FUNDAMENTAL AND APPLIED TOXICOLOGY 3 7 , 141 - 149 (1997)
ARTICLE NO. FA972323
Opposite Effects of 2,2',4,4',5,5'-Hexachlorobiphenyl and 2,3,7,8Tetrachlorodibenzo-p-dioxin on the Antibody Response
to Sheep Erythrocytes in Mice1
R. J. Smialowicz,2 M. J. DeVito, M. M. Riddle, W. C. Williams, and L. S. Birnbaum
National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina 27711
Received October 15, 1996; accepted April 21, 1997
munosuppressive dose of TCDD fails to suppress the PFC reOpposite Effects of 2,2',4,4',5,5'-Hexachlorobiphenyl and sponse relative to corn oil controls, while clearly suppressing it
2,3,7,8-Terrachlorodibenzo-p-dioxin on the Antibody Response to relative to the appropriate control, PCB153 alone, o iw sockty or
Sheep Erythrocytes in Mice. Smialowicz, R. J., DeVito, M. J., Rid- Toxicology.
dle, M. M., Williams, W. C, and Birnbaum, L. S. (1997). Fundam.
Appl. Toxicol. 37, 141-149.
The effect that cotreatment with 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
has on the antibody plaque-forming cell (PFC) response to sheep
red blood cells (SRBCs) was determined in female B6C3F1 mice.
Groups of eight mice per group were given a single oral dose of
PCB153 alone (0, 3.58, 35.8, or 358 mg/kg), TCDD alone (0, 0.1,
1, or 10 /xg/kg), and all possible combinations of these doses in corn
oil 7 days prior to immunization with SRBCs. Separate groups of
mice were given phenobarbital (PB) parenterally by intraperitoneal injection at a dosage of 160 mg/kg/day for 3 days. Four days
after intravenous immunization, body, spleen, thymus, and liver
weights and the PFC response to SRBCs were determined. Exposure to TCDD alone resulted in a dose-related suppression of the
PFC response, with significant suppression at 1 and 10 /xg/kg. In
contrast, exposure to PCB153 alone resulted in the enhancement
of the PFC response at 358 mg/kg. Combined exposure to 358 mg/
kg PCB153 and TCDD resulted in no change (PCB153 + 0.1 ixg/
kg TCDD) or suppression (PCB153 + 1 or 10 /xg/kg TCDD) of
the PFC response relative to PCB153 alone; however, the PFC
response was enhanced (PCB153 + 0.1 /zg/kg TCDD), unaffected
(PCB153 + 1 iig/kg TCDD), or suppressed (PCB153 + 10 /zg/kg
TCDD) relative to corn oil controls. PB did not affect the PFC
response to SRBCs, despite a 13-fold induction of hepatic pentoxyresorufin O-dealkylase (PROD) activity. These results suggest
that PCB153 enhancement of the PFC response is not related to
PROD induction and that it acts as a functional antagonist rather
than an aryl hydrocarbon receptor or dispositional antagonist.
By enhancing the PFC response to SRBCs, PCB153 raises the
"setpoint" response level. Consequently, cotreatment with an im-
' This report has been reviewed by the Environmental Protection
Agency's Office of Research and Development, and approved for publication. Approval does not signify that the contents necessarily reflect the
views and policies of the Agency nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
2
To whom correspondence and reprint requests should be addressed.
141
Halogenated aromatic hydrocarbons (HAHs) are a group
of industrial compounds or by-products that include the polychlorinated dioxins (PCDDs), biphenyls (PCBs), and dibenzofurans (PCDFs), which occur as complex mixtures in the
environment. A number of toxic and biochemical effects,
which are believed to require initial binding to the aryl hydrocarbon (Ah) receptor, are produced in experimental animals following exposure to HAHs of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent. One of
the most sensitive toxic endpoints of HAH exposure in mice
is suppression of the primary antibody response to sheep red
blood cells (SRBCs), as determined by the splenic plaqueforming cell (PFC) response, following a single exposure to
TCDD. The reproducibility and sensitivity of this T lymphocyte-dependent antibody response to suppression by TCDD
have been corroborated by a number of laboratories that
have demonstrated an ED50 of approximately 0.7 fj,g TCDD/
kg (Vecchi et al., 1980; Davis and Safe, 1988; Kerkvliet et
al, 1990; Smialowicz et al, 1994).
Certain structurally related HAHs which are approximate
isostereomers of TCDD (e.g., coplanar and mono-ortho coplanar PCB congeners), display Ah agonist activity and suppress the PFC response to SRBCs in mice (Silkworth et al,
1984; Davis and Safe, 1990). On the other hand, some complex PCB mixtures such as Aroclor 1242, 1248, 1254, and
1260 (Bannister et al, 1987; Davis and Safe, 1989), PCBs
such as 2,3,3',4,5'-penta- and 2,3,3',4,5,5'-hexachlorobiphenyl (Davis and Safe, 1990), and the 2,3,7,8-tetrachlorodibenzo-/7-furan (TCDF) derivative 6-methyl-l,3,8-trichlorodibenzofuran (Bannister et al, 1989) partially antagonize
the immunosuppressive effects of TCDD in mice. It has been
further hypothesized that there is a "window of antagonism"
that is dependent on the dose or concentration of antagonist
0272-0590/97 $25.00
Copyright © 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
142
SMIALOWICZ ET AL.
to TCDD (Safe, 1990). In addition, 2,2',4,4',5,5'-hexachlorobiphenyl or PCB153 has also been reported to antagonize
TCDD-induced immunosuppression of the PFC response to
SRBCs in mice as well as TCDD-induced cleft palate in mice
(Biegel et al., 1989). These observations have implications
relative to risk assessment, particularly since PCB153 is the
most prominent PCB congener present in human tissue (Jensen, 1987).
In this study, the potential for PCB 153 to antagonize
TCDD-induced suppression of the splenic PFC response to
SRBCs (Biegel et al., 1989) was reevaluated and an attempt
was made to identify the type of antagonism involved. There
are four types of antagonism that occur when two chemicals
are administered together, resulting in interference in each
of the chemical's actions or interference by one chemical
on the action of the other. These include the following:
(1) a functional antagonism occurs when "two chemicals
counterbalance each other by producing opposite effects on
the same physiological function"; (2) a chemical antagonism
occurs when "a chemical reaction between two compounds
produces a less toxic product"; (3) a dispositional antagonism occurs when "the disposition, that is, absorption, biotransformation, distribution, or excretion of the chemical, is
altered such that the concentration and/or duration of the
chemical at the target organ is diminished"; and (4) a receptor antagonism occurs when "two chemicals that bind to the
same receptor produce less of an effect when given together
than the addition of their separate effects . . . or when one
chemical antagonizes the effect of the second chemical" by
blocking the receptor (Klaassen and Eaton, 1991). As such,
dose-response data for both TCDD and PCB 153 effects on
the PFC response, following a single exposure of mice to
either TCDD or PCB 153 alone or in combination, were collected to better understand the interactions of these chemicals. In addition, since PCB 153 is considered to be a prototypical phenobarbital (PB)-type inducer (Safe, 1994) of hepatic pentoxyresorufin O-dealkylase (PROD) activity
(Parkinson et al, 1983; Van der Kolk et ai, 1992; Van
Birgelen et al., 1996), separate groups of mice were exposed
to PB to determine if there is an association between PROD
induction and the PFC response to SRBCs.
MATERIALS AND METHODS
Animals.
Female B6C3F1 mice, 8 weeks old, were obtained from
Charles River Laboratory (Raleigh, N Q and allowed to acclimate for 1
week prior to use. Animals were housed in shoebox-type polycarbonate
cages containing heat-treated pine shavings (Beta Chips, North Eastern
Products Inc., Warrensburg, NY) and given feed (Purina Lab Chow, Ralston
Purina Co., St. Louis, MO) and water ad libitum. An ambient temperature
of 22°C, relative humidity of 55 ± 5%, and 12-hr light-dark cycle were
provided. The mice were randomly assigned to treatment groups of eight
animals per group unless otherwise indicated. All procedures performed on
the animals were approved by the National Health and Environmental Effects Research Laboratory's Animal Care and Use Committee.
Chemicals and dosing. Dosing solutions of TCDD (>98% purity, Lot
MLB-15091-55, as determined by GC-MS, Radian Corp., Austin, TX) were
prepared from a stock solution containing 1 mg/kg body wt in 10 ml of
com oil (Sigma Chemical Co., St. Louis, MO). The stock solution was
prepared by dissolving TCDD in acetone, mixing the acetone solution with
com oil, and then removing the acetone by evaporation (DeVito el ai,
1993). PCB153 (>98% purity, Ultra Scientific, North Kingstown, RJ) dosing solutions were prepared in com oil. Mice were weighed and dosed with
TCDD alone (i.e., 0, 0.1, 1.0, or 10 /ig/kg body wt), PCB153 alone (i.e.,
0, 3.58, 35.8, or 358 mg/kg body wt), and all possible combinations, as a
single oral dose in a volume of 10 ml/kg. The doses of PCB 153 used in
this study were chosen based on the fact that these doses have been reported
to alter TCDD-induced responses (Bannister et al., 1987; Biegel et al,
1989; Morrissey et ai, 1992; DeJongh et al, 1995). It was impossible to
perform a single multimatrix experiment that included all TCDD and
PCB 153 doses alone as well as TCDD and PCB 153 dose combinations.
Consequently, individual experiments were performed in which data were
obtained for TCDD and PCB 153 alone dose responses, as well as for
individual doses of TCDD in combination with different PCB 153 doses
and vice versa.
In a separate experiment, sodium phenobarbital (Sigma) was prepared in
sterile saline at 160 mg/kg body wt, in a volume of 10 ml/kg, and administered via intraperitoneal injection on 3 consecutive days.
Antibody response. Seven days following chemical exposure, mice
were immunized with a single intravenous injection of 0.2 ml of 5% (i.e.,
2 x 1 0 " ) SRBCs (Environmental Diagnostics, Inc., Burlington, NC). Four
days following immunization the mice were weighed, a blood sample was
obtained, and thymus, spleen, and liver weights were determined. The primary antibody response to SRBCs was determined using the splenocyte
direct PFC assay as described (Smialowicz et ai, 1994). Both the mean
number of PFCs per 10s viable spleen cells and the mean number of PFCs
(XlO4) per spleen were calculated. Only the PFCs (XlO1) per spleen data
are reported, since essentially identical results were observed for the PFCs
per 106 spleen cells. Serum hemagglutination titers to SRBCs were also
determined at the time of PFC assay (Smialowicz et al, 1992).
Hepatic PROD activity. Hepatic PROD activity was determined in mice
24 hr following the third exposure to PB. Preparation of hepatic microsomal
fractions and the determination of PROD activity were performed as described by van der Kolk et al. (1992) and van Birgelen et al. (1996).
Data analysis. Data were analyzed by one-way analysis of variance
(ANOVA), with post hoc analysis using Dunnett's multiple comparison (
test or the Student-Newman-Keuls test (RS/1, 1988). Differences between
control and treatment groups were considered statistically significant when
p < 0.05. Data are presented as means ± standard errors of the means.
RESULTS
Body weights were not altered in mice dosed with either
TCDD or PCB 153 alone or in combination (Tables 1 and
2). Spleen and thymus weights were decreased, while liver
weights were increased in mice dosed at 1 or 10 fig TCDD/
kg. Liver weights were increased in mice exposed to 358
mg PCB153/kg (Tables 1 and 2). Spleen weights were decreased in mice exposed to 3.58 or 358 mg PCB153/kg +
10 fig TCDD/kg, and thymus weights were decreased at all
doses of PCB 153 in combination with 10 fig TCDD/kg (Table 2). Liver weights were increased in mice exposed to 358
mg PCB153/kg + all doses of TCDD, at 35.8 mg PCB153/
kg + 1 or 10 fig TCDD/kg, and at 3.58 mg PCB 153 + 10
fig TCDD/kg (Table 2).
143
EFFECTS OF PCB153/TCDD ON ANTIBODY RESPONSE
TABLE 1
Effect of Exposure to TCDD or PCB153 on Body, Spleen, Thymus, and Liver Weights of Female B6C3F1 Mice
Body weight
(g)
Treatment
TCDD (/ig/kg)
0
0.1
1.0
10.0
PCB153 (mg/kg)
0
3.58
35.8
358
Spleen weight*
(mg/g)
Thymus weight*
(mg/g)
Liver weight*
(mg/g)
23.9
23.5
24.1
23.8
±
±
±
±
0.3
0.4
0.4
0.4
4.1
3.8
3.4
3.0
± 0.1
± 0.1
±0.1**
±0.1**
2.3
2.1
1.8
1.3
± 0.1
± 0.1
± 0.1**
±0.1**
47.7
47.9
53.8
55.1
±
±
±
±
1.0
0.7
1.0**
1.0**
21.3
20.9
20.8
21.3
±
±
±
±
0.3
0.5
0.4
0.4
4.4
4.3
4.0
4.8
±
±
±
±
2.7
2.5
2.4
2.7
±
±
±
±
49.7
46.5
50.7
60.4
±
±
±
±
0.5
0.9
0.8
1.4**
0.2
0.2
0.1
0.2
0.1
0.2
0.1
0.2
Note. Values are means ± SEM.
° Relative organ weight-to-body weight ratio.
**p < 0.01, n = 8.
Figure 1 shows the effects that TCDD alone and PCB153
alone have on the PFC response to SRBCs. The PFC response was reduced in mice dosed at 1 or 10 ^.g TCDD/
kg to 52 and 30% of control, respectively (Fig. 1A). An
enhancement of the PFC response, to 162% of control, was
observed in mice dosed at 358 mg PCB153/kg (Fig. IB).
The effects of coadministration of a constant dose of
PCB 153 with different doses of TCDD are shown in Fig. 2.
Coadministration of 3.58 mg PCB153/kg with either 1 or
10 fig TCDD/kg resulted in a reduction in the PFC response
at 56 and 24% compared with the vehicle control and at 49
and 21% compared with the PCB153-alone control, respectively (Fig. 2A).
Figure 2B shows that the PFC response of mice dosed
with 35.8 mg PCB153/kg alone was 136% that of the vehicle
control. Suppression of the PFC response was observed only
TABLE 2
Effect of Exposure to PCB153 Alone or in Combiantion with TCDD on Body, Spleen, Thymus, and Liver Weights
of Female B6C3F1 Mice
Treatment
PCB 153
(mg/kg)
TCDD
0*g/kg)
0
3.58
3.58
3.58
3.58
0
35.8
35.8
35.8
35.8
0
358
358
358
358
0
0
0.1
1.0
10
0
0
0.1
1.0
10
0
0
0.1
1.0
10
Body weight
(g)
23.5
22.5
22.3
23.8
22.1
23.1
22.3
21.9
22.2
22.8
21.9
21.9
23.2
22.7
22.5
Note. Values are means ± SEM.
* Relative organ weight-to-body weight ratio.
* p < 0.05, **p < 0.01, n = 8.
± 0.6
± 0.2
± 0.3
± 0.2
± 0.2
± 0.7
± 0.4
±0.3
± 0.7
± 0.5
± 0.3
± 0.4
± 0.4
± 0.3
± 0.5
Spleen weight*
(mg/g)
4.4
4.7
4.7
4.4
3.6
3.8
4.1
4.4
3.8
3.3
4.3
4.5
4.7
4.3
3.6
± 0.1
± 0.2
± 0.1
± 0.2
±0.1**
± 0.1
± 0.3
± 0.2
± 0.2
± 0.3
± 0.2
± 0.2
± 0.3
± 0.2
±0.1*
Thymus weight*
(mg/g)
2.4
2.4
2.3
2.2
±
±
±
±
0.
0.
0.
0.
1.6
2.1
2.3
2.2
2.0
1.3
2.2
1.9
2.3
2.0
1.6
± 0 . **
±0.
± 0.
± 0.
± 0.
± 0. **
± 0.
± 0.2
± 0.2
± 0.2
± 0.1*
Liver weight*
(mg/g)
50.3
51.2
50.7
55.9
60.4
48.6
51.1
51.7
53.9
61.4
49.1
60.8
55.1
67.6
77.0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.4
1.2
0.6
1.5
0.4**
0.5
1.0
0.9
1.4**
1.4**
1.0
1.1**
1.3*
2.2**
2.0**
144
SMIALOWICZ ET AL.
1
A
30
Ul
UI
VI
20
15
u
u.
a.
1 1
25
I
(114)
•
V*
-
(38)
10
5
"" r
-
i
n
0
3.58
0
2
4
6
8
DOSE Gig/kg TCDD)
i
3.58
3.58
3.58
(mg/kg PCB)
0
0.1
1.0
10
(Mfl/kg TCDD)
35.8
35.B
35.8
35.8
(mg/kg PCB)
0.1
1.0
10
(/Jg/kg TCDD)
358
0.1
338
1.0
358
10
(mg/kg PCB)
0*g/kg TCDD)
in
•H
O
x
u
u_
0.
0
0
100
200
0
300
DOSE (mg/kg PCB 153)
FIG. 1. PFC response to SRBCs of mice dosed with TCDD alone (A)
or PCB 153 alone (B). p ( < ) 0.05 vs 0 (vehicle control), n = 8. Percentage
PFC response of control in parentheses
in mice coadministered 35.8 mg PCB153/kg and 10 fig
TCDD/kg (i.e., 24 that of the vehicle control and 18% that
of the PCB153-alone control, respectively). The PFC responses of mice coadministered 35.8 mg PCB 153 and 0.1
or 1.0 /j,g TCDD/kg, however, were not significantly reduced
compared with the vehicle control but were reduced compared with the PCB153-alone control.
PFC responses of mice dosed with either 358 mg
PCB153/kg alone or in combination with 0.1 /xg TCDD/
kg were enhanced to 193 and 186% that of the vehicle
control, respectively (Fig. 2C). Equivalent PFC responses
were observed in mice that were coexposed to 358 mg
0
0
358
0
FIG. 2. PFC response to SRBCs of mice dosed with 3.58 mg PCB 1537kg
(A), 35.8 mg PCB 153/kg (B), or 358 mg PCB 153/kg (C) alone or in combination
with different doses of TCDD. °p < 0.05 vs vehicle control. bp < 0.05 vs PCBaione control, n = 8. Percentage PFC response of vehicle control in parentheses.
Percentage PFC response of PCB-alone control in brackets.
145
EFFECTS OF PCB153/TCDD ON ANTIBODY RESPONSE
PCB153/kg and 1.0 p,g TCDD/kg compared with vehicle
control. PFC responses in mice coexposed to 358 mg
PCB153/kg and 10.0 ng TCDD/kg were reduced to 27
that of the vehicle control and 14% that of the PCB 153alone control (Fig. 2C).
Figure 3 shows the effects that coadministration of a constant dose of TCDD with different doses of PCB153 has on
the PFC response to SRBCs. A dose of 1.0 ng TCDD/kg
either alone or in combination with 3.58 mg or 35.8 mg
PCB153/kg resulted in suppression of the PFC response to
44, 45, and 45% that of the vehicle control, respectively
(Fig. 3A). In contrast, Fig. 3A shows that the combination
of 1.0 \ig TCDD/kg and 358 mg PCB153/kg did not signifi-
V)
TABLE 3
Effect of Exposure to Phenobarbital (PB) on Body, Spleen,
Thymus, and Liver Weights, on Hepatic PROD Activity, and on
the Antibody Plaque-Forming Cell Response to Sheep Red Blood
Cells of Female B6C3F1 Mice
Treatment
Parameter
Saline
Body weight (g)
Spleen :body weight (mg/g)
Thymus:body weight (mg/g)
Uver.body weight (mg/g)
PROD activity (pmol/mg/min)''
PFCs/106 cells
PFCs (X lOVspleen
Serum antibody titer Gog2)
22.1
5.1
2.1
49.0
61
1744
25.8
9.4
±
±
±
±
±
±
±
±
0.2
0.2
0.1
0.8
6
i$4
1.5
0.2
Phenobarbital"
21.2
5.8
1.6
54.5
819
1738
28.8
9.0
± 0.5
± 0.2*
± 0.1**
1.1**
±
± 51**
± 147
± 2.7
± 0.2
Note. Values are means ± SEM.
" Phenobarbital was administered intraperitoneally on 3 consecutive days
at a dosage of 160 mg/kg/day.
* Hepatic pentoxyresorufin O-dealkylase (PROD) activity was determined
in saline (n = 6)- and PB (n = 5)-exposed mice 24 hr after the last dosing
with PB.
* p < 0.05 or **p <0.01 vs saline control, n = 8, except for PROD
activity data.
-H
Q.
V)
O
Q_
0
o
0
1.0
3.58
i.o
35.8 358
1.0
1.0
(mg/kg PCB)
(MgAg TCDD)
0
0
0 3.SB 35.8 358 (mg/kg PCB)
10 10
10
10 Gig/kg TCDD)
FIG. 3. PFC response to SRBCs of mice dosed with I jig TCDD/kg
(A) or 10 /jg TCDD/kg (B) alone or in combination with different doses
of PCB 153. °p < 0.05 vs vehicle control. *p < 0.05 vs TCDD alone, n =
8. Percentage PFC response of vehicle control in parentheses. Percentage
PFC response of TCDD-alone control in brackets.
cantly reduce the PFC response compared with the vehicle
control. These data are consistent with the data presented in
Fig. 2C. Exposure of mice at a dose of 10 fig TCDD/kg in
the presence or absence of PCB 153 consistently suppressed
the PFC response to 22-23% that of the vehicle control
(Fig. 3B).
Since PCB 153, at a dose of 358 mg/kg, increases the
PFC response to SRBCs and is a potent inducer of hepatic
PROD activity (Parkinson et al., 1983; van der Kolk et
al., 1992; van Birgelen et al., 1996), it was of interest to
determine if enhancement of the PFC response by PCB 153
was related to induction of hepatic PROD activity. PB
was chosen for study because it is a potent inducer of
PROD activity in a variety of species (Paolini et al., 1995)
and because PCB 153 is often classified as a PB-type inducer. Mice dosed with 160 mg PB/kg/day for 3 days had
increased liver weights and a 13-fold increase in hepatic
PROD activity compared with saline controls (Table 3).
Spleen weights were also increased, whereas thymus
weights were decreased; however, the PFC response (Table 3) and serum antibody response were not different
between PB-exposed and control mice.
DISCUSSION
The results of this study indicate that both general toxicity
and the ability of mice to mount an immune response to the
T cell-dependent antigen SRBCs are subject not only to the
146
SMIALOWICZ ET AL.
dose of TCDD or PCB153 given separately, but also to exposed to PCB153; however, this increased activity was
the dose of TCDD and PCB153 given concomitantly. For determined not to be mediated via induction of CYP1A1,
example, while body weight was not affected by either chem- since the EROD activity was not associated with an inical given alone or in combination, spleen and thymus crease in CYP1A1 protein (De Jongh et al., 1995). Also,
weights were decreased at the intermediate and high TCDD PCB 153 does not induce cleft palate in mice (Birnbaum
doses. These decreases were ameliorated by cotreatment et al., 1985; Biegel et al., 1989), although it did ameliowith the intermediate but not high TCDD dose in combina- rate TCDD-induce cleft palate (Biegel et al., 1989); howtion with all of the PCB153 doses. In contrast, all doses of ever, PCB 153 induces hydronephrosis (Biegel et al.,
TCDD combined with the high dose of PCB153 resulted in 1989) and some doses that block TCDD-induced cleft
a dose-related increase in hepatic weight. This interactive palate actually enhance TCDD-induced hydronephrosis
effect of TCDD and PCB153 on increased liver weight has (Morrissey et al., 1992). While extremely high concentrations of this d\-ortho coplanar PCB competitively disbeen observed by others (DeJongh et al., 1995).
3
As expected (Smialowicz et al., 1994), mice exposed to placed [ H]TCDD from rat hepatic cytosolic Ah receptor
3
TCDD alone at 1 or 10 ^xg/kg had suppressed PFC responses. (Bandiera et al., 1982), it only minimally displaced [ H]In contrast, enhancement of the PFC response was observed TCDD from mouse hepatic cytosol receptor (Biegel et
specific binding of the radioliin mice dosed with 358 mg PCB153/kg alone. Coexposure al., 1989). Furthermore,
l25
gand
4,4'-[
I
]diiodo-2,2',5,5'-tetrachlorobiphenyl
to
2
to the intermediate dose of TCDD and the high dose of
murine
hepatic
Ah
receptor
or
other
cytosolic
proteins
PCB153 confirmed the results of Biegel et al. (1989), who
reported that this combination of PCB153 (i.e., 1000 /xmol did not occur (Biegel et al., 1989). In a companion study
PCB153/kg or 358 mg/kg) and immunosuppressive dose of to the work presented in this report using the same dose
TCDD (i.e., 3.7 nmol TCDD/kg or 1.1 fig/kg) did not sup- levels and dosing regimen, PCB 153 did not alter the
press the PFC response. In contrast to our results, however, TCDD-mediated induction of CYP1A1 protein in liver,
Biegel et al. (1989) did not observe enhancement of the PFC EROD activity in liver or lung, nor CYP1A2 activity or
response in mice dosed with 1000 /imol PCB153/kg alone. protein in liver (van Birgelen et al., 1996). Taken toThe reason for the discrepancy between the two studies for gether these data suggest that PCB 153 is at best an exthis important observation is not apparent, but may be related tremely weak antagonist of Ah receptor-mediated reto differences in sex and strain of mice (i.e., male C57BL/ sponses in mice and it seems unlikely that the antagonism
6J) and route of chemical exposure and immunization (i.e., of the TCDD-induced immunosuppression is due to alterboth by intraperitoneal injection) used by Biegel etal. (1989) ations in Ah receptor activation.
compared with the present study.
A second possible mechanism by which PCB 153 could
antagonize
the actions of TCDD is through dispositional
The high dose of PCB153 significantly enhanced the PFC
antagonism
[i.e., a situation in which the absorption, bioresponse relative to the corn oil control (Figs. IB and 2C),
transformation,
distribution, or excretion of a chemical is
and the data presented in Fig. 2B suggest that the intermedialtered
in
such
a
way that the concentration and/or duration
ate PCB153 dose also possesses immunoenhancing potenof
the
chemical
at
the target organ is diminished (Klaassen
tial, albeit not significant. These as well as the data that
and
Eaton,
1991)].
The interactions of TCDD and PCB 153
show that coadministration of the high PCB153 dose and 1
are
not
limited
to
pharmacodynamic
interactions. Previous
fig TCDD/kg (Figs. 2C and 3A) result in PFC responses
reports
indicate
that
the
antagonistic
interactions between
comparable to those of the vehicle control suggest that
PCB
153
and
TCDD-induced
cleft
palate
may be associated
PCB153 acts as a functional antagonist [i.e., the situation
with
a
40%
decrease
in
the
concentration
of TCDD in the
where "two chemicals counterbalance each other by producfetal
palate
compared
with
animals
treated
with
TCDD alone
ing opposite effects on the same physiological function"
(Biegel
et
al.,
1989).
PCB
153
increases
the
percentage
of
(Klaassen and Eaton, 1991)].
the
dose
of
TCDD
retained
in
the
liver
of
male
C57BI/6J
Evidence from studies of other biological endpoints
support our data which suggest that the amelioration of mice (DeJongh et al., 1995) and female B6C3F1 mice (van
TCDD-induced suppression of the PFC response in mice Birgelen et al., 1996). This pharmacokinetic interaction is
by PCB153 is not or at best minimally related to Ah caused in large part by the increase in liver size in mice
receptor involvement. For example, little or no change cotreated with TCDD and PCB 153 compared with mice
in the level of hepatic cytochrome P450a occurred in rats treated with TCDD alone. The increased sequestration of
exposed to PCB153 (Parkinson et al., 1983). Biegel et TCDD in the liver of mice cotreated with PCB 153 may
al. (1989) reported that exposure of mice to PCB153 result in decreased concentrations in the spleen, producing
did not result in a significant induction of hepatic 7- a dispositional antagonism of the immune suppression. Van
ethoxyresorufin (3-deethylase (EROD) activity. A slight Birgelen et al. (1996), however, demonstrated that neither
increase in hepatic EROD activity was observed in mice blood, splenic, nor thymic concentrations of TCDD were
147
EFFECTS OF PCB153/TCDD ON ANTIBODY RESPONSE
TABLE 4
PCB153/TCDD Dose Ratio, Concentrations of PB153 and TCDD in Liver and Spleen,
and PCB153/TCDD Concentration Ratios in Liver and Spleen"
Spleen
Liver
Concentration
PCB 153
(mg/kg)
3.58
3.58
3.58
35.8
35.8
35.8
358
358'
358
TCDD
(l»g/kg)
PCB 153/
TCDD*
0.1
1.0
10
0.1
1.0
10
0.1
1.0
10
35,800
3,580
358
358,000
35,800
3,580
3,580,000
358,000
35,800
TCDD
(ng/g)
0.4
6.9
72.4
0.4
5.8
77.9
0.4
5.9
69.8
±
±
±
±
±
±
±
±
±
0.04
0.5
2.4
0.04
0.6
7.6
0.02
0.3
7.9
Concentration
PCB153
tWg/g)
1.1
1.1
1.6
8.3
84
12.9
63.3
70.6
97.4
± 0.1
± 0.0
± 0.1
± 1 1
± 0.7
± 0.9
± 2.9
± 4.4
± 12.4
PCB 153/
TCDD'
TCDD
(ng/g)
2,821
160
22
21,015
1,443
166
161,579
12,007
1,395
0.03 ± 0.002
0.1
0.6
0.02
0.13
1.3
0.02
0.08
0.5
±0.01
± 0.02
± 0.002
± 0.01
± 0.3
± 0.002
± 0.008
±0.1
PCB153
(Pg/g)
0.37
0.28
0.34
2.82
3.9
3.36
26.8
32.7
22.9
±
±
±
±
±
±
±
±
±
0.05
0.002
0.04
0.4
0.8
0.3
4.2
3.0
3.0
PCB 153/
TCDD
ratio
13,801
2,386
559
131,776
30,469
2,584
1,654,320
389,286
45,347
"Adapted from data presented in van Birgelen el al. (1996).
* Ratio of administered dose.
e
Ratio of PCB153/TCDD tissue concentration (ng/g tissue) 7 days after dosing.
d
Antagonistic interaction observed for PFC response.
altered by PCB 153 in mice treated with the same dose levels
and dosing regimen used in the present study. Hence these
data suggest that the antagonistic interactions of PCB 153 and
TCDD are not mediated through dispositional antagonism.
It has been hypothesized that there is a ' 'window of antagonism" based on the antagonist/TCDD dose or concentration ratio (Safe, 1990) for some TCDD antagonists. The
ratios of doses used in the present study range from 358 to
3,580,000 (Table 4). Antagonism was consistently apparent
only at a dose ratio of 358,000 and only in the PFC response
to SRBCs and not for enzyme induction in liver or lung (van
Birgelen et al, 1996). The lack of antagonism of enzyme
induction in liver and lung may be due to altered distribution
of TCDD and PCB 153 between tissues. The PCB 153fTCDD
concentration ratio in the spleen, at combinations resulting
in antagonism, is approximately 389,000, which is similar
to the ratio of administered dose (Table 4). The PCB 153/
TCDD ratio is 30 times higher in spleen than it is in liver
and slightly lower than the ratio in the lung for this same
dose combination (Table 4). The data do not demonstrate a
consistent PCB153ATCDD ratio, based on either administered or tissue dose, where a "window of antagonism" will
occur. In addition, the PCB153/TCDD ratio in humans is
approximately 1000-10,000 (Schecter et al, 1994), suggesting that, if a "window of antagonism" exists for the
PCB 153 reversal of the immunosuppressive effects of
TCDD, humans are 35-350 times below those levels of
exposure.
The present study presents evidence that the antagonistic
interaction of PCB 153 is a functional antagonism and neither
a receptor nor dispositional antagonism. The differences in
the mechanism of antagonism may have significant effects
on estimation of risk from low-dose exposure to dioxins and
PCB 153. If antagonism were receptor mediated, then the
ratio ofdioxin-like chemicals to PCB 153 would be important
in that there should be a "window of antagonism" in which
at a specific ratio the dioxin response would be antagonized
while below that ratio the response would not be antagonized. On the other hand, if the antagonism is a functional
antagonism, then it is the concentration of PCB 153 that will
determine the antagonism. The dose levels used in the present study and in previous reports on the antagonistic effects
of PCB 153 on TCDD-induced toxicity (Davis and Safe,
1989, 1990; Biegel et al, 1989) are extremely high. Present
human body burdens of dioxin-like chemicals are 8 - 13 ng
TEQ/kg (DeVito et al, 1995) and the body burden of
PCB 153 is approximately 5000-7000 ng/kg (Schecter etal,
1994). Hence, present body burdens of PCB 153 are approximately 1000-100,000 times lower than the doses used in
the present study. Since the antagonistic interaction between
PCB 153 and TCDD is not receptor mediated, and there does
not appear to be a consistent "window of antagonism," the
antagonistic interactions described in this and other studies
(Biegel et al, 1989; Davis and Safe, 1990; Morrissey etal,
1992) would not appear to be relevant to present background
human exposures.
PCB 153 induces hepatic PROD activity, a marker of
CYP2B1, in both mice and rats (Parkinson et al, 1983;
van der Kolk et al, 1992; van Birgelen et al, 1996) and
as such is considered a prototypical PB-type inducer (Safe,
148
SMIALOWICZ ET AL.
1994). PCB153 produced a 16-fold induction of PROD at
the highest dose (i.e., 358 mg/kg) examined by van Birgelen et al. (1996). To determine if there is an association
between induction of PROD activity and the PFC response, a group of mice were treated with PB 160 mg/kg/
day for 3 days prior to immunization with SRBCs. A
different dosing regimen was used since PB has a much
shorter half-life than PCB153. Despite the difference in
dosing regimen, the administration of PB produced an
induction of PROD activity equivalent to that of mice
treated with 358 mg/kg PCB153; however, there was no
effect on the PFC response in mice, suggesting a lack of
concordance between PROD induction and enhancement
of the PFC response. In addition, these results corroborate
earlier work by Silkworth et al. (1984), who demonstrated
that the PFC response of male B6 mice was not affected
by a PB dosage of 88.7 mg/kg/day for 2 days.
In conclusion, the data suggest that rather than acting
as an Ah receptor antagonist of TCDD-induced immunosuppression, the high PCB153 dose alters the "setpoint"
or "baseline" response level of the PFC response to
SRBCs (i.e., up to 62-93% greater than that of the vehicle
control). Consequently, cotreatment with the high
PCB153 dose and an immunosuppressive dose of TCDD
(i.e., 1 fJ.g/kg) fails to reduce the response relative to that
of the vehicle control, yet TCDD still reduces the PFC
response compared with the PCB153-treated animals.
These observations emphasize the need for the generation
of dose-response data for both TCDD and PCBs or PCB
mixtures alone or in combination, to fully evaluate potential antagonisms.
ACKNOWLEDGMENTS
We thank C. Copeland and D. Ross for technical assistance, and Dr. R.
Luebke and Dr. D. Miller for review of this manuscript.
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