TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
51,
329-339
(1979)
2,3,7,8-Tetrachlorodibenzo-p-dioxin Tissue Distribution, Excretion, and Effects on Clinical Chemical Parameters in Guinea Pigs T. A. GASIEWICZ AND R. A. NEAL
Center in Toxicology, Vanderbilt University, Nashville,Tennessee 37232 ReceivedNovember15, 1978; acceptedJuly 23, I979 2,3,7,8-Tetrachlorodibenzo-p-dioxin Tissue Distribution, Excretion, and Effects on Clinical Chemical Parameters in Guinea Pigs. CASIEWICZ, T . A., AND NEAL, R. A. (1979). Toxicol. Appl. Pharmacol. 51, 329-339. The tissue distribution of Y-labeled 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in adult male guines pigs was studied up to 15 days following its ip injection (2.0 pg/kg). On Day 1, the highest levels of radioactivity (% of original dose/g tissue) were located in the adipose tissue, adrenals, liver, spleen, intestine, and skin. All other tissues examined contained less than 0.3%/g tissue. By Day 15, the level of radioactivity in the liver increased to nearly three times its initial value. An increase in radioactivity was also noted in the adrenals, kidneys, and lungs. These increases appeared to be due to the mobilization of fat stores and the subsequent redistribution of radioactivity contained in these stores to other organs. Following a single intraperitoneal dose of 0.5 pg [3H]TCDD/kg the excretion of 3H in the urine and feces appeared to be linear up to 23 days. Assuming the excretion of radioactivity would continue in a linear manner, the time for excretion of half the administered dose by way of the urine and feces was calculated to be 30.2f 5.8 days. The effect of TCDD (1.0 pg/kg) upon various clinical chemical parameters was determined periodically up to 14 days and compared to pairfed controls. Statistically significant increases in plasma albumin, total protein, iron, urea nitrogen, cholesterol, and triglycerides were observed in TCDD-treated pigs.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant in the synthesis of trichlorophenol used in the manufacture of 2,4,5-trichlorophenoxyacetic acid and related herbicides, and the germicide hexachlorophene (IARC, 1977). While other chlorinated dibenzo-p-dioxins may also occur in the environment, TCDD is the most toxic isomer thus far examined (Harris et al., 1973; McConnell et al., 1978a), and has been involved in accidental poisonings over the last 20 years (Kimbrough, 1974; Poland and Kende, 1976; Rawls and O’Sullivan, 1976; IARC, 1978). The acute LD50 of TCDD ranges from 0.6 pg TCDD/kg for male guinea pigs to 280 pug TCDD/kg for mice (Schwetz et al., 1973; McConnell et al.,
1978a). The signs and symptoms of TCDD toxicity are also shown by various chlorinated dibenzofurans (Moore et al., 1976) and certain polychlorinated biphenyls (Biocca et al., 1976). In experimental animals TCDD has been shown to produce chloracne (McConnell et al., 1978b), hepatic alterations (Norback and Allen, 1973 ; Jones and Butler, 1974), gastric ulcerations and hyperplasia (Allen and Carstens, 1967), hyperplasia of urinary tract epithelium (McConnell et al., 1978a), and reduced immunological response (Vos et al., 1973; Vos and Moore, 1974). TCDD has also been reported to produce alterations in the serum concentrations of bilirubin, glucose, proteins and cholesterol, as well as in 329 All
0041-008X/79/140329-11$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
330
GASIEWICZ
AND
the activity of alkaline phosphatase, SGOT, and SGPT in rats (Zinkl et al., 1973; Greig et al., 1973). Little data has been published concerning the effect of TCDD on clinical chemical parameters in other species. The tissue distribution of TCDD has been studied in rats (Piper et al., 1973; Allen et al., 1975; Van Miller et al., 1976; Rose et al., 1976) and nonhuman primates (Van Miller et al., 1976). A possible relation between the distribution of TCDD in rats and nonhuman primates and various pathological effects has been suggested (Van Miller et al., 1976). There is no published information on the tissue distribution of TCDD in guinea pigs. This information is of interest since the guinea pig appears to be the most sensitive mammalian species to TCDD toxicity and unlike the mouse or rat, hepatic damage in the guinea pig is minimal following TCDD treatment (Gupta et al., 1973; McConnell et al., 1978a). The objective of the work reported here was to determine the excretion and tissue distribution of radiolabeled TCDD in guinea pigs. In addition, the effects of TCDD upon various clinical chemical parameters have been examined.
NEAL
METHODS Male guinea pigs (Hartley strain) weighing 300350 g were purchased from Williams-Kentucky breeders (Fern Creek, KY.). The animals were housed under laboratory conditions for at least 1 week prior to use. They were fed Wayne guinea pig chow and offered water ad libitum. 14C-Labeled (40 mCi/mmol) and unlabeled ( > 98 ‘A pure) TCDD were purchased from KOR Isotopes (Cambridge, Mass.). [l,6-3H]TCDD (39 Ci/mmol) was a gift of Dr. Alan Poland. The radiochemical purity of TCDD was determined by high pressure liquid chromatography (hplc) using a modification of the method of Pfeiffer et al. (1978) and a Zorbax-ODS microparticulate column (E. I. DuPont de Nemours and Co., Wilmington, Del.). The column was eluted with 10% water in methanol at a flow rate of 60 ml/hr. Fractions were collected and the amount of radioactivity was determined by liquid scintillation counting. Figure 1 shows the separation of various chlorinated dioxin isomers (Analabs, Inc., North Haven, Conn.) by this technique. Using this procedure the 14C-TCDD was determined to be > 96 y0 pure. The major contaminant (3.8%) had an elution volume the same as 2,3,7-trichlorodibenzo-p-dioxin. The 3H-labeled material was found to be 92% 13H]TCDD with the remainder having the same elution volumes as the trichloro- and pentachloroisomers. The hplc method described above was utilized to further purify the [‘HJTCDD. The [3H]TCDD isolated by this procedure (>99% pure) was dissolved in 1,Cdioxane or anisole. Solutions of TCDD in anisole or dioxane were diluted with olive
.1.2,3,4,6.X8.(2.3.4.7.6.
2.3,7. . 1.2.4 1.6-
l
2,32.,-
l
I
I
1
, C5
*III4
Retention Ttme
IO
1 2c
3
(h)
FIG. 1. Separation of various chlorinated dibenzo-p-dioxin isomers by HPLC. A Zorbax-ODS microparticulate column, 4.6 x 25 cm, was used. The flow rate was 60 ml/hr of 10% water in methanol. The dioxins were detected by their absorbance at 245 nm.
TCDD
IN THE GUINEA
oil to a final concentration of 0.8 &ml. These solutions contained less than 20~1 of anisole or dioxane per milliliter. The solutions of TCDD in olive oil were administered to guinea pigs by ip injection. To determine the tissue distributions of [%]TCDD, animals (four per group) were housed in stainlesssteel cages and dosed by ip injection with 2.Opg [W]TCDD/kg. Due to the possible loss of animals succumbing to TCDD toxicity, groups to be sacrificed at later times consisted of five animals each. In this experiment three animals died between 10 and 15 days following TCDD administration. Three surviving animals from each group were sacrificed at the designated times and the various tissues examined for radioactivity. A portion of liver from the animals given [Y]TCDD was homogenized in 0.14 M KC1 in 0.05 M phosphate buffer, pH 7.4, and fractionated by centrifugation: 6OOg for 10 min, “crude nuclear” fraction; 15,OOOg for 5 min, “mitochondrial” fraction; lO5,OOOg for 60 min, “microsomal” fraction; 105,OOOg supernatant fraction. Each of the particulate fractions was resuspended in buffer prior to analysis for radioactivity. Tissues and subcellular fractions were solubilized in 2.0 ml of TS-1 tissue solubilizer (Research Products International, Elk Grove Village, Ill.) at 40°C 10 ml of scintillation cocktail was added followed by the addition of 100 ~1 acetic acid, and the samples were examined for radio-activity in a liquid scintillation spectrometer. The efficiency of counting was determined by the internal standard technique utilizing [Yltoluene. For the determination of the rate of excretion of TCDD by guinea pigs, PH]TCDD was administered at a dose of 0.5 pg/kg. Three animals were housed separately in stainless-steel cages designed for the separate collection of urine and feces. Urine and fecal samples were collected daily and separately prepared for 3H analysis utilizing a Packard Tri-Carb Model 306 tissue oxidizer. The “HZ0 was collected in 15 ml of Monophase 40 scintillation cocktail (Packard Instrument Co., Downers Grove, Ill.), and counted. Efficiencies for tissue oxidation and liquid scintillation counting were determined using rH]TCDD as a standard. Thus, [3H]TCDD was added to urine and fecal samples from untreated animals, the samples oxidized, counted, and the necessary corrections made to the results obtained with the experimental samples. To determine the effect of TCDD upon various clinical chemical parameters in guinea pigs the animals were divided into two groups: TCDD-treated (1.0 pg/kg ip) and pair-fed controls administered the appropriate volume of olive oil. These groups were further divided into subgroups containing five to seven animals. For each of the experimental groups food consumption and body weight change were measured
331
PIG
daily. The control animals were pair-fed the amount of food eaten by the experimental group on the previous day. Animals were observed up to 12 days following TCDD administration. Between 11 AM and 1 PM on the designated day following treatment, animals were sacrificed by exsanguination via heart puncture. The blood was allowed to coagulate at room temperature for 1 hr, the serum removed and frozen at -60°C for later analysis. Three of the twenty guinea pigs given a dose of 1.O pg/kg did not lose body weight; an invariable sign of TCDD’ toxicity. Analysis to the serum of these animals showed no significant differences from control animals. Only the results from those animals showing a significant loss in body weight along with the appropriate pair-fed controls are presented in the results section. Serum albumin, alkaline phosphatase activity, calcium, carbon dioxide, chloride, total cholesterol, creatinine, glucose, SGOT and SGPT activity, inorganic phosphorus, LDH activity, potassium, iron, sodium, total bilirubin, total protein, triglycerides, urea nitrogen, and uric acid were measured using the Technicon SMAC methods SG4-0030PC6, SG40006PC6, SG4-0003PC6, SG4-0037PC6, SG40005PC6, SG4-0026PC6, SG4-0011 PC6, SG4002PC6, SG4-OOlOPC6, SG4-0022PC6, SG40004PC6, SG4-0021 PC6, SG4-0034PC6, SG40025PC6, SG4-0033PC6, SG4-0018PC6, SG40014PC6, SG4-0023PC6, SG4-0001 PC6, and SG4-0013PC6, respectively (Technicon Instruments Corp., 1976). Statistical analysis for differences between groups was performed by the Student’s t test (Snedecor and Cochran, 1967).
RESULTS Distribution and Excretion of Radiolabeled TCDD One day following the administration of 2.0 pg [14C]TCDD/kg to guinea pigs, the highest concentrations (% dose/g) of radioactivity were located in adipose tissues, adrenals, liver, spleen, and intestine (Table 1). All other tissues examined contained less than 0.3 % ,of the original administered activity per gram of tissue. The higher concentrations of TCDD initially observed in some of the tissues, i.e., spleen and duodenum, may be a result of the administration of TCDD by ip injection. Calculated on the basis of the total weight of the tissue, nearly 25% of the administered dose of the
GASIEWICZ AND NEAL
332
TABLE DISTRIBUTION
1
OF RADIOACTIVITY IN GUINEA PIG TISSUES l-15 DAYS OF A SINGLE DOSE OF 2.0 lfg [Y]TCDD/kg
AFTER
ADMINISTRATION
14C activity (‘A of dose/g tissue) at Day Tissue
3
1
Perirenal adipose 3.2kl.o” 1.5kO.8 Epididymal adipose 1.450.3 Adrenal Liver 1.1+0.4 Liver’ 11.4L3.3 Spleen 0.7kO.3 0.4kO.2 Duodenum Pancreas 0.2kO.1 Stomach Testes 0.2*0.1 Kidneys 0.3fO.l Bone marrow 0.3kO.l Lungs 0.3fO.l Skin’ 13.8kO.7 Brain, heart, skeletal, muscle < 0.25
4.1 kO.4 3.8kO.5 1.4kO.2 1.5kO.4 15.5k3.3 0.5 +0.3 0.2+0.1 0.3kO.l 0.3fO.l 0.3kO.l 0.5kO.l 0.2+0.1 16.3kO.3
5
7
I1
15
2.1 kO.4 3.4kO.7 0.9kO.l 1.3kO.2 14.0+ 2.3 0.2+0.1 0.2kO.l 0.2kO.l 0.1+0.1 0.2kO.l 0.2kO.l 0.2kO.l 0.2kO.l 15.8 f 2.4
1.3kO.2 3.2kO.l 1.2kO.3 l.lkO.2 12.0* 1.9 0.4kO.2 0.2kO.l 0.5 + 0.3 0.2kO.l 0.3kO.l 0.4kO.l 0.4*0.1 0.4kO.l 6.5 +0.8
2.lkO.2 3.9b 2.lkO.9 2.2kO.2 21.2k2.3 0.4kO.2 0.2kO.l 0.4kO.3 0.3kO.l 0.3kO.l 0.8kO.4 0.4b 0.5 + 0.2 6.5 + 0.7
2.5+ 1.1 1.7kO.2 3.2kO.3 29.652.7 0.5kO.l 0.3 + 0.1 0.3 f 0. I 0.3kO.l 0.2kO.l 0.7kO.l 0.2kO.l 0.6kO.l 6.7 + 0.6
a Values are means f SE for groups of three animals, unless indicated otherwise. h Mean of two animals. c % of dose/g tissue.
[14C]TCDD was found in the skin and liver. By Day 15, while the level of radioactivity in SUBCELLMAR the total skin had declined to about 50% of GUINEA its value at Day 1, the radioactivity in the liver had increased to over twice its initial value. Smaller increasesin radioactivity also Dose were noted in the kidneys, lungs, and adren(KS als. These increases in concentration of TCDD/kg) radioactivity are likely due to the mobiliza- 7.0 (‘*C) tion of fat stores, a manifestation of TCDD toxicity, and the redistribution of radioactivity contained in these stores to other organs. By Day 15 all of the animals sacrificed had lost between 24 and 35 %, of body 2.0 (Y) weight. At necropsy, perirenal fat pads could not be found and epididymal fat stores were only 30-40x of their weight at Day 1. Subcutaneous fat stores were also sub- 0.3 (3H) stantially depleted. Table 2 shows the hepatic subcellular distribution
of radioactivity
at various
doses
and times following the administration of 14cor 3H-labeled TCDD. The highest levels of radioactivity were found in the
TABLE
2
DISTRIBUTION
OF
PIG LIVER AFTER RADIOLABELED
RADIOACTIVITY
A SINGLE
DOSE
IN OF
TCDD Percentage of total radioactivity on Day
Fraction
1
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
0.7*0.1”.h 35.624.4 9.5kO.2 47.4 + 3.7 7.6? 1.1
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
0.7+0.1b 20.1 f 1.5 12.9k2.5 40.7k3.1 26.4kO.9
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
l.lrtO.1” 31.3k5.1 9.5 +0.3 44.8k6.2 14.3kO.6
6 2.4&0.8b 16.7kO.3 9.9+ 1.0 7O.lkO.9 3.2kO.4
2.0* 0.2h 28.6& 1.6 9.6+ 1.5 49.2k2.6 12.6kO.7
0 All values are the mean + SE of three animals. h Values are expressed as o/0of dose/g tissue.
TCDD
IN THE GUINEA
crude nuclear and microsomal fractions. At a dose of 7.0 pg [14C]TCDD/kg, as the concentration of 14C in the liver increased with time after the administration of TCDD, the relative percentages of total radioactivity in the crude nuclear and soluble fractions were reduced by more than one-half. A similar, though less pronounced, increase in the concentration of radioactivity in the liver with time was observed at a dose of 0.3 ,ug rH]TCDD/kg. At this dose little or no body weight loss was observed in the TCDDtreated animals. Thus, less radioactivity from [3H]TCDD was apparently mobilized from adipose stores to the liver as compared to a toxic dose of 7.0 pg [14C]TCDD/kg. Figure 2 shows the cumulative excretion of 3H by guinea pigs following a single ip dose of 0.5 pug [3H]TCDD/kg. In these studies no signs of toxicity such as a decrease in body .weight were observed. At the dose administered, the appearance of 3H activity into urine and feces appeared to be linear for the total period of measurement (23 days). If the linearity of the excretion of 3H activity is
5
assumed and extrapolated, 50% of the administered dose would be excreted by 30.2 ri: 5.8 days. The majority (94.0 kO.8 %) of the jH excreted during each day was in the feces. The Effect of TCDD upon Various Chemical Parameters
Clinical
Figure 3a shows the time-related body weight changes of TCDD-treated guinea pigs and the pair-fed controls. A progressive weight loss was observed in the TCDDtreated animals, while the pair-fed controls essentially maintained body weight. One animal died at Day 9 following TCDD treatment and most animals sacrificed at Day 12 were moribund. Figures 3bi show the effect of TCDD upon various serum parameters in these animals. The increase in packed cell volume (b), plasma albumin (d) and total protein (c) in the TCDD-treated animals from Day 7 onward follow one another closely.and appear to reflect a general state of dehydration and subsequent hemoconcentration. Changes in serum iron (e) may also be due to the hemo-
IO
15 TIME
333
PIG
20
(DAYS)
FIG. 2. Excretion of 3H from the guinea pigs given a single ip dose of 0.5 pug/kg (39.0 Ci/mmol) 2,3,7,8-tetrachlorodibenzo-p-dioxin. Each value represents the mean f SE for three animals.
334
GASIEWICZ
AND
:* :
N
0
I
NEAL
:-
. :
i
,
0 6 P
no
N
0=
8 3nlvh
lOtllN03
B
u
%
N
‘(._ .
r
‘.., -4
‘..
‘.._ ‘.., ‘..,
.-
N
2
DoyDs
2
A-__
IU/L
4 DAY FOLLOWING
1278+327
4 DAY FOLLOWING
~_~
q.
~--
8 ADMINISTRbTTION
ADMINISTRATION
6 TCDD
TCDD
a
L
IO
.. ..
IO
--
-IL
,^
400
f
I
1
2
Day 0’
2
IO
IO
..1..,____... /” ;/’ ,...v , ”
6 8 TCDD ADMINISTRATION
4 6 8 DAY FOLLOWING TCDD ADMLNISTRAT!ON
L----.~
I--
,/ __.. i,/’
51OC 45mgldl
4 DAY FOLLOWlNG
-
--k-
e....
-A
*
I2
FIG. 3 (a-i) The effect of TCDD (1.0 pg/kg) upon body weight (a), packed ceh volume in blood, (b), serum concentrations of total protein (c), albumin (d), iron (e), urea nitrogen (f), SCOT activity (g), cholesterol (h), and triglycerides (i), in TCDD-treated (--) or pair-fed (-) controls at various days following treatment. Values of untreated control animals are given as Day 0 values. Results are expressed as the percentage of control value and are the mean and SE of the number ofanimals shown in parentheses in (a) for each day examined. Levels of significance as compared to pair-fed controls for each day are expressed as *,p
AND
APPLIED
PHARMACOLOGY
51,
329-339
(1979)
2,3,7,8-Tetrachlorodibenzo-p-dioxin Tissue Distribution, Excretion, and Effects on Clinical Chemical Parameters in Guinea Pigs T. A. GASIEWICZ AND R. A. NEAL
Center in Toxicology, Vanderbilt University, Nashville,Tennessee 37232 ReceivedNovember15, 1978; acceptedJuly 23, I979 2,3,7,8-Tetrachlorodibenzo-p-dioxin Tissue Distribution, Excretion, and Effects on Clinical Chemical Parameters in Guinea Pigs. CASIEWICZ, T . A., AND NEAL, R. A. (1979). Toxicol. Appl. Pharmacol. 51, 329-339. The tissue distribution of Y-labeled 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in adult male guines pigs was studied up to 15 days following its ip injection (2.0 pg/kg). On Day 1, the highest levels of radioactivity (% of original dose/g tissue) were located in the adipose tissue, adrenals, liver, spleen, intestine, and skin. All other tissues examined contained less than 0.3%/g tissue. By Day 15, the level of radioactivity in the liver increased to nearly three times its initial value. An increase in radioactivity was also noted in the adrenals, kidneys, and lungs. These increases appeared to be due to the mobilization of fat stores and the subsequent redistribution of radioactivity contained in these stores to other organs. Following a single intraperitoneal dose of 0.5 pg [3H]TCDD/kg the excretion of 3H in the urine and feces appeared to be linear up to 23 days. Assuming the excretion of radioactivity would continue in a linear manner, the time for excretion of half the administered dose by way of the urine and feces was calculated to be 30.2f 5.8 days. The effect of TCDD (1.0 pg/kg) upon various clinical chemical parameters was determined periodically up to 14 days and compared to pairfed controls. Statistically significant increases in plasma albumin, total protein, iron, urea nitrogen, cholesterol, and triglycerides were observed in TCDD-treated pigs.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) occurs as a contaminant in the synthesis of trichlorophenol used in the manufacture of 2,4,5-trichlorophenoxyacetic acid and related herbicides, and the germicide hexachlorophene (IARC, 1977). While other chlorinated dibenzo-p-dioxins may also occur in the environment, TCDD is the most toxic isomer thus far examined (Harris et al., 1973; McConnell et al., 1978a), and has been involved in accidental poisonings over the last 20 years (Kimbrough, 1974; Poland and Kende, 1976; Rawls and O’Sullivan, 1976; IARC, 1978). The acute LD50 of TCDD ranges from 0.6 pg TCDD/kg for male guinea pigs to 280 pug TCDD/kg for mice (Schwetz et al., 1973; McConnell et al.,
1978a). The signs and symptoms of TCDD toxicity are also shown by various chlorinated dibenzofurans (Moore et al., 1976) and certain polychlorinated biphenyls (Biocca et al., 1976). In experimental animals TCDD has been shown to produce chloracne (McConnell et al., 1978b), hepatic alterations (Norback and Allen, 1973 ; Jones and Butler, 1974), gastric ulcerations and hyperplasia (Allen and Carstens, 1967), hyperplasia of urinary tract epithelium (McConnell et al., 1978a), and reduced immunological response (Vos et al., 1973; Vos and Moore, 1974). TCDD has also been reported to produce alterations in the serum concentrations of bilirubin, glucose, proteins and cholesterol, as well as in 329 All
0041-008X/79/140329-11$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
330
GASIEWICZ
AND
the activity of alkaline phosphatase, SGOT, and SGPT in rats (Zinkl et al., 1973; Greig et al., 1973). Little data has been published concerning the effect of TCDD on clinical chemical parameters in other species. The tissue distribution of TCDD has been studied in rats (Piper et al., 1973; Allen et al., 1975; Van Miller et al., 1976; Rose et al., 1976) and nonhuman primates (Van Miller et al., 1976). A possible relation between the distribution of TCDD in rats and nonhuman primates and various pathological effects has been suggested (Van Miller et al., 1976). There is no published information on the tissue distribution of TCDD in guinea pigs. This information is of interest since the guinea pig appears to be the most sensitive mammalian species to TCDD toxicity and unlike the mouse or rat, hepatic damage in the guinea pig is minimal following TCDD treatment (Gupta et al., 1973; McConnell et al., 1978a). The objective of the work reported here was to determine the excretion and tissue distribution of radiolabeled TCDD in guinea pigs. In addition, the effects of TCDD upon various clinical chemical parameters have been examined.
NEAL
METHODS Male guinea pigs (Hartley strain) weighing 300350 g were purchased from Williams-Kentucky breeders (Fern Creek, KY.). The animals were housed under laboratory conditions for at least 1 week prior to use. They were fed Wayne guinea pig chow and offered water ad libitum. 14C-Labeled (40 mCi/mmol) and unlabeled ( > 98 ‘A pure) TCDD were purchased from KOR Isotopes (Cambridge, Mass.). [l,6-3H]TCDD (39 Ci/mmol) was a gift of Dr. Alan Poland. The radiochemical purity of TCDD was determined by high pressure liquid chromatography (hplc) using a modification of the method of Pfeiffer et al. (1978) and a Zorbax-ODS microparticulate column (E. I. DuPont de Nemours and Co., Wilmington, Del.). The column was eluted with 10% water in methanol at a flow rate of 60 ml/hr. Fractions were collected and the amount of radioactivity was determined by liquid scintillation counting. Figure 1 shows the separation of various chlorinated dioxin isomers (Analabs, Inc., North Haven, Conn.) by this technique. Using this procedure the 14C-TCDD was determined to be > 96 y0 pure. The major contaminant (3.8%) had an elution volume the same as 2,3,7-trichlorodibenzo-p-dioxin. The 3H-labeled material was found to be 92% 13H]TCDD with the remainder having the same elution volumes as the trichloro- and pentachloroisomers. The hplc method described above was utilized to further purify the [‘HJTCDD. The [3H]TCDD isolated by this procedure (>99% pure) was dissolved in 1,Cdioxane or anisole. Solutions of TCDD in anisole or dioxane were diluted with olive
.1.2,3,4,6.X8.(2.3.4.7.6.
2.3,7. . 1.2.4 1.6-
l
2,32.,-
l
I
I
1
, C5
*III4
Retention Ttme
IO
1 2c
3
(h)
FIG. 1. Separation of various chlorinated dibenzo-p-dioxin isomers by HPLC. A Zorbax-ODS microparticulate column, 4.6 x 25 cm, was used. The flow rate was 60 ml/hr of 10% water in methanol. The dioxins were detected by their absorbance at 245 nm.
TCDD
IN THE GUINEA
oil to a final concentration of 0.8 &ml. These solutions contained less than 20~1 of anisole or dioxane per milliliter. The solutions of TCDD in olive oil were administered to guinea pigs by ip injection. To determine the tissue distributions of [%]TCDD, animals (four per group) were housed in stainlesssteel cages and dosed by ip injection with 2.Opg [W]TCDD/kg. Due to the possible loss of animals succumbing to TCDD toxicity, groups to be sacrificed at later times consisted of five animals each. In this experiment three animals died between 10 and 15 days following TCDD administration. Three surviving animals from each group were sacrificed at the designated times and the various tissues examined for radioactivity. A portion of liver from the animals given [Y]TCDD was homogenized in 0.14 M KC1 in 0.05 M phosphate buffer, pH 7.4, and fractionated by centrifugation: 6OOg for 10 min, “crude nuclear” fraction; 15,OOOg for 5 min, “mitochondrial” fraction; lO5,OOOg for 60 min, “microsomal” fraction; 105,OOOg supernatant fraction. Each of the particulate fractions was resuspended in buffer prior to analysis for radioactivity. Tissues and subcellular fractions were solubilized in 2.0 ml of TS-1 tissue solubilizer (Research Products International, Elk Grove Village, Ill.) at 40°C 10 ml of scintillation cocktail was added followed by the addition of 100 ~1 acetic acid, and the samples were examined for radio-activity in a liquid scintillation spectrometer. The efficiency of counting was determined by the internal standard technique utilizing [Yltoluene. For the determination of the rate of excretion of TCDD by guinea pigs, PH]TCDD was administered at a dose of 0.5 pg/kg. Three animals were housed separately in stainless-steel cages designed for the separate collection of urine and feces. Urine and fecal samples were collected daily and separately prepared for 3H analysis utilizing a Packard Tri-Carb Model 306 tissue oxidizer. The “HZ0 was collected in 15 ml of Monophase 40 scintillation cocktail (Packard Instrument Co., Downers Grove, Ill.), and counted. Efficiencies for tissue oxidation and liquid scintillation counting were determined using rH]TCDD as a standard. Thus, [3H]TCDD was added to urine and fecal samples from untreated animals, the samples oxidized, counted, and the necessary corrections made to the results obtained with the experimental samples. To determine the effect of TCDD upon various clinical chemical parameters in guinea pigs the animals were divided into two groups: TCDD-treated (1.0 pg/kg ip) and pair-fed controls administered the appropriate volume of olive oil. These groups were further divided into subgroups containing five to seven animals. For each of the experimental groups food consumption and body weight change were measured
331
PIG
daily. The control animals were pair-fed the amount of food eaten by the experimental group on the previous day. Animals were observed up to 12 days following TCDD administration. Between 11 AM and 1 PM on the designated day following treatment, animals were sacrificed by exsanguination via heart puncture. The blood was allowed to coagulate at room temperature for 1 hr, the serum removed and frozen at -60°C for later analysis. Three of the twenty guinea pigs given a dose of 1.O pg/kg did not lose body weight; an invariable sign of TCDD’ toxicity. Analysis to the serum of these animals showed no significant differences from control animals. Only the results from those animals showing a significant loss in body weight along with the appropriate pair-fed controls are presented in the results section. Serum albumin, alkaline phosphatase activity, calcium, carbon dioxide, chloride, total cholesterol, creatinine, glucose, SGOT and SGPT activity, inorganic phosphorus, LDH activity, potassium, iron, sodium, total bilirubin, total protein, triglycerides, urea nitrogen, and uric acid were measured using the Technicon SMAC methods SG4-0030PC6, SG40006PC6, SG4-0003PC6, SG4-0037PC6, SG40005PC6, SG4-0026PC6, SG4-0011 PC6, SG4002PC6, SG4-OOlOPC6, SG4-0022PC6, SG40004PC6, SG4-0021 PC6, SG4-0034PC6, SG40025PC6, SG4-0033PC6, SG4-0018PC6, SG40014PC6, SG4-0023PC6, SG4-0001 PC6, and SG4-0013PC6, respectively (Technicon Instruments Corp., 1976). Statistical analysis for differences between groups was performed by the Student’s t test (Snedecor and Cochran, 1967).
RESULTS Distribution and Excretion of Radiolabeled TCDD One day following the administration of 2.0 pg [14C]TCDD/kg to guinea pigs, the highest concentrations (% dose/g) of radioactivity were located in adipose tissues, adrenals, liver, spleen, and intestine (Table 1). All other tissues examined contained less than 0.3 % ,of the original administered activity per gram of tissue. The higher concentrations of TCDD initially observed in some of the tissues, i.e., spleen and duodenum, may be a result of the administration of TCDD by ip injection. Calculated on the basis of the total weight of the tissue, nearly 25% of the administered dose of the
GASIEWICZ AND NEAL
332
TABLE DISTRIBUTION
1
OF RADIOACTIVITY IN GUINEA PIG TISSUES l-15 DAYS OF A SINGLE DOSE OF 2.0 lfg [Y]TCDD/kg
AFTER
ADMINISTRATION
14C activity (‘A of dose/g tissue) at Day Tissue
3
1
Perirenal adipose 3.2kl.o” 1.5kO.8 Epididymal adipose 1.450.3 Adrenal Liver 1.1+0.4 Liver’ 11.4L3.3 Spleen 0.7kO.3 0.4kO.2 Duodenum Pancreas 0.2kO.1 Stomach Testes 0.2*0.1 Kidneys 0.3fO.l Bone marrow 0.3kO.l Lungs 0.3fO.l Skin’ 13.8kO.7 Brain, heart, skeletal, muscle < 0.25
4.1 kO.4 3.8kO.5 1.4kO.2 1.5kO.4 15.5k3.3 0.5 +0.3 0.2+0.1 0.3kO.l 0.3fO.l 0.3kO.l 0.5kO.l 0.2+0.1 16.3kO.3
5
7
I1
15
2.1 kO.4 3.4kO.7 0.9kO.l 1.3kO.2 14.0+ 2.3 0.2+0.1 0.2kO.l 0.2kO.l 0.1+0.1 0.2kO.l 0.2kO.l 0.2kO.l 0.2kO.l 15.8 f 2.4
1.3kO.2 3.2kO.l 1.2kO.3 l.lkO.2 12.0* 1.9 0.4kO.2 0.2kO.l 0.5 + 0.3 0.2kO.l 0.3kO.l 0.4kO.l 0.4*0.1 0.4kO.l 6.5 +0.8
2.lkO.2 3.9b 2.lkO.9 2.2kO.2 21.2k2.3 0.4kO.2 0.2kO.l 0.4kO.3 0.3kO.l 0.3kO.l 0.8kO.4 0.4b 0.5 + 0.2 6.5 + 0.7
2.5+ 1.1 1.7kO.2 3.2kO.3 29.652.7 0.5kO.l 0.3 + 0.1 0.3 f 0. I 0.3kO.l 0.2kO.l 0.7kO.l 0.2kO.l 0.6kO.l 6.7 + 0.6
a Values are means f SE for groups of three animals, unless indicated otherwise. h Mean of two animals. c % of dose/g tissue.
[14C]TCDD was found in the skin and liver. By Day 15, while the level of radioactivity in SUBCELLMAR the total skin had declined to about 50% of GUINEA its value at Day 1, the radioactivity in the liver had increased to over twice its initial value. Smaller increasesin radioactivity also Dose were noted in the kidneys, lungs, and adren(KS als. These increases in concentration of TCDD/kg) radioactivity are likely due to the mobiliza- 7.0 (‘*C) tion of fat stores, a manifestation of TCDD toxicity, and the redistribution of radioactivity contained in these stores to other organs. By Day 15 all of the animals sacrificed had lost between 24 and 35 %, of body 2.0 (Y) weight. At necropsy, perirenal fat pads could not be found and epididymal fat stores were only 30-40x of their weight at Day 1. Subcutaneous fat stores were also sub- 0.3 (3H) stantially depleted. Table 2 shows the hepatic subcellular distribution
of radioactivity
at various
doses
and times following the administration of 14cor 3H-labeled TCDD. The highest levels of radioactivity were found in the
TABLE
2
DISTRIBUTION
OF
PIG LIVER AFTER RADIOLABELED
RADIOACTIVITY
A SINGLE
DOSE
IN OF
TCDD Percentage of total radioactivity on Day
Fraction
1
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
0.7*0.1”.h 35.624.4 9.5kO.2 47.4 + 3.7 7.6? 1.1
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
0.7+0.1b 20.1 f 1.5 12.9k2.5 40.7k3.1 26.4kO.9
Whole liver Crude nuclear Mitochondrial Microsomal Soluble
l.lrtO.1” 31.3k5.1 9.5 +0.3 44.8k6.2 14.3kO.6
6 2.4&0.8b 16.7kO.3 9.9+ 1.0 7O.lkO.9 3.2kO.4
2.0* 0.2h 28.6& 1.6 9.6+ 1.5 49.2k2.6 12.6kO.7
0 All values are the mean + SE of three animals. h Values are expressed as o/0of dose/g tissue.
TCDD
IN THE GUINEA
crude nuclear and microsomal fractions. At a dose of 7.0 pg [14C]TCDD/kg, as the concentration of 14C in the liver increased with time after the administration of TCDD, the relative percentages of total radioactivity in the crude nuclear and soluble fractions were reduced by more than one-half. A similar, though less pronounced, increase in the concentration of radioactivity in the liver with time was observed at a dose of 0.3 ,ug rH]TCDD/kg. At this dose little or no body weight loss was observed in the TCDDtreated animals. Thus, less radioactivity from [3H]TCDD was apparently mobilized from adipose stores to the liver as compared to a toxic dose of 7.0 pg [14C]TCDD/kg. Figure 2 shows the cumulative excretion of 3H by guinea pigs following a single ip dose of 0.5 pug [3H]TCDD/kg. In these studies no signs of toxicity such as a decrease in body .weight were observed. At the dose administered, the appearance of 3H activity into urine and feces appeared to be linear for the total period of measurement (23 days). If the linearity of the excretion of 3H activity is
5
assumed and extrapolated, 50% of the administered dose would be excreted by 30.2 ri: 5.8 days. The majority (94.0 kO.8 %) of the jH excreted during each day was in the feces. The Effect of TCDD upon Various Chemical Parameters
Clinical
Figure 3a shows the time-related body weight changes of TCDD-treated guinea pigs and the pair-fed controls. A progressive weight loss was observed in the TCDDtreated animals, while the pair-fed controls essentially maintained body weight. One animal died at Day 9 following TCDD treatment and most animals sacrificed at Day 12 were moribund. Figures 3bi show the effect of TCDD upon various serum parameters in these animals. The increase in packed cell volume (b), plasma albumin (d) and total protein (c) in the TCDD-treated animals from Day 7 onward follow one another closely.and appear to reflect a general state of dehydration and subsequent hemoconcentration. Changes in serum iron (e) may also be due to the hemo-
IO
15 TIME
333
PIG
20
(DAYS)
FIG. 2. Excretion of 3H from the guinea pigs given a single ip dose of 0.5 pug/kg (39.0 Ci/mmol) 2,3,7,8-tetrachlorodibenzo-p-dioxin. Each value represents the mean f SE for three animals.
334
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FIG. 3 (a-i) The effect of TCDD (1.0 pg/kg) upon body weight (a), packed ceh volume in blood, (b), serum concentrations of total protein (c), albumin (d), iron (e), urea nitrogen (f), SCOT activity (g), cholesterol (h), and triglycerides (i), in TCDD-treated (--) or pair-fed (-) controls at various days following treatment. Values of untreated control animals are given as Day 0 values. Results are expressed as the percentage of control value and are the mean and SE of the number ofanimals shown in parentheses in (a) for each day examined. Levels of significance as compared to pair-fed controls for each day are expressed as *,p
