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6J mice
10XIC0111/ ELSEVIER SCIENTIFIC PUBLISHERS IRELAND
Toxicology 86 (1994) 49-61
Immunologic and endocrine effects of the flameretardant pentabromodiphenyl ether (DE-71) in C57BL/6J mice Jeff R. Fowles a, Anne Fairbrother b, Linda Baecher-Steppan c, Nancy I. Kerkvliet *c "Toxicology Program, Oregon State University, Corvallis', OR 97331. USA hU.S. Environmental Protection Agency. Environmental Research Laboratory, 4 S W 35th Street. Corvallis. OR 97333, USA "Department of Agricultural Chemistry, ALSI039, Oregon State University, Corvallis. OR 97331-7301. USA
(Received 12 February 1993; accepted 27 May 1993)
Abstract
Polybrominated diphenyl ethers are manufactured for use as flame retardants in commercial plastics and textiles in Europe and North America. These studies investigated the acute and subchronic immunotoxicity and endocrine effects of a commercial pentabromodiphenyl ether mixture, DE-71, in female C57BL/6 mice. Mice were orally exposed to acute single doses of DE-71 of 0, 0.8, 4.0, 20, 100, or 500 mg/kg, or to subchronic daily doses totaling 0, 250, 500, or 1000 mg/kg over a 14 day period. Immunotoxicity was assessed by measuring the plaque-forming cell response to sheep erythrocytes (SRBC) and natural killer cell (NKC) activity (basal and poly I:C stimulated) to YAC-I target cells. Liver cytochrome P450 content and activities (ethoxyresorufin-o-deethylase (EROD) and pentoxyresorufin-o-deethylase (PROD)) as well as corticosterone (CS) and thyroxine (T4) concentrations were also measured. PROD activity was induced 3-5-fold in mice exposed acutely or subchronically to DE-71 at doses > 250 mg/kg. EROD activity and total microsomal cytochrome P450 content were significantly induced only in mice treated subchronically with DE-71; maximum induction of EROD was 3.3-fold. Total serum T4 concentrations were significantly lower in mice treated acutely with DE-71 at all doses except the 100 mg/kg dose, Total and free T4 concentrations were dose-dependently decreased in DE-71-treated mice following subchronic exposure. Plasma CS levels were elevated following subchronic exposure to DE-71. The elevation * Corresponding author. 0300-483X/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02636-U
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J.R. Fowles et al. / Toxicology 86 (1994) 49-61
of CS was correlatedwith order of capture at necropsy,suggestingan interactiveeffectof DE71 and stress. In regardto immunotoxicity,significantsuppressionof the anti-SRBC response was seen only in mice exposed subchronicallyto 1000 mg DE-71/kg, an exposure that also resulted in decreasedthymus weight. NKC activity was not altered by exposure to DE-71. Key words: Antibody response; Corticosterone; Immunotoxicity; Natural killer cell; Pentabromodiphenylether; Thyroxine
1. Introduction Brominated diphenyl ethers (BDE) are currently manufactured for use as flame retardant additives to various plastics, rubber, paints and resins (U.S. Environmental Protection Agency, 1991). This use has led to the appearance of residues of BDE, including pentabromodiphenyl ether (PBDE), in a variety of environments, both aquatic and terrestrial (U.S. Environmental Protection Agency, 1991). Pyrolysis of BDE can yield brominated dibenzofurans and dibenzodioxins of potential concern (Buser, 1986). Since there is potential for BDE to cause adverse environmental and human health effects, the U.S. Environmental Protection Agency has recommended that toxicity studies be conducted for commercially important BDE congeners and mixtures. Structurally-related chlorinated diphenyl ethers (CDE) have been shown to cause liver enzyme induction as well as immune suppression in laboratory mice (Howie et al., 1990). In these studies, the potency of specific CDE congeners to induce hepatic microsomal ethoxyresorufin-o-deethylase (EROD) activity correlated with their humoral immunotoxicity as measured by the antibody response to SRBC. Von-Meyerinck et al. (1990) have described a commercial PBDE mixture, Bromkal-70, as a mixed-type inducer, stimulating both EROD and benzphetamine-N-demethylase cytochrome P450 activities. The present studies examined the potency of another commercial PBDE mixture, DE-71, to induce mixed-function oxygenase activities and to alter anti-SRBC responses in C57BL/6 mice exposed to a range of acute and subchronic doses of DE-71. Induction of hepatic cytochrome P450IAI and IIB1 subfamilies was evaluated by EROD and pentoxyresorufin-o-deethylase (PROD) activities, respectively. Endocrine effects of BDE include hyperplasia of the thyroid gland in rats (U.S. Environmental Protection Agency, 1991). Thyroid hormones are endogenous iodinated diphenyl ethers bearing structural resemblance to PBDE and interference with normal thyroid hormone homeostasis has been proposed for structurally similar polychlorinated biphenyls (PCBs) (Brouwer, 1991). Halogenated compounds with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-Iike toxicity are also known to affect the thyroid in rats, causing
J.R. Fowles et al. /Toxicology 86 (1994) 49-61
51
reduced thyroxin (T4) and triiodothyronine levels in addition to thyroid hyperplasia (Collins et al., 1977; Allen-Rowlands et al., 1981; Pazdernik and Rozman, 1985). The mechanisms of reduction of thyroid hormone levels by TCDD-like compounds has been shown to be due to induced hepatic UDPglucuronyltransferase activity (Barter and Klaassen, 1992) as well as to effects at the level of the anterior pituitary and the thyroid (Gorski et al., 1988). Because natural killer cell (NKC) activity can be modulated by thyroid status (Kinoshita et al., 1991), NKC activity was examined in relation to circulating total and free T4 levels in DE-71-treated mice. Glucocorticoid (GC) hormone levels are also known to be modulated following exposure to various halogenated aromatic hydrocarbons including TCDD, presumably through a variety of mechanisms including altered metabolism (Gorski et al., 1988) and liver clearance through cytochrome P450 enzyme induction or suppression. Since GC hormones are also immunomodulatory, serum GC levels were monitored as possible indirect mediators of immunotoxicity of DE-71. 2. Materials and methods
2.1. Animals Adult (8-week-old) female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) from a colony sero-negative for murine hepatitis virus, were housed following National Institute of Health guidelines for the care and use of laboratory animals. Mice were housed six to a cage and maintained on a 12 h light cycle in a room with a laminar flow unit. Mice were provided with food (TEKLAD Rodent Chow, Madison, WI) and water ad libitum.
2.2. Experimental design Mice (6/group) were dosed once by gavage with 0, 0.8, 4, 20, 100, or 500 mg/kg body weight DE-71 (Great Lakes Chemical Co., West Lafayette, IN) for the acute study. For the subchronic study, oral doses were 0, 18, 36, or 72 mg/kg per day for 14 days (6-8 mice/group), for total doses of 0, 250, 500, or 1000 mg/kg. Mice in the subchronic study were not given injections of SRBC when measurements of NKC activity or other endocrine or P450 endpoints were made. Mice treated acutely with DE-71 were all injected with SRBC. Peanut oil was used as the vehicle for DE-71. Mice were weighed daily to the nearest 0.1 g. At the end of each experiment, mice were selected randomly from each cage, stratified across treatment groups and were killed by CO2 asphyxiation. Spleen, thymus, liver and body weights were measured. Following either acute or subchronic exposure to DE-71, spleen cells were assayed for antibody production to SRBC, serum for total T4 and livers for microsomal
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J.R. Fowles et al. / Toxicology 86 (1994) 49-61
cytochrome P450 enzyme content and specific activities. Statistical analyses were performed using Student's t-test with significant differences accepted at the P < 0.05 level. Multiple linear regression was used to analyze the interactive effects of DE-71 and stress in the CS data. Statview 512K software for the Macintosh computer was used for statistical analyses. 2.3. Plaque-forming cell (PFC) assay for the antibody response to S R B C Mice were sensitized by i.p. injection with 2.5 x 108 (Allen-Rowlands et al., 1981) SRBC 2 days after a single DE-71 exposure, or after 9 days of the 14 day chronic exposure regimen. Mice were killed 5 days after immunization in each study. Spleens were processed into single cell suspensions and the number of anti-SRBC plaques was determined using a modification (Deyo and Kerkvliet, 1990) of the method of Cunningham and Szenberg (Cunningham and Szenberg, 1968). Hanks' balanced salt solution with 2.5% fetal bovine serum (Hyclone, Logan, UT) with 10 mM HEPES buffer (pH = 7.4) was used as the medium throughout the assay. 2.4. Natural killer eell assay Splenic NKC cytotoxicity was measured in a 4 h chromium release assay using the murine target cell line YAC-1 as previously described (Kerkvliet et al., 1982) except that Ultraculture ® serum-free medium (Biowhittaker Labs, Walkersville, MD) was used throughout the assay. Thymocytes (Thy) from a control mouse were used as inactive effector cells to control for re-uptake of chromium. Poly(Inosinic:Cytidylic acid) (Poly I:C) (Sigma Chemical Co., St. Louis, MO) was added (25/~g/well) 2 h prior to addition of YAC-1 cells to stimulate NKC activity. Supernatants (100 ttl) were harvested after 4 h incubation and counted on a gamma counter. Effector:target ratios of 100, 50, 25 and 12.5:1 were tested. Comparisons from the 100:1 ratio were reported as specific lysis calculated from the counts/min of: (Test-Thy/MRSR) where SR was spontaneous release of chromium from YAC-1 cells incubated with media only and MR was YAC-I cells incubated with 0.5% sodium dodecyl sulfate. 2.5. Hormone measurements Total and free T4 and CS were measured in duplicate using commercial radioimmunoassay (RIA) kits (Diagnostic Products Corp. Los Angeles, CA). Lowest detectable limits for total T4, free T4 and CS were: 1.0/~g/dl, 1.0 pg/ml and 10 ng/ml, respectively. Mice were allowed to acclimate to the laboratory overnight before termination to lessen stress prior to necropsy. 2.6. Hepatic microsomal O'tochrome P450 activities Microsomes were prepared from livers that had been flash frozen in liquid nitrogen. Microsomes were prepared in phosphate buffer (0.1 M KH2PO4,
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
53
0.15 M KCI, 1 m M E D T A , 1 m M dithiothreitol, p H = 7.4) and stored at - 7 0 ° C until assayed. T o t a l p r o t e i n was d e t e r m i n e d using a bicinconic acid kit (Pierce, R o c k f o r d , IL). C y t o c h r o m e P450 c o n t e n t was d e t e r m i n e d on a scanning s p e c t r o p h o t o m e t e r using the m e t h o d s o f E s t a b r o o k et al. (1972). Substrates and s t a n d a r d s for the E R O D and P R O D e n z y m e assays were o b t a i n e d f r o m M o l e c u l a r P r o b e s (Eugene, OR). E n z y m e activities o f E R O D and P R O D were analyzed on a f l u o r o m e t e r using the m e t h o d s o f P r o u g h et al. (1978).
3. Results As s h o w n in T a b l e 1, the P F C response to S R B C was not significantly altered by a single acute dose o f DE-71 as high as 500 mg/kg. F o l l o w i n g subchronic exposure, the P F C response was modestly (63% o f control, P < 0.02) suppressed only in mice that received a total dose o f 1000 mg/kg DE-71. N e i t h e r resting n o r (poly I:C) induced N K C activities were affected in mice treated subchronically with DE-71. As s h o w n in Fig. 1, serum T 4 c o n c e n t r a t i o n s were decreased in a nond o s e - d e p e n d e n t m a n n e r following acute e x p o s u r e to DE-71. This effect was a p p a r e n t at the lowest dose o f DE-71 tested (0.8 mg/kg). S u b c h r o n i c exposure to DE-71 p r o d u c e d a similar suppression o f total serum T4 levels (Fig.
Table 1 Plaque-forming cell (PFC) number and natural killer cell (NKC) activity in C57BL/6 mice treated with a single dose or 14 daily doses of DE-71 ~ DE-71 (mg/kg)
PFC/106cells
NKC activity Basal
Induced b
1257 ± 126 999 ± 89
n.d. c n.d.
n.d. n.d.
1460 ± 1284 ± 1269 ± 981 ±
24.77 26.73 30.77 29.81
A cute
0 500 Subchronic d
0 250 500 1000
108 195 219 146"
± ± ± ±
0.74 2.15 3.41 3.15
The data are representative of 2 experimental trials. ~Data are presented as mean -4- S.E.M., n--6/dose. blnduced with Polyinosinic:cytidylic acid (Poly I:C). c n.d., not done dTotal dose is shown for subchronic treatment. *Significant difference from control P < 0.02.
41.62 44.48 48.79 43.77
+ ± ± +
1.48 2.72 2.86 3.98
54
J.R. Fowles et a l . / Toxicology 86 (1994) 49-61
60 5.0
..................................................................................
T ......................................................
4030-
o I-',
1
20-
----[3---
Acute
10-
Subchronic
@ O0
........
I
o
........
I
........
I
........
I
0
0
0
0
-
~
g
8O
DE-71
........
Dose (mg/kg)
Fig. 1. Total T4 in female C57BL/6 mice (5-8/dose) treated by gavage with 0, 0.8, 4.0, 20, 100, or 500 mg/kg DE-71 in an acute exposure, or with 0, 250, 500, or 1000 mg/kg over a 14 day period (1 experimental iteration for each exposure regime)• Data are presented as mean 4- S.E.M. * indicates a significant difference from control (represented by horizontal lines for mean 4- S.E.M. of 5.68 4- 0•32, n= 14.
0.25
~'
.
02
a
"r b
0.15C ~--"
O. 1
I,I. 0.05
0
I
I
I
I
250
500
750
1000
DE-71
1250
Dose (mg/kg)
Fig. 2. Serum free T4 in female C57BL/6 mice (6/dose) treated by gavage with DE-7I (0, 18, 36, or 72 mg/kg per day) for 14 days (total dose is shown). Data are presented as mean 4- S.E.M. The data are representative of 2 trials• Different letters indicate significant differences between treatment groups (P < 0•05).
55
J.R. Fowles et al./ Toxicology 86 (1994) 49-61
1), with maximum suppression to 60% of control at the 1000 mg/kg dose. Free serum T4 was also suppressed by DE-71 and paralleled the response seen for total T4 (Fig. 2). As shown in Fig. 3, corticosterone levels in the serum increased with increasing dosage and with order of kill. In addition, a highly significant (P < 0.01) statistical interaction of dose and order of kill, measured by multiple linear regression, was seen for the CS elevation. Liver weight/body weight ratios were dose-dependently increased compared to controls following subchronic exposure (Table 2). Liver weight/ body weight was also significantly increased in mice treated acutely with 500 mg/kg DE-71 compared to controls. Acute DE-71 treatments as high as 100 mg/kg had no effect on liver weight. Thymus weight was significantly decreased following subchronic treatment with 1000 mg DE-71/kg, while body and spleen weights were unchanged (Table 2). Hepatic microsomes exhibited cytochrome P450 enzyme induction in terms of elevated P R O D activity (2-fold) at the highest dose (500 mg/kg)
500
400
i23
622
i20 ..I E
300
19
1115
v
•1o
2OO-
•16
0 •2 100-
21 17
i 6,14,18
11,5.9,13
•
3
4 8
;
7,11
i
12
I
I
|
I
Control
250
500
1000
DE-71
Dose
(mg/kg)
Fig. 3. Serum corticosterone (CS) levels in female C57BL/6 mice treated subchronicallywith DE-71 (0, 18, 36, or 72 mg/kg per day). Total dose is shown. The number by each data point indicates the order in which the animal was killed at time of necropsy. The data was collected from a single experiment. A significant interaction was seen between order of kill, DE-71 treatment and elevation of CS levels using multiple linear regression Y-Intercept = -79.742 /3(order) = 44.903, /3(DE-71 dose) = 0.111, R 2= 0.478, S.E. = 95.381. Partial F-values were 14.067 for order, 4.456 for dose and 9.173 overall (P < 0.01).
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J.R. Fowles et al. / Toxicology 86 (1994) 49-61
Table 2 Liver, spleen, thymus and final body weights from female C57BL/6 mice treated with a single or 14 daily doses o f DE-71 a DE-71 (mg/kg)
Final BW (g)
Liver/BW (mg/g)
Thymus/BW (mg/g)
Spleen/BW (mg/g)
17.45 17.34 17.42 17.05 17.86 17.67
4- 0.14 ± 0.48 4- 0.32 ± 0.39 4- 0.18 4- 0.37
51.19 53.89 52.81 48.88 50.70 60.51
444± ± ±
1.77 1.25 0.91 0.74 1.69 1.06 ¢
2.46 2.93 2.89 2.03 3.21 2.91
4- 0.24 4- 0.36 4- 0.21 ± 0.31 4- 0.30 4- 0.18
4.30 4.26 4.13 3.55 4.36 4.42
4- 0.46 4- 0.21 4- 0.18 4- 0.22 4- 0.31 4- 0.24
17.30 17.22 16.74 17.28
4444-
48.45 53.96 58.19 62.13
4444-
1.25 0.62 ¢ 0.98 c 1.08 c
3.14 3.08 3.25 2.62
4444-
3.91 3.89 4.06 3.56
4444-
Acute
0 0.8 4.0 20 100 500 Subchronic b
0 250 500 1000
0.28 0.27 0.35 0.35
0.24 0.19 0.10 0.14 c
0.17 0.14 0.16 0.23
The data are representative o f 2 experimental trials. BW, Body weight. "Data are presented as means 4- S.E.M., n = 6-8/dose. bTotal dose is shown. CStatistically different from control (P < 0.05).
Table 3 Hepatic microsomal cytochrome P450 activities and content o f female C57BL/6 mice treated with a single or 14 daily doses o f DE-71 ~ DE-71 (mg/kg)
PROD (nmol/min/mg)
EROD (nmol/min/mg)
P450 (nmol/mg)
0.046 0.030 0.029 0.069 0.136
44444-
0.017 0.004 0.002 0.011 0.013 ~
0.118 0.134 0.113 0.109 0.147
44444-
0.010 0.020 0.023 0.018 0.017
0.664 0.760 0.660 0.672 0.644
44444-
0.068 0.064 0.065 0.128 0.076
0.104 0.484 0.478 0.389
± 444-
0.010 0.037 c 0.058 c 0.034 c
0.058 0.131 0.192 0.192
± + 44-
0.005 0.011 ~ 0.025 ¢ 0.003 c
0.619 0.760 0.787 0.882
4444-
0.027 0.020 c 0.033 c 0.034 ~
Acute
0 0.8 4 100 500 Subchronic b
0 250 500 1000
The data represents one experimental trial. aData are presented as means ± S.E.M., n = 6-8/dose. bTotal dose is shown. ¢Statistically different from control (P < 0.05),
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
57
following acute exposure (Table 3). Subchronic DE-71 exposure resulted in significant induction of PROD activity which was maximal with 250 mg DE71/kg (4.7-fold), and an induction of EROD activity which was maximal at 500 mg DE-71/kg (3.3-fold) (Table 3). Hepatic microsomal P450 content was dose-dependently increased up to 40% above controls in mice treated subchronically with DE-71. 4. Discussion
Induction of hepatic microsomal EROD activity is generally correlated with humoral immunotoxicity to the anti-SRBC response from exposure to halogenated aromatic hydrocarbons (HAH) such as: dioxins (Vecchi et al., 1983), furans (Davis and Safe, 1988), biphenyls (Silkworth et al., 1984) and CDEs (Howie et al., 1990) in C57BL/6 mice. The correlation of these effects is largely explained by the Ah-receptor model (Poland and Glover, 1980). However, Howie et al. (1990) observed marked congener-specific differences in potencies for EROD induction and immunotoxicity in CDEs, including some congeners which did not fit the correlation seen with the majority of CDEs. The congeners which did not show classic Ah-receptor-dependent effects in their study included 2,3 ',4,4',5,5'-hexaCDE, a congener which exhibited low EROD-inducing potential and a lack of a dose-response in humoral immunotoxicity. Other mono-ortho-substituted CDE congeners tested were highly potent for both EROD induction and immunotoxicity. The lack of classic Ah-receptor-dependent effects is similar to the modest EROD induction and poorly correlated immunotoxicity seen in our study using DE-71. Based on these results it is clear that EROD activity per se does not parallel immune suppression when maximal EROD induction is relatively modest. At equivalent doses, acute exposure to DE-71 did not yield as great an induction of P450 enzymes as did daily exposure for 14 days. This is likely due to the fact that acute exposure was given 8 days prior to necropsy, whereas subchronic exposure continued until the day prior to necropsy. Cytochrome P450 PROD and EROD activities were both significantly induced by subchronic exposure to DE-71, indicating that DE-71 induces both the IIB1 and IA1 forms of cytochrome P450 in mice. This is consistent with the results using Bromkal-70 in rats (Von Meyerinck et al., 1990) with the exception that, in our studies in mice, higher IIB1 type induction (using the PROD assay) and lower IA1 induction (using the EROD assay) was observed. The lack of highly pronounced induction of EROD activity in our study indicates either that rats are more readily induced by PBDE than mice, or that the DE-71 formulation of brominated diphenyl ethers is missing specific isomers that are present in the experiments using Bromkal-70 (Von
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J.R. Fowles et al. / Toxicology 86 (1994) 4 9 - 6 1
.0 t_ 0
,<
4
5
I
I
I
l
l
20:00
2 l :40
23:20
25:00
26:40
Time
(min)
Fig. 4. Gas chromatogram of DE-71 showing five major peaks corresponding to 1, tetrabromodiphenyl ether (33%); 2 and 3, pentabromodiphenyl ethers (58%); 4 and 5, hexabromodiphenyl ethers (6%). Other minor peaks make up 3% of the mixture.
Meyerinck et al., 1990). While the gas chromatographic analysis of DE-71 (Fig. 4) appears to be very similar to Bromkal-70 (Von Meyerinck et al., 1990), the isomeric make-up of the formulation is not known. It was apparent from our studies that the order in which mice were killed at the end of subchronic treatment correlated with CS levels, with higher CS levels seen in mice that remained in the cage the longest. This was likely due to stress caused by the repeated disturbance of the cage during captures. Interestingly, there was a significant interactive effect of DE-71 and the order of kill on elevated CS levels. These results suggest either an increase in CS production or a decreased clearance of CS by the liver in animals treated with DE-71 following acute stress. Further investigation into this phenomenon is warranted. Excluding the 100 mg/kg acute dose, total T4 levels were significantly suppressed following all doses of DE-71 given, both acutely and subchronically. Interestingly, the degree of suppression of T4 was as great at low acute doses as it was at higher subchronic doses. Because of the different exposure protocols, it is difficult to directly compare mechanistic bases for the suppression. However, it suggests that the effect of DE-71 on T4 is saturable at very low doses and persists for at least 7 days after exposure is terminated. Suppression of NKC activity has been reported in mice treated with T4
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
59
(Gupta et al., 1983; Stein-Streilein et al., 1987), or under conditions of hyperand hypothyroidism in C57BL/6 mice (Kinoshita et al., 1991). The suppressed T4 levels seen in our study did not result in decreased NKC activity, suggesting that a depressed circulating T4 concentration alone is not sufficient to cause suppression of NKC activity. Interestingly, ~25I-induced hypothyroidism has also been shown to have no effect upon NKC activity (Sharma et al., 1982). Given the close structural similarity of some BDE congeners that may be present in DE-71 to that of T4, DE-71 could have either agonistic or antagonistic properties to T4 receptors. The suppression of T4 levels seen in DE-71 exposure is unlikely due to competition for binding to carrier proteins in the plasma since total and free T4 were affected in a similar dose-dependent manner. The cause of the lowered hormone concentration may instead be at the thyroid gland itself. This is supported by previous reports of bromine levels 12 times that of normal controls found in the thyroid gland of rats exposed to PBDE in the diet for 28 days (U.S. Environmental Protection Agency, 1988.) The potential for structure-specific binding to T4 receptors for chlorinated aromatic hydrocarbons has been reviewed (McKinney, 1989). In conclusion, the DE-71 mixture of BDE congeners appears to be modestly immunotoxic to the anti-SRBC response but not NKC activity in C57BL/6 mice at a subchronic dose totaling 1000 mg/kg DE-71. This same dose results in the weak, saturated induction of EROD and PROD liver enzyme activity, induction of total microsomal P450 content, suppression of circulating T4 and elevation of CS levels. Acute exposures to DE-71, while not immunotoxic, resulted in a modest induction of liver PROD activity following a high dose (500 mg/kg) and suppression of total serum T4 at much lower doses (0.8 mg/kg).
5. Acknowledgments We wish to thank Susan Schiller (ManTech Environmental Technology Inc., Environmental Research Laboratory, Corvallis, OR) for assistance in microsome preparations, Steve Hennes and the laboratory of Dr. Donald Buhler (Toxicology Program, Oregon State University) for technical advice regarding microsome isolation and cytochrome P450 measurements and Dr. Donald Griffin (Environmental Health Sciences Center, Oregon State University) for GC-mass spectral analysis of the DE-71. The information in this document has been funded in part by the U.S. Environmental Protection Agency under cooperative agreement No. CR 15131 to Oregon State University. It has been subjected to the agency's peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.
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6, References Allen-Rowlands, C.F., Castracane, V.D., Hamilton, M.G. and Seifter, J. (1981) Effect of polybrominated biphenyls (PBB) on the pituitary-thyroid axis of the rat. Proc. Soc. Exp. Biol. Med. 166, 506-514. Barter, R.A. and Klaassen, C.D. (1992) UDP-glucuronyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol. Appl. Pharmacol. 113, 36-42. Brouwer, A. (1991) Role of biotransformation in PCB-induced alterations in vitamin A and thyroid hormone metabolism in laboratory and wildlife species. In: The role of enzymes in regulating the toxicity of xenobiotics. Biochem. Soc. Trans. 19, 731-737. Buser, H.R. (1986) Polybrominated dibenzofurans and dibenzo-p-dioxins: thermal reaction products of polybrominated diphenylether flame retardants. Environ. Sci. Technol. 20(4), 404-469. Collins, W.T., Capen, C.C., Kasza, L., Carter, C. and Dailey, R.E. (1977) Effect of polychlorinated biphenyl (PCB) on the thyroid gland of rats: Am. J. Pathol. 89, 119-136. Cunningham, A.J. and Szenberg, A. (1968) Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599-600. Davis, D. and Safe, S. (1988) lmmunosuppressive activities of polychlorinated dibenzofuran congeners: Quantitative structure activity relationships and interactive effects. Toxicol. Appl. Pharmacol. 94, 141-149. Deyo, J.A. and Kerkvliet, N.I. (1990) The immunotoxicity of the pyrrolizidine alkaloid monocrotaline following subchronic administration to C57BL/6 mice. Fundam. Appl. Toxicol. 14, 842-849. Estabrook, R.W., Peterson, J., Baron, J. and Hildebrandt, A.G. (1972) The spectrophotometric measurement of turbid suspensions of cytochromes associated with drug metabolism. In C.F. Chignell (Ed), Methods in Pharmacology, Vol. 2, Appleton-Century-Crofts, Meredith, NY, pp. 303-350. Gorski, J.R., Muzi, G., Weber, L.W., Pereira, D.W. Latropulos, M.J. and Rozman, K. (1988) Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Toxicology 53, 19-32 Gorski, J.R., Muzi, G., Weber, L.D., Pereira, D.W., Arceo, R.J., latropoulos, M.J. and Rozman, K.C. (1988) Some endocrine and morphological aspects of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Pathol. 16, 313-320 Gupta, M.K., Chiang, T. and Deodhar, S.D. (19831 Effect of thyroxine on immune response in C57BL/6J mice. Acta Endocrinol. 103, 76-80. Howie, L., Dickerson, R., Davis, D. and Safe, S. (1990) lmmunosuppressive and monooxygenase induction activities of polychlorinated diphenyl ether congeners in C57BL/6N mice: quantitative structure-activity relationships. Toxicol. Appl. Pharmacol. 105, 254-263. Kerkvliet, N.I., Baecher-Steppan, L., Claycomb, A.T., Craig, A.M. and Sheggeby, G.G. (1982) Immunotoxicity of technical pentachlorophenol (PCP-T): Depressed humoral immune responses to T-dependent and T-independent antigen stimulation in PCP-T exposed mice. Fundam. Appl. Toxicol. 2, 90-99. Kinoshita, S., Sone, S., Yamashita, T., Tsubura, E. and Ogura, T. (1991) Effects of experimental hyper- and hypothyroidism on natural defense activities against Lewis lung carcinoma and its spontaneous pulmonary metastases in C57BL/6 mice. Tokushima J. Exp. Med. 38, 25-35. McKinney, J. (1989) Multifunctional receptor model for dioxin and related compound toxic
J.R. Fowles et aL /Toxicology 86 (1994) 49-61
61
action: Possible thyroid hormone-responsive effector linked site. Environ. Health Perspect. 82, 323-336. Pazdernik, T.L. and Rozman, K.K. (1985) Effect of thyroidectomy and thyroxine on 2,3,7,8tetrachlorodibenzo-p-dioxin-induced immunotoxicty. Life Sci. 36, 695-703. Poland, A. and Glover, E. (1980) 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Segregation of toxicity with the Ah locus. Mol. Pharmacol. 17, 86-94. Prough, R.A., Burke, M.D. and Mayer, R.T. (1978) Direct fluorometric methods for measuring mixed-function oxidase activity. Methods Enzymol. 52, 372-377. Sharma, S.D., Tsai, V. and Proffitt, M.R. (1982) Enhancement of mouse natural killer cell activity by thyroxine. Cell. Immunol. 73, 83-97. Silkworth, J.B., Antrim, L. and Kaminsky, L.S. (1984) Correlations between polychlorinated biphenyl immunotoxicity, the aromatic hydrocarbon locus and liver microsomal enzyme induction in C57BL/6 and DBA/2 mice. Toxicol. Appl. Pharmacol. 75, 156-165. Stein-Streilein, J., Zakarija, M., Papic, M. and McKenzie, J.M. (1987) Hyperthyroxinemic mice have reduced natural killer cell activity. J. lmmunol. 139, 2502-2507. U.S. Environmental Protection Agency (1988) Information review: brominated diphenyl ethers (report No. IR-516), Washington DC, TSCA, lnteragency Testing Committee. U.S. Environmental Protection Agency (June 1991) Federal Register, 40 CFR parts 795, 798, 799. Vecchi, A., Sironi, M., Canegrati, M.A., Recchia, M. and Garattini, S. (1983) Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68, 434-441. Von-Meyerinck, L., Hufnagel, B., Schmoldt, A. and Benthe, H.F. (1990) Induction of rat liver microsomal cytochrome P450 by the pentabromodiphenyl ether Bromkal 70 and half-lives of its components in the adipose tissue. Toxicology 61,259-274.
Toxicology 86 (1994) 49-61
Immunologic and endocrine effects of the flameretardant pentabromodiphenyl ether (DE-71) in C57BL/6J mice Jeff R. Fowles a, Anne Fairbrother b, Linda Baecher-Steppan c, Nancy I. Kerkvliet *c "Toxicology Program, Oregon State University, Corvallis', OR 97331. USA hU.S. Environmental Protection Agency. Environmental Research Laboratory, 4 S W 35th Street. Corvallis. OR 97333, USA "Department of Agricultural Chemistry, ALSI039, Oregon State University, Corvallis. OR 97331-7301. USA
(Received 12 February 1993; accepted 27 May 1993)
Abstract
Polybrominated diphenyl ethers are manufactured for use as flame retardants in commercial plastics and textiles in Europe and North America. These studies investigated the acute and subchronic immunotoxicity and endocrine effects of a commercial pentabromodiphenyl ether mixture, DE-71, in female C57BL/6 mice. Mice were orally exposed to acute single doses of DE-71 of 0, 0.8, 4.0, 20, 100, or 500 mg/kg, or to subchronic daily doses totaling 0, 250, 500, or 1000 mg/kg over a 14 day period. Immunotoxicity was assessed by measuring the plaque-forming cell response to sheep erythrocytes (SRBC) and natural killer cell (NKC) activity (basal and poly I:C stimulated) to YAC-I target cells. Liver cytochrome P450 content and activities (ethoxyresorufin-o-deethylase (EROD) and pentoxyresorufin-o-deethylase (PROD)) as well as corticosterone (CS) and thyroxine (T4) concentrations were also measured. PROD activity was induced 3-5-fold in mice exposed acutely or subchronically to DE-71 at doses > 250 mg/kg. EROD activity and total microsomal cytochrome P450 content were significantly induced only in mice treated subchronically with DE-71; maximum induction of EROD was 3.3-fold. Total serum T4 concentrations were significantly lower in mice treated acutely with DE-71 at all doses except the 100 mg/kg dose, Total and free T4 concentrations were dose-dependently decreased in DE-71-treated mice following subchronic exposure. Plasma CS levels were elevated following subchronic exposure to DE-71. The elevation * Corresponding author. 0300-483X/94/$06.00 © 1994 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0300-483X(93)02636-U
50
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
of CS was correlatedwith order of capture at necropsy,suggestingan interactiveeffectof DE71 and stress. In regardto immunotoxicity,significantsuppressionof the anti-SRBC response was seen only in mice exposed subchronicallyto 1000 mg DE-71/kg, an exposure that also resulted in decreasedthymus weight. NKC activity was not altered by exposure to DE-71. Key words: Antibody response; Corticosterone; Immunotoxicity; Natural killer cell; Pentabromodiphenylether; Thyroxine
1. Introduction Brominated diphenyl ethers (BDE) are currently manufactured for use as flame retardant additives to various plastics, rubber, paints and resins (U.S. Environmental Protection Agency, 1991). This use has led to the appearance of residues of BDE, including pentabromodiphenyl ether (PBDE), in a variety of environments, both aquatic and terrestrial (U.S. Environmental Protection Agency, 1991). Pyrolysis of BDE can yield brominated dibenzofurans and dibenzodioxins of potential concern (Buser, 1986). Since there is potential for BDE to cause adverse environmental and human health effects, the U.S. Environmental Protection Agency has recommended that toxicity studies be conducted for commercially important BDE congeners and mixtures. Structurally-related chlorinated diphenyl ethers (CDE) have been shown to cause liver enzyme induction as well as immune suppression in laboratory mice (Howie et al., 1990). In these studies, the potency of specific CDE congeners to induce hepatic microsomal ethoxyresorufin-o-deethylase (EROD) activity correlated with their humoral immunotoxicity as measured by the antibody response to SRBC. Von-Meyerinck et al. (1990) have described a commercial PBDE mixture, Bromkal-70, as a mixed-type inducer, stimulating both EROD and benzphetamine-N-demethylase cytochrome P450 activities. The present studies examined the potency of another commercial PBDE mixture, DE-71, to induce mixed-function oxygenase activities and to alter anti-SRBC responses in C57BL/6 mice exposed to a range of acute and subchronic doses of DE-71. Induction of hepatic cytochrome P450IAI and IIB1 subfamilies was evaluated by EROD and pentoxyresorufin-o-deethylase (PROD) activities, respectively. Endocrine effects of BDE include hyperplasia of the thyroid gland in rats (U.S. Environmental Protection Agency, 1991). Thyroid hormones are endogenous iodinated diphenyl ethers bearing structural resemblance to PBDE and interference with normal thyroid hormone homeostasis has been proposed for structurally similar polychlorinated biphenyls (PCBs) (Brouwer, 1991). Halogenated compounds with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-Iike toxicity are also known to affect the thyroid in rats, causing
J.R. Fowles et al. /Toxicology 86 (1994) 49-61
51
reduced thyroxin (T4) and triiodothyronine levels in addition to thyroid hyperplasia (Collins et al., 1977; Allen-Rowlands et al., 1981; Pazdernik and Rozman, 1985). The mechanisms of reduction of thyroid hormone levels by TCDD-like compounds has been shown to be due to induced hepatic UDPglucuronyltransferase activity (Barter and Klaassen, 1992) as well as to effects at the level of the anterior pituitary and the thyroid (Gorski et al., 1988). Because natural killer cell (NKC) activity can be modulated by thyroid status (Kinoshita et al., 1991), NKC activity was examined in relation to circulating total and free T4 levels in DE-71-treated mice. Glucocorticoid (GC) hormone levels are also known to be modulated following exposure to various halogenated aromatic hydrocarbons including TCDD, presumably through a variety of mechanisms including altered metabolism (Gorski et al., 1988) and liver clearance through cytochrome P450 enzyme induction or suppression. Since GC hormones are also immunomodulatory, serum GC levels were monitored as possible indirect mediators of immunotoxicity of DE-71. 2. Materials and methods
2.1. Animals Adult (8-week-old) female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) from a colony sero-negative for murine hepatitis virus, were housed following National Institute of Health guidelines for the care and use of laboratory animals. Mice were housed six to a cage and maintained on a 12 h light cycle in a room with a laminar flow unit. Mice were provided with food (TEKLAD Rodent Chow, Madison, WI) and water ad libitum.
2.2. Experimental design Mice (6/group) were dosed once by gavage with 0, 0.8, 4, 20, 100, or 500 mg/kg body weight DE-71 (Great Lakes Chemical Co., West Lafayette, IN) for the acute study. For the subchronic study, oral doses were 0, 18, 36, or 72 mg/kg per day for 14 days (6-8 mice/group), for total doses of 0, 250, 500, or 1000 mg/kg. Mice in the subchronic study were not given injections of SRBC when measurements of NKC activity or other endocrine or P450 endpoints were made. Mice treated acutely with DE-71 were all injected with SRBC. Peanut oil was used as the vehicle for DE-71. Mice were weighed daily to the nearest 0.1 g. At the end of each experiment, mice were selected randomly from each cage, stratified across treatment groups and were killed by CO2 asphyxiation. Spleen, thymus, liver and body weights were measured. Following either acute or subchronic exposure to DE-71, spleen cells were assayed for antibody production to SRBC, serum for total T4 and livers for microsomal
52
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
cytochrome P450 enzyme content and specific activities. Statistical analyses were performed using Student's t-test with significant differences accepted at the P < 0.05 level. Multiple linear regression was used to analyze the interactive effects of DE-71 and stress in the CS data. Statview 512K software for the Macintosh computer was used for statistical analyses. 2.3. Plaque-forming cell (PFC) assay for the antibody response to S R B C Mice were sensitized by i.p. injection with 2.5 x 108 (Allen-Rowlands et al., 1981) SRBC 2 days after a single DE-71 exposure, or after 9 days of the 14 day chronic exposure regimen. Mice were killed 5 days after immunization in each study. Spleens were processed into single cell suspensions and the number of anti-SRBC plaques was determined using a modification (Deyo and Kerkvliet, 1990) of the method of Cunningham and Szenberg (Cunningham and Szenberg, 1968). Hanks' balanced salt solution with 2.5% fetal bovine serum (Hyclone, Logan, UT) with 10 mM HEPES buffer (pH = 7.4) was used as the medium throughout the assay. 2.4. Natural killer eell assay Splenic NKC cytotoxicity was measured in a 4 h chromium release assay using the murine target cell line YAC-1 as previously described (Kerkvliet et al., 1982) except that Ultraculture ® serum-free medium (Biowhittaker Labs, Walkersville, MD) was used throughout the assay. Thymocytes (Thy) from a control mouse were used as inactive effector cells to control for re-uptake of chromium. Poly(Inosinic:Cytidylic acid) (Poly I:C) (Sigma Chemical Co., St. Louis, MO) was added (25/~g/well) 2 h prior to addition of YAC-1 cells to stimulate NKC activity. Supernatants (100 ttl) were harvested after 4 h incubation and counted on a gamma counter. Effector:target ratios of 100, 50, 25 and 12.5:1 were tested. Comparisons from the 100:1 ratio were reported as specific lysis calculated from the counts/min of: (Test-Thy/MRSR) where SR was spontaneous release of chromium from YAC-1 cells incubated with media only and MR was YAC-I cells incubated with 0.5% sodium dodecyl sulfate. 2.5. Hormone measurements Total and free T4 and CS were measured in duplicate using commercial radioimmunoassay (RIA) kits (Diagnostic Products Corp. Los Angeles, CA). Lowest detectable limits for total T4, free T4 and CS were: 1.0/~g/dl, 1.0 pg/ml and 10 ng/ml, respectively. Mice were allowed to acclimate to the laboratory overnight before termination to lessen stress prior to necropsy. 2.6. Hepatic microsomal O'tochrome P450 activities Microsomes were prepared from livers that had been flash frozen in liquid nitrogen. Microsomes were prepared in phosphate buffer (0.1 M KH2PO4,
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
53
0.15 M KCI, 1 m M E D T A , 1 m M dithiothreitol, p H = 7.4) and stored at - 7 0 ° C until assayed. T o t a l p r o t e i n was d e t e r m i n e d using a bicinconic acid kit (Pierce, R o c k f o r d , IL). C y t o c h r o m e P450 c o n t e n t was d e t e r m i n e d on a scanning s p e c t r o p h o t o m e t e r using the m e t h o d s o f E s t a b r o o k et al. (1972). Substrates and s t a n d a r d s for the E R O D and P R O D e n z y m e assays were o b t a i n e d f r o m M o l e c u l a r P r o b e s (Eugene, OR). E n z y m e activities o f E R O D and P R O D were analyzed on a f l u o r o m e t e r using the m e t h o d s o f P r o u g h et al. (1978).
3. Results As s h o w n in T a b l e 1, the P F C response to S R B C was not significantly altered by a single acute dose o f DE-71 as high as 500 mg/kg. F o l l o w i n g subchronic exposure, the P F C response was modestly (63% o f control, P < 0.02) suppressed only in mice that received a total dose o f 1000 mg/kg DE-71. N e i t h e r resting n o r (poly I:C) induced N K C activities were affected in mice treated subchronically with DE-71. As s h o w n in Fig. 1, serum T 4 c o n c e n t r a t i o n s were decreased in a nond o s e - d e p e n d e n t m a n n e r following acute e x p o s u r e to DE-71. This effect was a p p a r e n t at the lowest dose o f DE-71 tested (0.8 mg/kg). S u b c h r o n i c exposure to DE-71 p r o d u c e d a similar suppression o f total serum T4 levels (Fig.
Table 1 Plaque-forming cell (PFC) number and natural killer cell (NKC) activity in C57BL/6 mice treated with a single dose or 14 daily doses of DE-71 ~ DE-71 (mg/kg)
PFC/106cells
NKC activity Basal
Induced b
1257 ± 126 999 ± 89
n.d. c n.d.
n.d. n.d.
1460 ± 1284 ± 1269 ± 981 ±
24.77 26.73 30.77 29.81
A cute
0 500 Subchronic d
0 250 500 1000
108 195 219 146"
± ± ± ±
0.74 2.15 3.41 3.15
The data are representative of 2 experimental trials. ~Data are presented as mean -4- S.E.M., n--6/dose. blnduced with Polyinosinic:cytidylic acid (Poly I:C). c n.d., not done dTotal dose is shown for subchronic treatment. *Significant difference from control P < 0.02.
41.62 44.48 48.79 43.77
+ ± ± +
1.48 2.72 2.86 3.98
54
J.R. Fowles et a l . / Toxicology 86 (1994) 49-61
60 5.0
..................................................................................
T ......................................................
4030-
o I-',
1
20-
----[3---
Acute
10-
Subchronic
@ O0
........
I
o
........
I
........
I
........
I
0
0
0
0
-
~
g
8O
DE-71
........
Dose (mg/kg)
Fig. 1. Total T4 in female C57BL/6 mice (5-8/dose) treated by gavage with 0, 0.8, 4.0, 20, 100, or 500 mg/kg DE-71 in an acute exposure, or with 0, 250, 500, or 1000 mg/kg over a 14 day period (1 experimental iteration for each exposure regime)• Data are presented as mean 4- S.E.M. * indicates a significant difference from control (represented by horizontal lines for mean 4- S.E.M. of 5.68 4- 0•32, n= 14.
0.25
~'
.
02
a
"r b
0.15C ~--"
O. 1
I,I. 0.05
0
I
I
I
I
250
500
750
1000
DE-71
1250
Dose (mg/kg)
Fig. 2. Serum free T4 in female C57BL/6 mice (6/dose) treated by gavage with DE-7I (0, 18, 36, or 72 mg/kg per day) for 14 days (total dose is shown). Data are presented as mean 4- S.E.M. The data are representative of 2 trials• Different letters indicate significant differences between treatment groups (P < 0•05).
55
J.R. Fowles et al./ Toxicology 86 (1994) 49-61
1), with maximum suppression to 60% of control at the 1000 mg/kg dose. Free serum T4 was also suppressed by DE-71 and paralleled the response seen for total T4 (Fig. 2). As shown in Fig. 3, corticosterone levels in the serum increased with increasing dosage and with order of kill. In addition, a highly significant (P < 0.01) statistical interaction of dose and order of kill, measured by multiple linear regression, was seen for the CS elevation. Liver weight/body weight ratios were dose-dependently increased compared to controls following subchronic exposure (Table 2). Liver weight/ body weight was also significantly increased in mice treated acutely with 500 mg/kg DE-71 compared to controls. Acute DE-71 treatments as high as 100 mg/kg had no effect on liver weight. Thymus weight was significantly decreased following subchronic treatment with 1000 mg DE-71/kg, while body and spleen weights were unchanged (Table 2). Hepatic microsomes exhibited cytochrome P450 enzyme induction in terms of elevated P R O D activity (2-fold) at the highest dose (500 mg/kg)
500
400
i23
622
i20 ..I E
300
19
1115
v
•1o
2OO-
•16
0 •2 100-
21 17
i 6,14,18
11,5.9,13
•
3
4 8
;
7,11
i
12
I
I
|
I
Control
250
500
1000
DE-71
Dose
(mg/kg)
Fig. 3. Serum corticosterone (CS) levels in female C57BL/6 mice treated subchronicallywith DE-71 (0, 18, 36, or 72 mg/kg per day). Total dose is shown. The number by each data point indicates the order in which the animal was killed at time of necropsy. The data was collected from a single experiment. A significant interaction was seen between order of kill, DE-71 treatment and elevation of CS levels using multiple linear regression Y-Intercept = -79.742 /3(order) = 44.903, /3(DE-71 dose) = 0.111, R 2= 0.478, S.E. = 95.381. Partial F-values were 14.067 for order, 4.456 for dose and 9.173 overall (P < 0.01).
56
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
Table 2 Liver, spleen, thymus and final body weights from female C57BL/6 mice treated with a single or 14 daily doses o f DE-71 a DE-71 (mg/kg)
Final BW (g)
Liver/BW (mg/g)
Thymus/BW (mg/g)
Spleen/BW (mg/g)
17.45 17.34 17.42 17.05 17.86 17.67
4- 0.14 ± 0.48 4- 0.32 ± 0.39 4- 0.18 4- 0.37
51.19 53.89 52.81 48.88 50.70 60.51
444± ± ±
1.77 1.25 0.91 0.74 1.69 1.06 ¢
2.46 2.93 2.89 2.03 3.21 2.91
4- 0.24 4- 0.36 4- 0.21 ± 0.31 4- 0.30 4- 0.18
4.30 4.26 4.13 3.55 4.36 4.42
4- 0.46 4- 0.21 4- 0.18 4- 0.22 4- 0.31 4- 0.24
17.30 17.22 16.74 17.28
4444-
48.45 53.96 58.19 62.13
4444-
1.25 0.62 ¢ 0.98 c 1.08 c
3.14 3.08 3.25 2.62
4444-
3.91 3.89 4.06 3.56
4444-
Acute
0 0.8 4.0 20 100 500 Subchronic b
0 250 500 1000
0.28 0.27 0.35 0.35
0.24 0.19 0.10 0.14 c
0.17 0.14 0.16 0.23
The data are representative o f 2 experimental trials. BW, Body weight. "Data are presented as means 4- S.E.M., n = 6-8/dose. bTotal dose is shown. CStatistically different from control (P < 0.05).
Table 3 Hepatic microsomal cytochrome P450 activities and content o f female C57BL/6 mice treated with a single or 14 daily doses o f DE-71 ~ DE-71 (mg/kg)
PROD (nmol/min/mg)
EROD (nmol/min/mg)
P450 (nmol/mg)
0.046 0.030 0.029 0.069 0.136
44444-
0.017 0.004 0.002 0.011 0.013 ~
0.118 0.134 0.113 0.109 0.147
44444-
0.010 0.020 0.023 0.018 0.017
0.664 0.760 0.660 0.672 0.644
44444-
0.068 0.064 0.065 0.128 0.076
0.104 0.484 0.478 0.389
± 444-
0.010 0.037 c 0.058 c 0.034 c
0.058 0.131 0.192 0.192
± + 44-
0.005 0.011 ~ 0.025 ¢ 0.003 c
0.619 0.760 0.787 0.882
4444-
0.027 0.020 c 0.033 c 0.034 ~
Acute
0 0.8 4 100 500 Subchronic b
0 250 500 1000
The data represents one experimental trial. aData are presented as means ± S.E.M., n = 6-8/dose. bTotal dose is shown. ¢Statistically different from control (P < 0.05),
J.R. Fowles et al. / Toxicology 86 (1994) 49-61
57
following acute exposure (Table 3). Subchronic DE-71 exposure resulted in significant induction of PROD activity which was maximal with 250 mg DE71/kg (4.7-fold), and an induction of EROD activity which was maximal at 500 mg DE-71/kg (3.3-fold) (Table 3). Hepatic microsomal P450 content was dose-dependently increased up to 40% above controls in mice treated subchronically with DE-71. 4. Discussion
Induction of hepatic microsomal EROD activity is generally correlated with humoral immunotoxicity to the anti-SRBC response from exposure to halogenated aromatic hydrocarbons (HAH) such as: dioxins (Vecchi et al., 1983), furans (Davis and Safe, 1988), biphenyls (Silkworth et al., 1984) and CDEs (Howie et al., 1990) in C57BL/6 mice. The correlation of these effects is largely explained by the Ah-receptor model (Poland and Glover, 1980). However, Howie et al. (1990) observed marked congener-specific differences in potencies for EROD induction and immunotoxicity in CDEs, including some congeners which did not fit the correlation seen with the majority of CDEs. The congeners which did not show classic Ah-receptor-dependent effects in their study included 2,3 ',4,4',5,5'-hexaCDE, a congener which exhibited low EROD-inducing potential and a lack of a dose-response in humoral immunotoxicity. Other mono-ortho-substituted CDE congeners tested were highly potent for both EROD induction and immunotoxicity. The lack of classic Ah-receptor-dependent effects is similar to the modest EROD induction and poorly correlated immunotoxicity seen in our study using DE-71. Based on these results it is clear that EROD activity per se does not parallel immune suppression when maximal EROD induction is relatively modest. At equivalent doses, acute exposure to DE-71 did not yield as great an induction of P450 enzymes as did daily exposure for 14 days. This is likely due to the fact that acute exposure was given 8 days prior to necropsy, whereas subchronic exposure continued until the day prior to necropsy. Cytochrome P450 PROD and EROD activities were both significantly induced by subchronic exposure to DE-71, indicating that DE-71 induces both the IIB1 and IA1 forms of cytochrome P450 in mice. This is consistent with the results using Bromkal-70 in rats (Von Meyerinck et al., 1990) with the exception that, in our studies in mice, higher IIB1 type induction (using the PROD assay) and lower IA1 induction (using the EROD assay) was observed. The lack of highly pronounced induction of EROD activity in our study indicates either that rats are more readily induced by PBDE than mice, or that the DE-71 formulation of brominated diphenyl ethers is missing specific isomers that are present in the experiments using Bromkal-70 (Von
58
J.R. Fowles et al. / Toxicology 86 (1994) 4 9 - 6 1
.0 t_ 0
,<
4
5
I
I
I
l
l
20:00
2 l :40
23:20
25:00
26:40
Time
(min)
Fig. 4. Gas chromatogram of DE-71 showing five major peaks corresponding to 1, tetrabromodiphenyl ether (33%); 2 and 3, pentabromodiphenyl ethers (58%); 4 and 5, hexabromodiphenyl ethers (6%). Other minor peaks make up 3% of the mixture.
Meyerinck et al., 1990). While the gas chromatographic analysis of DE-71 (Fig. 4) appears to be very similar to Bromkal-70 (Von Meyerinck et al., 1990), the isomeric make-up of the formulation is not known. It was apparent from our studies that the order in which mice were killed at the end of subchronic treatment correlated with CS levels, with higher CS levels seen in mice that remained in the cage the longest. This was likely due to stress caused by the repeated disturbance of the cage during captures. Interestingly, there was a significant interactive effect of DE-71 and the order of kill on elevated CS levels. These results suggest either an increase in CS production or a decreased clearance of CS by the liver in animals treated with DE-71 following acute stress. Further investigation into this phenomenon is warranted. Excluding the 100 mg/kg acute dose, total T4 levels were significantly suppressed following all doses of DE-71 given, both acutely and subchronically. Interestingly, the degree of suppression of T4 was as great at low acute doses as it was at higher subchronic doses. Because of the different exposure protocols, it is difficult to directly compare mechanistic bases for the suppression. However, it suggests that the effect of DE-71 on T4 is saturable at very low doses and persists for at least 7 days after exposure is terminated. Suppression of NKC activity has been reported in mice treated with T4
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(Gupta et al., 1983; Stein-Streilein et al., 1987), or under conditions of hyperand hypothyroidism in C57BL/6 mice (Kinoshita et al., 1991). The suppressed T4 levels seen in our study did not result in decreased NKC activity, suggesting that a depressed circulating T4 concentration alone is not sufficient to cause suppression of NKC activity. Interestingly, ~25I-induced hypothyroidism has also been shown to have no effect upon NKC activity (Sharma et al., 1982). Given the close structural similarity of some BDE congeners that may be present in DE-71 to that of T4, DE-71 could have either agonistic or antagonistic properties to T4 receptors. The suppression of T4 levels seen in DE-71 exposure is unlikely due to competition for binding to carrier proteins in the plasma since total and free T4 were affected in a similar dose-dependent manner. The cause of the lowered hormone concentration may instead be at the thyroid gland itself. This is supported by previous reports of bromine levels 12 times that of normal controls found in the thyroid gland of rats exposed to PBDE in the diet for 28 days (U.S. Environmental Protection Agency, 1988.) The potential for structure-specific binding to T4 receptors for chlorinated aromatic hydrocarbons has been reviewed (McKinney, 1989). In conclusion, the DE-71 mixture of BDE congeners appears to be modestly immunotoxic to the anti-SRBC response but not NKC activity in C57BL/6 mice at a subchronic dose totaling 1000 mg/kg DE-71. This same dose results in the weak, saturated induction of EROD and PROD liver enzyme activity, induction of total microsomal P450 content, suppression of circulating T4 and elevation of CS levels. Acute exposures to DE-71, while not immunotoxic, resulted in a modest induction of liver PROD activity following a high dose (500 mg/kg) and suppression of total serum T4 at much lower doses (0.8 mg/kg).
5. Acknowledgments We wish to thank Susan Schiller (ManTech Environmental Technology Inc., Environmental Research Laboratory, Corvallis, OR) for assistance in microsome preparations, Steve Hennes and the laboratory of Dr. Donald Buhler (Toxicology Program, Oregon State University) for technical advice regarding microsome isolation and cytochrome P450 measurements and Dr. Donald Griffin (Environmental Health Sciences Center, Oregon State University) for GC-mass spectral analysis of the DE-71. The information in this document has been funded in part by the U.S. Environmental Protection Agency under cooperative agreement No. CR 15131 to Oregon State University. It has been subjected to the agency's peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.
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6, References Allen-Rowlands, C.F., Castracane, V.D., Hamilton, M.G. and Seifter, J. (1981) Effect of polybrominated biphenyls (PBB) on the pituitary-thyroid axis of the rat. Proc. Soc. Exp. Biol. Med. 166, 506-514. Barter, R.A. and Klaassen, C.D. (1992) UDP-glucuronyltransferase inducers reduce thyroid hormone levels in rats by an extrathyroidal mechanism. Toxicol. Appl. Pharmacol. 113, 36-42. Brouwer, A. (1991) Role of biotransformation in PCB-induced alterations in vitamin A and thyroid hormone metabolism in laboratory and wildlife species. In: The role of enzymes in regulating the toxicity of xenobiotics. Biochem. Soc. Trans. 19, 731-737. Buser, H.R. (1986) Polybrominated dibenzofurans and dibenzo-p-dioxins: thermal reaction products of polybrominated diphenylether flame retardants. Environ. Sci. Technol. 20(4), 404-469. Collins, W.T., Capen, C.C., Kasza, L., Carter, C. and Dailey, R.E. (1977) Effect of polychlorinated biphenyl (PCB) on the thyroid gland of rats: Am. J. Pathol. 89, 119-136. Cunningham, A.J. and Szenberg, A. (1968) Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599-600. Davis, D. and Safe, S. (1988) lmmunosuppressive activities of polychlorinated dibenzofuran congeners: Quantitative structure activity relationships and interactive effects. Toxicol. Appl. Pharmacol. 94, 141-149. Deyo, J.A. and Kerkvliet, N.I. (1990) The immunotoxicity of the pyrrolizidine alkaloid monocrotaline following subchronic administration to C57BL/6 mice. Fundam. Appl. Toxicol. 14, 842-849. Estabrook, R.W., Peterson, J., Baron, J. and Hildebrandt, A.G. (1972) The spectrophotometric measurement of turbid suspensions of cytochromes associated with drug metabolism. In C.F. Chignell (Ed), Methods in Pharmacology, Vol. 2, Appleton-Century-Crofts, Meredith, NY, pp. 303-350. Gorski, J.R., Muzi, G., Weber, L.W., Pereira, D.W. Latropulos, M.J. and Rozman, K. (1988) Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Toxicology 53, 19-32 Gorski, J.R., Muzi, G., Weber, L.D., Pereira, D.W., Arceo, R.J., latropoulos, M.J. and Rozman, K.C. (1988) Some endocrine and morphological aspects of the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Pathol. 16, 313-320 Gupta, M.K., Chiang, T. and Deodhar, S.D. (19831 Effect of thyroxine on immune response in C57BL/6J mice. Acta Endocrinol. 103, 76-80. Howie, L., Dickerson, R., Davis, D. and Safe, S. (1990) lmmunosuppressive and monooxygenase induction activities of polychlorinated diphenyl ether congeners in C57BL/6N mice: quantitative structure-activity relationships. Toxicol. Appl. Pharmacol. 105, 254-263. Kerkvliet, N.I., Baecher-Steppan, L., Claycomb, A.T., Craig, A.M. and Sheggeby, G.G. (1982) Immunotoxicity of technical pentachlorophenol (PCP-T): Depressed humoral immune responses to T-dependent and T-independent antigen stimulation in PCP-T exposed mice. Fundam. Appl. Toxicol. 2, 90-99. Kinoshita, S., Sone, S., Yamashita, T., Tsubura, E. and Ogura, T. (1991) Effects of experimental hyper- and hypothyroidism on natural defense activities against Lewis lung carcinoma and its spontaneous pulmonary metastases in C57BL/6 mice. Tokushima J. Exp. Med. 38, 25-35. McKinney, J. (1989) Multifunctional receptor model for dioxin and related compound toxic
J.R. Fowles et aL /Toxicology 86 (1994) 49-61
61
action: Possible thyroid hormone-responsive effector linked site. Environ. Health Perspect. 82, 323-336. Pazdernik, T.L. and Rozman, K.K. (1985) Effect of thyroidectomy and thyroxine on 2,3,7,8tetrachlorodibenzo-p-dioxin-induced immunotoxicty. Life Sci. 36, 695-703. Poland, A. and Glover, E. (1980) 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Segregation of toxicity with the Ah locus. Mol. Pharmacol. 17, 86-94. Prough, R.A., Burke, M.D. and Mayer, R.T. (1978) Direct fluorometric methods for measuring mixed-function oxidase activity. Methods Enzymol. 52, 372-377. Sharma, S.D., Tsai, V. and Proffitt, M.R. (1982) Enhancement of mouse natural killer cell activity by thyroxine. Cell. Immunol. 73, 83-97. Silkworth, J.B., Antrim, L. and Kaminsky, L.S. (1984) Correlations between polychlorinated biphenyl immunotoxicity, the aromatic hydrocarbon locus and liver microsomal enzyme induction in C57BL/6 and DBA/2 mice. Toxicol. Appl. Pharmacol. 75, 156-165. Stein-Streilein, J., Zakarija, M., Papic, M. and McKenzie, J.M. (1987) Hyperthyroxinemic mice have reduced natural killer cell activity. J. lmmunol. 139, 2502-2507. U.S. Environmental Protection Agency (1988) Information review: brominated diphenyl ethers (report No. IR-516), Washington DC, TSCA, lnteragency Testing Committee. U.S. Environmental Protection Agency (June 1991) Federal Register, 40 CFR parts 795, 798, 799. Vecchi, A., Sironi, M., Canegrati, M.A., Recchia, M. and Garattini, S. (1983) Immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in strains of mice with different susceptibility to induction of aryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68, 434-441. Von-Meyerinck, L., Hufnagel, B., Schmoldt, A. and Benthe, H.F. (1990) Induction of rat liver microsomal cytochrome P450 by the pentabromodiphenyl ether Bromkal 70 and half-lives of its components in the adipose tissue. Toxicology 61,259-274.