed in T L-E rats after intraperitoneal dosing (Pohjanvirta et al., 1990). In contrast to other tissues, concentrations and, to a lesser extent, AUCs of TCDD in liver of both substrains were surprisingly similar, though concentrations declined more rapidly in livers of CR L-E rats as a result of faster growth rate. It seems likely that the liver TCDD concentrations during the first few days after dosing are less relevant for toxicity than the later concentrations. Peak liver concentrations were very similar to those measured by Pohjanvirta et al. (1990) in the sensitive T L-E rats and also in the resistant Han/Wistar (Kuopio) rats after intraperitoneal injection, but were twice as high as in male Sprague–Dawley rats (Weber et al., 1993) which have intermediate sensitivity to TCDD (oral LD50 43 mg/kg; Stahl et al., 1992). Therefore, liver TCDD concentrations and AUCs do not correlate with the sensitivity of different rat strains, suggesting either that the
FIG. 3. Urinary, fecal, and combined excretion of [14C]TCDD-associated radioactivity from Charles River (open symbols) and Turku/AB (solid symbols) Long–Evans rats (32-day groups). Group means { SE, n Å 6. Disposition half-lives calculated from total excretion rates (on Days 0–32) were 28.9 days (exponential curve fit) for CR L-E rats and 20.0 days (linear regression) for T L-E rats.
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liver is not a critical target organ in these rat strains or that the crucial liver lesions have different substrain sensitivity. Recent observations by Tuomisto et al. (1995) provide support for the former notion. A good correlation with sensitivity is apparent with WAT TCDD concentrations and AUCs. The AUCs were 5.8 times higher in T L-E than in CR L-E rats. In comparison, Sprague–Dawley rats (Weber et al., 1993) are intermediate both in terms of sensitivity and AUCs. BAT, which contains large numbers of lipid droplets, followed the same pattern. Among tissues with clearly higher concentrations or AUCs in the sensitive T L-E rats were other organs typically affected by TCDD treatment, like thymus (Gupta et al., 1973; Kociba et al., 1976), skin (Schwetz et al., 1973), adrenals (Gupta et al., 1973), testes (Kociba et al., 1976), thyroids (Gupta et al., 1973; Rozman et al., 1986), and pancreas (Rozman et al., 1986; Gorski et al., 1988). Also brain peak TCDD concentrations were 2.6 times higher in T L-E than in CR L-E rats, and this difference increased toward the end of the study, being 7.9-fold on Day 32. Although neither mechanisms of action nor critical target organ(s) can be deduced from toxicokinetic data alone, it is a remarkable coincidence, if it is one, that the liver does not, but all other known target organs of TCDD do reflect the sensitivity difference in terms of corresponding differences in concentrations and AUCs. An important determinant of tissue distribution of TCDD is the expression of proteins that bind TCDD with high affinity, especially cytochrome P450 1A2 (CYP1A2), which is induced by TCDD in liver, but not in extrahepatic tissues (Van den Berg et al., 1994; Goldstein and Linko, 1984). There are no data available about the induction of CYP1A2 in the livers of these rat substrains. It is, however, likely that the liver concentrations of TCDD as measured in these rat strains accurately reflect the expression of CYP1A2 and possibly other high-affinity binding sites for TCDD. Although concentration of radioactivity decreased faster in CR L-E than in T L-E rats, the opposite was true for its fecal and urinary excretion. This is indicative of a faster rate of TCDD metabolism and/or nonbiliary intestinal excretion in T L-E than in CR L-E rats, both of which are likely secondary to higher tissue concentrations of TCDD in T L-E than in CR L-E rats. Retarded excretion of radioactivity in CR L-E during the latter part of the observation period is most likely a consequence of dilution by growth. In T L-E rats the faster excretion rate due to very slow growth dilution was not, however, sufficient to counterbalance the more profound dilution by growth in CR L-E rats. The present study demonstrates the significance of dilution by growth in toxicokinetics of compounds with long half-lives. This phenomenon is especially important for dioxins, which affect body weight development. At toxic doses, which cause growth retardation and body weight loss, toxi-
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cokinetics are modified by the outcome of toxicity, resulting in decreased volume of distribution and, subsequently, redistribution of TCDD (Weber et al., 1993; Van den Berg et al., 1994). In conclusion, the toxicokinetic profile of TCDD in the two L-E substrains provides a likely explanation for the fivefold greater sensitivity of T L-E than CR L-E rats to TCDD, since the observed differences in tissue concentrations and AUC values (especially thymus, WAT, BAT, and adrenals) are in good agreement with the difference in susceptibility with the exception of the liver. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics between these two rat substrains is a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the different susceptibility of these rat substrains to TCDD. ACKNOWLEDGMENTS We thank Dr. Linda S. Birnbaum (U.S. EPA, Research Triangle Park, Chapel Hill, NC) for generously providing supplies and the use of a sample oxidizer for the analysis of fecal samples. Ed Brown (Department of Biometry, University of Kansas Medical Center, Kansas City, KS) performed the calculation on kinetic constants and AUCs with the SAS software package. This study was supported by the GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Germany, and the Academy of Finland, Research Council for Environmental Sciences (Grant 5410/4011/89). Bernhard Stahl was supported by a fellowship of the Deutsche Forschungsgemeinschaft (Sta 300/3-3).
REFERENCES Beatty, P. W., Vaughn, W. K., and Neal, R. A. (1978). Effect of alteration of rat hepatic mixed-function oxidase (MFO) activity on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Appl. Pharmacol. 45, 513–519. Fan, F., and Rozman, K. (1994). Relationship between acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and disturbance of intermediary metabolism in the Long–Evans rat. Arch. Toxicol. 69, 73–78. Goldstein, J. A., and Linko, P. (1984). Differential induction of two 2,3,7,8tetrachlorodibenzo-p-dioxin-inducible forms of cytochrome P450 in extrahepatic versus hepatic tissues. Mol. Pharmacol. 25, 185–191. Gorski, J. R., Muzi, G., Weber, L. W. D., Pereira, D. W., Arceo, R. J., Iatropoulos, M. J., and Rozman, K. (1988). Some endocrine and morpho-
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logical aspects of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Pathol. 16, 313–320. Gupta, B. N., Vos, J. G., Moore, J. A., Zinkl, J. G., and Bullock, B. C. (1973). Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ. Health Perspect. 5, 125–140. Kociba, R. J., Keeler, P. A., Park, C. N., and Gehring, P. J. (1976). 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD): Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35, 553–574. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., and Dittenber, D. A. (1978). Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46, 279–303. Li, X., Weber, L. W. D., and Rozman, K. K. (1995). Toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female Sprague–Dawley rats including placental and lactational transfer to fetuses and neonates. Fundam. Appl. Toxicol. 27, 70–76. Neal, R. A., Olson, J. R., Gasiewicz, T. A., and Geiger, L. E. (1982). The toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian systems. Drug Metab. Rev. 13, 355–385. Pohjanvirta, R., Unkila, M., and Tuomisto, J. (1993). Comparative acute lethality of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin in the most TCDD susceptible and the most TCDD resistant rat strain. Pharmacol. Toxicol. 73, 52–56. Pohjanvirta, R., Vartiainen, T., Uusi-Rauva, A., Mo¨nkko¨nen, J., and Tuomisto, J. (1990). Tissue distribution, metabolisms, and excretion of 14CTCDD in a TCDD-susceptible and a TCDD-resistant rat strain. Pharmacol. Toxicol. 66, 93–100. Rozman, K., Pereira, D., and Iatropoulos, M. J. (1986). Histopathology of interscapular brown adipose tissue, thyroid, and pancreas in 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)-treated rats. Toxicol. Appl. Pharmacol. 82, 551–559. Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe, V. K., Gehring, P. J., Emerson, J. L., and Gebrig, C. G. (1973). Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5, 87–99. Stahl, B. U., Kettrup, A., and Rozman, K. (1992). Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. Part I: Acute toxicity and toxic equivalent factors (TEFs). Arch. Toxicol. 66, 471–477. Tuomisto, J., Andrzejewski, W., Unkila, M., Pohjanvirta, R., Linden, J., Vartiainen, T., and Tuomisto, L. (1995). Modulation of TCDD-induced wasting syndrome by portocaval anastomosis and vagotomy in Long– Evans and Han/Wistar rats. Eur. J. Pharmacol. 292, 277–285. Van den Berg, M., De Jongh, J., Poiger, H., and Olson, J. R. (1994). The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit. Rev. Toxicol. 24, 1–74. Weber, L. W. D., Ernst, S. W., Stahl, B. U., and Rozman, K. (1993). Tissue distribution and toxicokinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats after intravenous injection. Fundam. Appl. Toxicol. 21, 523–534.
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0090
Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection MATTI VILUKSELA,*,† THANG V. DUONG,* BERNHARD U. STAHL,*,‡ XUELIN LI,* JOUKO TUOMISTO,† AND KARL K. ROZMAN*,‡ *Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417; †Department of Toxicology, National Public Health Institute, Kuopio, Finland; and ‡Section of Environmental Toxicology, GSF-Institut fu¨r Toxikologie, 85758 Neuherberg, Federal Republic of Germany Received July 27, 1995; accepted January 29, 1996
Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in Two Substrains of Male Long–Evans Rats after Intravenous Injection. VILUKSELA, M., DUONG, T. V., STAHL, B. U., LI, X., TUOMISTO, J., AND ROZMAN, K. K. (1996). Fundam. Appl. Toxicol. 31, 184–191. Toxicokinetics of a nontoxic intravenous dose of 14C-labeled TCDD were studied in two substrains of Long–Evans (L-E) rats with a fivefold difference in sensitivity in terms of TCDD-induced mortality. The Turku/AB Long–Evans rat (T L-E) is the most sensitive rat strain with an oral LD50 of 17.7 mg/kg, whereas the Charles River Long–Evans rat (CR L-E) is a more resistant strain (oral LD50 95.2 mg/kg). Samples of 18 tissues were collected 1, 2, 4, 8, 16, and 32 days after dosing and analyzed for radioactivity. Body weight and fecal and urinary excretion of radioactivity were monitored daily during the 32-day study period. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% in 32 days compared with only 16% in T L-E rats. This difference was not caused by toxicity, because the weight gain was identical in control and TCDD-treated rats of both substrains. Tissue concentrations of [14C]TCDD-associated radioactivity and area under the curve (AUC) values were lower in CR L-E than in T L-E rats. The most pronounced differences were found in thymus, white adipose tissue, brown adipose tissue, and adrenals. The decrease of TCDD concentration in tissues was faster in CR L-E than in T L-E rats, whereas fecal and urinary excretion was faster in T L-E than in C L-E rats. Elimination half-life was 20.0 days in T L-E rats and 28.9 days in CR L-E rats. Differential toxicokinetics of TCDD in the two L-E substrains provide a likely explanation for the greater sensitivity of the T L-E strain, since observed differences in tissue concentrations and AUC values are in good agreement with the difference in susceptibility. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics seems to be a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the difference in susceptibility. More rapid excretion of TCDD in T L-E rats than in CR L-E rats is clearly a result of higher tissue concentrations in T L-E rats. However, this faster excretion rate is not sufficient to counterbalance the much slower dilution by growth in T L-E rats than in CR L-E rats. Thus, dilution by growth can be a more 0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.
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important factor in determining the toxicokinetics and toxicity of TCDD in rodents than is excretion. q 1996 Society of Toxicology
TCDD is extremely toxic to some animal species, with huge inter- and intraspecies variations in sensitivity regarding acute toxicity (reviewed by Pohjanvirta and Tuomisto, 1994). For example, a more than a 1000-fold sensitivity difference has been reported between two rat strains (Pohjanvirta et al., 1993). The Han/Wistar (Kuopio), a substrain of the Wistar rat, is probably the most TCDD-resistant laboratory animal. It has been impossible to determine the LD50 for this strain in an acute study because of solubility limitations of TCDD (Pohjanvirta et al., 1993). The most sensitive rat strain is the Turku/AB Long–Evans substrain, being only slightly less sensitive than the guinea pig, which is the most TCDD-sensitive species. Extreme sensitivity to TCDD, however, is not a specific feature of the Long–Evans strain, since the Charles River substrain seems to be about five times more resistant to TCDD than the Turku/AB substrain (oral LD50 in male CR L-E rats is 95.2 mg/kg vs 17.7 mg/ kg in male T L-E rats) (Fan and Rozman, 1994; Pohjanvirta et al., 1993). Toxicokinetic factors clearly can explain only partially the differential sensitivity of various species to TCDD (Van den Berg et al., 1994). In fact, kinetic factors were reported to have at most a contributory impact on the intraspecies difference in TCDD-induced lethality between the most sensitive (T L-E) and the most resistant (Han/Wistar, Kuopio) rat strains (Pohjanvirta et al., 1990). On the other hand, recent studies from this laboratory suggested that a relatively small sensitivity difference between male and female Sprague–Dawley rats (cf. Beatty et al., 1978; Kociba et al., 1976, 1978) is likely to be due to toxicokinetics alone rather than to gender-specific physiological differences (Weber et al., 1993; Li et al., 1995). To reduce the confounding effects of physiological dissimilarities between species or even strains, we have utilized in the present study the fivefold intrastrain sensitivity differ-
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ence to examine the role of toxicokinetics in the toxicity of TCDD. Intravenous route of administration was chosen to allow an accurate evaluation of the toxicokinetic profile as well as to permit comparisons with two previous kinetic studies from this laboratory with the same route of administration and a comparable study design (Weber et al., 1993; Li et al., 1995). MATERIALS AND METHODS Animals. Male outbred CR L-E were obtained from Charles River (Boston, MA). Male inbred L-E (Turku/AB) rats (T L-E), originally obtained from the National Laboratory Animal Center of the University of Kuopio (Kuopio, Finland), were bred at the Laboratory Animal Resources of the University of Kansas Medical Center (Kansas City, KS). Rats were about 8 weeks old at the beginning of the experiments. They were kept individually in stainless steel wire-bottom cages or polycarbonate metabolism cages for urine and feces collection (32-day time point). Their maintenance consisted of Purina 5001 rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum. Their room was artificially illuminated from 6 AM to 6 PM and air-conditioned with a Honeywell DeltaNet W1044 computer controller (Minneapolis, MN) programmed to maintain the ambient temperature at 21–227C and relative humidity at 40–60%. Rats were acclimated to experimental conditions for 1 week before dosing. Emulsions. 14C-labeled TCDD (ED-902H, sp act 122 mCi/mmol) was obtained from Cambridge Isotope Laboratories Inc. (Woburn, MA). The solvent (toluene) was evaporated and [14C]TCDD redissolved in corn oil (Sigma) by stirring for 5 days. Thirty milligrams of cholic acid was dissolved in 9 ml of Ringer’s solution with the aid of 1 N NaOH, after which 30 mg of lecithin (Sigma) was added. The pH was adjusted to 8, and 1 ml of corn oil containing [14C]TCDD was added and shaken and the emulsion sonicated (Sonifier 185, micro-tip, Branson Sonic Power, Danbury, CT). The final emulsion was stored in a refrigerator and found to be stable for at least 3 months. Treatment. Animals were randomly allocated into groups of four or six (groups sampled on Day 32) and given a slow (1 min) intravenous injection of 14C-labeled TCDD emulsion into the lateral tail vein using Quick-Cath over-the-needle Teflon catheters (22 gauge, Baxter, Deerfield, IL). CR L-E rats received a single dose of 5.6 mg/kg (2.12 mCi/kg) and T L-E rats 2.0 mg/kg (0.76 mCi/kg) of [14C]TCDD in a volume of 4 ml/kg. The doses were selected to be as low as possible (to avoid toxicity), but still sufficient to obtain a reasonable level of detection. Body weights, urine volumes and dry feces weights were recorded daily for the 32-day groups. Rats were killed by decapitation 1, 2, 4, 8, 16, and 32 days after dosing. Tissue collection. Serum (from trunk blood), liver, kidney, thymus, heart, pancreas, lung, spleen, brain, thyroids, adrenals, and testes were weighed and duplicate samples (except for thyroids and adrenals) of about 100–200 mg prepared for analysis. Duplicate samples of the same size were also taken from epididymal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), proximal large intestine, small intestine (jejunum), muscle, and dorsal skin. The intestines were flushed with saline and the fat was scraped off from the skin with a scalpel. Tissue samples were digested overnight by 1 ml of Soluene-350 tissue solubilizer (Packard Instrument Co., Downers Grove, IL) at 507C. Radioactivity was determined, after the addition of 10 ml scintillation cocktail (Hionic Fluor, Packard Instrument Co.), in a TriCarb-1900AC liquid scintillation counter (Packard Instrument Co.). Duplicate aliquots of 1 ml were procured from serum and urine, and radioactivity was measured after addition of 10 ml scintillation cocktail. Feces were dried and ground, and radioactivity was determined after combustion of about 100 mg duplicate samples in a sample oxidizer (Packard 306B biological oxidizer).
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FIG. 1. Body weight development of the [14C]TCDD-treated Charles River (open circles) and Turku/AB (solid circles) Long–Evans rats (32-day groups). The dotted lines represent untreated control groups of both strains. (CR L-E control rats were slightly older and thus heavier than the TCDD-treated rats.) Group means { SE, n Å 6, except for controls, n Å 4.
Calculations. Radioactivity found in various tissues was converted to percentage of total dose recovered per gram of tissue (or total tissue) or percentage of total dose excreted per day (urine, feces). Uptake rate constants were calculated by linear regression. Rate constants of elimination (one-compartment model), areas under the curve (AUC), and half-lives were calculated on a mainframe computer using the SAS software package. Sigma-minus plots (log percentage of administered dose remaining to be excreted vs day) were constructed for fecal, urinary, and combined excretions.
RESULTS
Body weight development of the 32-day groups and their controls is shown in Fig. 1. CR L-E rats grew significantly faster than T L-E rats, increasing their body weight by 60% (146 g) compared to 16% (41 g) in T L-E rats. The difference in growth rate was not caused by toxicity, since untreated control rats gained weight similar to those treated with TCDD of both substrains. Percentages of dose (radioactivity) recovered per gram of tissue are depicted in Tables 1A and 1B, absolute organ weights in Tables 2A and 2B, and percentages of dose per total tissue in Tables 3A and 3B. The only remarkable changes in absolute organ weights (Tables 2A and 2B) were the same reported also by others, i.e., increased liver weight (more prominent in CR L-E rats) and decreased thymus weights (more pronounced in T L-E rats). The time course of [14C]TCDD-associated radioactivity in selected tissues is illustrated in Fig. 2. The tissue distribution profile of TCDD was distinctly and consistently different in the two substrains. All tissue concentrations were lower in CR L-E than in T L-E rats with peak concentrations being typically reached between 1 and 2 days (CR L-E) versus 4 to 8 days (T L-E) after dosing (Fig. 2; Tables 1A, 1B, 3A, and 3B). It appears that this pattern of tissue distribution is at least partly due to slow release of TCDD from the emulsion (vehicle) trapped in the lymphatic system and/or to redistribution of TCDD from highly perfused to poorly perfused tissues.
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TABLE 1A Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [11000]:a Charles River Long–Evans Rats Time after injection Tissue Adrenals BAT Brain Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT Liver/WAT a
1 day 101.5 { 231.1 { 8.4 { 35.9 { 31.3 { 17.9 { 1507.1 { 43.3 { 6.4 { 28.7 { 6.8 { 32.3 { 28.1 { 38.8 { 7.0 { 46.0 { 60.5 { 183.2 { 8.2
2 days 10.4 21.3 0.4 12.4 2.6 1.0 80.8 6.2 0.6 2.8 0.3 1.3 2.0 9.3 0.6 3.2 12.1 15.2
73.9 { 257.5 { 6.9 { 29.6 { 31.0 { 22.5 { 1777.5 { 52.0 { 5.8 { 21.9 { 8.7 { 40.6 { 25.4 { 59.9 { 5.8 { 43.0 { 43.5 { 290.6 { 6.1
4 days
21.6 13.7 0.5 7.5 3.8 4.1 211.0 10.9 0.3 2.9 1.8 3.4 3.9 23.2 0.6 6.1 5.2 63.2
72.9 { 211.4 { 7.3 { 25.4 { 27.2 { 14.0 { 1759.8 { 37.2 { 5.0 { 19.2 { 7.4 { 41.0 { 23.8 { 33.3 { 5.1 { 43.6 { 21.1 { 299.2 { 5.9
8 days
5.0 12.5 1.4 9.1 2.7 1.0 241.0 5.9 0.2 1.7 0.8 2.7 1.9 6.1 0.2 2.7 3.5 18.4
53.0 { 128.6 { 5.2 { 22.8 { 25.0 { 11.0 { 1635.0 { 34.3 { 3.6 { 13.3 { 5.3 { 23.8 { 15.0 { 52.6 { 4.7 { 25.6 { 19.6 { 262.5 { 6.2
9.6 11.7 1.4 7.3 3.1 0.6 152.0 8.1 0.2 1.5 0.7 1.6 1.3 22.4 0.8 0.7 3.3 9.9
16 days 32.5 92.2 4.1 21.9 16.9 8.6 841.2 28.9 3.0 9.1 3.2 14.1 11.0 32.5 2.8 24.1 19.7 171.0
{ 2.4 { 4.1 { 1.2 { 10.0 { 4.9 { 0.8 { 106.2 { 8.6 { 0.4 { 0.8 { 0.5 { 1.0 { 1.7 { 13.8 { 0.1 { 2.7 { 4.1 { 14.0 4.9
32 days 22.2 { 47.2 { 2.7 { 19.3 { 13.2 { 4.8 { 234.2 { 26.1 { 2.1 { 5.3 { 0.9 { 4.5 { 6.5 { 41.1 { 1.7 { 12.5 { 12.3 { 77.4 { 3.0
2.4 2.3 0.4 7.5 4.5 0.4 31.2 9.0 0.3 0.3 0.1 0.6 0.5 18.3 0.1 0.9 2.7 2.2
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined.
AUCs were also lower in CR L-E rats than in T L-E rats (Table 4). In both substrains the highest tissue concentrations and AUCs were measured in liver, followed by WAT, BAT,
and adrenals. The most remarkable differences in peak concentrations between the two substrains were found in thymus (8-fold) and WAT (4.9-fold). Skin, adrenals, testes, BAT,
TABLE 1B Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [11000]:a Turku/AB Long–Evans Rats Time after injection Tissue Adrenals BAT Brain Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT Liver/WAT a
1 day 369.4 { 699.4 { 22.2 { 70.2 { 99.2 { 48.0 { 1227.5 { 114.3 { 17.4 { 69.0 { 10.7 { 66.2 { 64.6 { 95.9 { 15.9 { 313.8 { 113.8 { 424.1 { 2.9
33.3 17.6 3.1 16.2 17.7 4.2 164.1 24.7 1.9 3.6 1.2 4.6 5.1 20.3 1.3 32.2 11.4 43.8
2 days 372.0 { 731.5 { 17.1 { 45.6 { 85.9 { 45.2 { 1438.2 { 93.4 { 14.8 { 60.2 { 12.0 { 123.5 { 58.8 { 65.8 { 15.6 { 239.7 { 143.6 { 712.6 { 2.0
36.7 52.7 1.7 3.9 6.8 3.4 103.1 7.9 1.8 2.9 1.4 8.9 6.5 4.7 1.0 13.5 22.4 52.7
4 days 419.4 { 889.4 { 19.1 { 53.4 { 78.2 { 51.3 { 1691.1 { 109.4 { 20.5 { 75.6 { 13.6 { 172.5 { 66.2 { 69.7 { 19.0 { 368.5 { 195.4 { 1158.1 { 1.5
8 days
26.6 63.5 1.7 5.1 4.0 4.0 107.0 10.4 0.9 10.9 1.4 18.3 5.3 11.4 1.0 31.1 8.2 92.7
366.6 { 702.1 { 15.9 { 35.6 { 71.7 { 48.9 { 2149.4 { 95.8 { 14.2 { 61.8 { 16.5 { 168.6 { 63.4 { 52.8 { 19.0 { 281.3 { 86.1 { 1451.2 { 1.5
11.5 26.2 0.6 1.3 1.7 3.3 195.9 3.5 0.3 4.3 1.1 23.4 2.0 2.8 1.5 25.4 23.2 59.7
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined.
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16 days 242.4 { 538.1 { 14.4 { 62.0 { 63.1 { 35.3 { 1513.6 { 96.6 { 14.7 { 46.3 { 8.1 { 99.1 { 50.1 { 95.4 { 13.6 { 242.9 { 68.1 { 1167.9 { 1.3
18.7 45.5 2.8 25.1 12.7 3.2 22.4 22.3 0.8 6.6 0.2 16.0 3.6 27.2 1.2 32.1 19.3 29.8
32 days 175.8 { 314.6 { 21.3 { 54.9 { 52.2 { 39.0 { 819.1 { 79.3 { 20.6 { 65.5 { 5.5 { 45.4 { 42.9 { 102.8 { 24.9 { 169.6 { 272.4 { 667.1 { 1.2
22.6 9.3 3.4 18.9 14.7 2.4 58.7 22.3 2.5 10.1 0.5 3.8 3.7 32.9 2.9 20.0 28.0 20.1
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TABLE 2A Absolute Organ Weights (g):a Charles River Long–Evans Rats Time after injection Tissue
1 day
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids a
0.052 0.240 1.875 1.024 2.685 13.477 1.153 0.884 0.647 3.036 0.590 0.018
{ { { { { { { { { { { {
2 days
0.006 0.014 0.066 0.032 0.097 0.617 0.048 0.059 0.028 0.155 0.055 0.001
0.047 0.262 1.792 0.984 2.477 15.008 1.113 0.774 0.630 3.221 0.423 0.015
{ { { { { { { { { { { {
4 days
0.005 0.025 0.048 0.052 0.045 0.866 0.077 0.069 0.034 0.083 0.050 0.001
0.057 0.139 1.854 1.034 2.669 16.807 1.132 0.962 0.705 3.353 0.459 0.018
{ { { { { { { { { { { {
8 days
0.003 0.013 0.054 0.075 0.210 1.240 0.030 0.057 0.068 0.164 0.034 0.001
0.053 0.106 1.880 1.148 2.270 17.427 1.510 0.879 0.643 3.332 0.372 0.017
{ { { { { { { { { { { {
16 days
0.001 0.009 0.053 0.156 0.206 0.868 0.112 0.066 0.038 0.083 0.021 0.000
0.052 0.158 1.811 1.352 2.558 17.174 1.859 0.775 0.637 3.391 0.403 0.024
{ { { { { { { { { { { {
32 days
0.003 0.016 0.051 0.215 0.180 1.139 0.580 0.100 0.041 0.116 0.032 0.002
0.054 0.135 1.632 1.158 2.558 15.561 1.328 0.815 0.626 3.448 0.469 0.019
{ { { { { { { { { { { {
0.002 0.007 0.296 0.069 0.182 1.125 0.111 0.087 0.035 0.120 0.048 0.002
Mean { SE, n Å 4 (6 for the 32-day group).
thyroids, muscle, and kidney showed 3.2- to 4.3-fold differences. The most pronounced differences in terms of AUC values were found in thymus (10.2-fold), adrenals (6.9-fold), skin (6.2-fold), WAT (5.8-fold), and BAT (5-fold). Most notably, the substrain differences in liver peak concentrations and AUCs were only 1.2- and 1.5-fold, respectively. The decrease in TCDD tissue concentrations was faster in CR L-E than in T L-E rats (Table 5). Tissue half-lives of [14C]TCDD-associated radioactivity in CR L-E and T L-E rats were 9.4 and 13.3 days for serum, 10.5 and 16.9 days for liver, and 14.5 and 22.4 days for WAT, respectively. In contrast to the disappearance of TCDD from tissues, its fecal and urinary excretion occurred at a lower rate in CR L-E rats than in T L-E rats (Fig. 3). In T L-E rats the excretion followed a strictly
linear pattern (r Å 0.987) on a semilogarithmic plot during 32 days, whereas in CR L-E rats fecal and total (fecal / urinary) excretion curves flattened out, indicating retarded elimination during the second half of the observation period. The elimination half-life of TCDD, calculated from total excretion using linear regression, was 20.0 days in T L-E rats. Because of retarded excretion of TCDD in CR L-E rats a curve fit was obtained according to the equation 1.2
y Å a(1 0 b 1 e0c1t ), where a Å 41.658, b Å 01.3715, and c Å 0.035237, which described accurately (r Å 0.999) the total elimination curve
TABLE 2B Absolute Organ Weights (g):a Turku/AB Long–Evans Rats Time after injection Tissue
1 day
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids a
0.049 0.095 1.568 0.795 2.194 9.488 1.005 0.707 0.422 2.612 0.232 0.014
{ { { { { { { { { { { {
2 days
0.003 0.007 0.030 0.028 0.151 0.279 0.030 0.074 0.028 0.089 0.014 0.001
0.052 0.095 1.602 0.749 2.040 10.045 1.001 0.523 0.449 2.542 0.223 0.012
{ { { { { { { { { { { {
4 days
0.001 0.004 0.004 0.015 0.030 0.249 0.029 0.038 0.018 0.023 0.012 0.001
0.053 0.115 1.571 0.770 2.181 10.852 1.030 0.657 0.404 2.578 0.225 0.013
{ { { { { { { { { { { {
0.001 0.007 0.017 0.019 0.090 0.371 0.033 0.048 0.064 0.037 0.015 0.002
8 days 0.051 0.092 1.605 0.777 2.058 10.949 1.012 0.702 0.487 2.701 0.185 0.013
{ { { { { { { { { { { {
0.0005 0.008 0.041 0.026 0.053 0.268 0.021 0.066 0.021 0.071 0.007 0.001
16 days 0.056 0.089 1.561 0.856 2.198 11.451 1.170 0.598 0.481 2.618 0.135 0.014
Mean { SE, n Å 4 (6 for the 32-day group).
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{ { { { { { { { { { { {
0.001 0.008 0.054 0.028 0.071 0.639 0.114 0.051 0.025 0.190 0.006 0.001
32 days 0.056 0.084 1.679 0.870 2.431 11.353 1.181 0.619 0.502 2.902 0.110 0.014
{ { { { { { { { { { { {
0.002 0.007 0.017 0.037 0.035 0.319 0.050 0.037 0.013 0.036 0.005 0.001
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TABLE 3A Percentage of Dose (Radioactivity) Recovered per Total Tissue [11000]:a Charles River Long–Evans Rats Time after injection Tissue
1 day
2 days
4 days
8 days
16 days
32 days
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids WATb
0.053 { 0.008 0.555 { 0.069 0.157 { 0.009 0.368 { 0.124 0.843 { 0.079 204 { 19 0.506 { 0.086 0.258 { 0.040 0.254 { 0.064 0.211 { 0.022 0.274 { 0.035 0.011 { 0.003 4218
0.032 { 0.009 0.671 { 0.065 0.123 { 0.009 0.286 { 0.062 0.764 { 0.084 264 { 30 0.587 { 0.151 0.166 { 0.017 0.394 { 0.175 0.186 { 0.019 0.179 { 0.024 0.006 { 0.000 6910
0.042 { 0.003 0.293 { 0.032 0.135 { 0.026 0.276 { 0.119 0.717 { 0.066 287 { 20 0.422 { 0.071 0.183 { 0.015 0.231 { 0.045 0.171 { 0.015 0.201 { 0.023 0.004 { 0.001 7474
0.028 { 0.005 0.137 { 0.017 0.097 { 0.024 0.295 { 0.139 0.583 { 0.111 281 { 11 0.536 { 0.165 0.115 { 0.010 0.359 { 0.176 0.157 { 0.025 0.096 { 0.007 0.003 { 0.0005 7088
0.017 { 0.002 0.147 { 0.020 0.073 { 0.020 0.268 { 0.109 0.416 { 0.102 141 { 10 0.491 { 0.137 0.069 { 0.008 0.199 { 0.076 0.095 { 0.004 0.098 { 0.015 0.005 { 0.001 5118
0.012 { 0.001 0.063 { 0.003 0.046 { 0.011 0.232 { 0.100 0.321 { 0.097 35.7 { 4.4 0.340 { 0.126 0.044 { 0.006 0.248 { 0.105 0.058 { 0.005 0.059 { 0.007 0.002 { 0.001 2717
a b
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined. Values for WAT calculated by assuming that the carcass fat content is 9% of body weight (Pohjanvirta et al., 1990).
in CR L-E rats. According to this the disposition half-life of TCDD was 28.9 days in CR L-E rats. The excreted radioactivity (94.1% CR L-E and 90.2% T L-E) was found in feces, whereas urine contributed only 5.9 and 9.8% to the total excretion, respectively. In 32 days CR L-E and T L-E rats excreted 52.4 and 69.7% of the total radioactivity, respectively.
tivity to TCDD. This approach was undertaken to clarify the role of toxicokinetics in the sensitivity difference between genetically closely related substrains. The doses of TCDD administered were nontoxic for both substrains as shown by their body weight development. The doses were 9 and 17 times lower than the corresponding oral LD50 in T L-E and CR L-E rats, respectively (Pohjanvirta et al., 1993; Fan and Rozman, 1994). T L-E rats received the lowest possible dose that would allow accurate detection of radioactivity in tissues with the lowest concentrations. The most distinct feature in the body weight development was a
DISCUSSION
This study was performed to compare the toxicokinetics of TCDD in two substrains of L-E rats with different sensi-
TABLE 3B Percentage of Dose (Radioactivity) Recovered per Total Tissue [11000]:a Turku/AB Long–Evans Rats Time after injection Tissue
1 day
2 days
4 days
8 days
16 days
32 days
Adrenals BAT Brain Heart Kidney Liver Lung Pancreas Spleen Testis Thymus Thyroids WATb
0.181 { 0.018 0.662 { 0.049 0.347 { 0.048 0.564 { 0.139 2.155 { 0.390 117 { 19 1.149 { 0.250 0.490 { 0.066 0.396 { 0.073 0.416 { 0.043 0.727 { 0.082 0.016 { 0.003 9703
0.192 { 0.021 0.694 { 0.054 0.274 { 0.027 0.343 { 0.035 1.748 { 0.117 144 { 9 0.930 { 0.062 0.311 { 0.007 0.295 { 0.023 0.397 { 0.023 0.532 { 0.026 0.016 { 0.003 16,569
0.220 { 0.012 1.015 { 0.080 0.299 { 0.026 0.409 { 0.032 1.708 { 0.127 183 { 10 1.126 { 0.105 0.496 { 0.075 0.298 { 0.086 0.488 { 0.024 0.825 { 0.080 0.025 { 0.003 26,844
0.188 { 0.006 0.643 { 0.065 0.254 { 0.005 0.277 { 0.018 1.475 { 0.045 235 { 19 0.970 { 0.041 0.431 { 0.040 0.256 { 0.009 0.513 { 0.035 0.525 { 0.063 0.012 { 0.003 33,667
0.136 { 0.011 0.481 { 0.073 0.223 { 0.040 0.543 { 0.228 1.393 { 0.302 173 { 8 1.200 { 0.406 0.282 { 0.056 0.465 { 0.141 0.359 { 0.050 0.333 { 0.055 0.010 { 0.003 28,252
0.097 { 0.011 0.264 { 0.028 0.357 { 0.056 0.476 { 0.166 1.268 { 0.355 92.8 { 6.5 0.931 { 0.256 0.409 { 0.077 0.510 { 0.154 0.720 { 0.079 0.189 { 0.028 0.038 { 0.004 18,111
a b
Mean { SE, n Å 4 (6 for the 32-day group). Highest value underlined. Values for WAT calculated by assuming that the carcass fat content is 9% of body weight (Pohjanvirta et al., 1990).
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TABLE 4 Area under the Curves (AUCs)a for [14C]TCDD in Tissues of Male Turku/AB Long–Evans (T L-E) and Charles River Long– Evans (CR L-E) Rats AUC Tissueb
T L-E
CR L-E
TL-E/CRL-E ratio
Adrenals BAT Kidney Liver Lung Serum Skin Small intestine Thymus WAT
8.57 17.04 1.82 44.88 2.54 0.31 3.35 1.70 7.66 32.58
1.24 3.42 0.52 29.49 0.77 0.11 0.54 0.39 0.75 5.67
6.91 4.98 3.50 1.52 3.30 2.82 6.20 4.36 10.21 5.75
a Unit is ‘‘percentage of dose per gram tissue 1 days’’ over the 32-day observation period. b Only tissues suitable for analysis in both substrains were included.
were higher in T L-E rats than in CR L-E rats. Peak concentrations of most tissues were also reached at later time points in T L-E rats, facilitating the detection of an uptake phase for TABLE 5 Kinetic Rate Constants for [14C]TCDD in Tissues of Male Turku/AB Long–Evans (T L-E) and Charles River Long–Evans (CR L-E) Rats FIG. 2. Time courses of [14C]TCDD-associated radioactivity in some tissues of Charles River (open symbols) and Turku/AB (solid symbols) Long–Evans rats. (A) Liver, kidney, adrenals, and serum. (B) White adipose tissue (WAT), large intestine, thymus, and brain. Group means { SE, n Å 4 or 6 (32-day groups).
considerably slower growth rate of T L-E rats compared to CR L-E rats, although their body weights were very similar at the time of dosing (Fig. 1). From the toxicokinetic point of view this is a crucial difference, because distribution, tissue concentrations and elimination are all critically influenced by growth rate which leads to dilution by growth. Tissue distribution of TCDD in the L-E substrains followed the general pattern observed in rodents (cf. Neal et al., 1982; Van den Berg et al., 1994) in that the highest concentrations were found in liver and WAT. Similar to previous studies (Neal et al., 1982; Pohjanvirta et al., 1990; Weber et al., 1993; Li et al., 1995) tissues with lowest concentrations were brain, serum, testis, and muscle. Also, elimination half-lives (calculated from combined urinary and fecal excretion) and the relative proportions of fecal and urinary excretion were within the range previously reported in other rat strains. In general, tissue concentrations and related AUCs of TCDD
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T L-E
CR L-E
Tissue
Uptakea
Eliminationb
Adrenals Brain BAT Heart Kidney Large intestine Liver Lung Muscle Pancreas Serum Skin Small intestine Spleen Testis Thymus Thyroids WAT
0.0002 —c 0.0008 —c
0.0362 —c 0.0424 —c 0.0293 —c 0.0410 0.0139 —c —c 0.0522 0.0490 0.0165 —c —c 0.0281 —c 0.0309
—c 0.0013 —c —c 0.0004 —c —c 0.0003 —c 0.0024
Uptake
0.0019
0.0001
0.0004
Elimination 0.0701 0.0522 0.0662 0.0330 0.0471 0.0532 0.0658 0.0404 0.0450 0.0830 0.0735 0.1009 0.0546 —c 0.0500 0.0470 0.1007 0.0479
a Uptake rate constant calculated by linear regression. The unit is ‘‘days01.’’ Open space means no uptake phase was detected. b Elimination rate constant calculated by SAS one-compartment model. The unit is days01. Negative sign omitted for clarity. c No rate constant could be calculated.
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many tissues in this substrain. In most tissues, however, TCDD concentrations of T L-E rats were clearly higher already on Day 1 after dosing, when body weights of the substrains were equal and no dilution by growth had occurred yet. Therefore, it is concluded that tissue uptake of TCDD is more efficient in T L-E than in CR L-E rats. The faster growth rate of CR L-E rats resulting in faster dilution of TCDD tissue concentrations is also likely to have partially contributed to the earlier appearance of peak tissue concentrations in this substrain compared with T L-E rats. This is also the most likely explanation for the faster tissue elimination of TCDD in CR L-E than in T L-E rats. Sprague–Dawley rats seem to have a faster tissue uptake of TCDD than the L-E substrains. Intravenous administration of TCDD to Sprague–Dawley rats resulted in most cases in peak tissue concentrations within 1 day (Weber et al., 1993; Li et al., 1995), whereas a tissue uptake very similar to the present data was observed in T L-E rats after intraperitoneal dosing (Pohjanvirta et al., 1990). In contrast to other tissues, concentrations and, to a lesser extent, AUCs of TCDD in liver of both substrains were surprisingly similar, though concentrations declined more rapidly in livers of CR L-E rats as a result of faster growth rate. It seems likely that the liver TCDD concentrations during the first few days after dosing are less relevant for toxicity than the later concentrations. Peak liver concentrations were very similar to those measured by Pohjanvirta et al. (1990) in the sensitive T L-E rats and also in the resistant Han/Wistar (Kuopio) rats after intraperitoneal injection, but were twice as high as in male Sprague–Dawley rats (Weber et al., 1993) which have intermediate sensitivity to TCDD (oral LD50 43 mg/kg; Stahl et al., 1992). Therefore, liver TCDD concentrations and AUCs do not correlate with the sensitivity of different rat strains, suggesting either that the
FIG. 3. Urinary, fecal, and combined excretion of [14C]TCDD-associated radioactivity from Charles River (open symbols) and Turku/AB (solid symbols) Long–Evans rats (32-day groups). Group means { SE, n Å 6. Disposition half-lives calculated from total excretion rates (on Days 0–32) were 28.9 days (exponential curve fit) for CR L-E rats and 20.0 days (linear regression) for T L-E rats.
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liver is not a critical target organ in these rat strains or that the crucial liver lesions have different substrain sensitivity. Recent observations by Tuomisto et al. (1995) provide support for the former notion. A good correlation with sensitivity is apparent with WAT TCDD concentrations and AUCs. The AUCs were 5.8 times higher in T L-E than in CR L-E rats. In comparison, Sprague–Dawley rats (Weber et al., 1993) are intermediate both in terms of sensitivity and AUCs. BAT, which contains large numbers of lipid droplets, followed the same pattern. Among tissues with clearly higher concentrations or AUCs in the sensitive T L-E rats were other organs typically affected by TCDD treatment, like thymus (Gupta et al., 1973; Kociba et al., 1976), skin (Schwetz et al., 1973), adrenals (Gupta et al., 1973), testes (Kociba et al., 1976), thyroids (Gupta et al., 1973; Rozman et al., 1986), and pancreas (Rozman et al., 1986; Gorski et al., 1988). Also brain peak TCDD concentrations were 2.6 times higher in T L-E than in CR L-E rats, and this difference increased toward the end of the study, being 7.9-fold on Day 32. Although neither mechanisms of action nor critical target organ(s) can be deduced from toxicokinetic data alone, it is a remarkable coincidence, if it is one, that the liver does not, but all other known target organs of TCDD do reflect the sensitivity difference in terms of corresponding differences in concentrations and AUCs. An important determinant of tissue distribution of TCDD is the expression of proteins that bind TCDD with high affinity, especially cytochrome P450 1A2 (CYP1A2), which is induced by TCDD in liver, but not in extrahepatic tissues (Van den Berg et al., 1994; Goldstein and Linko, 1984). There are no data available about the induction of CYP1A2 in the livers of these rat substrains. It is, however, likely that the liver concentrations of TCDD as measured in these rat strains accurately reflect the expression of CYP1A2 and possibly other high-affinity binding sites for TCDD. Although concentration of radioactivity decreased faster in CR L-E than in T L-E rats, the opposite was true for its fecal and urinary excretion. This is indicative of a faster rate of TCDD metabolism and/or nonbiliary intestinal excretion in T L-E than in CR L-E rats, both of which are likely secondary to higher tissue concentrations of TCDD in T L-E than in CR L-E rats. Retarded excretion of radioactivity in CR L-E during the latter part of the observation period is most likely a consequence of dilution by growth. In T L-E rats the faster excretion rate due to very slow growth dilution was not, however, sufficient to counterbalance the more profound dilution by growth in CR L-E rats. The present study demonstrates the significance of dilution by growth in toxicokinetics of compounds with long half-lives. This phenomenon is especially important for dioxins, which affect body weight development. At toxic doses, which cause growth retardation and body weight loss, toxi-
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KINETICS OF TCDD IN TWO RAT SUBSTRAINS
cokinetics are modified by the outcome of toxicity, resulting in decreased volume of distribution and, subsequently, redistribution of TCDD (Weber et al., 1993; Van den Berg et al., 1994). In conclusion, the toxicokinetic profile of TCDD in the two L-E substrains provides a likely explanation for the fivefold greater sensitivity of T L-E than CR L-E rats to TCDD, since the observed differences in tissue concentrations and AUC values (especially thymus, WAT, BAT, and adrenals) are in good agreement with the difference in susceptibility with the exception of the liver. In addition to the more efficient tissue uptake of TCDD in T L-E rats than in CR L-E rats, the major contributing factor to differences in toxicokinetics between these two rat substrains is a differential growth rate (dilution by growth), which in turn appears to provide an explanation for the different susceptibility of these rat substrains to TCDD. ACKNOWLEDGMENTS We thank Dr. Linda S. Birnbaum (U.S. EPA, Research Triangle Park, Chapel Hill, NC) for generously providing supplies and the use of a sample oxidizer for the analysis of fecal samples. Ed Brown (Department of Biometry, University of Kansas Medical Center, Kansas City, KS) performed the calculation on kinetic constants and AUCs with the SAS software package. This study was supported by the GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Germany, and the Academy of Finland, Research Council for Environmental Sciences (Grant 5410/4011/89). Bernhard Stahl was supported by a fellowship of the Deutsche Forschungsgemeinschaft (Sta 300/3-3).
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