FUNDAMENTAL AND APPLIED TOXICOLOGY 3 1 , 184 -191 (1996)

ARTICLE NO. 0090

Toxicokinetics of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in TwoSubstrains of Male Long-Evans Rats after Intravenous Injection

MATTI VlLUKSELA,*f THANG V. DUONG,* BERNHARD U. STAHL,*i: XUELIN Ll,*JOUKO TuOMISTO.f AND KARL K. ROZMAN*t

*Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417;^Department of Toxicology, National Public Health Institute, Kuopw, Finland; and tSection of Environmental Toxicology,

GSF-lnstitut fiir 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 IntravenousInjection. 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-labeledTCDD were studied in two substrains of Long-Evans (L-E) ratswith a fivefold difference in sensitivity in terms of TCDD-inducedmortality. The Turku/AB Long-Evans rat (T L-E) is the mostsensitive rat strain with an oral LD50 of 17.7 £ig/kg, whereas theCharles River Long-Evans rat (CR L-E) is a more resistant strain(oral LD50 95.2 //g/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 weremonitored daily during the 32-day study period. CR L-E rats grewsignificantly faster than T L-E rats, increasing their body weightby 60% in 32 days compared with only 16% in T L-E rats. Thisdifference was not caused by toxicity, because the weight gain wasidentical in control and TCDD-treated rats of both substrains.Tissue concentrations of [14C]TCDD-associated radioactivity andarea under the curve (AUC) values were lower in CR L-E than inT L-E rats. The most pronounced differences were found in thy-mus, white adipose tissue, brown adipose tissue, and adrenals. Thedecrease of TCDD concentration in tissues was faster in CR L-Ethan in T L-E rats, whereas fecal and urinary excretion was fasterin T L-E than in C L-E rats. Elimination half-life was 20.0 daysin T L-E rats and 28.9 days in CR L-E rats. Differential toxicoki-netics of TCDD in the two L-E substrains provide a likely explana-tion for the greater sensitivity of the T L-E strain, since observeddifferences in tissue concentrations and AUC values are in goodagreement with the difference in susceptibility. In addition to themore efficient tissue uptake of TCDD in T L-E rats than in CRL-E rats, the major contributing factor to differences in toxicoki-netics seems to be a differential growth rate (dilution by growth),which in turn appears to provide an explanation for the differencein susceptibility. More rapid excretion of TCDD in T L-E rats thanin CR L-E rats is clearly a result of higher tissue concentrations inT L-E rats. However, this faster excretion rate is not sufficient tocounterbalance the much slower dilution by growth in T L-E ratsthan in CR L-E rats. Thus, dilution by growth can be a more

0272-0590/96 SI8.00 1 8 4

Copyright © 1996 by the Society of Toxicology.All rights of reproduction in any form reserved.

important factor in determining the toxicokinetics and toxicity ofTCDD in rodents than is excretion. © 19% society of Toxicology

TCDD is extremely toxic to some animal species, withhuge inter- and intraspecies variations in sensitivity regard-ing acute toxicity (reviewed by Pohjanvirta and Tuomisto,1994). For example, a more than a 1000-fold sensitivitydifference has been reported between two rat strains (Poh-janvirta et al, 1993). The Han/Wistar (Kuopio), a substrainof the Wistar rat, is probably the most TCDD-resistant labo-ratory animal. It has been impossible to determine the LD50for this strain in an acute study because of solubility limita-tions of TCDD (Pohjanvirta et al, 1993). The most sensitiverat strain is the Turku/AB Long-Evans substrain, being onlyslightly less sensitive than the guinea pig, which is the mostTCDD-sensitive species. Extreme sensitivity to TCDD, how-ever, is not a specific feature of the Long-Evans strain,since the Charles River substrain seems to be about fivetimes more resistant to TCDD than the Turku/AB substrain(oral LD50 in male CR L-E rats is 95.2 //g/kg vs 17.7 figlkg in male T L-E rats) (Fan and Rozman, 1994; Pohjanvirtaet al, 1993).

Toxicokinetic factors clearly can explain only partiallythe differential sensitivity of various species to TCDD (Vanden Berg et al., 1994). In fact, kinetic factors were reportedto have at most a contributory impact on the intraspeciesdifference in TCDD-induced lethality between the most sen-sitive (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 relativelysmall sensitivity difference between male and femaleSprague-Dawley rats (cf. Beatty et al, 1978; Kociba et al,1976, 1978) is likely to be due to toxicokinetics alone ratherthan to gender-specific physiological differences (Weber etal, 1993; Li et al, 1995).

To reduce the confounding effects of physiological dis-similarities between species or even strains, we have utilizedin the present study the fivefold intrastrain sensitivity differ-

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KINETICS OF TCDD IN TWO RAT SUBSTRAINS 185

ence to examine the role of toxicokinetics in the toxicity ofTCDD. Intravenous route of administration was chosen toallow an accurate evaluation of the toxicokinetic profile aswell as to permit comparisons with two previous kineticstudies from this laboratory with the same route of adminis-tration 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 ob-tained from the National Laboratory Animal Center of the University ofKuopio (Kuopio, Finland), were bred at the Laboratory Animal Resourcesof the University of Kansas Medical Center (Kansas City, KS). Rats wereabout 8 weeks old at the beginning of the experiments. They were keptindividually in stainless steel wire-bottom cages or polycarbonate metabo-lism cages for urine and feces collection (32-day time point). Their mainte-nance consisted of Purina 5001 rodent chow (Ralston Purina, St. Louis.MO) and tap water ad libitum. Their room was artificially illuminated from6 AM to 6 PM and air-conditioned with a Honeywell DeltaNet W1044computer controller (Minneapolis, MN) programmed to maintain the ambi-ent temperature at 21-22°C and relative humidity at 40-60%. Rats wereacclimated to experimental conditions for 1 week before dosing

Emulsions. "C-labeled TCDD (ED-902H, sp act 122 mCi/mmol) wasobtained from Cambridge Isotope Laboratories Inc. (Woburn, MA) Thesolvent (toluene) was evaporated and [14C]TCDD redissolved in corn oil(Sigma) by stirring for 5 days. Thirty milligrams of cholic acid was dis-solved in 9 ml of Ringer's solution with the aid of 1 N NaOH, after which30 mg of lecithin (Sigma) was added. The pH was adjusted to 8, and 1 mlof corn oil containing [I4C]TCDD was added and shaken and the emulsionsonicated (Sonifier 185, micro-tip, Branson Sonic Power, Danbury. CT)The final emulsion was stored in a refrigerator and found to be stable forat least 3 months.

Treatment. Animals were randomly allocated into groups of four orsix (groups sampled on Day 32) and given a slow (1 min) intravenousinjection of 14C-labeled TCDD emulsion into the lateral tail vein usingQuick-Cath over-the-needle Teflon catheters (22 gauge, Baxter, Deerfield,IL). CR L-E rats received a single dose of 5.6 /xg/kg (2.12 /zCi/kg) and TL-E rats 2.0 ^g/kg (0.76 //Ci/kg) of [I4C]TCDD in a volume of 4 ml/kg.The doses were selected to be as low as possible (to avoid toxicity), butstill sufficient to obtain a reasonable level of detection. Body weights, urinevolumes and dry feces weights were recorded daily for the 32-day groupsRats 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 wereweighed and duplicate samples (except for thyroids and adrenals) of about100-200 mg prepared for analysis. Duplicate samples of the same sizewere also taken from epididymal white adipose tissue (WAT), interscapularbrown adipose tissue (BAT), proximal large intestine, small intestine (JeJu'num), muscle, and dorsal skin. The intestines were flushed with saline andthe fat was scraped off from the skin with a scalpel. Tissue samples weredigested overnight by I ml of Soluene-350 tissue solubilizer (Packard In-strument Co., Downers Grove, IL) at 50°C. Radioactivity was determined,after the addition of 10 ml scintillation cocktail (Hionic Fluor, PackardInstrument Co.), in a TriCarb-1900AC liquid scintillation counter (PackardInstrument Co.). Duplicate aliquots of 1 ml were procured from serum andurine, and radioactivity was measured after addition of 10 ml scintillationcocktail. Feces were dried and ground, and radioactivity was determinedafter combustion of about 100 mg duplicate samples in a sample oxidizer(Packard 306B biological oxidizer).

360

**

260

22010 14 It 22

Time after Dosing (Days)

26 34

FIG. 1. Body weight development of the [l4C]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-Econtrol rats were slightly older and thus heavier than the TCDD-treated rats.)Group means ± SE, n = 6, except for controls, n = 4.

Calculations. Radioactivity found in various tissues was converted topercentage of total dose recovered per gram of tissue (or total tissue) orpercentage of total dose excreted per day (urine, feces). Uptake rate constantswere calculated by linear regression. Rate constants of elimination (one-com-partment model), areas under the curve (AUC), and half-lives were calculatedon a mainframe computer using the SAS software package. Sigma-minus plots(log percentage of administered dose remaining to be excreted vs day) wereconstructed for fecal, urinary, and combined excretions.

RESULTS

Body weight development of the 32-day groups and theircontrols is shown in Fig. 1. CR L-E rats grew significantlyfaster than T L-E rats, increasing their body weight by 60%(146 g) compared to 16% (41 g) in T L-E rats. The differencein growth rate was not caused by toxicity, since untreatedcontrol rats gained weight similar to those treated withTCDD of both substrains.

Percentages of dose (radioactivity) recovered per gram oftissue are depicted in Tables 1A and IB, absolute organweights in Tables 2A and 2B, and percentages of dose pertotal tissue in Tables 3A and 3B. The only remarkablechanges in absolute organ weights (Tables 2A and 2B) werethe same reported also by others, i.e., increased liver weight(more prominent in CR L-E rats) and decreased thymusweights (more pronounced in T L-E rats). The time courseof [14C]TCDD-associated radioactivity in selected tissues isillustrated in Fig. 2. The tissue distribution profile of TCDDwas distinctly and consistently different in the two sub-strains. All tissue concentrations were lower in CR L-E thanin T L-E rats with peak concentrations being typicallyreached between 1 and 2 days (CR L-E) versus 4 to 8 days(T L-E) after dosing (Fig. 2; Tables 1A, IB, 3A, and 3B).It appears that this pattern of tissue distribution is at leastpartly due to slow release of TCDD from the emulsion (vehi-cle) trapped in the lymphatic system and/or to redistributionof TCDD from highly perfused to poorly perfused tissues.

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186 VILUKSELA ET AL.

TABLE 1A

Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [XlOOO]:" Charles River Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLarge intestineLiverLungMusclePancreasSerumSkinSmall intestineSpleenTestisThymusThyroidsWATLiver/WAT

1 day

101.5 ± 10.4231.1 ± 21.3

!U ± 0.435.9 ± 12.431.3 ± 2.617.9 ± 1.0

1507.1 ± 80.843.3 ± 6.2

6.4 ± 0.628.7 ± 2.86.8 ± 0.3

32.3 ± 1.328.1 ± 2.038.8 ± 9.3

7.0 ± 0.646.0 ± 3.260.5 ± 12.1

183.2 ± 15.28.2

2 days

73.9 ± 21.6257.5 ± 13.7

6.9 ± 0.529.6 ± 7.531.0 ± 3.822.5 ± 4.1

1777.5 ± 211.052.0 ± 10.9

5.8 ± 0.321.9 ± 2.9

8/7 ± 1.840.6 ± 3.425.4 ± 3.959.9 ± 23.2

5.8 ± 0.643.0 ± 6.143.5 ± 5.2

290.6 ± 63.26.1

Time after

4 days

72.9 ± 5.0211.4 + 12.5

7.3 ± 1.425.4 ± 9.127.2 ± 2.714.0 ± 1.0

1759.8 + 241.037.2 ± 5.9

5.0 ± 0.219.2 ± 1.77.4 ± 0.8

41.0 ± 2.723.8 ± 1.933.3 ± 6.1

5.1 ± 0.243.6 ± 2.721.1 ± 3.5

299.2 ± 18.45.9

injection

8 days

53.0 ± 9.6128.6 ± 11.7

5.2 ± 1.422.8 ± 7.325.0 ± 3.111.0 ± 0.6

1635.0 ± 152.034.3 ± 8.1

3.6 ± 0.213.3 ± 1.55.3 ± 0.7

23.8 ± 1.615.0 ± 1.352.6 ± 22.44.7 ± 0.8

25.6 ± 0.719.6 ± 3.3

262.5 ± 9.96.2

16 days

32.5 ± 2.492.2 ± 4.1

4.1 ± 1.221.9 ± 10.016.9 ± 4.98.6 ± 0.8

841.2 ± 106.228.9 ± 8.6

3.0 ± 0.49.1 ± 0.83.2 ± 0.5

14.1 ± 1.011.0 ± 1.732.5 ± 13.8

2.8 ± 0.124.1 ± 2.719.7 ± 4.1

171.0 ± 14.04.9

32 days

22.2 ± 2.447.2 ± 2.3

2.7 ± 0.419.3 ± 7.513.2 ± 4.54.8 ± 0.4

234.2 ±31 .226.1 ± 9 . 02.1 ± 0.35.3 ± 0.30.9 ± 0.14.5 ± 0.66.5 ± 0.5

41.1 ± 18.31.7 ± 0.1

12.5 ± 0.912.3 ± 2.777.4 ± 2.2

3.0

' Mean ± SE, n = 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 con-

centrations between the two substrains were found in thymus

(8-fold) and WAT (4.9-fold). Skin, adrenals, testes, BAT,

TABLE IB

Percentage of Dose (Radioactivity) Recovered per Gram of Tissue [Xl000]:° Turku/AB Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLarge intestineLiverLungMusclePancreasSerumSkinSmall intestineSpleenTestisThymusThyroidsWATLiver/WAT

1 day

369.4 ± 33.3699.4 ± 17.6

22.2 ± 3.170.2 ± 16.299.2 ± 17.748.0 ± 4.2

1227.5 ±164.1114.3 ± 24.7

17.4 ± 1.969.0 ± 3.610.7 ± 1.266.2 ± 4.664.6 ± 5.195.9 ± 20.315.9 ± 1.3

313.8 ± 32.2113.8 ± 11.4424.1 ± 43.8

Z9

2 days

372.0 ± 36.7731.5 ± 52.7

17.1 ± 1.745.6 ± 3.985.9 ± 6.845.2 ± 3 4

1438.2 ± 103.193.4 ± 7.914.8 ± 1.860.2 ± 2.912.0 ± 1.4

123.5 ± 8.958.8 ± 6.565.8 ± 4.715.6 ± 1.0

239.7 ± 13.5143.6 ± 22.4712.6 + 52.7

2.0

Time after

4 days

419.4 ± 26.6889.4 ± 63.5

19.1 ± 1.753.4 ± 5.178.2 ± 4.051.3 ± 4.0

1691.1 ± 107.0109.4 ± 10.420.5 ± 0.975.6 ± 10.913.6 ± 1.4

172.5 ± 18.366.2 ± 5.369.7 ±11 .419.0 ± 1.0

368.5 ± 31.1195.4 ± 8.2

1158.1 + 92.71.5

injection

8 days

366.6 ± 11.5702.1 ± 26.2

15 9 ± 0.635.6 ± 1.371.7 ± 1.748.9 ± 3.3

2149.4 ± 195.995.8 ± 3.514.2 ± 0.361 8 ± 4.316.5 ± 1.1

168.6 ± 23.463.4 ± 2.052.8 ± 2.819.0 ± 1.5

281.3 ± 25.486.1 ± 23.2

1451.2 ± 59.71.5

16 days

242.4 ± 18.7538.1 ±45.5

14.4 ± 2.862.0 ± 25.163.1 + 12.735.3 ± 3.2

1513.6 ± 22.496.6 ± 22.314.7 ± 0.846.3 ± 6.6

8.1 + 0.299.1 ± 16.050.1 ± 3.695.4 ± 27.213.6 ± 1.2

242.9 ± 32.168.1 ± 19.3

1167.9 ± 29.81.3

32 days

175.8 ± 22.6314.6 ± 9.3

21.3 ± 3.454.9 ± 18.952.2 ± 14.739.0 ± 2 4

819.1 ± 58.779.3 ± 22.320.6 ± 2.565.5 ±10.1

5.5 ± 0.545.4 ± 3.842.9 ± 3.7

102.8 ± 32.924.9 ± 2.9

169.6 ± 20.0272.4 ± 28.0667.1 ± 20.1

1.2

1 Mean ± SE, n = 4 (6 for the 32-day group). Highest value underlined.

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KINETICS OF TCDD IN TWO RAT SUBSTRAINS 187

TABLE 2AAbsolute Organ Weights (g):° Charles River Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLiverLungPancreasSpleenTestisThymusThyroids

1

0.0520.2401.8751.0242.685

13.4771.1530.8840.6473.0360.5900.018

day

± 0.006± 0.014± 0.066± 0.032± 0.097± 0.617± 0.048± 0.059± 0.028±0.155± 0.055± 0.001

2 days

0.047 ± 0.0050.262 ± 0.0251.792 ± 0.0480.984 ± 0.0522.477 ± 0.045

15.008 ± 0.8661.113 ±0.0770.774 ± 0.0690.630 ± 0.0343.221 ± 0.0830.423 ± 0.0500.015 ± 0.001

Time after

4 days

0.057 ± 0.0030.139 ± 0.0131.854 ± 0.0541.034 ± 0.0752.669 ± 0.210

16.807 ± 1.2401.132 ± 0.0300.962 ± 0.0570.705 ± 0.0683.353 ± 0.1640.459 ± 0.0340.018 ± 0.001

injection

8 days

0.053 ± 0.0010.106 ± 0.0091.880 ± 0.0531.148 ± 0.1562.270 ± 0.206

17.427 ± 0.8681.510 ± 0.1120.879 ± 0.0660.643 ± 0.0383.332 ± 0.0830.372 ± 0.0210.017 ± 0.000

16 days

0.052 ± 0.0030.158 ± 0.0161.811 ± 0.0511.352 ±0.2152.558 ±0.180

17.174 ± 1.1391.859 ± 0.5800.775 ±0.1000.637 ± 0.0413.391 ± 0.1160.403 ± 0.0320.024 ± 0.0o_

32 days

0.054 ± 0.0020.135 ± 0.0071.632 ± 0.2961.158 ± 0.0692.558 ±0.182

15.561 ± 1.1251.328 ±0.1110.815 ± 0.0870.626 ± 0.0353.448 ± 0.1200.469 ± 0.0480.019 ± 0.002

' Mean ± SE, n = 4 (6 for the 32-day group).

thyroids, muscle, and kidney showed 3.2- to 4.3-fold differ-ences. The most pronounced differences in terms of AUCvalues were found in thymus (10.2-fold), adrenals (6.9-fold),skin (6.2-fold), WAT (5.8-fold), and BAT (5-fold). Mostnotably, the substrain differences in liver peak concentra-tions and AUCs were only 1.2- and 1.5-fold, respectively.

The decrease in TCDD tissue concentrations was faster inCR 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 ratswere 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 tothe disappearance of TCDD from tissues, its fecal and urinaryexcretion occurred at a lower rate in CR L-E rats than in TL-E rats (Fig. 3). In T L-E rats the excretion followed a strictly

linear pattern (r = 0.987) on a semilogarithmic plot during 32days, whereas in CR L-E rats fecal and total (fecal + urinary)excretion curves flattened out, indicating retarded eliminationduring the second half of the observation period. The elimina-tion half-life of TCDD, calculated from total excretion usinglinear regression, was 20.0 days in T L-E rats. Because ofretarded excretion of TCDD in CR L-E rats a curve fit wasobtained according to the equation

y = a(l - b X e"cx ' '2),

where a = 41.658, b = -1.3715, and c = 0.035237, whichdescribed accurately (r = 0.999) the total elimination curve

TABLE 2BAbsolute Organ Weights (g):" Turku/AB Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLiverLungPancreasSpleenTestisThymusThyroids

1 day

0.049 ± 0.0030.095 ± 0.0071.568 ± 0.0300.795 ± 0.0282.194 ± 0.1519.488 ± 0.2791.005 ± 0.0300.707 ± 0.0740.422 ± 0.0282.612 ± 0.0890.232 ± 0.0140.014 ± 0.001

2 days

0.052 ± 0.0010.095 ± 0.0041.602 ± 0.0040.749 ±0.0152.040 ± 0.030

10.045 ± 0.2491.001 ± 0.0290.523 ± 0.0380.449 ±0.0182.542 ± 0.0230.223 ±0.0120.012 ± 0.001

Time

4 days

0.053 ± 0.0010.115 ± 0.0071.571 ± 0.0170.770 ± 0.0192.181 ± 0.090

10.852 ± 0.3711.030 ±0.0330.657 ± 0.0480.404 ± 0.0642.578 ± 0.0370.225 ± 0.0150.013 ± 0.002

after injection

8 days

0.051 ± 0.00050.092 ± 0.0081.605 ± 0.0410.777 ± 0.0262.058 ± 0.053

10.949 ± 0.2681.012 ±0.0210.702 ± 0.0660.487 ± 0.0212.701 ± 0.0710.185 ± 0.0070.013 ± 0.001

16 days

0.056 ± 0.0010.089 ± 0.0081.561 ± 0.0540.856 ± 0.0282.198 ± 0.071

11.451 ±0.6391.170 ± 0.1140.598 ± 0.0510.481 ± 0.0252.618 ± 0.1900.135 ± 0.0060.014 ± 0.001

32 days

0.056 ± 0.0020.084 ± 0.0071.679 ± 0.0170.870 ± 0.0372.431 ± 0.035

11.353 ± 0.3191.181 ± 0.0500 619 ± 0.0370.502 ± 0.0132.902 ± 0.0360.110 + 0.0050.014 ± 0.001

° Mean ± SE, n = 4 (6 for the 32-day group).

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188 VILUKSELA ET AL.

TABLE 3APercentage of Dose (Radioactivity) Recovered per Total Tissue [XlOOO]:" Charles River Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLiverLungPancreasSpleenTestisThymusThyroidsWAT*

1 day

0.053 ± 0.0080.555 ± 0.0690.157 ± 0.0090.368 ±0 .1240.843 ± 0.079

204 ± 190.506 ± 0.0860.258 ± 0.0400.254 ± 0.0640.211 ± 0.0220.274 ± 0.0350.011 ± 0.003

4218

2 days

0.032 ± 0.0090.671 ± 0.0650.123 ± 0.0090.286 ± 0.0620.764 ± 0.084

264 ± 300.587 ± 0.1510.166 ± 0.0170.394 ±0.1750.186 ± 0.0190 179 ± 0.0240.006 ± 0.000

6910

Time

4 days

0.042 ± 0.0030.293 ± 0.0320.135 ± 0.0260.276 ±0.1190.717 ± 0.066

287 ± 200.422 + 0.0710 183 ± 0.0150.231 + 0.0450 171 ± 0.0150 201 ± 0.0230.004 ± 0.001

7474

after injection

8 days

0.028 ± 0.0050.137 ± 00170 097 ± 0.0240.295 ±0.1390.583 ±0.111

281 ± 110 536 ± 0.1650.115 ± 0.0100.359 ±0.1760.157 ± 0 0250.096 ± 0.0070.003 ± 0.0005

7088

16 days

0.017 ± 0.0020.147 ± 0.0200.073 ± 0.0200.268 ±0.1090.416 ± 0.102

141 ± 100.491 ±0.1370.069 ± 0.0080.199 ± 0.0760.095 ± 0.0040.098 ±0.0150.005 ± 0.001

5118

32 days

0.012 ± 0.0010.063 ± 0.0030.046 ± 0.0110.232 ±0.1000.321 ± 0.097

35.7 ± 4.40.340 ±0.1260 044 ± 0.0060.248 ± 0 1050.058 ± 0.0050.059 ± 0.0070.002 ± 0.001

2717

° Mean ± SE, n = 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-lifeof TCDD was 28.9 days in CR L-E rats. The excreted radio-activity (94.1% CR L-E and 90.2% T L-E) was found infeces, whereas urine contributed only 5.9 and 9.8% to thetotal excretion, respectively. In 32 days CR L-E and TL-E rats excreted 52.4 and 69.7% of the total radioactivity,respectively.

DISCUSSION

This study was performed to compare the toxicokineticsof TCDD in two substrains of L-E rats with different sensi-

tivity to TCDD. This approach was undertaken to clarify therole of toxicokinetics in the sensitivity difference betweengenetically closely related substrains.

The doses of TCDD administered were nontoxic for bothsubstrains as shown by their body weight development. Thedoses were 9 and 17 times lower than the corresponding oralLD50 in T L-E and CR L-E rats, respectively (Pohjanvirta etal., 1993; Fan and Rozman, 1994). T L-E rats received thelowest possible dose that would allow accurate detection ofradioactivity in tissues with the lowest concentrations. Themost distinct feature in the body weight development was a

TABLE 3BPercentage of Dose (Radioactivity) Recovered per Total Tissue [X1000]:" Turku/AB Long-Evans Rats

Tissue

AdrenalsBATBrainHeartKidneyLiverLungPancreasSpleenTestisThymusThyroidsWAT*

1 day

0.181 ± 00180.662 ± 0.0490 347 ± 0.0480.564 ±0 .1392.155 ± 0.390

117 ± 191.149 ± 0.2500.490 ± 0.0660.396 ± 0.0730.416 ± 0.0430.727 ± 0.0820.016 ± 0.003

9703

2 days

0.192 ± 0.0210.694 ± 0.0540.274 ± 0.0270.343 ± 0.0351.748 ±0.117

144 ± 90.930 ± 0.0620.311 ± 0.0070.295 ± 0.0230.397 ± 0.0230.532 ± 0.0260.016 ± 0.003

16,569

Time after

4 days

0.220 ± 0.0121.015 ± 0.0800.299 ± 0.0260 409 ± 0.0321.708 ± 0 127

183 ± 101.126 ± 0.1050.496 ± 0.0750.298 ± 0.0860.488 ± 0.0240.825 ± 0.0800.025 ± 0.003

26,844

injection

8 days

0.188 ± 0.0060.643 ± 0.0650.254 ± 0.0050.277 ± 0.0181 475 ± 0.045

235 ± 190.970 + 0.0410 431 ± 0.0400.256 ± 0.0090.513 ± 0.0350.525 ± 0.0630.012 ± 0.003

33.667

16 days

0.136 ± 0.0110.481 ± 0.0730.223 ± 0.0400.543 ± 0.2281.393 ± 0.302

173 ± 81.200 ± 0.4060.282 ± 0.0560.465 ±0.1410.359 ± 0.0500.333 ± 0.0550.010 ± 0.003

28,252

32 days

0.097 ± 0.01 10.264 ± 0.0280.357 ± 0.0560.476 ±0.1661.268 ± 0.35592.8 ± 6.5

0.931 ± 0.2560 409 ± 0.0770.510 ± 0.1540.720 ± 0.0790.189 ±0.0280.038 ± 0.004

18.111

' Mean ± SE. n = 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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KINETICS OF TCDD IN TWO RAT SUBSTRAINS 189

12 16 20 24Time after Dosing (Days)

28 32

n° L

10'

\0>

103

0 4 ) 12 14 20 24 28 32Time after Dosing (Days)

FIG. 2. Time courses of [l4C]TCDD-associated radioactivity in sometissues of Charles River (open symbols) and Turku/AB (solid symbols)Long-Evans rats. (A) Liver, kidney, adrenals, and serum. (B) White adiposetissue (WAT), large intestine, thymus, and brain. Group means ± SE, n =4 or 6 (32-day groups).

considerably slower growth rate of T L-E rats compared toCR L-E rats, although their body weights were very similarat the time of dosing (Fig. 1). From the toxicokinetic pointof view this is a crucial difference, because distribution,tissue concentrations and elimination are all critically influ-enced by growth rate which leads to dilution by growth.

Tissue distribution of TCDD in the L-E substrains fol-lowed the general pattern observed in rodents (cf. Neal etal., 1982; Van den Berg et al., 1994) in that the highestconcentrations were found in liver and WAT. Similar toprevious studies (Neal et al., 1982; Pohjanvirta et al., 1990;Weber et al., 1993; Li et al., 1995) tissues with lowest con-centrations were brain, serum, testis, and muscle. Also, elim-ination half-lives (calculated from combined urinary and fe-cal excretion) and the relative proportions of fecal and uri-nary excretion were within the range previously reported inother rat strains.

In general, tissue concentrations and related AUCs of TCDD

TABLE 4Area under the Curves (AUCs)° for [14C]TCDD in Tissues of

Male Turku/AB Long-Evans (T L-E) and Charles River Long-Evans (CR L-E) Rats

AUC

Tissue*

AdrenalsBATKidneyLiverLungSerumSkinSmall intestineThymusWAT

TL-E

8.5717.04

1.8244.88

2.540.313.351.707.66

32.58

CR L-E

1.243.420.52

29.490.770.110.540.390.755.67

TL-E/CRL-E ratio

6.914.983.501.523.302.826.204.36

10.215.75

" Unit is "percentage of dose per gram tissue x days" over the 32-dayobservation period.

* Only tissues suitable for analysis in both substrains were included.

were higher in T L-E rats than in CR L-E rats. Peak concentra-tions of most tissues were also reached at later time points inT L-E rats, facilitating the detection of an uptake phase for

TABLE 5Kinetic Rate Constants for [I4C]TCDD in Tissues of Male

Turku/AB Long-Evans (T L-E) and Charles River Long-Evans(CR L-E) Rats

Tissue

AdrenalsBrainBATHeartKidneyLarge intestineLiverLungMusclePancreasSerumSkinSmall intestineSpleenTestisThymusThyroidsWAT

Uptake"

0.0002'

0.0008:'

—'0.0013

— '.—c

0.0004

—c

c

0.0003—c

0.0024

TL-E

Elimination*

0.0362

0.0424—c

0.0293—'

0.04100.0139

c

, . , , „„ .**

0.05220.04900.0165

c

c

0.0281—''

0.0309

Uptake

0.0019

0.0001

0.0004

CRL-E

Elimination

0.07010.05220 06620.03300.04710.05320.06580.04040.04500.08300.07350.10090.0546

c

0.05000.04700.10070.0479

° Uptake rate constant calculated by linear regression. The unit is"days"' ." Open space means no uptake phase was detected.

b Elimination rate constant calculated by SAS one-compartment model.The unit is days"1. Negative sign omitted for clarity.

c No rate constant could be calculated.

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ifO VILUKSELA ET AL.

many tissues in this substrain. In most tissues, however, TCDDconcentrations of T L-E rats were clearly higher already onDay 1 after dosing, when body weights of the substrains wereequal and no dilution by growth had occurred yet. Therefore,it is concluded that tissue uptake of TCDD is more efficientin T L-E than in CR L-E rats. The faster growth rate of CRL-E rats resulting in faster dilution of TCDD tissue concentra-tions is also likely to have partially contributed to the earlierappearance of peak tissue concentrations in this substrain com-pared with T L-E rats. This is also the most likely explanationfor the faster tissue elimination of TCDD in CR L-E than inT L-E rats.

Sprague-Dawley rats seem to have a faster tissue uptakeof TCDD than the L-E substrains. Intravenous administrationof TCDD to Sprague-Dawley rats resulted in most cases inpeak tissue concentrations within 1 day (Weber et al., 1993;Li et al., 1995), whereas a tissue uptake very similar to thepresent data was observed in T L-E rats after intraperitonealdosing (Pohjanvirta et al., 1990).

In contrast to other tissues, concentrations and, to a lesserextent, AUCs of TCDD in liver of both substrains weresurprisingly similar, though concentrations declined morerapidly in livers of CR L-E rats as a result of faster growthrate. It seems likely that the liver TCDD concentrations dur-ing the first few days after dosing are less relevant for toxic-ity than the later concentrations. Peak liver concentrationswere very similar to those measured by Pohjanvirta et al.(1990) in the sensitive T L-E rats and also in the resistantHan/Wistar (Kuopio) rats after intraperitoneal injection, butwere twice as high as in male Sprague-Dawley rats (Weberet al., 1993) which have intermediate sensitivity to TCDD(oral LD50 43 //g/kg; Stahl et al., 1992). Therefore, liverTCDD concentrations and AUCs do not correlate with thesensitivity of different rat strains, suggesting either that the

100

80

60

2012 16 20

Time after Dosing (Days)

FIG. 3. Urinary, fecal, and combined excretion of [HC]TCDD-associ-ated radioactivity from Charles River (open symbols) and Turku/AB (solidsymbols) Long-Evans rats (32-day groups). Group means ± SE, n - 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 (linearregression) for T L-E rats.

liver is not a critical target organ in these rat strains or thatthe crucial liver lesions have different substrain sensitivity.Recent observations by Tuomisto et al. (1995) provide sup-port for the former notion.

A good correlation with sensitivity is apparent with WATTCDD concentrations and AUCs. The AUCs were 5.8 timeshigher in T L-E than in CR L-E rats. In comparison,Sprague-Dawley rats (Weber et al., 1993) are intermediateboth in terms of sensitivity and AUCs. BAT, which containslarge numbers of lipid droplets, followed the same pattern.Among tissues with clearly higher concentrations or AUCsin the sensitive T L-E rats were other organs typically af-fected 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 peakTCDD concentrations were 2.6 times higher in T L-E thanin CR L-E rats, and this difference increased toward the endof the study, being 7.9-fold on Day 32. Although neithermechanisms of action nor critical target organ(s) can bededuced from toxicokinetic data alone, it is a remarkablecoincidence, if it is one, that the liver does not, but allother known target organs of TCDD do reflect the sensitivitydifference in terms of corresponding differences in concen-trations and AUCs.

An important determinant of tissue distribution of TCDDis the expression of proteins that bind TCDD with highaffinity, especially cytochrome P450 1A2 (CYP1A2), whichis 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 CYP1A2in the livers of these rat substrains. It is, however, likelythat the liver concentrations of TCDD as measured in theserat strains accurately reflect the expression of CYP1A2 andpossibly other high-affinity binding sites for TCDD.

Although concentration of radioactivity decreased fasterin CR L-E than in T L-E rats, the opposite was true for itsfecal and urinary excretion. This is indicative of a faster rateof TCDD metabolism and/or nonbiliary intestinal excretionin T L-E than in CR L-E rats, both of which are likelysecondary to higher tissue concentrations of TCDD in TL-E than in CR L-E rats. Retarded excretion of radioactivityin CR L-E during the latter part of the observation periodis most likely a consequence of dilution by growth. In TL-E rats the faster excretion rate due to very slow growthdilution was not, however, sufficient to counterbalance themore profound dilution by growth in CR L-E rats.

The present study demonstrates the significance of dilu-tion by growth in toxicokinetics of compounds with longhalf-lives. This phenomenon is especially important fordiox-ins, 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 191

cokinetics are modified by the outcome of toxicity, resultingin decreased volume of distribution and, subsequently, redis-tribution of TCDD (Weber et al, 1993; Van den Berg etal., 1994).

In conclusion, the toxicokinetic profile of TCDD in thetwo L-E substrains provides a likely explanation for thefivefold greater sensitivity of T L-E than CR L-E rats toTCDD, since the observed differences in tissue concentra-tions and AUC values (especially thymus, WAT, BAT, andadrenals) are in good agreement with the difference in sus-ceptibility with the exception of the liver. In addition to themore efficient tissue uptake of TCDD in T L-E rats than inCR L-E rats, the major contributing factor to differences intoxicokinetics between these two rat substrains is a differen-tial growth rate (dilution by growth), which in turn appearsto provide an explanation for the different susceptibility ofthese rat substrains to TCDD.

ACKNOWLEDGMENTS

We thank Dr. Linda S. Birnbaum (U.S. EPA, Research Triangle Park, ChapelHill, NC) for generously providing supplies and the use of a sample oxidizerfor the analysis of fecal samples. Ed Brown (Department of Biometry, Univer-sity of Kansas Medical Center. Kansas City, KS) performed the calculationon kinetic constants and AUCs with the SAS software package. This studywas supported by the GSF-Forschungszentrum fur Umwelt und Gesundheit,Germany, and the Academy of Finland, Research Council for EnvironmentalSciences (Grant 5410/4011/89). Bernhard Stahl was supported by a fellowshipof the Deutsche Forschungsgemeinschaft (Sta 300/3-3).

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