The acute toxicity of 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) in the male Fischer rat

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

( 1988)

l&236-249

The Acute Toxicity of 2,3,4,7,8-Pentachlorodibenzofuran in the Male Fischer Rat DAVID *Systemic

W.

BREWSTER,*T~,’

LINDA

C.

URAIH,$

AND

LINDA

(4PeCDF)

S. BIRNBAUM*,~

Toxicology Branch and $.Chemical Pathology Branch, National Institute of Environmental Sciences, Research Triangle Park, North Carolina 2 7709 and tCurriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27514

Received

The Acute Toxicity BREWSTER,

D. W.,

September

28, 1987:

accepted

of2,3,4,7,8-Pentachlorodibenzofuran URAIH,

L. C., AND

BIRNBAUM,

March

Health

8. 1988

(4PeCDF) in the Male Fischer Rat.

L. S. Fundam.

Appl.

Toxicol.

11,236-249.

Polychlorinated dibenzofurans are ubiquitous environmental pollutants which have great potential for human exposure. To characterize the toxicity of 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), male F344 rats were administered a single oral dose of 0, 100, 250, 500, 1000, or 2000 pg 4PeCDF/kg. A progressive and dose-dependent loss of body weight was evident by 3 days after treatment. Signs of toxicity included piloerection, hair loss, hypoactivity, morbidity, and death. Death occurred as soon as 14 days after treatment and continued throughout the 3% day observation period. The LD50/35 was estimated to be 916 &kg with a 95% confidence interval of 565-1484 pg/kg. Dose-dependent increases were observed in serum cholesterol, triglyceride, and bile acid concentrations and in sorbitol dehydrogenase and aspartate aminotransferase activities. The hematocrit, hemoglobin, mean corpuscular volume. and mean corpuscular hemoglobin concentrations were depressed in a dose-dependent fashion. Hepatic ethoxyresorufin-O-deethylase (EROD) activity was increased in all treatment groups approximately 25 times above that of control animals. Lymphoid depletion in the thymus and spleen was observed in the three highest doses and thymic atrophy was present at all dose levels. Absolute liver weight and the 1iver:body weight ratio were significantly increased above controls. Hepatotoxicity was dose-dependent and was characterized by lipid accumulation resulting in hepatocytomegaly. Epithelial hyperplasia and focal ulcerations ofthe forestomach was observed in animals administered 500 pg 4PeCDF/kg. Spontaneous cardiomyopathy was exacerbated by treatment with 2000 pg/kg. Since 4PeCDF and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produce a similiar spectrum of toxic effects, the biochemical mechanism(s) of toxicity for these chemicals may be similar. 0 1988 Society ofToxicology.

ators (Czuczwa and Hites, 1984). PCDFs are produced in high temperature combustion processes or during the synthesis of chemicals from trichlorophenolic compounds (Rappe et al., 1979a). PCDFs may also be present as contaminants in disinfectants and antiseptics or as by-products in the wood, leather, and glue preservation processes (Jensen and Renberg, 1973). PCDFs were involved in the human poisoning episodes in Japan and Taiwan in which ingestion of rice oil contaminated

Polychlorinated dibenzofurans (PCDFs), related in structure and toxicity to polychlorinated dibenzodioxins (PCDDs), have been detected worldwide as environmental pollutants. Their presence in the environment has increased since 1940 as a result of the increased combustion of chlorinated organic products in municipal and industrial inciner’ Supported by Grant 5T32ES0712, Curriculum in Toxicology, University of North Carolina at Chapel Hill. ’ To whom reprint request should be addressed. 0272-0590/88

$3.00

Copyright 0 1988 by the Society of Toxicology. All rights of reproduction in any form reserved.

236

TOXICITY

OF 4PeCDF IN THE RAT

with PCDFs and polychlorinated biphenyls (PCBs) occurred (Kashimoto et al., 198 1; Masuda and Yoshimura, 1984). Toxic effects included chloracne, fatigue, gastrointestinal disorders, nausea, joint pain, weight loss, anorexia, hyperpigmentation of the nails and skin, and swelling and discharges of the Meibomian glands (Okumura, 1984; Yoshimura and Hayabuchi, 1985). The clinical signs of toxicity were persistent and were more closely associated with total PCDF than PCB intake. The long duration of toxicity was believed to be related to minimal excretion of PCDFs, especially 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF) from the body (Kashimoto et al., 1981; Masuda and Yoshimura, 1984; Rappe et al., 1979b). Because 4PeCDF has a very long biological half-life in rodents, monkeys, and humans (Brewster and Birnbaum, 1987; Brewster et al., 1988b; Chen et al., 1985) and because of its high toxicity when administered to Rhesus monkeys (Brewster et al., 1988b), the objective of this study was to characterize the toxicity of 4PeCDF in the rodent model. MATERIALS

AND

METHODS

Animals and treatment. Male Fischer 344 rats (250300 g) were obtained from Charles River Breeding Laboratory (Raleigh, NC) and were provided food (NIH 3 I rat chow) and tap water ad libitum. The animals were housed in shoebox cages and were maintained at constant temperature (23 + 2°C) and humidity (50 f 5%) and kept on a 12-hr light: I2-hr dark cycle. The animals were acclimated for 2 weeks before use. 4PeCDF (purity > 97% as determined with CC/MS) was obtained from Chemsyn Science Laboratories (Lenexa, KS) and dissolved in acetone:corn oil (1:9). After removal of the acetone by evaporation under vacuum, 4PeCDF was administered by gavage to five groups of animals (eight animals/group) at doses of 2000, 1000. 500,250. and 100 &kg at a volume of 5 ml corn oil/kg. An additional vehicle control group of eight animals was administered corn oil only. The rats were observed daily for signs of toxicity and body weights were recorded. After 35 days the surviving animals were killed with COZ, blood was collected from the retroorbital sinus, and the liver, thymus, spleen, stomach, heart, bladder, kidneys, lungs, and testicles were weighed and tissue samples were taken for histo-

231

pathological examination. Liver S-9 homogenates were prepared in 0.15 M KC1 and frozen at -70°C for analysis of ethoxyresorufin-O-deethylase (EROD) activity. During the 35-day observation period moribund animals were killed by CO2 asphyxiation and their tissues sampled for pathological analysis. Histopathology. Tissues were fixed in buffered 10% formalin, trimmed, processed, and embedded, then cut and mounted in paraffin blocks. Sections were stained with hematoxylin and eosin and examined for pathological alterations. Additional liver sections were stained with 0~0, (Luna, 1968) to determine the presence of fat. Clinical pathology. Seven days prior to dosing and 7, 2 I, and 35 days after the administration of 4PeCDF, the animals were lightly anesthetized with COZ and blood was collected from the retroorbital sinus for evaluation of clinical pathology. Samples were collected into EDTAcoated tubes and analyzed with an ORTHO ELT-8/ds Hematology Analyzer (ORTHO Diagnostics, Westwood, MA). Hematology parameters examined included white and red blood cell count (WBC, RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet number (PLT). A second sample of blood was obtained and allowed to clot at room temperature for 20 min. The serum obtained after centrifugation (20 min, 3000g) was analyzed using a Centrifichem 400 Autoanalyzer (Baker Inst. Co., Allentown, PA). The following clinical chemistry parameters were examined: Blood urea nitrogen (BUN), creatinine (CREA). albumin (ALB), total protein (PRO), bile acid (B. ACID), cholesterol (CHOL), and triglyceride (TRIG) concentrations, sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP), alanine aminotransferasc (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and 5’nucleotidase (5’ND) activities. Ethoxyresorujin - 0- deethylase induction. Hepatic EROD activity was measured in S-9 homogenates as described by Pohl and Fouts ( 1980). The presence of 10 pM dicumarol in the reaction buffer prevented DT-diaphorase activity (Lubet et al., 1985). Fluorescence of resorufin was measured after incubation of the homogenate mixture at 37°C for 20 min using a Perkin-Elmer (Oak Park, IL) fluorometer. Under these conditions production of resorufin from ethoxyresorutin was in the linear range of the standard curve. The fluorescence of resorufin in the supernatant ofthe reaction buffer after the addition of methanol was used as a standard. Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as a standard. Resorufin was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI), ethoxyresorufin from Pierce Chemical Co. (Rockford, IL), and all other biochemicals from Sigma Chemical Corp. (St. Louis, MO). All solvents were analytical grade and of the highest purity available. Statistical analysis. The LD50 determination was made using the Litchfield and Wilcoxon (1949) graphical

238

BREWSTER,

URAIH,

FIG. 1. Changes in the body weight of rats (expressed as % of initial body weight) as a function of time after oral administration of 0 (0) 100 (Cl), 250 (A), 500 (m). 1000 (V), and 2000 (0) rg 4PeCDF/kg. All points are the mean of eight animals unless otherwise indicated (error bars omitted for clarity).

AND BIRNBAUM

from 18 to 35 days depending on dose (Table 1). Twelve days after administration, rats in the 1000 and 2000 pg/kg group began displaying increased signs of toxicity including piloerection, splayed and hunched posture, fur loss, and hypoactivity. The animals appeared to have stopped grooming and normal cage activity ceased. Tremors and lacrimation were noticed in one animal at the highest dose. Four of the animals given 2000 pg/kg became moribund 19 days after administration (Table 2). The earliest time to death was 14 days after administration of 2000 pg/kg. Hemorrhages under the nails were observed in 4 of the 13 animals that died. The LD50 was estimated to be 9 16 pg/kg (Table 1). Tissue Weights

estimation method. Comparisons of body weight, organ weights, EROD activity, and hematology data were made by Fisher’s LSD test (p < 0.01) after factorial ANOVA. Variances in the clinical chemistry data were stabilized by logarithmic transformation; then comparisons were made by Fisher’s LSD test after ANOVA. Clinical pathology data from each group of 4PeCDF-treated animals were compared to control rats sampled the same day and to baseline data obtained on Day -7.

RESULTS Body Weight Loss and Toxicity Rats given the three highest doses of 4PeCDF (500, 1000, and 2000 pg/kg) began to show a loss of body weight 3 days after administration (Fig. 1). This loss in body weight and reduction in body weight gain, compared to control animals, followed a dose-response pattern and was observed in rats administered a dose as low as 250 pg/kg. The survivors in the groups administered 250,500, and 1000 pg/kg were 96, 83, and 74% of control, respectively (Table 1). In animals which had died before the termination date of the experiment, the final body weight as a percentage of the initial body was 85, 60, and 56% that of the controls (500, 1000, or 2000 pg/kg, respectively). The mean time to death varied

The absolute liver weight in animals given 100, 250, or 500 pg 4PeCDF/kg was significantly increased above that from control animals. Livers from rats administered 1000 or 2000 pg/kg weighed approximately 50% less than controls (Table 3). Liver weight as a percentage of body weight in rats administered 100, 250, or 500 pg/kg was significantly increased relative to control animals. A significant dose-related decrease in thymic weight was seen at all dose levels (Table 3). Weight of the thymus from animals administered 1000 or 2000 pg/kg was - 10% that of control animals. In 6 of the 13 animals that died, no thymic tissue was detectable. Absolute and relative tissue weights for spleen, heart, kidney, lungs, testes, and stomach are also provided in Table 3. In general, 500, 1000, or 2000 pg 4PeCDF/kg reduced tissue weights compared to those from control animals and most of the relative increases in tissue weights at the higher doses were a result of general toxic insult and the loss of body weight. Clinical Pathology Serum cholesterol and bile acid concentrations and enzymatic activity of SDH and

TOXICITY

239

OF 4PeCDF IN THE RAT TABLE I

MORTALITY

Dose b&cd

BODY WEIGHT 35 DAYS AFTER ADMINISTRATION (MEAN + SD)

AND BODY WEIGHT AS PERCENTAGE OF INITIAL OF~P~CDFORATTHETIMEOFDEATH

No. survivors Day 35

0

8

100 250 500 1000 2000

8 8 7 4 0

Final body weight Day 35 (‘% initial) I 17.6 f 117.8 f 1 12.8 f 98.2 k 86.8 + -

No. dead Day 35

Final body weight at time of death (W initial)

0 0 0 I 4 8

99.6* 70.5 + 8.0* 66.5 f 4. I*

5.2 4.3 3.7* 14.8; 11.9*

Mean time to death -

35 31*3 18+2

Note. LD50 = 916 rg/kg (565-1484) by Litchfield-Wilcoxon graphical estimation. * Significantly different from control after ANOVA and LSD, p < 0.05.

AST were significantly increased in a dosedependent manner after administration of 4PeCDF (Fig. 2). Serum cholesterol concentrations were nearly doubled in all groups 7 days after administration and remained elevated throughout the observation period. On Day 35, serum cholesterol was increased 1.6-, 1.8-, I .9-, and 2.2-fold (100, 250, 500, and 1000 pg/kg, respectively) above control animals. SDH activity was also doubled by 7 days after treatment with 500, 1000, or 2000 pg/kg, but no changes were observed in ani-

mals given the lower doses. On Day 21 bile acid concentrations and AST activity in animals administered 500 pg/kg were increased relative to controls. This change occurred sooner (7 days) at the higher doses. In contrast, serum triglyceride concentrations were significantly increased in all animals 7 days after treatment, compared to controls, except in those rats administered 2000 pg/kg (Fig. 3). This transitory increase was followed by a decline at the later time periods.

TABLE 2 CLINICALSYMPTOMSOFTOXICITY Dose

WW 0 100 250 500

No. dead/ No. treated

INTHE

RATAFTER

EXPOSURETO~P~CDF

Clinical toxicity

‘318

None None

018

None

l/8

Day 12-piloerection one animal. Day 30-l dead. Day 35-piloerection, lacrimation. and moribund,” one animal. Day 18-wasting, all animals. Day 26-death, one animal. Day 29-hypoactivity, six animals. Day 3 I-piloerection, all animals. Day 31-moribund. two animals. Day 34-death. one animal. Day IO-wasting, all animals. Day 14-death. one animal. Day 16-piloerection, changes in posture, and hypoactivity, all animals. Day 16-death. one animal. Day 17-moribund. two animals. Day l9-death, three animals. moribund, one animal.

O/8

1000

4/8

2000

818

’ Moribund animals killed by CO1 asphyxiation.

240

BREWSTER,

URAIH,

AND BIRNBAUM

TABLE 3 TISSUE WEIGHTS(~)

AND RELATIVE

100,250,500,1000,

TISSUE WEIGHTS

(‘4%BODY WEIGHT)

35 DAYS AFER

or 2000 rg4PeCDF/kg OR AT TIME OF DEATH (MEAN

ORAL EXPOSURE + SD. n = 4)

TO 0,

Dose Wk)

Tissue

0

100

250

500

1000

2000

Body wt (9) Liver %Bodywt Thymus % Body wt Spleen % Body wt Heart % Body wt Kidneys % Body wt Lungs % Body wt Testes % Body wt Stomach” % Body wt

311.25 k6.13 10.2 kO.3 3.3 kO.1 0.31 + 0.03 0.60 + 0.01 0.58 + 0.05 0.19*0.01 0.89 f 0.03 0.29 + 0.0 1 2.01 + 0.08 0.65 + 0.03 1.29&0.15 0.42 f 0.04 2.79 f 0.10 0.90 f 0.02 1.31 * 0.11 0.42 + 0.03

321.25 f12.47 13.9 ?0.8* 4.3 f 0.21 0.22 f 0.04* 0.07 f 0.01 0.63 AZ0.04 0.20 + 0.01 0.89 + 0.04 0.28 f 0.02 2.24 f 0.14* 0.70 f 0.03 1.31 kO.07 0.4 1 * 0.04 2.91 f 0.06 0.91 f 0.02 1.50 + 0.20 0.47 f 0.07

303.50 k25.04 13.9 f 1.6; 4.6 +0.2* 0.2 1 f 0.05* 0.07 * 0.01 0.65 + 0.03 0.21 + 0.01 0.85 + 0.05 0.28 + 0.02 2.20 + 0.18* 0.73 f 0.04 1.25 + 0.08 0.41 f 0.05 2.88 + 0.19 0.95 + 0.04 1.39kO.18 0.46 f 0.03

258.25 ?54.67* 11.7 +4.2 4.4 f 0.8* 0.10 f 0.07* 0.03 zk 0.02* 0.51 * 0.15 0.20 L 0.03 0.74*0.12* 0.29 + 0.02 1.98 + 0.01 0.80 k 0.20* 1.22+0.15 0.48 f 0.08 2.30 f 0.90 0.86 z!z0.22 1.33 f 0.09 0.53*0.12

182.50 *18.70* 5.0 +0.5* 2.8 20.1 0.05 2 0.03* 0.03 f 0.02* 0.36 f 0.05* 0.20 f 0.04 0.57 k 0.06* 0.31 +0.01 1.46f0.19* 0.80 f 0.04* 0.96 + O.lO* 0.53 f 0.03* 1.75 + 0.75* 0.94 k 0.36 1.14f0.19* 0.63 f 0.11

157.33 *10.41* 5.6 +0.3* 3.6 kO.4 0.03 f 0.06* 0.02 + 0.04* 0.19 f 0.05* 0.12*0.03* 0.50 f 0.01* 0.32 f 0.02* 1.56 f 0.02* 0.99 f 0.06* 0.88 zk 0.19* 0.56 + 0.12* 1.53 + 0.29* 0.97*0.13 1.12+0.13* 0.7 1 f 0.07*

’ Empty. * Significantly different from control.

No significant changes were noted in serum CREA concentration or in serum CK, ALT, or ALP activity (data not shown). BUN concentration was elevated, 64% above controls, and albumin and total protein were depressed, 32 and 34%, respectively, only in the high dose animals when moribund (data not shown). Dose-dependent decreases in MCH, MCV, HGB, and HCT were observed beginning between 7 and 21 days after administration of 4PeCDF (Fig. 4). Changes in WBC number were difficult to evaluate due to animal variability, and there were no significant changes in RBC concentration, MCHC, or PLT number (data not shown). EROD Activity Hepatic EROD activity, measured in S-9 homogenates, was induced approximately

25-fold by all doses of 4PeCDF 35 days after administration (Table 4). Average induction was 25-fold. Pathology Dose-dependent hepatotoxicity was characterized by cytoplasmic lipid accumulation which resulted in hepatocytomegaly (Fig. 5). The fatty change in livers from animals administered 100 and 250 pg/kg was in a centrilobular distribution, while entire lobes were involved from animals treated with higher doses of 4PeCDF. Fatty accumulation was confirmed by staining with 0~0~ and electron microscopic examination (data not shown). Biliary hyperplasia was present in livers of rats administered 500, 1000, or 2000 &kg. Epithelial hyperplasia of the nonglandular stomach, characterized by acanthosis and hy-

TOXICITY

OF 4PeCDF IN THE RAT

241

CHOL 160

=L!!!+h &ACIDS

400

AST

1

i

V

300

1

/

T 5 200 E 9 100

I/

,.’

/

/

0 -7

P

.f :*. 1-.--- -W i....

/

7

21

5o-i Days

FIG. 2. Serum cholesterol and bile acid concentrations and serum SDH and AST activities 7 days prior to dosing and 7,2 1, and 35 days after oral administration of0 (0), 100 (A), 250 (0),500 (m), 1000 (0), or 2000 (v) rg 4PeCDF/kg. *Significantly different from control animals on the same day and from baseline values (Day -7) of corresponding dose group (p < 0.05). Error bars omitted for clarity. Data (mean f SD of four to eight animals) analyzed by Fischer’s LSD test after ANOVA.

perkeratosis, was present in animals given the three higher doses. In animals administered 2000 pg/kg forestomach hyperplasia was associated with focal ulceration, active inflammation, and edema (Fig. 6). This may

FIG. 3. Serum triglyceride concentration 7 days previous to and 7,2 1, and 35 days after oral administration of 0 (0), 100 (A), 250 (O), 500 (U), 1000 (0), and 2000 (v) pg 4PeCDF/kg. Each point is the mean of four to eight animals. *Significantly different from control animals on the same day and from baseline values (Day -7) of corresponding dose group (p < 0.05). Error bars omitted for clarity.

have been related to the general debilitation observed at this dose. Lymphoid depletion in the spleen and thymus was present in animals given 500, 1000, or 2000 pgfkg. Thymic atrophy was characterized by lack of corticomedullary differentiation (Fig. 7). Spontaneous cardiomyopathy, characterized by multifocal hyaline degeneration of cardiac muscle fibers, sarcolemma cell proliferation, fibrosis, and mononuclear leukocytic infiltration was present in control animals and was exacerbated by the debilitated condition of the rats following treatment with 2000 pg 4PeCDF/kg (Fig. 8). No treatment-related histological changes were observed in the kidneys, urinary bladder, testes, or lungs. DISCUSSION The objective of this study was to evaluate the toxicity of 4PeCDF in the adult male rat

242

BREWSTER,

URAIH,

AND BIRNBAUM

HCT

-7

7

21

35

Days

HGE

IL

-i

i

Days

MCH

il

i5

-i

i

Dayt

il

3i

FIG. 4. Hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in rats 7 days before and 7,2 1, and 35 days after oral administration of 0 (0), 100 (a), 250 (o), 500 (O), 1000 (0), and 2000 (7) fig 4PeCDF/kg. Each point is the mean of four to eight animals. *Significantly different from control animals on the same day and from baseline values (Day -7) of corresponding dose group (p < 0.05). Error bars omitted for clarity.

after a single oral exposure. The clinical signs of toxicity and the pathological lesions produced by 4PeCDF are similar to those produced by 2,3,7,8-tetrachlorodibenzo-p-di-

TABLE 4 HEPATIC ETHOXYRE~~RUFIN-~DETHYLASE (EROD) ACTIVITY IN SURVIVING ANIMALS 35 DAYS AFTER TREATMENTWITH 4PeCDF (MEAN f SD OFFOURTO EIGHTANIMALS)

Dose M/kg) 0

100 250 500 1000 *Significantly
EROD (nmol/mg S-9 protein/min) 1.13 kO.54 27.85 f 4.87* 16.27 f 5.94* 24.90 f 6.10* 3 I .86 + 6.02* different from control with LSD, p

oxin (TCDD) (Gupta et al., 1973; Harris et al., 1973; Kociba and Schwetz, 1982; Poland and Knutson, 1982; Zinkl et al., 1973). Animals administered 4PeCDF displayed a progressive and dose-dependent loss in body weight, significant alterations in organ weights, increased serum cholesterol and triglyceride concentrations, increased bile acids concentration and serum SDH, and AST activities, declined MCH, HGB, and HCT, hepatomegaly with hepatocellular fatty change, gastric epithelial hyperplasia, and an average 25-fold increase in hepatic EROD activity. 4PeCDF produced a profound loss of thymic tissue. TCDD is speculated to cause thymic atrophy by altering the pattern of differentiation of the thymic epithelial target cells (Greenlee et al., 1984). 4PeCDF may act in a similar manner. The increase in liver weight at the lower doses can be attributed both to fatty accumu-

TOXICITY

OF 4PeCDF IN THE RAT

243

FIG. 5. Liver from a control rat (top) and from a rat administered 500 pg 4PeCDF/kg 35 days previously (bottom). In the 4PeCDF-treated animals fatty accumulation (large arrow), biliary hyperplasia (small arrow), focal inflammation (double arrows), and necrosis were observable. These alterations were dose-dependent.

lati Ion and to an increase in microsomal proten IS, reflected in the change in EROD activity. The elevated levels of serum AST and SD IH are indicative of hepatic damage which

occurred at all doses as revealed by histcIpathology. At the higher doses 4PeCDF cat .tsed a decline in liver weight as a result of gen (era1 toxic insult. This was also observed for

244

BREWSTER,

URAIH,

AND BIRNBAUM

FIG. 6. Sections of stomach from a control rat (A) and from a rat administered 500 (B) or 2OOCl(C) pg 4PeCDF/kg 35 days previously. Hyperplasia consisting of acanthosis (large arrow) and hyperkeratosis (small arrow) was present in the nonglandular portion of the stomach as well as edema (E) and chronic active inflammation (see B). Ulcerations (arrows) were present in the epithelial lining of the stomach from animals given the higher doses (see C).

spleen, heart, kidneys, lungs, stomach, and testes. The heart, kidneys, and lungs are less sensitive to this general toxicity since the or-

gan body weight ratios are increased. The increase in absolute kidney weight at the lower doses could be reflective of enzyme induction

TOXICITY

OF

4PeCDF

IN

THE

RAT

245

FIG. &-Continued.

(Poland et al., 1974), although that possibility was not examined in this study. The increase in relative stomach weight at the higher doses is reflective of the gastric mucosal edema and coincided with observable morphological alterations. The most sensitive change in the clinical pathology parameters evaluated in this study was the increase in serum cholesterol which could be the result of increased hepatic cholesterol synthesis as well as decreased hepatic low density lipoprotein binding observed in guinea pigs and rats after exposure to TCDD (Bombick et al., 1984). Elevated levels of serum cholesterol have been observed in industrial workers after exposure to these types of polychlorinated aromatic hydrocarbons (Oliet al., ver, 1975; Pazderova-Vejlupkova 1981). The serum triglyceride concentration underwent a transitory increase at Day 7 followed by a rapid decline at later time periods. Studies with TCDD have shown that serum triglyceride concentrations did not change 10 (Brewster and Matsumura, 1988) or 13 days (Albro et al., 1978) after TCDD treatment, while they were increased threefold 2 and 8

days after TCDD administration (Christian et al., 1985). These apparently conflicting results suggest that TCDD produces a transitory pattern of hypertriglyceridemia in the rat similar to that produced by 4PeCDF in the present study. Of course, a reduction in food intake, which does occur after prolonged exposure to this class of compounds, can influence serum triglyceride concentration. The rabbit, which shows little weight loss after TCDD administration and little reduction in food intake, does display continued high levels of serum triglycerides (Brewster et al., 1988a). The reported LD50 for TCDD in male Fischer rats is - 160 pg/kg (Walden and Schiller, 1985). Data from this study indicate that in the rat, 4PeCDF is - 5 times less toxic than TCDD in terms of a single oral LD50/35. Recent studies from our laboratory (Brewster et al., 1988b) indicate the toxicity of 4PeCDF in the Rhesus monkey is similar to that of TCDD, which has been estimated to be ~70 pg/kg (McConnell et al., 1978). Although the spectrum of toxic effects in the rat and monkey after exposure to 4PeCDF is similar (Table 5), several differ-

246

BREWSTER,

URAIH,

AND BIRNBAUM

FIG. 7. Thymus from a control rat (top) and from a rat administered 1000 pg 4PeCDF/kg 35 days previously (bottom). Lymphoid depletion and a Lossofthe corticomedullary differentiation were evident.

ences are apparent. The monkey is 15-20 times more sensitive than is the rat to the lethal effects of 4PeCDF as calculated by the

estimated LD50 values. Although the rat less sensitive, death occurs after 2-4 wee while death in the monkey occurred 5

TOXICITY

247

OF 4PeCDF IN THE RAT

FIG. 8. Sections ofthe heart from a 4PeCDF-treated rat I9 days after administration of 2000 pg 4PeCDF/ kg. Cardiomyopathy consisted of multifocal hyaline degeneration, sarcolemma cell proliferation, fibrosis. and mononuclear leukocytic infiltration.

weeks after administration of a single lethal dose. The changes in the clinical pathology between the two species are striking and re-

semble the differences seen between rats and monkeys after administration of TCDD. As previously demonstrated (McConnell et al.,

TABLE 5

Dose (&cd LD50 (r&z) Clinical toxicity Time to death (days) Clinical pathology

Rat

Monkey

500-2000 -900 Loss of body weight, wasting, piloerection, some hair loss, decreased activity. 14-30 Increased chol, SDH, AST, and transitory increase in TG followed by decrease in TG. Decreased MCH, HGB, HCT, and trend of decreased WBC. Thymic atrophy, loss of fat stores. Hepatomegaly, hyperplasia of nonglandular stomach, lymphoid depletion of spleen and thymus. Exacerbation of cardiomyopathy.

34 -50” Body weight loss, wasting, alopecia, loss of finger and toenails. muscular atrophy. lacrimation, diarrhea. 40-50 Increased TG, B. Acid, WBC. Decreased RBC, HGB, HCT, protein, chol, albumin.

p Two of three monkeys died 40 and 48 days after administration Oral absorption was -70% therefore 34 rg/kg iv - 50 &kg po.

Muscular and thymic atrophy. Loss of fat stores. Hypertrophy of the gastric epithelium, gall bladder, eyelid, ear canal, and nail beds. of 34 fig 4PeCDF/kg, iv (Brewster et af., 1988).

248

BREWSTER,

URAIH,

1978), monkeys display more dramatic signs of toxicity (hair loss, nail loss, respiratory involvement) than do rats, but the liver of the monkey is less sensitive to these types of compounds than is that of the rat. Furthermore, 4PeCDF produced a different type of gastric lesion in the rat than was earlier observed in the monkey (Brewster et al., 1988b). In the monkey there was cystic gland formation in the mucosa, with downgrowth of the glands into the submucosa and replacement of the parietal epithelium with columnar mucous cells. In the rat, 4PeCDF produced epithelial hyperplasia in the nonglandular portion of the stomach and was associated with acanthosis and hyperkeratosis. TCDD produces similar types of lesions. The significance of the enhanced cardiomyopathy is not known and may be related to the debilitated condition of the rats. In conclusion, the results from this study indicate that 4PeCDF produces a similar spectrum of toxicity in the rat as does TCDD. Although a greater than fivefold increase in dose is needed to produce this toxicity, the fact that the spectrum of toxic effects is similar indicates that these compounds, and probably the entire class of dibenzofurans and dibenzodioxins, act in a similar biochemical manner. ACKNOWLEDGMENTS The authors express their appreciation to Morrow Thompson, D.V.M., Ralph Wilson, and Pat Blair for the clinical pathology analysis, and to Fred Talley for his technical assistance. The statistical assistance of Dr. J. Haseman was also greatly appreciated.

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