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Effects of a polybrominated biphenyl mixture in the rat and mouse
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
68,
Effects of a Polybrominated
I- 18 ( 1983)
Biphenyl Mixture in the Rat and Mouse
I. Six-Month
Exposure
B. N. GuPTA,‘,’ E. E. MCCONNELL,’ J. A. GOLDSTEIN,’ M. W. HARRIS,’ AND J. A. MOORE’ National
Toxicology
Program, National Instittrfe Re.yearch Triangle Park, North
Received
January
qf Environmental Carolina 27709
5. 1981; acrepled
November
Health
Sciences,
I, 1982
Effects of a Polybrominated Biphenyl Mixture in the Rat and Mouse. 1. Six-Month Exposure. GUPTA,
B. N.,
MCCONNELL,
E. E., GOLDSTEIN,
J. A.,
HARRIS,
M.
W.,
AND
MOORE,
J. A.
I-18. A 1973 environmental accident in Michigan resulted in exposure of humans via the food chain to polybrominated biphenyl (PBB). To better characterize the toxicity of the halogenated polycyclic aromatic hydrocarbon class of chemicals, rodents were dosed with PBB and their target organs examined for morphological, histological. biochemical, and selected endocrine changes. Male and female rats and mice were given I25 po doses of PBB over a B-month period at 0.1, 0.3, 1.0. 3.0, and 10.0 mg/kg of body weight/day (5 days/week). There was a dose-related decrease in body weight gain in both male and female rats and male mice. Thymus weights were significantly decreased in all rats exposed to 0.3 mg/ kg or more of PBB. Dose-related hepatotoxic effects were observed in both rats and mice characterized by marked increase in liver weight with accentuation of hepatic lobular markings. Microscopically, there were moderate to marked swelling, disorganization, and single cell necrosis of hepatocytes, fatty infiltration, bile duct proliferation, and presence of atypical hyperplastic foci. Hepatic porphyrin levels were markedly increased in both rats and mice primarily in females. There was a significant decrease in serum thyroxine (T4) and triiodothyronine (Tj) suggesting that PBB may interfere with thyroid hormone secretion. There was a significant dose-related increase in serum cholesterol and y-glutamyl transpeptidase, and a decrease in serum glucose. (1983).
Toxicol.
Appt. Pharmacol.
68,
The accidental addition of polybrominated biphenyls3 (PBB) instead of magnesium oxide to animal feed resulted in widespread contamination in Michigan in 1973 (Cook et al., 1978; Jackson and Halbert, 1974; Mercer et al., 1976). This error subsequently resulted in PBB exposure to farmers and their families, and later, other consumers of contaminated meat, dairy products, and eggs (DiCarlo et al., 1978; Humphrey and Hayner, 1975; Kay,
1977). Although PBB was reported to have a relatively low toxicity with a single po dose (LD50-2 1.5 g/kg of body weight) in the laboratory rat (Anon, 197 1, 1976; Hilltop4), it was more toxic when given in smaller doses over a 30-day period and the animals were observed for 90 days (Gupta and Moore, 1979). In another report, the po LD50 of octabromobiphenyl for female rats was stated to be greater than 2000 mg/kg body weight (Aftosmis et al., 1972). Several subsequent studies have shown that the toxicity of PBB was similar to other halogenated polycyclic hy-
’ National Toxicology Program, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC. 27709. * To whom all correspondence should be addressed. 3 Firemaster BP-6, Michigan Chemical Corp., St. Louis, Mich.
4 Hill Top Research Inc. (1970). Acute toxicity and irritation studies on Firemaster BP-6 hexabromobiphenyl for Michigan Chemical Corporation. I
0041-008X/83
$3.00
Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reqwd.
GUPTA
2
drocarbons in laboratory animals (Kasza et al., 1978b; Harris et al., 1978a). Rats fed a diet containing 10 ppm of PBB for 30 days developed a slight hepatotoxicosis, and pups nursing treated dams also had microscopic and ultrastructural hepatic lesions (Sleight and Sanger, 1976). Hepatic changes, such as necrosis, fibrosis, and neoplastic nodules, were seen in rats 10 months after they were given a single po dose (1 .O g/kg of body weight) of PBB (Kimbrough et al., 1977, 1978). Similar atypical foci in the liver were observed in rats as early as 6 months when they were exposed to 22 daily doses of PBB at 30.0 or 100.0 mg/kg body weight (Gupta and Moore, 1979). PBB was also reported to be embryotoxic and teratogenic to rat embryo (Beaudoin, 1977; Corbett et al., 1975; Harris et al., 1978b). Other studies have shown that PBB is transferred to the fetus (Aftosmis et al., 1972; Detering et al., 1975; Rickert et al., 1978) and is excreted in the milk (Fries and Marrow, 1975; Fries, 1978; Gutenmann and Lisk, 1975; Moore et al., 1978). The current study was designed primarily to determine the pathobiologic effects of the commercial polybrominated biphenyl mixture exposure in the rat and mouse during a 6-month period. METHODS A polybrominated biphenyl (PBB) mixture5 (Firemaster BP-6 and 2% calcium trisilicate, Lot No. 1312 Ff, Batch 03) was mixed with corn oil and given to Fischer 344/N (CDF) rats6 (51 rats/dose/sex) and B&F, mice’ (50 mice/dose/sex). Both rats and mice were intubated on 5 consecutive days per week for 25 consecutive weeks ( 125 doses over a 6-month period). PBB was given to rats and mice at 0 (control), 0.1, 0.3, 1.0, 3.0, and 10.0 mgl kg body weight/day. The concentration of the solutions in corn oil was adjusted weekly based on the mean body weight so that rats and mice received a constant volume of 0.2 and 0.1 ml/day, respectively. Control animals received equal amounts of corn oil. ’ Midwest Research Institute, Kansas City, MO. 6 Charles River Breeding Laboratories, Inc., Wilmington, Mass. ’ Harlan Industries, Indianapolis, Ind.
ET AL. Three rats or five mice of each sex were housed in one polycarbonate cage (45 X 20 X 20 cm for rats and 30 X 17 X 12 cm for mice). Food* and water were provided ad lib&m. Ground corn cob’ was the bedding for rats and hard wood chipslo for mice. A 12-hr light-dark cycle was provided, and temperature was maintained at 21 + 1°C and 50 f 5% was the relative humidity. All animals were observed daily for clinical signs. Each animal was weighed weekly, and food consumption was recorded once a week. For weight gain, organ weight, hematology. and clinical chemistry data, the Kruskal-Wallis nonparametric analysis of variance (Hollander and Wolfe, 1973) was used to determine the significance of among-group differences. The significance of dose-response trends was assessedby Jonckheere’s test (Jonckheere, 1954). If an overall effect was detected, then pairwise comparisons were made by Mann-Whitney U tests (Hollander and Wolfe, 1973). At the end of the 6-month exposure, groups of 10 randomly selected rats or mice/dose/sex were killed. Rats were killed by exposure to high levels of CO2 until respiration ceased, and mice were killed by placing them in a glass desiccator containing cotton gauze sponges soaked with methoxyfluorane” until deep anesthesia was achieved. Blood samples were collected from rats via orbital sinus and from mice by cardiac puncture for hematologic and clinical chemistry evaluation. Clinical pathologic examination of blood included total erythrocyte (RBC), leukocyte (WBC), hemoglobin (Hb) concentration, packed cell volume (PCV), platelet counts, and differential counts of leukocytes. Clinical chemistry examination of serum included serum glutamic pyruvic transaminase (SGPT), y-glutamyl transpeptidase (GGTP), alkaline phosphatase (AP). blood (serum) urea nitrogen (BUN), glucose, triglycerides, cholesterol, and total protein. Serum thyroxine (T4) concentrations and triiodothyronine (T,) uptake were determined with TETRA-TAB and TRI-TAB T1 uptake kits.‘* The test was performed as recommended by the manufacturer except that 20 ~1 of bovine serum was used for T4 determination instead of 10 ~1. Tr was determined by the Serulate Total T-3 KitI with the following modifications: The T, antiserum was diluted to provide approximately 40% retention of ‘3’I-labeled TS on the column in the absence of nonra-
s Open Formula Rat and Mouse Ration (NIH-3 I), Ziegler Brothers Inc., Gardners, Penn. 9 Bed-O’Cobs, The Andersons, Maumee, Ohio. lo Betta Chip Bedding, Granvtlle Milling Co., Creedmoor, N.C. ‘I Metofane, Pitman-Moore, Inc., Washington Crossing, N.J. I2 Nuclear Medical Labs, Dallas, Tex. ” Ames Co., Division of Miles Laboratories, Elkhart, Ind.
POLYBROMINATED Male
q
260
BIPHENYL
30
IN RAT AND MOUSE
3
rat
control
fZII0mg 0 3.0mg I 10.0 mg
QOlmg 803mg
t 8m260 t
TOXICITY
60
90 -uays
FIG. 1. Histogram showing difference in body weight gain in male rats exposed to PBB, a total of 125 doses, at the end of a 6-month exposure period. dioactive T3 (zero standard), and the incubation period was changed from 2 to 1 hr. Weights of lung, heart, liver, spleen, thymus, right kidney, right adrenal gland, right gonad, both thyroid glands, uterus, and brain were recorded, and organ to body weight ratios were calculated. In addition to the organs weighed, tissue samples from pinna, eye, trachea, urinary bladder, sternum, quadriceps muscle, accessory male and female sex organs, six levels of gastrointestinal tract, lumbar spinal cord, sciatic nerve, spinal ganglion, salivary glands, mesenteric and thoracic lymph nodes, mammary glands, ear canal, and nasal turbinates were fixed in buffered neutral 10% formalin for histopathologic examination. These specimens were embedded in paraffin, sectioned 6 pm thick, and stained with hematoxylin and eosin. Cryostat prepared sections (10 pm thick) of formalin-fixed liver were also stained with oil red 0 for examination for neutral lipids. Paraffin sections of liver and spleen from six rats and mice each of both sexes given 10.0 mg/kg of body weight of PBB and control (a total of 48 animals) were also stained with Mallory’s method for iron (Luna, 1968). Portions of liver from two male and two female rats (exposed to highest dose and control) were also fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde in cacodylate buffer (pH 7.4) for 6 hr. These specimens were washed with cacodylate buffer (pH 7.4) for 2 hr, dehydrated in alcohol and propylene oxide, and embedded in Epon 812. Thick sections (0.5 pm) for light microscopy were cut with an ultramicrotome and stained with tolu-
idine blue. Thin sections (400 to 600 A) were stained with uranyl acetate and Reynold’s lead citrate (Reynolds, 1963). At the time of necropsy, liver, sternum, bones, and incisor teeth of all rats and mice were examined under long wavelength (366 nm) ultraviolet (uv) lightI for reddish pink fluorescence as an indication of excess accumulation of porphyrin. A portion of liver from rats and mice was frozen for determination of total hepatic porphyrins (Abritti and de Matteis, 1971, 1972).
RESULTS
Clinical Observation During the 6-month dosing period, animals were clinically normal in appearance. No treatment related deaths were observed. There were depressed rates of body weight gain in rats as a function of time and dose (Figs. 1 and 2). The body weight gain in female mice (10.0 mg/kg/day) was higher, whereas in the male mice, it was depressed (Fig. 3). There was no significant difference in the body weight I4 Model B-100 Blak-Ray Ultraviolet Lamp, Ultraviolet Products Inc., San Gabriel, Calif.
GUPTA 140 a 0”
Female rat Cl control 610.1 mg ElO3mg
ET AL.
El I.Omg El 3.0 mg I IOOmg
120
90
60
Days FIG. 2. Histogram showing difference in doses, at the end of a 6-month period.
body
weight gain in female rats exposed to PBB. a total of I25
gain in other groups of treated mice and control. Food Consumption There was no significant difference in food consumption either in the rat or mouse. However, there was a slight decrease in food intake in the female rats exposed to 10.0 mg/kg/day of PBB during the last 6 weeks of exposure.
No significant differences in thymus weights were observed. Spleen weight was increased only in higher dose female mice. Another notable effect was a decrease in uterine weights of the 10.0 mg/kg dose. Significant weight differences were not observed in other organs. Hematologic
Findings
The Hb and PCV values were decreased in both male and female rats at the two highest dosages (Table 4). Similarly, mean corpusOrgan Weights cular volume (MCV) and mean corpuscular (MCH) were also decreased. There was a dose-related increase in the hemoglobin absolute and relative liver weights in ail treated However, this decrease was more pronounced in males compared with females at the same groups of female rats, and in males receiving 0.3 mg/kg/day or more of PBB (Tables 1 dose levels. Total serum protein was slightly lower primarily in female rats exposed to 1.O, and 2). Thymus weights were significantly 3.0, and 10.0 mg/kg of PBB. The WBC count (p < 0.05) decreased in rats given 0.3 mg/kg or more of PBB. The weight of the spleen was was significantly (p < 0.05) increased in treated increased in those rats given 1.0 mg/kg or female rats at dose levels of 1.O, 3.0, and 10.0 mg/kg due to an increase in both neutrophils higher of PBB (Tables 1 and 2). Significant @ < 0.05) increases in liver and lymphocytes. However, this trend was not weights were also observed in female mice observed in treated male rats. given 0.3 mg/kg or more, and in male mice There was a significant (s, < 0.01) dose-regiven 1.O mg/kg and more of PBB (Table 3). lated increase in the RBC count in male and
POLYBROMINATED
I
BIPHENYL
0 0 mgQ mouse KllOmgQ
R 0 mgd R IO mgd
mouse
0
TOXICITY
30
5
IN RAT AND MOUSE
mouse mouse
so
60
120
150
180
Days FIG. 3. Histogram showing changes in the body weights of male and female mice exposed to PBB at 10.0 mg/kg, 125 total doses during a 6-month period.
female mice. The MCV was decreased in both male and female mice in a dose-related fashion. However, significant differences in PCV and Hb concentration in the treated and control mice were not observed. Platelet counts were significantly QI < 0.01) decreased in the female mice given 3.0 and 10.0 mg/kg of PBB but there was no difference in treated and control male mice. There was leukocytosis due primarily to an increase in lymphocytes in female mice treated with the highest dose level.
Clinical Chemistry
Total serum protein was decreased in a dose-related fashion in female rats primarily due to the dose-related decrease in albumin. There was a significant @ < 0.05) increase in the serum levels of GGTP in female rats given the 10.0 mg/kg dose (Table 5). Serum glucose was significantly 0, < 0.05) decreased in 10.0 mg/kg treated female rats only. There was a marked decrease in the serum triglyceride level
TABLE ABSOLUTE AND RELATIVE WEIGHTS POLYBROMINATED BIPHENVL
I
OF LIVER, SPLEEN, AND THYMUS OF MALE RATS TREATED (125 DOSES) AND RECORDED AT THE END OF 6 MONTHS~
Liver weight
Spleen weight
Thymus weight
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
Absolute (g)
0.1
13.2 f 0.14 13.9 + 0.43
0.3
15.8 f 0.27’
1.0
18.8 + 0.32’ 21.4 + 0.51’ 20.8 f 0.34’
0.58 0.57 0.63 0.64 0.77 0.76
0.14 2 0.01 0.14 f 0.01 0.16 f 0.01 0.16 f 0.01’ 0.21 f 0.01’ 0.25 + 0.01’ p < 0.01
0.14 0.13 0.12 0.11
3.0
3.2 3.4 4.0 4.8 5.9 6.7
0
10.0 Dose-response
p < 0.01
+ + f 2 + +
0.03 0.08’ 0.15 0.04’ 0.08’ 0.05’
p < 0.01
f + f + f -+
0.03 0.02 0.02 0.02b 0.03’ 0.04’
p < 0.01
“Dataareexpressedas~~SEMof10rat.s. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
WITH
-c 0.01 f 0.01 + 0.016 + o.OOc 0.07 + 0.0 1c 0.04 + 0.00’ p <
0.01
Relative (70) 0.034 0.033 0.030 0.029 0.020 0.012
2 0.001 f 0.002
+ 0.001 + 0.00 1c + o.Oc2c
+ O.OOlC
p < 0.01
GUPTA
6
ET AL.
TABLE2 ABSOLUTE AND RELATIVE WEIGHTS OF LIVER, SPLEEN, AND THYMUS OF FEMALE RATS TREATED POLYBROMINATED BIPHENYL (125 DOSES) AND RECORDEDAT THE END OF 6 MONTHS“ Liver weight Dose bwWday) 0 0.1 0.3 1.0 3.0 10.0 Dose-response
Spleen weight
WITH
Thymus weight
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
6.2 + 0.12 6.8 t 0.23" 6.6 * 0.216 7.1 f 0.27’ 7.8 f 0.22' 8.6 k 0.24' p < 0.01
2.9 + 0.06 3.1 f 0.07b 3.3 f 0.11’ 3.4 + 0.09' 4.3 f 0.10’ 5.6 rt_0.09' p < 0.01
0.38 * 0.01 0.38 r 0.01 0.39 k 0.01 0.41 -t 0.01 0.49 F 0.02' 0.43 k 0.02' p < 0.01
0.18 + 0.01 0.17 * 0.01 0.19 * 0.01 0.20 t- 0.01* 0.27 t 0.01’ 0.28 + 0.01’ p < 0.01
0.14 + 0.00 0.13 + 0.01 0.12 + 0.01’ 0.11 + 0.01’ 0.07 + 0.00' 0.04 + 0.01 c p < 0.01
0.070 +- 0.002 0.06 1 + 0.002 0.056 f 0.002' 0.052 -+ 0.002' 0.037 * 0.002' 0.024 f 0.004' p < 0.01
a Data are expressed as X f SEM of 10 rats. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
in treated male rats except at the lowest dose (0.1 mg/kg). There was an apparently doserelated increase in the serum levels of cholesterol in both male and female rats (Table 5). There were no significant differences in SGPT, AP, and BUN of treated and control rats of either sex. Serum levels of GGTP tended to be slightly increased in both male and female mice given 10.0 mg/kg of PBB. There was a five- to sixfold increase in the activity of SGPT in both male and female mice in 10.0 mg/kg groups.
AP was also increased in mice given the highest dose of PBB. Serum glucose was significantly (p < 0.0 1) decreased only in female mice exposed to 10.0 mg/kg of PBB. At the high dose, PBB decreased serum thyroxine values to 40% of control rats (Table 6). The decrease was dose related and significant decreases were seen at doses of 0.3 mg/ kg of PBB in males, and 1.O mg/kg in females. Serum triiodothyronine (T3) levels were also decreased by PBB, but the effect was somewhat less and only seen at high doses. The T3
TABLE3 ABSOLUTE WEIGHT (g) AND RELATIVE (%) LIVER TO BODY WEIGHT OF MALE AND FEMALE MICE TREATED POLYBROMINATED BIPHENYL (125 DOSES) AND RECORDEDAT THE END OF 6 MONTHS’ Liver weight of male mice Dose b.Odday) 0 0.1 0.3 1.0 3.0 10.0 Dose-response
Liver weight of female mice
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
1.84 f 0.06 1.95 -t 0.03 1.81 + 0.05 2.12 f O.OSb 2.53 k 0.07' 4.32 f 0.10’ p < 0.01
4.96 4.89 4.88 5.55 6.42 12.38
1.23 * 1.27 f I .34 f 1.57 k 1.89 f 4.56 f
4.48 4.51 4.89 5.68 6.68 15.03
f 0.15 + 0.08 + 0.11 f 0.166 _+ 0.17’ + 0.32’ p < 0.01
a Data are expressed as X f SEM of 10 mice. b Significantly (p < 0.05) different compared with control. ‘Significantly (p -C 0.01) different compared with control.
0.04 0.03 0.03 b 0.06' 0.03 0.10’ p < 0.01
+ 0.08 + 0.07 f 0.07' f 0.19’ t 0.09 f 0.29' p < 0.01
WITH
POLYBROMINATED
BIPHENYL
TOXICITY
7
IN RAT AND MOUSE
TABLE 4 HEMATOL~GIC
Treatment Male Male Female Female
Control PBB Control PBB
VALUE OF RATS TREATED WITH POLYBROMINATED BIPHENYL (PBB) AT 10.0 mp/kg BODY WEIGHT/DAY DURING A ~-MONTH PERIOD”
Hemoglobin (ti1~ ml) 16.9 ?z 0.3 13.6 ? 0.2’ 17.8 + 0.3 16.3 f 0.6’
Packed cell volume (%) 50.6 39.5 50.0 46.2
f f f f
Red blood cells ( I 06/cmm)
0.94 1.05 1.44 1.50
5.79 5.55 5.44 6.09
k f + +
0.09 0.11 0.16 0.29
Mean corpuscular volume (pm)) 85.9 71.2 92.0 76.1
+ f k f
1.3 1.8’ 1.9 1.8’
125 DOSES Mean corpuscular hemoglobin (p& 29.2 24.5 32.8 26.9
f 0.5 + 0.2* k 0.7 zt 0.9’
a Values represent X + SEM. * Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.0 1) different compared with control.
uptake in male rats given the highest dose females of both species. Males, however, were (10.0 mg/kg) was significantly (p < 0.01) de- affected much less than females. creased compared with the controls, but it was slightly increased in female rats given 1.O mg/ ~~~~~~~~ Findings kg of PBB (Table 6). PBB produced dose-related hepatic porLiver, bone, and teeth of female rats given phyria in rats and mice of both sexes (Table 1.O to 10.0 mg/kg of PBB had slight to marked 7). At the highest doses, hepatic porphyrin reddish pink fluorescence under uv light delevels were increased several hundred-fold in noting excess porphyrin accumulation. The TABLE 5 CLINICAL
CHEMISTRY
Dose OWWday) Male
0 0.1 0.3 1.0 3.0 10.0 Dose-response
Female
0 0.1 0.3 1.0 3.0 10.0 Dose-response
*p cp
VALUES OF RATS ADMINISTERED POLYBROMINATED AND RECORDED AT THE END OF 6 MONTHS’
y-Glutamyl transpeptidase (IU/liter) 3.1 3.3 2.6 3.0 3.5 3.9
k 0.7 + 0.8 f 0.6 k 0.5 f 0.3 + 0.6 NS
3.3 + 3.2 + 2.6 + 2.2 f 4.5 + 7.9 +
0.7 0.5 0.4 0.4 0.6 1.2b
p < 0.05
Serum
BIPHENYL
( 125 Doss)
Serum triglyceride (mg/lOO ml)
Serum cholesterol (mg/lOO ml)
135 + 12 133 f 13 138 + 12 143 +- 10 120+ 4 122+ 6 NS
335 + 146 324 AZ139 155 k 15 119+ 8’ 152 +- 12* 155 f 18
89+ 12 110+21 94+ 6 116+ 5
131 f 118& 120+ 113~ 116+ 101 +
109* llO-+ 122 & 91 + 115 +107+ NS
glUCOS.2
(m&l00
ml)
13 3 8 6 4 10
p < 0.05
< 0.05, significantly ditRrent compared with control of same sex. < 0.01, significantly different compared with control of same sex.
p < 0.05
7 13 32 19 10 5
160 + 18”
243 + 15” p < 0.01
123+ 5 136 of: 10 141 f 6 146k 6* 181 ? lib 147 + 12
p < 0.01
GUPTA
8
ET AL.
TABLE 6 EFFECTOF ~-MONTH ADMINISTRATION OF POLYBROMINATED BIPHENYL (I 25 TOTAL DOSES) ON SERUM THYROID HORMONES IN RATS’
T4 h/l~ Do= bg/W Control 0.1 0.3 1.0 3.0 10.0 Dose-response
T, (ng/lOO ml)
ml)
T, uptake
Male
Female
Male
Female
Male
Female
5.3 f 0.2 4.5 f 0.8 4.4 f 0.3b 4.0 f o.3b 3.0 f 0.3’ 2.1 + 0.1’ p < 0.01
3.1 f 0.2 3.6 f 0.3 3.3 + 0.4 2.1 f 0.2’ 1.7 f 0.2’ 1.5 * 0.2’ p < 0.01
126+ 7 99-c 15 111 _+ 13 133 f 11 107 f 10 104+ 3 NS
123 f 14 106+ 9 112+ 8 86? 10 8Ok 8* 77k 96 p < 0.01
61.9 + 0.9 62.6 ‘-e 1.4 59.9 ” 0.6 58.9 I!I 0.7 59.1 z!I 1.4 56.1 I!Y0.5’ p < 0.01
58.1 58.4 58.7 61.7 59.2 60.3
f + + f k f
1.0 0.7 0.5 0.6’ 0.6 0.6
p c 0.02
LIValues represent X f SEM of 10 rats. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
number of positive samples and intensity of red fluorescence was less in treated male rats as compared with treated females. These tissues from all female mice exposed to 10.0 mg/ kg of PBB showed slight to marked tluorescence whereas only 8 of 10 male mice were positive at the same dosage. The livers of treated rats and mice of both sexes were moderately to markedly enlarged, especially in higher dose levels. The edge of the liver of treated rats extended much farther posteriorly from the edge of the rib cage as compared with the control animals. The livers
were pale or slightly yellow and mottled. Two male rats (one given 0.3 mg/kg and the other 10.0 mg/kg) had several grayish white nodules (about 5 mm in diameter) in the liver. One female rat of 10 given 10.0 mg/kg of PBB had a grayish white space occupying mass involving the interior of urinary bladder. The thymus of treated rats (10.0 mg/kg) appeared smaller compared with the control. No lesions were observed in other organs. Eight male and one female of 10 mice per sex in the 10.0 mg/kg groups contained several small grayish white foci in the liver. One
TABLE 7 EFFECT OF POLYBROMINATED BIPHENYL ADMINISTRATION (125 TOTAL DOSESDURING A 6-MONTH PERIOD) ON HEPATIC PORPHYRINLEVELS (ccg/g) IN THE RAT AND MOUSE” Rats Dose Ow/W
Male
Control 0.1 0.3 1.0 3.0 10.0 Dose-response
1.0 f 0.2 0.8 f 0.1 2.0 f 0.3’ 15.3 + 4.2’ 36.1 + 8.3’ 45.3 XL 11.W p < 0.01
Mice Female 0.7 0.5 15.8 1002.0 1369.0 517.0
f 0.1 iz 0.1 + 8.2’ f 65.0’ + 65.0’ f 80.0’ p < 0.01
’ Values represent X -t SEM of 10 animals. b Significantly (p < 0.05) increased compared with controls. ‘Significantly (p < 0.01) increased compared with controls.
Male 1.0 f 1.0 f 1.2 f 1.7 -t 18.7 f 58.0 2
0.1 0.1 O.lb 0.1’ 4.3 10.0’ p < 0.01
Female 1.1 0.7 0.9 3.5 97.0 435.0
f 0.2 + 0.1 f 0.1 f 2.0 k 23.0’ * 20.0’ p < 0.01
POLYBROMINATED
BIPHENYL
TOXICITY
IN RAT AND MOUSE
male mouse of 10 controls also showed a similar lesion.
9
foci invariably contained golden brown granular pigment which was positive for iron (Mallory’s method for iron; Luna, 1968). Occasionally, the reacting cells in the center of Histopathologic Findings these granulomas formed rosette-like structures around these hemosiderin granules. Slight to marked histopathologic dose-re- Small clefts of fatty acids were occasionally lated changes in the liver were observed in all observed in these foci. Single cell hepatocelgroups of treated male rats but only in the lular necrosis with infiltration of neutrophils 1.0, 3.0, and 10.0 mg/kg female groups. The was also observed. There was slight to modmicroscopic appearance of these livers was erate focal or diffuse bile duct proliferation in characterized by disorganization of trabecular the liver (Fig. 5). cords, moderate to marked enlargement of Atypical foci were observed in the livers of hepatocytes some of which were multinucle3 of 100 (3.0%) treated rats. These foci were ated, moderate fatty infiltration (oil red 0 well delineated groups of hypertrophic hestain), and hyalinization of the cytoplasm of patocytes with enlarged nuclei containing other hepatocytes (Fig. 4). Some of the he- prominent nucleoli and foamy or eosinopatocytes had a foamy appearance. Lipid ac- philic ground glass cytoplasm (Fig. 6). On ultrastructural examination of the liver, cumulation was more prominent in male than there was an increase in the amount of rough female rats and was primarily located around endoplasmic reticulum (RER) and lipid globthe central vein. Numerous granulomatous foci with occa- ules of different sizes in the hepatocytes of sional multinucleated giant cells were ob- male rats given the highest dose of PBB. In served primarily near the central vein. These the treated female rats (highest dose), the
FIG. 4. Photomicrograph of the liver of a female rat given PBB at 10.0 mg/kg, 125 total doses during a 6-month period. Notice three multinucleated giant hepatocytes. H and E stain: X750.
10
GUPTA
ET AL.
FIG. 5. Bile duct proliferation and fibrosis in the liver of a male rat treated with PBB, 125 total doses at 10.0 mg/kg. Note a few bile ducts with hyperchromic cells. H and E stain; X600.
changes in the liver were much more severe as denoted by marked proliferation and disorganization of RER, dilated cristae, and dense matrix of mitochondria (Figs. 7 and 8). Occasionally, intramitochondrial flocculent deposits were also observed. The space occupying mass in the urinary bladder of the female rat in the 10.0 mg/kg group was a squamous cell carcinoma. This neoplastic mass protruded into the lumen and was covered with one to several layers of atypical flattened epithelium. It was composed of islands of squamous epithelium some of which contained concentric laminations of keratin. The connective tissue stroma was heavily infiltrated with inflammatory cells. Although the neoplastic cells infiltrated into the deeper muscular wall of the urinary bladder, no metastases were observed.
Although there was no significant difference in the weights of thyroid glands, slight to moderate morphologic changes were observed primarily in male rats exposed to 10.0 mg/kg of PBB. Microscopic changes in the thyroid gland were characterized by thin, sparse, or bluish colloid with basophilic stippling. Some follicles were lined with columnar epithelium and also contained a few epithelial papillary projections. Changes in the thyroid gland of exposed female rats (highest dose) were not remarkable compared with the female control. The kidneys of male rats treated with 10.0 mg/kg dose level of PBB consistently showed atrophy of a few glomerular tufts with marked dilatation of Bowman’s capsule which contained amorphous eosinophilic staining material. A few glomerular tufts also appeared
POLYBROMINATED
BIPHENYL
TOXICITY
IN
RAT
AND
MOUSE
11
12
GUPTA
FIG. 7. Electron photomicrograph of a portion PBB at 10.0 mg/kg body weight. Note the marked of mitochondria. Uranyl acetate and lead citrate;
ET AL.
of a hepatocyte from a female rat given 125 doses of increase and disorganization of RER, and dense matrix X 19,350.
edematous. Some renal tubules in both cortical and medullary regions were dilated and contained either serous fluid or proteinaceous casts. Significant lesions were not observed in other organs.
Microscopic alterations in the livers of male and female mice (10.0 mg/kg dose levels) were marked swelling of hepatocytes primarily around the central veins, foamy or vacuolated cytoplasm with hyaline bodies, and focal co-
POLYBROMINATED
BIPHENYL
TOXICITY
IN RAT AND MOUSE
13
FIG. 8. Electron photomicrograph of a portion of a hepatocyte from a female rat given the highest dose of PBB. Note intramitochondrial flocculent deposits (arrows) and dilated and vesicular cristae in several mitochondria. Uranyl acetate and lead citrate; X30,638.
agulative necrosis or scattered single cell necrosis of hepatocytes. Only slight to moderate swelling of hepatocytes was observed in mice treated with 1.0 and 3.0 m&kg of PBB, and no lesions were observed at lower doses. Atypical hepatocellular foci (Fig. 9) were present in nine male and two female treated mice of 100 examined (I 1.O%), and in 1 of 20 (5.0%)
control mice. These foci were similar to those described in the rat. Iron-positive granules, probably hemosiderin, were observed primarily in the red pulp of the spleen of all treated and control animals. However, these granules were more prominent in the spleen of treated female rats than treated males and both more so than the
14
GUPTA
ET AL.
FIG. 9. Delineated area of foamy or vacuolated hepatocytes in the liver of a male mouse exposed to PBB at 10.0 mg/kg ( I25 total doses). Notice slight compression of hepatocytes at the margin of this atypical focus. H and E stain; X240.
controls. There was no difference in the spleens of treated and control male mice. However, the spleen of control female mice tended to have more iron-positive granules compared with the PBB-treated female mice.
DISCUSSION The LD50 of PBB was mathematically determined to be 65 mg/kg/day (total 1.43 g/kg) for F344 female rats and 149 mg/kg/day (total
POLYBROMINATED
BIPHENYL
3.28 g/kg) for the males (Gupta and Moore, 1979). These results were obtained from rats exposed to 22 multiple doses of PBB during a l-month period and then observed for 90 days. There was 100 and 38% mortality in female and male rats, respectively, given doses of PBB at 100 mg/kg, and no deaths in the 30 mg/kg/dose groups. On the basis of the above observations (mortality, body weight gain effect, thymic atrophy, and hepatotoxicity), 10.0 mg/kg of PBB was selected as the highest dose level for a 6-month study. The lower four doses were selected on a logarithmic basis, i.e., 10.0, 3.0, 1.0,0.3, and 0.1 mg/ kg/day. In this 6-month exposure to PBB there was no significant difference in the food consumption among PBB-treated rats and mice and controls, yet there was a persistent decrease in body weight gain in treated male and female rats and treated male mice. This decrease suggests that PBB may cause poor feed utilization. Similar weight gain effects which are only partially related to feed intake have been observed in monkeys exposed to PBBs (Allen et al., 1978), and in other species of animals (rats, monkeys, and cattle) with structurally similar halogenated aromatic hydrocarbons such as PCBs (Allen and Abrahamson, 1973) and the dibenzodioxins (McConnell et al., 1978, 1980). Interestingly, there was an increase in body weight of female mice exposed to PBB at 10.0 m&kg/day ( 125 total doses) during a 6-month period. Similar gains of body weight were observed in female mice exposed to PBB in our previous experiment (Gupta et al., 198 1). The explanation for this disparity is not clear; however, body weight gain may be related to fluid accumulation in different tissues of the body. Extracellular fluid (subcutaneous edema, ascites, and hydropericardium) is often found in mice dying from acute intoxication to these classes of compounds (McConnell and Moore, 1979), and, in fact, mice showing this lesion do actually gain weight just prior to death.
TOXICITY
IN
RAT
AND
MOUSE
15
Weights of the thymus glands were decreased in rats administered PBB (0.3 mg/kg or higher) but not in mice. Administration of PBB to rats and mice caused suppression of both humoral and particularly cell-mediated immunity (Luster et al., 1978). In a 6-month exposure of PBB in rats and mice, this chemical was not a potent immunosuppressant in rodents since high doses were required to elicit immune effects (Luster et al., 1980). Increase in the absolute and relative liver weights of rats and mice, and microscopic alterations were expressions of hepatoxicity. The increase in liver weights was primarily due to hepatocytomegaly, increase in endoplasmic reticulum, and excess lipid accumulation (Gupta et al., 1981). At the end of 6 months, only 3.0% of the treated rats, 11 .O% of the treated mice, and 5.0% of the control mice developed atypical hepatocellular foci. Since the incidence of atypical foci in the liver was so low at the 6-month observation, the potential hepatocarcinogenicity of PBB could not be evaluated. Chronic administration of hexachlorobenzene, PCBs, or TCDD to rats produces hepatic porphyria characterized by a delayed onset and massive accumulation of uroporphyrins in the liver (Ockner and Schmid, 196 1; Goldstein et al., 1973, 1974, 1976). In the present study, PBBs also produced hepatic porphyria at doses as low as 0.3 mg/kg/day. The rat was more sensitive than the mouse and, in both species, the female was much more susceptible than the male. This sex difference has been reported previously for the porphyrogenic action of hexachlorobenzene in rats (San Martin de Viale et al., 1970). However, this finding is the first report of a similar sex difference in mice. The porphyrogenic effects of PBBs in the rat may be of particular importance since an equivalent condition has been reported in humans through accidental consumption of hexachlorobenzene in Turkey in 1956 (Ockner and Schmid, 1961). Frank hepatic porphyria has not been found in people exposed to PBBs in
16
GUPTA
Michigan; however, an alteration in the urinary porphyrin pattern which may reflect preliminary changes has been reported (Strik et al., 1979). Pigment (hemosiderin) accumulation probably represents by-products of a breakdown of erythrocytes which in turn are phagocytized by the reticuloendothelial cells of the liver and spleen. Breakdown of RBCs would also explain the observed anemia. Slight decrease in MCV and MCH demonstrated that the anemia was micro&c and hypochromic. There was no indication of an increase in the number of circulating nucleated red blood cells. These findings suggest a direct depression of red blood precursors. Anemia and bone marrow depression have been observed in all species of animals exposed to toxic levels of polychlorinated dioxins and furans, especially in long-term studies (World Health Organization, 1978). The mildly increased activities of serum GGTP in both rats and mice (highest dose only) and marked increases in the concentration of SGPT and AP in the mouse only were further indication of hepatic injury. Although clinical chemistry is a useful noninvasive tool in determining hepatotoxicity in rodents, morphologic changes in hepatocytes appeared to be the most sensitive parameters in this study. Dose-related microscopic alterations in the liver were observed in all groups of treated male rats but only in the 1.0, 3.0, and 10.0 mg/kg female groups; however, on clinical chemistry evaluation, serum GGTP values were increased only in the higher dosages (Table 5). In the present study, PBB decreased the serum thyroxine concentrations. The absence of an effect on uptake indicates that this effect was not due to alterations in binding to plasma protein. The binding of thyroid hormone to serum proteins was not affected by PBB as indicated by the T3 uptakes. Microscopic changes in the thyroid glands were observed but only at the higher doses. Morphologic changes in the thyroid glands were also observed in male rats exposed to PBB (Kasza et
ET AL.
al., 1978a; Gupta and Moore, 1979), but serum levels of thyroid hormones were not measured. Norris et al. ( 1975) found evidence of thyroid hyperplasia in rats fed octabromobiphenyl for 30 days at 8.0 mg/kg/day. TCDD and PCB also decrease serum thyroxine levels and produce goiters in rats (Bastomsky, 1974, 1977a,b). The goiters were more evident in animals on low iodine diet. The effect of TCDD and PCB on serum thyroxine concentrations appears to be secondary to an increase in the hepatic glucuronidation and biliary excretion of thyroxine. Interestingly, there has been a report of primary hypothyroidism in four men (11.4% of those examined) employed in a plant which produced the PBBs, decabromobiphenyl and decabromobiphenyl oxide (Bahn et al., 1980). ACKNOWLEDGMENTS The authors appreciate the excellent technical help of Mses. J. D. Allen and D. L. Myers and Messrs. R. E. Wilson and F. A. Talley. We also thank Dr. J. K. Haseman for statistical analyses.
REFERENCES ABRITTI, G., AND DE MATTEIS, F. (197 1, 1972). Decreased levels of cytochrome P-450 and catalase in hepatic porphyria caused by substituted acetamides and barbiturates. Chem.-Biol. Interact. 4, 28 l-286. AFTOSMIS, J. G., CULIK, R., LEE, K. P., SHERMAN, H., AND WARITZ, R. S. (1972). Toxicology of brominated biphenyls. I. Oral toxicity and embryotoxicity. Toxicol. Appl. Pharmacol. 22, 3 16 (Abstr.). ALLEN, J. R., AND ABRAHAMSON, L. J. (1973). Morphological and biochemical changes in the liver of rats fed polychlorinated biphenyls. Arch. Environ. Cont. Toxicol. 1, 265-280. ALLEN, J. R., LAMBRECHT, L. K., ANDBARXXTI, D. A. (1978). Effects of polybrominated biphenyls in nonhuman primates. J. Amer. Vet. Med. Assoc. 173, 14851489.
ANONYMOUS (197 1). Firemaster BP-6. A new frame retardant additive from Michigan Chemical Corp. 2nd revision, April 26, I97 1. ANONYMOUS MICHIGAN STATE UNIVER~I~ AGRICULTURAL EXPERIMENT STATION ( 1976). MSU Research on PBBs. Mich. Sci. Action, May 1976. BAHN, A. K., MILLS, J. L., SNYDER,P. J., GANN, P. H., HOUTEN, L., BIALIK, O., HOLLMANN, L., AND UTIGER,
POLYBROMINATED
BIPHENYL
R. D. (1980). Hypothyroidism in workers exposed to polybrominated biphenyls. New England J. Med. 302, 31-33. BASTOMSKY, C. H. (1974). Effect of a polychlotinated biphenyl mixture (Aroclor 1254) and DDT on biliary thyroxine excretion in rats. Endocrinology 95, 11501155. BASTOMSKY, C. H. (1977a). Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzo-pdioxin. Endocrinology 101, 292-296.
BASTOMSKY, C. H. (1977b). Goiters in rats fed polychlorinated biphenyls. Canad. J. Phys. Pharmacol. 55,288292. BEAUDOIN, A. R. (1977). Teratogenicity of polybrominated biphenyls in rats. Environ. Res. 14, 8 l-86. COOK, H., HELLAND, D. R., VANDERWEELE, B. H., AND DJZJONG,R. J. (1978). Histotoxic effects of polybrominated biphenyls in Michigan dairy cattle. Environ. Res. 15, 82-89. CORBETT, T. H., BEAUDOIN, A. R., CORNELL, R. G., ANVER, M. R., SCHUMACHER, R., ENDRES, J., AND SZWABOWSKA,M. (1975). Toxicity of polybrominated biphenyls (Firemaster BP-6) in rodents. Environ. Res. 10, 390-396. DETERING, C. N., PREWITT, L. R., COOK, R. M., AND FRIES, G. F. (1975). Placental transfer of polybrominated biphenyl by Holstein cows. J. Dairy Sci. 58, 764 (Abstr.). DICARLO, F. J., SEIFFER,J., AND DECARLO, V. J. (1978). Assessment of the hazards of polybrominated biphenyls. U.S. Environmental Protection Agency, Washington, DC. Technical Report No. EPA-560/6-77-037. FRIES, G. F. (1978). Distribution and kinetics of polybrominated biphenyls and selected chlorinated hydrocarbons in farm animals. J. Amer. Vet. Med. Assoc. 173, 1479-1484. FRIES, G. F., AND MARROW, G. S. (1975). Excretion of polybrominated biphenyls into the milk of cows. J. Dairy Sci. 58, 947-95 1. GOLDSTEIN, J. A., HICKMAN, P., BERGMAN, H., AND VOS, J. G. (1973). Hepatic porphyria induced by 2,3,7,8tetrachlorodibenzo-pdioxin in the mouse Res. Commun. Chem. Pathol. Pharmacol. 6, 919-928. GOLDSTEIN, J. A., HICKMAN, P., AND JUE, D. L. (1974). Experimental hepatic porphyria induced by polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 27, 437448. GOLDSTEIN, J. A., HICKMAN, P., AND BERGMAN, H. (1976). Induction of hepatic porphyria and drug-metabolizing enzymes by 2,3,7,8,-tetrachlorodibenzo-p dioxin (TCDD). Fed. Proc. 35, 708 (Abstr.). GUPTA, B. N., MCCONNELL, E. E., HARRIS, M. W., AND MOORE, J. A. (1981). Polybrominated biphenyl toxicosis in the rat and mouse. Toxicol. Appl. Pharmacol. 57, 99-l 18. GUPTA, B. N., AND MOORE, J. A. (1979). Toxicologic
TOXICITY
IN RAT AND MOUSE
17
assessmentsof a commercial polybrominated biphenyl mixture in the rat. Amer. J. Vet. Res. 40, 1458-1468. GUTENMANN, W. H., AND LISK, D. J. ( 1975). Tissue storage and excretion in milk of polybrominated biphenyls in ruminants. J. Agric. Food Chem. 23, 1005-1007. HARRIS, S. J., CECIL, H. C., AND BITMAN, J. (1978a). Effects of feeding a polybrominated biphenyl flame retardant (Firemaster BP-6) to male rats. Bull. Environ. Contam.
Toxicol.
19, 692-696.
HARRIS, S. J., CECIL, H. C., AND BITMAN, J. (1978b). Embryotoxic effectsof polybrominated biphenyls (PBB) in rats. Environ. Health Perspect. 23, 295-300. HOLLANDER, M., AND WOLFE, D. A. (1973). Nonparametric Statistical Methods. Wiley, New York. HUMPHREY, H. E. B., AND HAYNER, N. S. (1975). Polybrominated biphenyls: An agricutural incident and its consequences. II. An epidemiological investigation of human exposure. In Trace Substances in Environmental Health IX (D. D. Hemphill), pp. 57-63. Univ. of Missouri Press, Columbia. JACKSON, T. F., AND HALBERT, F. L. (1974). A toxic syndrome associated with the feeding of polybrominated biphenyl contaminated protein concentrate to dairy cattle. J. Amer. Vet. Med. Assoc. 165, 437-439. JONCKHEERE,A. R. (1954). A distribution-free K-sample test against ordered alternatives. Biometrika 41, 133145. KASZA, L., COLLINS, W. T., CAPEN, C. C., GARTHOFF, L. H., AND FRIEDMAN, L. (1978a). Comparative toxicity of polychlorinated biphenyl and polybrominated biphenyl in the rat thyroid gland: Light and electron microscopic alterations after subacute dietary exposure. J. Environ.
Path.
Toxicol.
1, 587-599.
KASZA, L., WEINBERGER,M. A., HINTON, D. E., TRUMP, B. F., PATEL, C., FRIEDMAN, L., AND GARTHOFF, L. H. (1978b). Comparative toxicity of polychlorinated biphenyl and polybrominated biphenyl in the rat liver: Light and electron microscopic alterations after subacute dietary exposure. J. Environ. Path. Toxicol. 1, 241-257. KAY, K. (1977). Polybrominated biphenyls (PBB) environmental contamination in Michigan, 1973-1976. Environ.
Res. 13, 74-93.
KIMBROUGH, R. D., BURSE, V. W., AND LIDDLE, J. A. (1978). Persistent liver lesions in rats after a single oral dose of polybrominated biphenyls (Firemaster FF- 1) and concomitant PBB tissue levels. Environ. Health Perspect.
23, 265-273.
K~MBROUGH, R. D., BURSE, V. W., LIDDLE, J. A., AND FRIES, G. F. (1977). Toxicity of polybrominated biphenyl. Lancet 2, 602-603. LUNA, L. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology 3rd Ed., pp. 183-184. McGraw-Hill, New York. LUSTER, M. I., BOORMAN, G. A., HARRIS, M. W., AND MOORE, J. A. (1980). Laboratory studies on polybrominated biphenyl-induced immune alterations following
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low-level chronic or pre/postnatal exposure. Int. J. Immunopharmacol..
2, 69-80.
LUSTER, M. I., FAITH, R. E.. AND MOORE, J. A. (1978). Effectsof polybrominated biphenyls (PBB) on immune response in rodents. Environ. Health Perspect. 23,227232.
MCCONNELL, E. E., AND MOORE, J. A. (1979). Toxicopathology characteristics of the halogenated aromatics. Ann. NY Acad.
Sri. 320, 138-150.
MCCONNELL, E. E., MOORE, J. A., AND DALGARD, D. W. (1978). Toxicity of 2,3,7.%tetrachlorodibenzop-dioxin in rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. Appl. Pharmacol. 43, 175-187.
MCCONNELL, E. E.. MOORE, 3. A., GUPTA, B. N., RAKES, A. H., LUSTER, M. I., GOLDSTEIN, J. A., HASEMAN, J. K., AND PARKER, C. E. (1980). The chronic toxicity of technical and analytical pentachlorophenol in cattle. I. Clinicopathology. Toxicol. Appl. Pharmacol. 52,468490.
MERCER, H. D., TESKE, R. H., CONDON, R. J., FURR, A., MEERDINK, G., BUCK, W., AND FRIES, G. (1976). Herd health status of animals exposed to polybrominated biphenyls (PBB). J. Toxicol. Environ. Health 2, 335-349.
MOORE, R. W., DANNAN, G. A., ANDAUST, S. D. (1978). Induction of drug metabolizing enzymes in polybrominated biphenyls-fed lactating rats and their pups. Environ.
Health
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23, 159-165.
NORRIS, J. M., KOCIBA, R. J., SCHWETZ, B. A., ROSE, J. Q., HUMISTON, C. G., JEWETT, G. L., GEHRING, P. J., AND MAILHES, J. B. (1975). Toxicology of octabromobiphenyl and decabromodiphenyl oxide. Environ. Health Perspect. 11, 153-161.
ET AL. OCKNER, R. K.. AND SCHMID, R. (1961). Acquired porphyria in man and rat due to hexachlorobenzene intoxication. Nature (London) 189, 499. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RICKERT. D. E.. DENT. J. G., CAGEN, S. J.. MCCORMACK. K. M.. MELROSE, P.. AND GIBSON. J. E. (1978). Distribution of polybrominated biphenyls after dietary exposure in pregnant and lactating rats and their offspring. Environ. Health Perspect. 23, 63-66. SAN MARTIN DE VIALE, I. C., NACHT. S., AND GRINSTEIN. M. (1970). Experimental porphyria induced in rats by hexachlorobenzene: A study of the porphyrins excreted by urine. Clin. Chim. Acta 28, 13-23. SLEIGHT. S. D., AND SANGER, V. L. (1976). Pathologic features of polybrominated biphenyl toxicosis in the rat and guinea pig. J. Amer. Vet. Med. Assoc. 169, 1231-1235. STRIK, J. J. T. W. A., Doss, M., SCHRAA, G., ROBERTSON, L. W., TIEPERMANN, R., AND HARMSEN, E. G. M. (1979). Coproporphyrinuria and chronic hepatic porphyria type A found in farm families from Michigan (USA) exposed to polybrominated biphenyls (PBB). In Chemical Porphyria in Man. (J. J. T. W. A. Strik and J. H. Koeman, eds.), pp. 29-53. Elsevier/ North-Holland Biomedical Press, Amsterdam, The Netherlands.
WORLD HEALTH ORGANIZATION, International Agency for Research on Cancer (1978). Long-term hazards of polychlorinated dibenzodioxins and polychlorinated dibenzofurans. IARC Internal Technical Report 781001, Lyon, France.
AND
APPLIED
PHARMACOLOGY
68,
Effects of a Polybrominated
I- 18 ( 1983)
Biphenyl Mixture in the Rat and Mouse
I. Six-Month
Exposure
B. N. GuPTA,‘,’ E. E. MCCONNELL,’ J. A. GOLDSTEIN,’ M. W. HARRIS,’ AND J. A. MOORE’ National
Toxicology
Program, National Instittrfe Re.yearch Triangle Park, North
Received
January
qf Environmental Carolina 27709
5. 1981; acrepled
November
Health
Sciences,
I, 1982
Effects of a Polybrominated Biphenyl Mixture in the Rat and Mouse. 1. Six-Month Exposure. GUPTA,
B. N.,
MCCONNELL,
E. E., GOLDSTEIN,
J. A.,
HARRIS,
M.
W.,
AND
MOORE,
J. A.
I-18. A 1973 environmental accident in Michigan resulted in exposure of humans via the food chain to polybrominated biphenyl (PBB). To better characterize the toxicity of the halogenated polycyclic aromatic hydrocarbon class of chemicals, rodents were dosed with PBB and their target organs examined for morphological, histological. biochemical, and selected endocrine changes. Male and female rats and mice were given I25 po doses of PBB over a B-month period at 0.1, 0.3, 1.0. 3.0, and 10.0 mg/kg of body weight/day (5 days/week). There was a dose-related decrease in body weight gain in both male and female rats and male mice. Thymus weights were significantly decreased in all rats exposed to 0.3 mg/ kg or more of PBB. Dose-related hepatotoxic effects were observed in both rats and mice characterized by marked increase in liver weight with accentuation of hepatic lobular markings. Microscopically, there were moderate to marked swelling, disorganization, and single cell necrosis of hepatocytes, fatty infiltration, bile duct proliferation, and presence of atypical hyperplastic foci. Hepatic porphyrin levels were markedly increased in both rats and mice primarily in females. There was a significant decrease in serum thyroxine (T4) and triiodothyronine (Tj) suggesting that PBB may interfere with thyroid hormone secretion. There was a significant dose-related increase in serum cholesterol and y-glutamyl transpeptidase, and a decrease in serum glucose. (1983).
Toxicol.
Appt. Pharmacol.
68,
The accidental addition of polybrominated biphenyls3 (PBB) instead of magnesium oxide to animal feed resulted in widespread contamination in Michigan in 1973 (Cook et al., 1978; Jackson and Halbert, 1974; Mercer et al., 1976). This error subsequently resulted in PBB exposure to farmers and their families, and later, other consumers of contaminated meat, dairy products, and eggs (DiCarlo et al., 1978; Humphrey and Hayner, 1975; Kay,
1977). Although PBB was reported to have a relatively low toxicity with a single po dose (LD50-2 1.5 g/kg of body weight) in the laboratory rat (Anon, 197 1, 1976; Hilltop4), it was more toxic when given in smaller doses over a 30-day period and the animals were observed for 90 days (Gupta and Moore, 1979). In another report, the po LD50 of octabromobiphenyl for female rats was stated to be greater than 2000 mg/kg body weight (Aftosmis et al., 1972). Several subsequent studies have shown that the toxicity of PBB was similar to other halogenated polycyclic hy-
’ National Toxicology Program, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC. 27709. * To whom all correspondence should be addressed. 3 Firemaster BP-6, Michigan Chemical Corp., St. Louis, Mich.
4 Hill Top Research Inc. (1970). Acute toxicity and irritation studies on Firemaster BP-6 hexabromobiphenyl for Michigan Chemical Corporation. I
0041-008X/83
$3.00
Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reqwd.
GUPTA
2
drocarbons in laboratory animals (Kasza et al., 1978b; Harris et al., 1978a). Rats fed a diet containing 10 ppm of PBB for 30 days developed a slight hepatotoxicosis, and pups nursing treated dams also had microscopic and ultrastructural hepatic lesions (Sleight and Sanger, 1976). Hepatic changes, such as necrosis, fibrosis, and neoplastic nodules, were seen in rats 10 months after they were given a single po dose (1 .O g/kg of body weight) of PBB (Kimbrough et al., 1977, 1978). Similar atypical foci in the liver were observed in rats as early as 6 months when they were exposed to 22 daily doses of PBB at 30.0 or 100.0 mg/kg body weight (Gupta and Moore, 1979). PBB was also reported to be embryotoxic and teratogenic to rat embryo (Beaudoin, 1977; Corbett et al., 1975; Harris et al., 1978b). Other studies have shown that PBB is transferred to the fetus (Aftosmis et al., 1972; Detering et al., 1975; Rickert et al., 1978) and is excreted in the milk (Fries and Marrow, 1975; Fries, 1978; Gutenmann and Lisk, 1975; Moore et al., 1978). The current study was designed primarily to determine the pathobiologic effects of the commercial polybrominated biphenyl mixture exposure in the rat and mouse during a 6-month period. METHODS A polybrominated biphenyl (PBB) mixture5 (Firemaster BP-6 and 2% calcium trisilicate, Lot No. 1312 Ff, Batch 03) was mixed with corn oil and given to Fischer 344/N (CDF) rats6 (51 rats/dose/sex) and B&F, mice’ (50 mice/dose/sex). Both rats and mice were intubated on 5 consecutive days per week for 25 consecutive weeks ( 125 doses over a 6-month period). PBB was given to rats and mice at 0 (control), 0.1, 0.3, 1.0, 3.0, and 10.0 mgl kg body weight/day. The concentration of the solutions in corn oil was adjusted weekly based on the mean body weight so that rats and mice received a constant volume of 0.2 and 0.1 ml/day, respectively. Control animals received equal amounts of corn oil. ’ Midwest Research Institute, Kansas City, MO. 6 Charles River Breeding Laboratories, Inc., Wilmington, Mass. ’ Harlan Industries, Indianapolis, Ind.
ET AL. Three rats or five mice of each sex were housed in one polycarbonate cage (45 X 20 X 20 cm for rats and 30 X 17 X 12 cm for mice). Food* and water were provided ad lib&m. Ground corn cob’ was the bedding for rats and hard wood chipslo for mice. A 12-hr light-dark cycle was provided, and temperature was maintained at 21 + 1°C and 50 f 5% was the relative humidity. All animals were observed daily for clinical signs. Each animal was weighed weekly, and food consumption was recorded once a week. For weight gain, organ weight, hematology. and clinical chemistry data, the Kruskal-Wallis nonparametric analysis of variance (Hollander and Wolfe, 1973) was used to determine the significance of among-group differences. The significance of dose-response trends was assessedby Jonckheere’s test (Jonckheere, 1954). If an overall effect was detected, then pairwise comparisons were made by Mann-Whitney U tests (Hollander and Wolfe, 1973). At the end of the 6-month exposure, groups of 10 randomly selected rats or mice/dose/sex were killed. Rats were killed by exposure to high levels of CO2 until respiration ceased, and mice were killed by placing them in a glass desiccator containing cotton gauze sponges soaked with methoxyfluorane” until deep anesthesia was achieved. Blood samples were collected from rats via orbital sinus and from mice by cardiac puncture for hematologic and clinical chemistry evaluation. Clinical pathologic examination of blood included total erythrocyte (RBC), leukocyte (WBC), hemoglobin (Hb) concentration, packed cell volume (PCV), platelet counts, and differential counts of leukocytes. Clinical chemistry examination of serum included serum glutamic pyruvic transaminase (SGPT), y-glutamyl transpeptidase (GGTP), alkaline phosphatase (AP). blood (serum) urea nitrogen (BUN), glucose, triglycerides, cholesterol, and total protein. Serum thyroxine (T4) concentrations and triiodothyronine (T,) uptake were determined with TETRA-TAB and TRI-TAB T1 uptake kits.‘* The test was performed as recommended by the manufacturer except that 20 ~1 of bovine serum was used for T4 determination instead of 10 ~1. Tr was determined by the Serulate Total T-3 KitI with the following modifications: The T, antiserum was diluted to provide approximately 40% retention of ‘3’I-labeled TS on the column in the absence of nonra-
s Open Formula Rat and Mouse Ration (NIH-3 I), Ziegler Brothers Inc., Gardners, Penn. 9 Bed-O’Cobs, The Andersons, Maumee, Ohio. lo Betta Chip Bedding, Granvtlle Milling Co., Creedmoor, N.C. ‘I Metofane, Pitman-Moore, Inc., Washington Crossing, N.J. I2 Nuclear Medical Labs, Dallas, Tex. ” Ames Co., Division of Miles Laboratories, Elkhart, Ind.
POLYBROMINATED Male
q
260
BIPHENYL
30
IN RAT AND MOUSE
3
rat
control
fZII0mg 0 3.0mg I 10.0 mg
QOlmg 803mg
t 8m260 t
TOXICITY
60
90 -uays
FIG. 1. Histogram showing difference in body weight gain in male rats exposed to PBB, a total of 125 doses, at the end of a 6-month exposure period. dioactive T3 (zero standard), and the incubation period was changed from 2 to 1 hr. Weights of lung, heart, liver, spleen, thymus, right kidney, right adrenal gland, right gonad, both thyroid glands, uterus, and brain were recorded, and organ to body weight ratios were calculated. In addition to the organs weighed, tissue samples from pinna, eye, trachea, urinary bladder, sternum, quadriceps muscle, accessory male and female sex organs, six levels of gastrointestinal tract, lumbar spinal cord, sciatic nerve, spinal ganglion, salivary glands, mesenteric and thoracic lymph nodes, mammary glands, ear canal, and nasal turbinates were fixed in buffered neutral 10% formalin for histopathologic examination. These specimens were embedded in paraffin, sectioned 6 pm thick, and stained with hematoxylin and eosin. Cryostat prepared sections (10 pm thick) of formalin-fixed liver were also stained with oil red 0 for examination for neutral lipids. Paraffin sections of liver and spleen from six rats and mice each of both sexes given 10.0 mg/kg of body weight of PBB and control (a total of 48 animals) were also stained with Mallory’s method for iron (Luna, 1968). Portions of liver from two male and two female rats (exposed to highest dose and control) were also fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde in cacodylate buffer (pH 7.4) for 6 hr. These specimens were washed with cacodylate buffer (pH 7.4) for 2 hr, dehydrated in alcohol and propylene oxide, and embedded in Epon 812. Thick sections (0.5 pm) for light microscopy were cut with an ultramicrotome and stained with tolu-
idine blue. Thin sections (400 to 600 A) were stained with uranyl acetate and Reynold’s lead citrate (Reynolds, 1963). At the time of necropsy, liver, sternum, bones, and incisor teeth of all rats and mice were examined under long wavelength (366 nm) ultraviolet (uv) lightI for reddish pink fluorescence as an indication of excess accumulation of porphyrin. A portion of liver from rats and mice was frozen for determination of total hepatic porphyrins (Abritti and de Matteis, 1971, 1972).
RESULTS
Clinical Observation During the 6-month dosing period, animals were clinically normal in appearance. No treatment related deaths were observed. There were depressed rates of body weight gain in rats as a function of time and dose (Figs. 1 and 2). The body weight gain in female mice (10.0 mg/kg/day) was higher, whereas in the male mice, it was depressed (Fig. 3). There was no significant difference in the body weight I4 Model B-100 Blak-Ray Ultraviolet Lamp, Ultraviolet Products Inc., San Gabriel, Calif.
GUPTA 140 a 0”
Female rat Cl control 610.1 mg ElO3mg
ET AL.
El I.Omg El 3.0 mg I IOOmg
120
90
60
Days FIG. 2. Histogram showing difference in doses, at the end of a 6-month period.
body
weight gain in female rats exposed to PBB. a total of I25
gain in other groups of treated mice and control. Food Consumption There was no significant difference in food consumption either in the rat or mouse. However, there was a slight decrease in food intake in the female rats exposed to 10.0 mg/kg/day of PBB during the last 6 weeks of exposure.
No significant differences in thymus weights were observed. Spleen weight was increased only in higher dose female mice. Another notable effect was a decrease in uterine weights of the 10.0 mg/kg dose. Significant weight differences were not observed in other organs. Hematologic
Findings
The Hb and PCV values were decreased in both male and female rats at the two highest dosages (Table 4). Similarly, mean corpusOrgan Weights cular volume (MCV) and mean corpuscular (MCH) were also decreased. There was a dose-related increase in the hemoglobin absolute and relative liver weights in ail treated However, this decrease was more pronounced in males compared with females at the same groups of female rats, and in males receiving 0.3 mg/kg/day or more of PBB (Tables 1 dose levels. Total serum protein was slightly lower primarily in female rats exposed to 1.O, and 2). Thymus weights were significantly 3.0, and 10.0 mg/kg of PBB. The WBC count (p < 0.05) decreased in rats given 0.3 mg/kg or more of PBB. The weight of the spleen was was significantly (p < 0.05) increased in treated increased in those rats given 1.0 mg/kg or female rats at dose levels of 1.O, 3.0, and 10.0 mg/kg due to an increase in both neutrophils higher of PBB (Tables 1 and 2). Significant @ < 0.05) increases in liver and lymphocytes. However, this trend was not weights were also observed in female mice observed in treated male rats. given 0.3 mg/kg or more, and in male mice There was a significant (s, < 0.01) dose-regiven 1.O mg/kg and more of PBB (Table 3). lated increase in the RBC count in male and
POLYBROMINATED
I
BIPHENYL
0 0 mgQ mouse KllOmgQ
R 0 mgd R IO mgd
mouse
0
TOXICITY
30
5
IN RAT AND MOUSE
mouse mouse
so
60
120
150
180
Days FIG. 3. Histogram showing changes in the body weights of male and female mice exposed to PBB at 10.0 mg/kg, 125 total doses during a 6-month period.
female mice. The MCV was decreased in both male and female mice in a dose-related fashion. However, significant differences in PCV and Hb concentration in the treated and control mice were not observed. Platelet counts were significantly QI < 0.01) decreased in the female mice given 3.0 and 10.0 mg/kg of PBB but there was no difference in treated and control male mice. There was leukocytosis due primarily to an increase in lymphocytes in female mice treated with the highest dose level.
Clinical Chemistry
Total serum protein was decreased in a dose-related fashion in female rats primarily due to the dose-related decrease in albumin. There was a significant @ < 0.05) increase in the serum levels of GGTP in female rats given the 10.0 mg/kg dose (Table 5). Serum glucose was significantly 0, < 0.05) decreased in 10.0 mg/kg treated female rats only. There was a marked decrease in the serum triglyceride level
TABLE ABSOLUTE AND RELATIVE WEIGHTS POLYBROMINATED BIPHENVL
I
OF LIVER, SPLEEN, AND THYMUS OF MALE RATS TREATED (125 DOSES) AND RECORDED AT THE END OF 6 MONTHS~
Liver weight
Spleen weight
Thymus weight
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
Absolute (g)
0.1
13.2 f 0.14 13.9 + 0.43
0.3
15.8 f 0.27’
1.0
18.8 + 0.32’ 21.4 + 0.51’ 20.8 f 0.34’
0.58 0.57 0.63 0.64 0.77 0.76
0.14 2 0.01 0.14 f 0.01 0.16 f 0.01 0.16 f 0.01’ 0.21 f 0.01’ 0.25 + 0.01’ p < 0.01
0.14 0.13 0.12 0.11
3.0
3.2 3.4 4.0 4.8 5.9 6.7
0
10.0 Dose-response
p < 0.01
+ + f 2 + +
0.03 0.08’ 0.15 0.04’ 0.08’ 0.05’
p < 0.01
f + f + f -+
0.03 0.02 0.02 0.02b 0.03’ 0.04’
p < 0.01
“Dataareexpressedas~~SEMof10rat.s. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
WITH
-c 0.01 f 0.01 + 0.016 + o.OOc 0.07 + 0.0 1c 0.04 + 0.00’ p <
0.01
Relative (70) 0.034 0.033 0.030 0.029 0.020 0.012
2 0.001 f 0.002
+ 0.001 + 0.00 1c + o.Oc2c
+ O.OOlC
p < 0.01
GUPTA
6
ET AL.
TABLE2 ABSOLUTE AND RELATIVE WEIGHTS OF LIVER, SPLEEN, AND THYMUS OF FEMALE RATS TREATED POLYBROMINATED BIPHENYL (125 DOSES) AND RECORDEDAT THE END OF 6 MONTHS“ Liver weight Dose bwWday) 0 0.1 0.3 1.0 3.0 10.0 Dose-response
Spleen weight
WITH
Thymus weight
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
6.2 + 0.12 6.8 t 0.23" 6.6 * 0.216 7.1 f 0.27’ 7.8 f 0.22' 8.6 k 0.24' p < 0.01
2.9 + 0.06 3.1 f 0.07b 3.3 f 0.11’ 3.4 + 0.09' 4.3 f 0.10’ 5.6 rt_0.09' p < 0.01
0.38 * 0.01 0.38 r 0.01 0.39 k 0.01 0.41 -t 0.01 0.49 F 0.02' 0.43 k 0.02' p < 0.01
0.18 + 0.01 0.17 * 0.01 0.19 * 0.01 0.20 t- 0.01* 0.27 t 0.01’ 0.28 + 0.01’ p < 0.01
0.14 + 0.00 0.13 + 0.01 0.12 + 0.01’ 0.11 + 0.01’ 0.07 + 0.00' 0.04 + 0.01 c p < 0.01
0.070 +- 0.002 0.06 1 + 0.002 0.056 f 0.002' 0.052 -+ 0.002' 0.037 * 0.002' 0.024 f 0.004' p < 0.01
a Data are expressed as X f SEM of 10 rats. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
in treated male rats except at the lowest dose (0.1 mg/kg). There was an apparently doserelated increase in the serum levels of cholesterol in both male and female rats (Table 5). There were no significant differences in SGPT, AP, and BUN of treated and control rats of either sex. Serum levels of GGTP tended to be slightly increased in both male and female mice given 10.0 mg/kg of PBB. There was a five- to sixfold increase in the activity of SGPT in both male and female mice in 10.0 mg/kg groups.
AP was also increased in mice given the highest dose of PBB. Serum glucose was significantly (p < 0.0 1) decreased only in female mice exposed to 10.0 mg/kg of PBB. At the high dose, PBB decreased serum thyroxine values to 40% of control rats (Table 6). The decrease was dose related and significant decreases were seen at doses of 0.3 mg/ kg of PBB in males, and 1.O mg/kg in females. Serum triiodothyronine (T3) levels were also decreased by PBB, but the effect was somewhat less and only seen at high doses. The T3
TABLE3 ABSOLUTE WEIGHT (g) AND RELATIVE (%) LIVER TO BODY WEIGHT OF MALE AND FEMALE MICE TREATED POLYBROMINATED BIPHENYL (125 DOSES) AND RECORDEDAT THE END OF 6 MONTHS’ Liver weight of male mice Dose b.Odday) 0 0.1 0.3 1.0 3.0 10.0 Dose-response
Liver weight of female mice
Absolute (g)
Relative (%)
Absolute (g)
Relative (%)
1.84 f 0.06 1.95 -t 0.03 1.81 + 0.05 2.12 f O.OSb 2.53 k 0.07' 4.32 f 0.10’ p < 0.01
4.96 4.89 4.88 5.55 6.42 12.38
1.23 * 1.27 f I .34 f 1.57 k 1.89 f 4.56 f
4.48 4.51 4.89 5.68 6.68 15.03
f 0.15 + 0.08 + 0.11 f 0.166 _+ 0.17’ + 0.32’ p < 0.01
a Data are expressed as X f SEM of 10 mice. b Significantly (p < 0.05) different compared with control. ‘Significantly (p -C 0.01) different compared with control.
0.04 0.03 0.03 b 0.06' 0.03 0.10’ p < 0.01
+ 0.08 + 0.07 f 0.07' f 0.19’ t 0.09 f 0.29' p < 0.01
WITH
POLYBROMINATED
BIPHENYL
TOXICITY
7
IN RAT AND MOUSE
TABLE 4 HEMATOL~GIC
Treatment Male Male Female Female
Control PBB Control PBB
VALUE OF RATS TREATED WITH POLYBROMINATED BIPHENYL (PBB) AT 10.0 mp/kg BODY WEIGHT/DAY DURING A ~-MONTH PERIOD”
Hemoglobin (ti1~ ml) 16.9 ?z 0.3 13.6 ? 0.2’ 17.8 + 0.3 16.3 f 0.6’
Packed cell volume (%) 50.6 39.5 50.0 46.2
f f f f
Red blood cells ( I 06/cmm)
0.94 1.05 1.44 1.50
5.79 5.55 5.44 6.09
k f + +
0.09 0.11 0.16 0.29
Mean corpuscular volume (pm)) 85.9 71.2 92.0 76.1
+ f k f
1.3 1.8’ 1.9 1.8’
125 DOSES Mean corpuscular hemoglobin (p& 29.2 24.5 32.8 26.9
f 0.5 + 0.2* k 0.7 zt 0.9’
a Values represent X + SEM. * Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.0 1) different compared with control.
uptake in male rats given the highest dose females of both species. Males, however, were (10.0 mg/kg) was significantly (p < 0.01) de- affected much less than females. creased compared with the controls, but it was slightly increased in female rats given 1.O mg/ ~~~~~~~~ Findings kg of PBB (Table 6). PBB produced dose-related hepatic porLiver, bone, and teeth of female rats given phyria in rats and mice of both sexes (Table 1.O to 10.0 mg/kg of PBB had slight to marked 7). At the highest doses, hepatic porphyrin reddish pink fluorescence under uv light delevels were increased several hundred-fold in noting excess porphyrin accumulation. The TABLE 5 CLINICAL
CHEMISTRY
Dose OWWday) Male
0 0.1 0.3 1.0 3.0 10.0 Dose-response
Female
0 0.1 0.3 1.0 3.0 10.0 Dose-response
*p cp
VALUES OF RATS ADMINISTERED POLYBROMINATED AND RECORDED AT THE END OF 6 MONTHS’
y-Glutamyl transpeptidase (IU/liter) 3.1 3.3 2.6 3.0 3.5 3.9
k 0.7 + 0.8 f 0.6 k 0.5 f 0.3 + 0.6 NS
3.3 + 3.2 + 2.6 + 2.2 f 4.5 + 7.9 +
0.7 0.5 0.4 0.4 0.6 1.2b
p < 0.05
Serum
BIPHENYL
( 125 Doss)
Serum triglyceride (mg/lOO ml)
Serum cholesterol (mg/lOO ml)
135 + 12 133 f 13 138 + 12 143 +- 10 120+ 4 122+ 6 NS
335 + 146 324 AZ139 155 k 15 119+ 8’ 152 +- 12* 155 f 18
89+ 12 110+21 94+ 6 116+ 5
131 f 118& 120+ 113~ 116+ 101 +
109* llO-+ 122 & 91 + 115 +107+ NS
glUCOS.2
(m&l00
ml)
13 3 8 6 4 10
p < 0.05
< 0.05, significantly ditRrent compared with control of same sex. < 0.01, significantly different compared with control of same sex.
p < 0.05
7 13 32 19 10 5
160 + 18”
243 + 15” p < 0.01
123+ 5 136 of: 10 141 f 6 146k 6* 181 ? lib 147 + 12
p < 0.01
GUPTA
8
ET AL.
TABLE 6 EFFECTOF ~-MONTH ADMINISTRATION OF POLYBROMINATED BIPHENYL (I 25 TOTAL DOSES) ON SERUM THYROID HORMONES IN RATS’
T4 h/l~ Do= bg/W Control 0.1 0.3 1.0 3.0 10.0 Dose-response
T, (ng/lOO ml)
ml)
T, uptake
Male
Female
Male
Female
Male
Female
5.3 f 0.2 4.5 f 0.8 4.4 f 0.3b 4.0 f o.3b 3.0 f 0.3’ 2.1 + 0.1’ p < 0.01
3.1 f 0.2 3.6 f 0.3 3.3 + 0.4 2.1 f 0.2’ 1.7 f 0.2’ 1.5 * 0.2’ p < 0.01
126+ 7 99-c 15 111 _+ 13 133 f 11 107 f 10 104+ 3 NS
123 f 14 106+ 9 112+ 8 86? 10 8Ok 8* 77k 96 p < 0.01
61.9 + 0.9 62.6 ‘-e 1.4 59.9 ” 0.6 58.9 I!I 0.7 59.1 z!I 1.4 56.1 I!Y0.5’ p < 0.01
58.1 58.4 58.7 61.7 59.2 60.3
f + + f k f
1.0 0.7 0.5 0.6’ 0.6 0.6
p c 0.02
LIValues represent X f SEM of 10 rats. b Significantly (p < 0.05) different compared with control. ’ Significantly (p < 0.01) different compared with control.
number of positive samples and intensity of red fluorescence was less in treated male rats as compared with treated females. These tissues from all female mice exposed to 10.0 mg/ kg of PBB showed slight to marked tluorescence whereas only 8 of 10 male mice were positive at the same dosage. The livers of treated rats and mice of both sexes were moderately to markedly enlarged, especially in higher dose levels. The edge of the liver of treated rats extended much farther posteriorly from the edge of the rib cage as compared with the control animals. The livers
were pale or slightly yellow and mottled. Two male rats (one given 0.3 mg/kg and the other 10.0 mg/kg) had several grayish white nodules (about 5 mm in diameter) in the liver. One female rat of 10 given 10.0 mg/kg of PBB had a grayish white space occupying mass involving the interior of urinary bladder. The thymus of treated rats (10.0 mg/kg) appeared smaller compared with the control. No lesions were observed in other organs. Eight male and one female of 10 mice per sex in the 10.0 mg/kg groups contained several small grayish white foci in the liver. One
TABLE 7 EFFECT OF POLYBROMINATED BIPHENYL ADMINISTRATION (125 TOTAL DOSESDURING A 6-MONTH PERIOD) ON HEPATIC PORPHYRINLEVELS (ccg/g) IN THE RAT AND MOUSE” Rats Dose Ow/W
Male
Control 0.1 0.3 1.0 3.0 10.0 Dose-response
1.0 f 0.2 0.8 f 0.1 2.0 f 0.3’ 15.3 + 4.2’ 36.1 + 8.3’ 45.3 XL 11.W p < 0.01
Mice Female 0.7 0.5 15.8 1002.0 1369.0 517.0
f 0.1 iz 0.1 + 8.2’ f 65.0’ + 65.0’ f 80.0’ p < 0.01
’ Values represent X -t SEM of 10 animals. b Significantly (p < 0.05) increased compared with controls. ‘Significantly (p < 0.01) increased compared with controls.
Male 1.0 f 1.0 f 1.2 f 1.7 -t 18.7 f 58.0 2
0.1 0.1 O.lb 0.1’ 4.3 10.0’ p < 0.01
Female 1.1 0.7 0.9 3.5 97.0 435.0
f 0.2 + 0.1 f 0.1 f 2.0 k 23.0’ * 20.0’ p < 0.01
POLYBROMINATED
BIPHENYL
TOXICITY
IN RAT AND MOUSE
male mouse of 10 controls also showed a similar lesion.
9
foci invariably contained golden brown granular pigment which was positive for iron (Mallory’s method for iron; Luna, 1968). Occasionally, the reacting cells in the center of Histopathologic Findings these granulomas formed rosette-like structures around these hemosiderin granules. Slight to marked histopathologic dose-re- Small clefts of fatty acids were occasionally lated changes in the liver were observed in all observed in these foci. Single cell hepatocelgroups of treated male rats but only in the lular necrosis with infiltration of neutrophils 1.0, 3.0, and 10.0 mg/kg female groups. The was also observed. There was slight to modmicroscopic appearance of these livers was erate focal or diffuse bile duct proliferation in characterized by disorganization of trabecular the liver (Fig. 5). cords, moderate to marked enlargement of Atypical foci were observed in the livers of hepatocytes some of which were multinucle3 of 100 (3.0%) treated rats. These foci were ated, moderate fatty infiltration (oil red 0 well delineated groups of hypertrophic hestain), and hyalinization of the cytoplasm of patocytes with enlarged nuclei containing other hepatocytes (Fig. 4). Some of the he- prominent nucleoli and foamy or eosinopatocytes had a foamy appearance. Lipid ac- philic ground glass cytoplasm (Fig. 6). On ultrastructural examination of the liver, cumulation was more prominent in male than there was an increase in the amount of rough female rats and was primarily located around endoplasmic reticulum (RER) and lipid globthe central vein. Numerous granulomatous foci with occa- ules of different sizes in the hepatocytes of sional multinucleated giant cells were ob- male rats given the highest dose of PBB. In served primarily near the central vein. These the treated female rats (highest dose), the
FIG. 4. Photomicrograph of the liver of a female rat given PBB at 10.0 mg/kg, 125 total doses during a 6-month period. Notice three multinucleated giant hepatocytes. H and E stain: X750.
10
GUPTA
ET AL.
FIG. 5. Bile duct proliferation and fibrosis in the liver of a male rat treated with PBB, 125 total doses at 10.0 mg/kg. Note a few bile ducts with hyperchromic cells. H and E stain; X600.
changes in the liver were much more severe as denoted by marked proliferation and disorganization of RER, dilated cristae, and dense matrix of mitochondria (Figs. 7 and 8). Occasionally, intramitochondrial flocculent deposits were also observed. The space occupying mass in the urinary bladder of the female rat in the 10.0 mg/kg group was a squamous cell carcinoma. This neoplastic mass protruded into the lumen and was covered with one to several layers of atypical flattened epithelium. It was composed of islands of squamous epithelium some of which contained concentric laminations of keratin. The connective tissue stroma was heavily infiltrated with inflammatory cells. Although the neoplastic cells infiltrated into the deeper muscular wall of the urinary bladder, no metastases were observed.
Although there was no significant difference in the weights of thyroid glands, slight to moderate morphologic changes were observed primarily in male rats exposed to 10.0 mg/kg of PBB. Microscopic changes in the thyroid gland were characterized by thin, sparse, or bluish colloid with basophilic stippling. Some follicles were lined with columnar epithelium and also contained a few epithelial papillary projections. Changes in the thyroid gland of exposed female rats (highest dose) were not remarkable compared with the female control. The kidneys of male rats treated with 10.0 mg/kg dose level of PBB consistently showed atrophy of a few glomerular tufts with marked dilatation of Bowman’s capsule which contained amorphous eosinophilic staining material. A few glomerular tufts also appeared
POLYBROMINATED
BIPHENYL
TOXICITY
IN
RAT
AND
MOUSE
11
12
GUPTA
FIG. 7. Electron photomicrograph of a portion PBB at 10.0 mg/kg body weight. Note the marked of mitochondria. Uranyl acetate and lead citrate;
ET AL.
of a hepatocyte from a female rat given 125 doses of increase and disorganization of RER, and dense matrix X 19,350.
edematous. Some renal tubules in both cortical and medullary regions were dilated and contained either serous fluid or proteinaceous casts. Significant lesions were not observed in other organs.
Microscopic alterations in the livers of male and female mice (10.0 mg/kg dose levels) were marked swelling of hepatocytes primarily around the central veins, foamy or vacuolated cytoplasm with hyaline bodies, and focal co-
POLYBROMINATED
BIPHENYL
TOXICITY
IN RAT AND MOUSE
13
FIG. 8. Electron photomicrograph of a portion of a hepatocyte from a female rat given the highest dose of PBB. Note intramitochondrial flocculent deposits (arrows) and dilated and vesicular cristae in several mitochondria. Uranyl acetate and lead citrate; X30,638.
agulative necrosis or scattered single cell necrosis of hepatocytes. Only slight to moderate swelling of hepatocytes was observed in mice treated with 1.0 and 3.0 m&kg of PBB, and no lesions were observed at lower doses. Atypical hepatocellular foci (Fig. 9) were present in nine male and two female treated mice of 100 examined (I 1.O%), and in 1 of 20 (5.0%)
control mice. These foci were similar to those described in the rat. Iron-positive granules, probably hemosiderin, were observed primarily in the red pulp of the spleen of all treated and control animals. However, these granules were more prominent in the spleen of treated female rats than treated males and both more so than the
14
GUPTA
ET AL.
FIG. 9. Delineated area of foamy or vacuolated hepatocytes in the liver of a male mouse exposed to PBB at 10.0 mg/kg ( I25 total doses). Notice slight compression of hepatocytes at the margin of this atypical focus. H and E stain; X240.
controls. There was no difference in the spleens of treated and control male mice. However, the spleen of control female mice tended to have more iron-positive granules compared with the PBB-treated female mice.
DISCUSSION The LD50 of PBB was mathematically determined to be 65 mg/kg/day (total 1.43 g/kg) for F344 female rats and 149 mg/kg/day (total
POLYBROMINATED
BIPHENYL
3.28 g/kg) for the males (Gupta and Moore, 1979). These results were obtained from rats exposed to 22 multiple doses of PBB during a l-month period and then observed for 90 days. There was 100 and 38% mortality in female and male rats, respectively, given doses of PBB at 100 mg/kg, and no deaths in the 30 mg/kg/dose groups. On the basis of the above observations (mortality, body weight gain effect, thymic atrophy, and hepatotoxicity), 10.0 mg/kg of PBB was selected as the highest dose level for a 6-month study. The lower four doses were selected on a logarithmic basis, i.e., 10.0, 3.0, 1.0,0.3, and 0.1 mg/ kg/day. In this 6-month exposure to PBB there was no significant difference in the food consumption among PBB-treated rats and mice and controls, yet there was a persistent decrease in body weight gain in treated male and female rats and treated male mice. This decrease suggests that PBB may cause poor feed utilization. Similar weight gain effects which are only partially related to feed intake have been observed in monkeys exposed to PBBs (Allen et al., 1978), and in other species of animals (rats, monkeys, and cattle) with structurally similar halogenated aromatic hydrocarbons such as PCBs (Allen and Abrahamson, 1973) and the dibenzodioxins (McConnell et al., 1978, 1980). Interestingly, there was an increase in body weight of female mice exposed to PBB at 10.0 m&kg/day ( 125 total doses) during a 6-month period. Similar gains of body weight were observed in female mice exposed to PBB in our previous experiment (Gupta et al., 198 1). The explanation for this disparity is not clear; however, body weight gain may be related to fluid accumulation in different tissues of the body. Extracellular fluid (subcutaneous edema, ascites, and hydropericardium) is often found in mice dying from acute intoxication to these classes of compounds (McConnell and Moore, 1979), and, in fact, mice showing this lesion do actually gain weight just prior to death.
TOXICITY
IN
RAT
AND
MOUSE
15
Weights of the thymus glands were decreased in rats administered PBB (0.3 mg/kg or higher) but not in mice. Administration of PBB to rats and mice caused suppression of both humoral and particularly cell-mediated immunity (Luster et al., 1978). In a 6-month exposure of PBB in rats and mice, this chemical was not a potent immunosuppressant in rodents since high doses were required to elicit immune effects (Luster et al., 1980). Increase in the absolute and relative liver weights of rats and mice, and microscopic alterations were expressions of hepatoxicity. The increase in liver weights was primarily due to hepatocytomegaly, increase in endoplasmic reticulum, and excess lipid accumulation (Gupta et al., 1981). At the end of 6 months, only 3.0% of the treated rats, 11 .O% of the treated mice, and 5.0% of the control mice developed atypical hepatocellular foci. Since the incidence of atypical foci in the liver was so low at the 6-month observation, the potential hepatocarcinogenicity of PBB could not be evaluated. Chronic administration of hexachlorobenzene, PCBs, or TCDD to rats produces hepatic porphyria characterized by a delayed onset and massive accumulation of uroporphyrins in the liver (Ockner and Schmid, 196 1; Goldstein et al., 1973, 1974, 1976). In the present study, PBBs also produced hepatic porphyria at doses as low as 0.3 mg/kg/day. The rat was more sensitive than the mouse and, in both species, the female was much more susceptible than the male. This sex difference has been reported previously for the porphyrogenic action of hexachlorobenzene in rats (San Martin de Viale et al., 1970). However, this finding is the first report of a similar sex difference in mice. The porphyrogenic effects of PBBs in the rat may be of particular importance since an equivalent condition has been reported in humans through accidental consumption of hexachlorobenzene in Turkey in 1956 (Ockner and Schmid, 1961). Frank hepatic porphyria has not been found in people exposed to PBBs in
16
GUPTA
Michigan; however, an alteration in the urinary porphyrin pattern which may reflect preliminary changes has been reported (Strik et al., 1979). Pigment (hemosiderin) accumulation probably represents by-products of a breakdown of erythrocytes which in turn are phagocytized by the reticuloendothelial cells of the liver and spleen. Breakdown of RBCs would also explain the observed anemia. Slight decrease in MCV and MCH demonstrated that the anemia was micro&c and hypochromic. There was no indication of an increase in the number of circulating nucleated red blood cells. These findings suggest a direct depression of red blood precursors. Anemia and bone marrow depression have been observed in all species of animals exposed to toxic levels of polychlorinated dioxins and furans, especially in long-term studies (World Health Organization, 1978). The mildly increased activities of serum GGTP in both rats and mice (highest dose only) and marked increases in the concentration of SGPT and AP in the mouse only were further indication of hepatic injury. Although clinical chemistry is a useful noninvasive tool in determining hepatotoxicity in rodents, morphologic changes in hepatocytes appeared to be the most sensitive parameters in this study. Dose-related microscopic alterations in the liver were observed in all groups of treated male rats but only in the 1.0, 3.0, and 10.0 mg/kg female groups; however, on clinical chemistry evaluation, serum GGTP values were increased only in the higher dosages (Table 5). In the present study, PBB decreased the serum thyroxine concentrations. The absence of an effect on uptake indicates that this effect was not due to alterations in binding to plasma protein. The binding of thyroid hormone to serum proteins was not affected by PBB as indicated by the T3 uptakes. Microscopic changes in the thyroid glands were observed but only at the higher doses. Morphologic changes in the thyroid glands were also observed in male rats exposed to PBB (Kasza et
ET AL.
al., 1978a; Gupta and Moore, 1979), but serum levels of thyroid hormones were not measured. Norris et al. ( 1975) found evidence of thyroid hyperplasia in rats fed octabromobiphenyl for 30 days at 8.0 mg/kg/day. TCDD and PCB also decrease serum thyroxine levels and produce goiters in rats (Bastomsky, 1974, 1977a,b). The goiters were more evident in animals on low iodine diet. The effect of TCDD and PCB on serum thyroxine concentrations appears to be secondary to an increase in the hepatic glucuronidation and biliary excretion of thyroxine. Interestingly, there has been a report of primary hypothyroidism in four men (11.4% of those examined) employed in a plant which produced the PBBs, decabromobiphenyl and decabromobiphenyl oxide (Bahn et al., 1980). ACKNOWLEDGMENTS The authors appreciate the excellent technical help of Mses. J. D. Allen and D. L. Myers and Messrs. R. E. Wilson and F. A. Talley. We also thank Dr. J. K. Haseman for statistical analyses.
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