~~x~c~L~GYANDAPPLIEDPHARMACOLOGY~~,

The

209-226( 1976)

Fate of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Single and Repeated Oral Doses to the

following Rat1

J. Q. ROSE,~ J. C. RAMSEY, T. H. WENTZLER, R. A. HUMMEL AND P. J. GEHRING Dow Chemical U.S.A., Midland, Michigan 48640 Received July 24,1975; accepted December I,1975

The Fate of 2,3,7,8-Tetrachlorodibenzo-p-Dioxinfollowing Single and RepeatedOral Dosesto the Rat. ROSE, J. Q., RAMSEY, J. C., WENTZLER, T. H., HUMMEL, R. A. and GEHRING, P. J. (1976)Toxicol. Appl. Pharmacol. 36, 209-226. Rats were given a singleoral doseof 1.0 pg of [r4C]-2,3,7,8Tetrachlorodibenzo-p-dioxin ([14C]TCDD)/kg/day, or repeated oral dosesof 0.01, 0.1, or 1.0 c(g of [“C]TCDD/kg/day Monday through Friday for 7 weeks.Following a singleoral doseof 1.O,ugof [r4C]TCDD/kg, r4Cactivity could bedetectedonly in feces,but not in urine. The half-life of r4Cactivity in the body was31 f 6 days. Twenty-two days after the single oral dose,concentrationsof 14Cactivity werelocatedprincipallyin liver and fat. Following repeatedoral dosesthe major route of excretion was via the feces;urine contained3-18% of the cumulativedoseof 14Cactivity by 7 weeks.The half-life of 14Cactivity in the body of theserats was23.7 days. Assuminga one compartmentopen model, 76.2% of steady-stateconcentrations were achieved in the whole body after 7 weeks. 14Cactivity in liver and fat approachedsteady-statevaluesat a rate similar to the whole body. Radioactivity in the liver wasidentified asTCDD by gaschromatography-massspectrometry and could be extracted from liver tissuewith organic solvents. The results of this study in rats indicate that TCDD approachessteady-stateconcentrationsin the body within 13weeks,andthe rate constant defining the approach to steady-state concentrations is independentof the dosageof TCDD over the doserangeof 0.01-l .Opg of TCDD/kg/day. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic compound formed as an unwanted contaminant in the manufacture of 2,4,5-trichlorophenol. Use of this trichlorophenol to manufacture 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) may result in contamination of the latter with TCDD. The single oral-dose LD50 of TCDD ranges from 0.6 pugof TCDDjkg for male guinea pigs to 22 pg of TCDDjkg and 115 pg of TCDD/kg for male rats and rabbits, respectively (Schwetz et al., 1973). A variety of toxic manifestations have been associated with exposure to TCDD. In man and rabbits, dermal contact with TCDD produces chloracne (Schwetz et al., 1973; Hofman, 1957; Kimmig and Schulz, 1957). The compound is embryotoxic and 1A preliminaryreport of thispublicationwaspresented at the FourteenthAnnual Meetingof the Societyof Toxicology,Williamsburg,Virginia, March g-13,1975. * Presentaddress:Departmentof Pharmacokinetics, Schoolof Pharmacy,State University of NewYork, Buffalo,N.Y. 14214. Copyright 0 1976 by Academic Press, Inc. 209 All rights of reproduction in any form reserved. Printed

in Great

Britain

210

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ET AL.

fetotoxic in mice and rats (Neubert et al., 1973). A condition referred to as chick edema disease has also been associated with TCDD (Schwetz et al., 1973). Recently, a study was conducted in which rats were given 0.001, 0.01, 0. I, or I .O /(g of TCDD/kg/day by gavage Monday through Friday for 13 weeks (Kociba et a!.. 1976). No untoward effects were seen in rats receiving 0.001 and 0.01 pg of TCDDjkgi day. In rats receiving repeated oral doses of 0.1 or 1.O pg of TCDD/kg/day, pathological changes were observed principally in liver and thymus; deviations in the values of various associated clinical chemistry parameters were also observed. Deaths occurred in rats receiving the highest dose level. To further assess the potential hazard of long-term repeated exposure to low levels of TCDD, a knowledge of the fate of TCDD in the body is necessary. In a previous study the half-life (t+) for the apparent first order excretion of 14C activity by rats given a single oral dose of 50 pg of [14C]TCDD/kg was 17.4 f 4.6 days (Piper et al., 1973). The 14C activity was excreted primarily in the feces. Small amounts of i4C activity were also found in the urine and expired air, suggesting that TCDD was metabolized to some degree. Since this dose is twice the LD50 for rats and since the fate of the compound may be dependent on the dose level, the fate of lower sublethal doses of TCDD should be determined. The objectives of the work reported herein are to elucidate the fate of TCDD in rats given a single oral dose of 1.O pg of TCDD/kg and repeated oral doses of 0.01, 0. I, or 1.O pg of [14C]TCDD/kg/day, Monday through Friday for 7 weeks, and to assess the propensity of TCDD to accumulate in the body and its tissues. METHODS

Dosematerial andpreparation for administration. i4C-labeled TCDD was synthesized by Dr. A. S. Kende of the University of Rochester by the condensation of 4,5-dichlorocatechol with [ring-UL-14C]-1 ,2,4,5-tetrachlorobenzene (Kende and Wade, 1973). The synthesized [i4C]TCDD had a specific activity of 148 mCi/mmol(O.46 mCi/mg). The concentrations of suspectedimpurities in the sampleof [14C]TCDD were determined by gaschromatography using electron capture detection. The gaschromatograph was equipped with a 3 m x 2 mm (i.d.) glasscolumn packed with 0.1 y0 QF-1 silicone and 0.1% OV-17 silicone on 100/120 mesh GLC 100 glassbeads; the temperature of the column was maintained at 175°C. Becausepure standards for trichlorodibenzo-pdioxin(s) and pentachlorodibenzo-p-dioxin(s) were unavailable, the response of the electron capture detector was assumedproportional to the number of chlorines in the molecule. To establishthe retention times of trichlorodibenzo-p-dioxin, 2,3,7,8-TCDD, and pentachlorodibenzo-p-dioxin, a solution containing these materials was analyzed (Muelder and Shadoff, 1973). For the [14C]TCDD sample, the pentachlorodibenzo-pdioxin signal was lessthan 0.5 % of the TCDD peak and did not constitute a significant responsebecausethe signal-to-noiseratio waslessthan 2.5 to 1. The trichlorodibenzo-pdioxin responserelative to the 2,3,7,8-TCDD response indicated that 2% trichlorodibenzo-p-dioxin was present. No other substancesresponsive to the electron capture detector were observed. Therefore, the purity of this sampleof [14C]TCDD was estimated to be 98 % by gaschromatography. The radiochemical purity wasgreater than 99 % as determined by thin-layer chroma-

FATE OF TCDD

211

tography using 0.25-mm Brinkman silica gel plates and three different solvent systems: (a) cyclohexane, (b) hexane, and (c) chloroform: acetone (1: 1). To detect 14C activity, l-cm2 sections of silica gel from the plates were added to 3.5 ml of HZ0 and 11.5 ml of Aquasol (New England Nuclear, Boston, Mass.), and these samples were analyzed by liquid scintillation counting. For administration of 1 pg of TCDD/kg, [r4C]TCDD was dissolved in acetone and mixed with corn oil 1: 25 (v : v) to provide a solution of 1 pg/ml. Serial dilutions of this solution were made with 1:24 acetone: corn oil to prepare solutions containing 0.01 and 0.1 ,ug/ml; these solutions were used to administer 0.01 and 0.1 pg/kg. The solutions containing TCDD were administered by gavage using a l-ml syringe fitted with a feeding needle. To determine the homogeneity and stability of the [14]TCDD in the acetone-corn oil solution, a test solution containing 1 pg of [‘4C]TCDD/ml was prepared, and triplicate aliquots were analyzed for 14C activity after 0, 1, 2, 3, 4, 5, 9, 15, and 30 days. For analysis, l-ml aliquots were added directly to 15 ml of a liquid scintillation solution containing 4.0 g of 2,5-diphenyloxazole and 0.05 g of p-bis-2-(5-diphenyloxazolyl) benzene per liter of toluene. The concentration of 14C activity in the solution remained constant (100.0 f 0.8 % of the original determination at Day 0) over the 30 days. The test solution of [r4C]TCDD was analyzed for the concentration of TCDD immediately after formulation and 30 days later by combination gas chromatography-mass spectrometry (Shadoff and Hummel, 1975). No difference in the concentration of TCDD was detected within the reproducibility of the method (k20 % of the amount detected). Animals. Sprague-Dawley strain rats were purchased from Spartan Research Animals, Inc., Haslett, Michigan. To determine the fate of TCDD following a single oral dose, three male (155-161 g) and three female (135-145 g) rats were given 1 pg of [14C]TCDD/kg. The rats were maintained in glass Roth-type metabolism cages designed for the separate collection of urine, feces, and respired CO, in a room with controlled humidity (45-55 %) and temperature (70-75°F) and a 12-hr light-dark cycle. Room air was drawn through the cages at a rate of 300-500 cm3/min, and subsequently through CO, scrubbers containing 3 : 7 (v : v) ethanolamine : 2-methoxyethanol. The rats were acclimated for 1 week prior to the administration of TCDD and were given Purina Laboratory Chow and water ad Iibitum throughout the experiment. To determine the fate of TCDD upon repeated oral administration, 27 male (280334 g) and 27 female (215-240 g) rats were divided randomly into three groups of three rats per sex per dose level and were given 0.01, 0.1, or 1.0 pg of [14C]TCDD/kg/day, Monday through Friday, weekly. Three male and three female rats at each dose level were sacrificed at 1 and at 3 weeks to determine the concentrations of 14C activity in selected tissues at these times. Excreta from a third group of three male and three female rats at each dose level were collected throughout a 7-week period. At the end of this time, the concentrations of 14C activity in selected tissues of these rats were also determined. All of these rats were housed in stainless steel cages designed for the separate collection of urine and feces. Food and water were provided ad libitum, and the rats were acclimated to the environment for 1 week prior to the administration of [14C]TCDD. Sample collection and analyses. For the experiment in which rats were given a single oral dose of [14C]TCDD, urine, feces, and CO, trapped from expired air were collected

212

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ET AL.

every 24 hr for 22 days. Excreta and tissues were analyzed for isC activity by liquid scintillation counting. Samples of urine, 0.25 ml, were analyzed directly for 14C activity following the addition of 1.O ml of HZ0 and 15 ml of Aquasol. Spleen, thymus, fat. and 50% aqueous homogenates of feces, liver, kidney, and carcass were oxidized in a Beckman Biological Materials Oxidizer. The 14C0, resulting from the oxidation was trapped as [‘4C]2-hydroxyethylcarbamic acid in a solution containing 8 ml of 3: 7 (v:v) ethanolamine:Zmethoxyethanol and counted after the addition of 10 ml of a scintillation solution containing 125 ml of Liquifluor, (New England Nuclear, Boston, Mass.) 220ml of 2-methoxyethanol, and 655 ml of toluene. 14C0, expired during each 24-hr period was trapped in 225 ml of 3 : 7 (v: v) ethanolamine: 2-methoxyethanol, and 1 ml was added to 15 ml of XDC liquid scintillation cocktail (Bruno and Christian, 1961) and counted. For experiments in which rats were given repeated oral doses, urine and feces were collected each day for the first 7 days. Thereafter, urine and feces were collected each day, Monday through Friday, and pooled. Individual daily collections of urine and feces were made on Saturday and Sunday and maintained separately for analyses. At the end of week 7, the animals were sacrificed by anesthesia with diethyl ether. Tissues were removed and stored frozen together with the remaining carcass until analyzed. To determine what fraction of the 14C activity in liver could be attributed to [‘“ClTCDD, liver homogenates were analyzed for TCDD by gas chromatography-mass spectrometry (GC/MS) using the method of Shadoff and Hummel (1975). In this method an aqueous ethanol solution of potassium hydroxide is added to the liver sample and heated. TCDD is then extracted with hexane. After cleanup, the TCDD is quantitated by measuring its M and M + 2 ions at m/e 320 and 322. An LKB 9000s GUMS equipped with a 6-ft x 3-mm glass column containing 3 % OV-3 silicone was used. The [14C]TCDD contained sufficient 14C to alter the isotopic ratios of TCDD, so a solution of the [14C]TCDD was used as a standard for the GC/MS measurements. This solution of [14C]TCDD was calibrated against a solution of nonlabeled TCDD by gas chromatography using electron capture detection. The mean recoveries of TCDD from liver samples spiked to contain 0.02 or 0.2 ppm were 80 and 85 %, respectively, with a reproducibility of *lo%. When concentrations greater than 0.1 ppm TCDD in liver were detected by gas chromatography-mass spectrometry, the analysis was checked using a Hewlett Packard Model 5700 gas chromatograph equipped with an electron capture (ec) detector and the 5709A linear ec control. The 3-mm x lo-ft glass column containing 3 % OV-210 silicone was maintained at 210°C and a 5 % methane in argon carrier gas at a flow rate of 30 ml/min was used. To determine the extractability of TCDD from liver, a modification of the method of Vinopal and Casida (1973) was used. In a separate study, rats were given 1.O pg of [14C]TCDD/kg/day, Monday through Friday, for 6 weeks. An equal volume of Sorensen’s buffer (pH 7.4) was added to 50% homogenates of livers from these rats. After lyophilization, the homogenates were extracted with hexane overnight using a Soxhlet extractor and reextracted with 2 : 1 (v : v) diethylether : ethanol the following night. At ambient temperature, aliquots of the two extracts were evaporated to dryness in scintihation vials under N,. After addition of a toluene scintillation cocktail, the 14C

FATE OF TCDD

213

activity was determined. Samples of liver from control rats were spiked with [14C]TCDD and carried through the same procedure. Extracted residues were analyzed for 14C activity by combustion. Because the aforementioned procedure did not extract all of the 14C activity, two additional methods were used to characterize further the propensity of TCDD to bind to liver tissue. In the first method, 0.25-g samples of liver, prepared and extracted as described above, were added to 7 ml of 0.6 M trichloroacetic acid and shaken for 10 min. This preparation was extracted four times in 50-ml centrifuge tubes with 10 ml of diethyl ether. After each extraction, the samples were centrifuged to separate the phases and the organic phase was collected for analysis. One-milliliter samples of the organic phase from each extraction were evaporated under N, at ambient temperature, a toluene scintillation cocktail was added, and the samples were analyzed for 14C activity. The remaining aqueous phase was filtered, and the precipitate and filter paper were oxidized as described above to determine the 14C activity in the precipitate. One-milliliter samples of the filtrates were also analyzed for the nonextractable 14C activity. In the second method, 0.35 g of a 50% homogenate of liver was added to 20 ml of 8 M urea, and this solution was extracted four times with 10 ml of hexane and then six times with 10 ml of diethyl ether. The extractions were centrifuged to separate the phases. 14C activity in the organic phase was determined as described previously. To determine the 14C activity in a l-ml aliquot of the remaining aqueous phase, 11.5 ml of Aquasol and 3.5 ml of H,O were added. All samples were analyzed for 14C radioactivity in a Nuclear Chicago Mark II liquid scintillation counter. The counting efficiency was determined by the external standard technique, and the disintegrations per minute were calculated by correcting net counts per minute for counting efficiency. 14C activity twice that of background was considered significant. Statistical anulysis. For animals given a single oral dose of TCDD, maximum likelihood parameter estimates were obtained for the elimination rate constant, k, and the fraction of dose absorbed,J These estimates were obtained using a conjugate gradient search technique and an IBM 370/155 computer. Cumulative body burden data for rats given repeated oral doses of TCDD were used to obtain the maximum likelihood estimates ofSand k for these rats. In the analysis, the assumptions made were that TCDD is rapidly absorbed relative to its elimination rate from the body and that the overall elimination of 14C activity is an apparent first order process. The unbiased, minimum variance estimates off and k for rats given repeated oral doses were also obtained by a conjugate gradient search of the likelihood function. RESULTS Fate qf 14C activity after a single oral dose of 1.0 pg of [‘4C]TCDD/kg. In Fig. 1, the percentage of the dose of 14C activity remaining in the body of three male and three female rats given a single oral dose of 1.Opg of [14C]TCDD/kg is plotted semilogarithmically as a function of time in days. To determine the percentage of the dose remaining in the body (body burden) for each day, the cumulative percentage of the dose excreted in the urine, feces, and respired air was subtracted from 100%. Since 14C activity was

214

ROSE ET AL.

SOL-------0 2

4

6

8

10

12

Time,

14

16

18

20

22

24

Days

FIG. 1. Percentage of the 14C activity remaining in the body (body burden) of rats given a single oral dose of 1 .O pg of [‘%]TCDD/kg.

detected in feces but not in either urine or expired air, fecal excretion accounted for most if not all of the elimination of TCDD and/or its metabolites. The percentage of the dose remaining in the body as a function of time followed apparent first-order kinetics. The fraction, f, of the dose absorbed by each rat and the rate constant, k, for elimination of 14C activity together with the corresponding half-life are shown in Table 1. There were no statistical differences (p < 0.05) in the rate constants for male versus female rats, The average f value was 0.83, the elimination rate constant 0.023 days’, and the corresponding half-life 31 days. TABLE CONSTANTS

Rat number 1 2 3 4 5 6

1

FOR THE EQUATION DESCRIBING THE BODY BURDEN ACTIVITY IN RATS GIVEN A SINGLE ORAL DOSE OF 1 .O pg OF [W]TCDD/kg“, b

Sex Male Male Male Female Female Female

f 0.66 0.77 0.91 0.93 0.87 0.91 0.84 i- O.lld

k (days-‘) 0.026 0.018 0.021 0.022 0.019 0.033 0.023

IL-0.001’ * 0.001 + 0.000 + 0.001 + 0.001 rt 0.002 & 0.006

OF

14C

t3 (days) 21 39 33 32 36 21 31 +6

‘f, The fraction of the dose absorbed; k, the elimination rate constant; t+, the body burden half-life. b Body burden = f @ose)e-kz. c Confidence limits 95%. d Mean + SD.

The concentration of 14C activity in organs and tissues was determined 22 days after a single oral dose. The concentrations of 14C activity in liver, fat, thymus, spleen, and kidney are shown in Table 2. Liver and fat contained similar concentrations of 14C activity while other selected tissues contained lesser amounts. A total of 97 f 8 y0

FATb OF TCDD

215

TABLE 2 THE CONCENTRATIONS OF 14CACTIVITY IN SELECTED TISSUES OF RATS 22 DAYS AFTER A SINGLE ORAL DOSE OF 1 .O pg OF

[14C]TCDD/kg Tissue

Percentage of dose/g of tissue

Liver Kidney Fat Thymus Spleen

1.26 +- 0.31” 0.06 f 0.06 1.25 + 1.14 0.09 + 0.05 0.02 -t 0.01

’ Mean +_SD of three male and three female rats. (mean + SD) of the 14C activity administered as [14C]TCDD was recovered in excreta, tissues, and carcasses. Fate of 14Cactivity after repeated oral dosesof 0.01, 0.1, or 1.0 pg qf [‘4C]TCDD/kg/ day Monday through Friday, weekly. In rats receiving repeated oral doses of [14C]TCDD,

the 14C activity was excreted primarily in the feces. However, significant amounts of 14C activity were also found in urine. For both male and female rats the percentage of the dose excreted in the urine relative to that excreted in the feces tended to increase with time. Females excreted more 14C activity in the urine than males. Male rats given 1.O pg/kg/day for 7 weeks excreted 3.1 f. 0.2 ‘A of the cumulative dose in the urine. Female rats given the same dose excreted 12.5 I 5.1% of the cumulative dose in the urine. The one female rat that died during the seventh week excreted 17.8% of the cumulative dose in the urine. A similar comparison was not possible for rats that received 0.01 or 0.1 /‘g/kg/day, Monday through Friday, because the urinary excretion of 14C activity was often below the limits of detection. In Fig. 2, the body burden of 14C activity in rats given 0.1 or 1.Oyg/kg/day, Monday through Friday of each week, is plotted semilogarithmically as a function of time in days. To determine the body burden, the cumulative amounts of 14C activity excreted in both the urine and feces were subtracted from the cumulative amounts of 14C activity administered orally as [14C]TCDD, and that difference was divided by the body weight to give the concentration of 14C activity in the body. The average overall recovery of 14C expressed as percentage of the cumulative dose for the 12 rats comprising the O.l- and I.O+g/kglday dose levels was 97.7 f 7.9%. The average body burdens calculated from the cumulative excretion data and the average body burdens observed in the carcasses at the end of the experiment, both expressed as percentage of the cumulative dose of 14C, were as follows: 0.1 pg/kg/day talc. 48.3 + 3.9 %, obs. 47.7 + 8.8%; 1.0 pg/kg/day talc. 41 .O k 4.3 %, obs. 37.1 + 7.5 “/o. For rats receiving 0.01 pg/kg/day, the amount of 14C activity excreted per day was too low to allow accurate determinations of the body burden. The best estimates for the fraction of the dose absorbed, J; and for the rate constant for elimination, k, over the ‘I-week study were obtained by fitting the body burden versus time data for each rat to a one compartment open model exhibiting an apparent first-order rate of elimination. The parameter estimates forfand k are shown in Table 3.

216

ROSE H-AL.

14C-TCDDI kg/Day

L -

5 0z

!?I ::_

. 0.1

-

; m

-,,I*.:

2 :

jig Equir.

“C.TCDD/ kg/Day

it, ’ Male ’ Female @Dead

I. ;

0.1 0

I

I

I

I

5

10

15

20

I

I

I

I,

25 30 Days

I

35

40

45

50

55

FIG. 2. The concentration of [%]TCDD microgram equivalents in the body of rats given 1.O or 0.1 pugof [‘%]TCDD/kg/day, MondaythroughFriday, for 7 weeks.

TABLE 3 AVERAGE PHARMACOKINETIC PARAMETER ESTIMATES FOR THE ELIMINATION OF 14CACTIVITY FROMMALEANDFEMALERATSADMINISTERED 0.1 OR l.Opg OF [14C]TCDD/kg/DAY, MONDAY THROUGHFRIDAY,FOR 7 WEEKS'

k (days-l)b

f

Rat sex

1.Oshdday

0.1 ~tzlkgldw

1.O~glkglday

0.1 ,&k/day

Male Female

0.0256 + 0.0050 0.0356 + 0.0031

0.0286 + 0.0213 0.0273 + 0.0021

0.733 f 0.104 0.931 + 0.023

0.870 + 0.061 0.910 + 0.013

(1Each parameter is expressed as the mean f SD of three rats. b Elimination rate constant.

cThe fractionabsorbed. To test for statistically significant differences between the constants of the different groups, the Mann-Whitney nonparametric test was employed (Siegel, 1956). No statistically significant differences due to sex or dose level were found (p < 0.05). Pooling the constantsfand k from all rats, the parameter estimatesare k = 0.0293 + 0.0079 days-’ andJ= 0.861 + 0.122 (mean f SD). The overall rate constant for elimination corresponds to a half-life of 23.7 days.

217

FATE OF TCDD

The steady-state body burden ip rats given daily doses of D, bg/kg) can be calculated whenf and k are known (Eqs. [A41 and [A14], Appendix). Using the values off and k determined in this study, the steady-state body burden was calculated to be 21.3 D, for rats given a daily dose of D,, 5 consecutive days per week for an infinite numbers of weeks. If D, were administered every day, the steady-state body burden would be 29.0 Do. The fraction of the ultimate steady-state body burden attained within a specified time can also be calculated. After 7 weeks of administration, 76.2% of the ultimate steady-state body burden (2 1.3 D,) was attained as determined by calculation using the designated values of,f and k. When the body burdens determined by 14C analysis at

t 0.1000

-

& > i .? E k F z

0.1

o.oloo

-

2 Y J 2

/

,~ylkylday

y------! 0.01

iiyikyhy .

7 0 0010

-

0.0001

t L

;------: Lime

1

,

Of

ihtection

I

FIG. 3. The concentration of [14C]TCDD microgram equivalents in the liver of rats given single oral doses of 0.01, 0.1, or 1.O pg of [14C]TCDD/kg, Monday through Friday, for 1, 3, or 7 weeks.

the end of 7 weeks were divided by the calculated ultimate steady-state body burden, a mean value of 73.8 y0 was found. The time required to reach 90% of the ultimate steady-state body burden was calculated to be 78.5 days (Eqs. [A81 or [A16], Appendix). Thus, after 78.5 days of a regimen in which rats are given a dose of D, pg of TCDD/kg/day, 5 consecutive days per week, the concentration of TCDD retained in the body becomes essentially constant. If the same dose were given daily instead of 5 days/week, the same fraction of the ultimate steady-state body burden would be attained at 78.5 days; however, the absolute value would be somewhat greater (26.1 D, instead of 19.2 D,). Accumulation of TCD D in tissue. For rats given 0.01, 0.1, or 1.O pg of [14C]TCDD/ kg/day, Monday through Friday, the concentrations of 14C activity in the liver are shown in Fig. 3, as the logarithm of the microgram equivalents of [14C]TCDD per gram of liver versus time in days. These data demonstrate that the microgram equiv of TCDD/g of liver approaches steady-state concentrations as treatment continues. When the concentrations of 14C activity found in the liver are divided by the dose to

218

ROSE ET AL.

normalize the data, the data demonstrate that the accumulation of 14C activity in the liver is not dose-dependent within the range of dose levels administered in this study (Fig. 4). The results shown in Figs. 3 and 4 suggest that the accumulation of [14C]TCDD in the liver follows apparent first-order kinetics. Concentrations of 14C activity in the liver as a function of time are defined by the equation C, = C,,( 1 - ePkt). In this equation, C, is the concentration of 14C activity in the liver at time f, C,, is the concentration of 14C activity in the liver at steady state, and k is the elimination rate constant from the liver. The constants, k and C,,, were obtained by a conjugate gradient search of the maximized likelihood function using the observed concentrations (C) and the time (t) when the observations were made. Values of C,, = 0.250 + 0.000 pg equiv TCDD/g of

FIG. 4. Dose-normalized concentrations of [L4C]TCDD in the liver of rats given 0.01, 0.1, or 1.Ofig of [ldC]TCDD/kg/day, Monday through Friday, for 1,3, or 7 weeks.

liver/pg dose and k = 0.026 + 0.000 days-’ (mean ? SD) were obtained. The normalized curve in Fig. 4 was generated from the above equation and the estimates fork and G. For the same rats, the concentration of 14C activity in the fat, pg equiv TCDD/g, and the concentrations normalized for dose are shown as a function of time in Figs. 5 and 6, respectively. Again, assuming apparent first-order kinetics for accumulation, the parameters C,, and k were estimated for fat to be 0.058 rf: 0.003 pg equiv TCDD/g of fat/pg dose and 0.029 f 0.001 days-‘, respectively. Figure 6 demonstrates the lack of a dose-dependent rate of accumulation within the range of doses administered. The rates of accumulation of 14C activity were similar in fat and liver as well as the whole body. However, the concentration in the liver is about five times greater than that in fat. The concentrations of 14C activity in both tissues approached plateau levels with continuous administration. Other tissues examined from rats sacrificed after 1,3, or 7 weeks were thymus, kidney, and spleen. The concentrations of 14C activity (pg equiv TCDD/g of tissue) are given in

219

FATE OF TCDD

Llmlt 0

Of

Detec,ion

1

I

I

7

21

40

0 000,

TII,,P,

FIG. 5. The concentration of [‘%JTCDD doses of 0.01, 0.1, 1 .O yg of [%]TCDD/kg,

60

Dqr

microgram equivalents in the fat of rats given single:oral Monday through Friday, for 1,3, or 7 weeks.

s0

I1.00 !&kg/day 0 0.10

Irg/kg/day

l

pg/kghlay

0.01

0

110 ” C-TCDD

Equdg

Farlpg

Dose

FIG. 6. Dose normalized concentrations of [W]TCDD in the fat of rats given 0.01, 0.1, or 1.Opg of [%]TCDD/kg/day, Monday through Friday, for 1,3, or 7 weeks.

Table 4 for liver, fat, kidney, thymus, and spleen. Concentrations found in kidney, thymus, and spleen were 1/12th to 1/50th those found in the liver. The low concentrations of 14C activity in these tissues resulted in considerable variability. The concentrations of 14C activity were often below the limits of detection in kidneys of rats receiving 0.1 pg/kg/day and in kidneys, thymus, and spleen of rats receiving 0.01 wdkldw. Since liver is the principal site of metabolism of many foreign compounds, and since liver contained the highest concentrations of 14C activity in this study, liver samples

220

ROSE ET AL.

TABLE 4 THE CONCENTRATION EQUIVALENTS OF

OF

Y

ACTIVITY IN SELECTED TISSUES EXPRESSED AS MICR~CRAM TCDD PER GRAM OF TISSUEFOR RATS GIVEN 0.01, 0.1, OR 1 .O pg OF [“‘C]TCDD/kg/Dau, MONDAY THROUGH FRIDAY, FOR 1,3, OR 7 WEEKS“

1 week

3 weeks

7 weeks -

Fat Liver Thymus Spleen Kidney

0.0100f 0.0030 0.0495f 0.0036 0.0009i- 0.0002 0.0004+ O.oool 0.0009* 0.0002

0.0235-t 0.0075

Fat Liver Thymus Spleen Kidney

0.0009+ 0.0006 0.0039rtr0.0011 ND” ND ND

0.0027+ 0.0008

0.0045f 0.0007

0.0118 + 0.0022 0.0010 + 0.0006

0.0198 + 0.0031

Fat Liver Thymus Spleen Kidney

ND ND ND ND ND

0.1102 + 0.0371

0.0073+ 0.0045 0.0016+ 0.0098 0.0019 + 0.0009

0.0614f 0.0367 0.2040L-0.0522 0.0069+ 0.0025 0.0019+ 0.0011 0.0055rt 0.0051

0.1 adkzlday

0.0006+ 0.0004 ND

0.0006+ 0.0002 0.00035 O.oool ND

0.01dkglday 0.0003(2) 0.0008+ 0.0001 0.0006+ 0.0001 0.00064 0.0003 ND

0.0003f 0.0001(4) 0.0016+ 0.0005 ND ND ND

a The mean + SD of three male and three female rats. * Not detected. c Indicates the number of animals with detectable concentrations of Y

activity.

were analyzed for concentrations of TCDD by combination gaschromatography-mass spectrometry and gaschromatography (Table 5). The concentrations of TCDD found in the liver with these methods were not statistically different from those calculated using the 14Cactivity for the samplesand the specific activity of [14C]TCDD, p < 0.05 with the paired t-test. Therefore, the r4C activity in liver (and probably fat) is predominantly [14C]TCDD. This result indicates that what has heretofore been referred to as 14Cactivity in the liver is TCDD rather than degradation products of TCDD. When the liver of rats given 1 pg of [r4C]TCDD/kg/day Monday through Friday for 7 weeks was continuously extracted with hexane and subsequently with diethyl ether: ethanol (2: l), 94.4 + 2.1 ‘A of the r4C activity was recovered. The sameprocedure extracted 99.7% of the 14Cactivity from control samplesof liver homogenate spiked with [14C]TCDD. From the extracted liver residue, 99.9 + 0.1 % of the 14C activity was extracted following treatment with 0.6 M trichloroacetic acid. From spiked control samples, 100% recovery wasattained. A total of 104 f 3 % of the 14Cactivity was extracted from liver tissuetreated with 8 M urea and no significant amounts of 14Cactivity remained in the homogenate. The recovery of 14Cactivity from samplesof liver spiked with [‘“ClTCDD was 102 + 0% after treatment with 8 M urea.

221

FATE OF TCDD

TABLE 5 CONCENTRATION OF TCDD FOUND IN THE LIVER OF RATS GIVEN 0.1 OR 1.Opg OF [14C]TCDD/kg/DAY, MONDAY THROUGH FRIDAY, FOR 7 WEEKS USING

GAS CHROMATOGRAPHY-MASS

SPECXROMETRY, AND 14C Acrrvrrv

GC/MS 1.O,4Wday

0.1 flglkglday

1.8 x 1.7 x 2.0 x 2.1 x 2.9 x 1.4 x

GAS CHROMATOGRAPHY

Micrograms of TCDD/gram of liver 14Cactivity EC/GC

10-r 10-l 10-l 10-t 10-l’ 10-t

2.1 x 2.2 x 2.0 x 3.0 x 1.5 x

1.6 x lO-2

-b -

2.1 1.8 1.2 1.8 1.8

x x x x x

10-z 1O-2 1o-2 lo-’ lo-’

10-l 10-l 10-l 10-r IO-’

1.59 x 10-l 2.16 x 10-l 2.20 x 10-t 2.04 x 10-t 2.92 x 10-l 1.33 x 10-I 2.11 2.31 1.80 2.16

x x x x 2.03 x 1.44 x

10-a 1O-2 1O-z 1O-2 1O-2 10-a

’ Rat died during Week 7 of the study. b Concentrations were not determined by EC/GC since the results would be near the limit of detection. DISCUSSION

The objectives of the study reported herein were to elucidate further the fate of TCDD in rats given oral doses of this toxic chemical. Specifically the dynamics were determined (A) for the excretion of TCDD and/or its metabolites following a single oral dose of 1 pg of [r4C]TCDD/kg, and (B) for the excretion and buildup of TCDD and/or metabolites in the whole body and selected tissues of rats given repeated oral doses of 0.01, 0.1, or 1.O pg of [14CjTCDD/kg/day, 5 days/week. The results of this study agree with findings from earlier, less extensive studies, Piper et al. (1973) reported a half-life of 17.4 f 5.6 days for the body burden of 14C activity from rats given a single oral dose of 50 ,ng/kg. As reported herein, the body burden half-life values varied from 21 to 39 days for rats given a single oral dose of 1 pg/kg. Although the dose used in the study by Piper et al. (1973) exceeded that necessary to cause death, the kinetics of elimination of TCDD and/or metabolites were not markedly different from those found in the present study. Fries and Marrow (1975) calculated a body burden half-life of 12 to 15 days for rats given 7 or 20 ppb (0.5 or 1.5 pg/kg/day) [r4C]TCDD for 42 days in the diet. In the present study, half-life values ranged from 16 to 35 days for rats given repeated oral doses of 0.01, 0.1, or 1.O ,ug/kg/day, 5 days/week, for 7 weeks. Although these values are somewhat greater, they are consistent with those reported previously because even within this study there was considerable variation between individuals. It is noteworthy that those rats demonstrating overt untoward effects excreted the 14C activity as readily as those not showing overt signs of toxicity.

222

ROSE

ET AL.

The most important information gleaned from this study has been the definition of the propensity of TCDD to accumulate in the body and individual tissues. Because TCDD is a highly toxic compound, considerable concern has been expressed that its chemical nature may allow it to accumulate toxic amounts in the body of animals exposed to extremely low levels in the diet. The results of this study indicate that such accumulation is not likely. By Week 7 of repeated daily administration of 0.01, 0.1, or 1.0 pg/kg/day, Monday through Friday, the concentrations in the body in toto had reached 74 f 4% of the ultimate steady state value. It was calculated that after 13 weeks of this regimen, 93 % of the ultimate steady-state value would be attained. In a toxicological evaluation of rats given oral doses of 0.001, 0.01, 0.1, or 1.O pg of TCDD/kg/day, Monday through Friday, for 13 weeks Kociba et al. (1976) found no perceptible untoward effects in rats given 0.001 or 0.01 icg/kg/day. Repeated oral doses of 0.1 and 1.O pg/kg/day caused untoward effects, including hepatic pathologic and functional changes, atrophy of the thymus, and hematologic alterations, with death in some rats given 1.Opg/kg/day. The results of the present study indicate clearly that those rats which had received 0.01 pg/kg/day in the study of Kociba et al. (1976) would not continue to accumulate TCDD in tissues to concentrations associated with the toxicity seen in rats receiving either 0.1 or 1.O pg/kg/day. Another important finding gleaned from the results of this study is that the rate of accumulation in the whole body is similar to the rate of accumulation in liver and fat. This was expected because most of the body burden was found in liver and fat. Liver and fat contained 50 and 10 time more 14C activity, respectively, than any other tissue examined. The rate constants of elimination of TCDD from the total body, liver, and fat were calculated to be 0.0293, 0.029, and 0.026 days-‘, respectively. At steady state the concentration of TCDD expected in the body of rats given a daily dose of D,, Monday through Friday, was calculated to be 21.3 Do. The concentrations expected in liver and fat are 0.250 and 0.058 Llg of TCDD/g of tissue/pg dose, respectively. The results of this study indicate that TCDD may be altered chemically prior to clearance from the body. The 14C activity found in the liver was unchanged TCDD. However, preliminary work indicates that materials other than TCDD constitute a significant fraction of the 14C activity excreted in the feces. That [14C]TCDD is metabolized is consistent with the fact that 14C activity is excreted in the urine of rats in this study and in the urine and expired air of studies performed in rats by Piper et al. (1973). However, tritium activity was not detected in the urine of mice given 130 fig of [3H]TCDD/kg, (Vinopal and Casida, 1973). While much of the administered 14C activity is localized in the liver and the 14C activity in the liver has been identified as [14C]TCDD, small amounts of polar metabolites may be formed in the liver and readily excreted in the bile and urine, or excreted in the bile and subsequently reabsorbed and excreted in the urine. In this study, female rats excreted more 14C activity in the urine than males, and one female rat which died during week 1 of dosing had excreted as much as 18 % of the total cumulative dose of 14C activity in the urine. The larger urinary excretion of 14C activity by females than males may be secondary to the toxic effects of TCDD. In this study and that of Kociba et al. (1976) female rats were more adversely affected by repeated administration of

223

FATE! OF TCDD

TCDD. The toxic effects of TCDD on the liver may result in a less efficient mechanism for the excretion of the more polar metabolite(s) of TCDD via the bile and a greater excretion of these polar products via the urine. Such a hypothesis is supported in part by studies in the rat showing that TCDD inhibits the rate of hepatobiliary excretion of indocyanine green (Hwang, 1973). The results of this study demonstrates that TCDD and/or metabolites approach a constant concentration in the whole body and fat and liver within 13 weeks. The rate constant defining this approach to steady state concentrations is independent of the dose level administered over the dose range of 0.001-1.0 pg/kg/day.

APPENDIX

The equation describing the cumulative body burden resulting from repetitive dosing of a compound is essentially that given by Wagner (1971). The derivation is based on the assumption that the organism under study can be treated pharmacokinetically as a single compartment and that the overall elimination rate of the compound from the body can be described by a single first-order elimination rate constant. It is implicit in the derivation that the dose, if administered orally, must be absorbed at a rate appreciably greater than the overall elimination rate from the body. The following is a derivation of the equation describing the cumulative body burden as a function of time with repetitive dosing at a constant,dose interval. Then an equation is developed that describes cumulative body burden as a function of time for the specific regimen of this experiment, i.e., when the animals were dosed daily, Monday through Friday, each week. Also, the equations describing the steady-state body burden and the number of doses necessary to reach a specified percent of steady state are developed for each of these dosing regimens. The symbols used in the subsequent mathematical expressions are: D,, dose administered (pg cmpd/kg body wt) ; 5 fraction of the dose absorbed; C,(i), concentrati

The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat.

~~x~c~L~GYANDAPPLIEDPHARMACOLOGY~~, The 209-226( 1976) Fate of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Single and Repeated Oral Doses to the following...
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