FUNDAMENTAL AND APPLIED TOXICOLOGY 3 5 , 1-8 (1997)
ARTICLE NO. FA96225 1
Inhibition of 3,3',4,4',5-Pentachlorobiphenyl-lnduced Chicken
Embryotoxicity by 2,2',4,4',5,5'-Hexachlorobiphenyl
FENG ZHAO,* KITTANE MAYURA,* NATHAN KOCUREK,* JOHN F. EDWARDS,! LEON F. KUBENA,$
STEPHEN H. SAFE,§ AND TIMOTHY D. PHILLIPS* 1
*Department of Veterinary Anatomy and Public Health, ^Department of Veterinary Physiology and Pharmacology, and jDepartment of Veterinary
Pathobiology, Texas A&M University, College Station, Texas 77843-4458; and tll.S.D.A. Agricultural Research Service, College Station, Texas 77843
Received November 9. 1995: accepted September 10, 1996
Inhibition of 3,3',4,4',5-Pentachlorobiphenyl-Induced Chicken
TEQ = 2 [PCDD], X TEF, + 2 [PCDF], X TEF,
Embryotoxicity by 2,2',4,4',5,5'-Hexachlorobiphenyl. ZHAO, F.,
MAYURA, K., KOCUREK, N., EDWARDS, J. F., KUBENA, L. F.,
SAFE, S. H., AND PHILLIPS, T. D. (1997). Fundam. Appl. Toxicol.
This relationship is based on the mechanism of action of
PCDDs and PCDFs which involves initial binding to the
35, 1-8.
aryl hydrocarbon (Ah) receptor. Thus, the rank order structure-Ah
receptor binding relationships for PCDDs/PCDFs
3,3',4,4',5-Pentachlorobiphenyl (pentaCB) caused a dose-deparallel
their
structure-activity relationships and the potenpendent induction of chicken embryolethality, malformations,
cies
or
TEF
values
for the 2,3,7,8-substituted congeners relaedema, and liver lesions at doses ranging from 0.5 to 12.0 /xg/kg.
In contrast, no embryotoxicity was observed after treatment with tive to 2,3,7,8-TCDD have been determined for diverse Ah
10, 25, or 50 mg/kg 2,2',4,4',5,5'-hexaCB. In eggs cotreated with receptor-mediated biochemical and toxic responses (Safe,
2.0 Mg/kg, 3,3',4,4',5-pentaCB plus 10, 25, or 50 mg/kg 1990). Although a range of TEF values have been deter2,2',4,4',5,5'-hexaCB, there was significant protection from mined for each individual congener, regulatory agencies
3,3',4,4',5-pentaCB-induced embryo malformations, edema, and
have selected single TEFs for the 2,3,7,8-substituted PCDDs
liver lesions, whereas no inhibition of embryolethality was oband PCDFs (Bellin and Barnes, 1989; Ahlborg et al, 1988).
served. These results further extend the response-specific nonaddiSeveral polychlorinated biphenyl (PCB) congeners,
tive interactions of binary mixtures of polychlorinated biphenyls
including
3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, and 3,3',
(PCBs) and should be considered in the development of ap4,4',5,5'-hexaCB
and their monoortho-substituted analogs,
proaches for hazard assessment of PCB mixtures and related compounds. © 1997 Society of Toxicology.
bind to the Ah receptor and elicit Ah receptor-mediated toxic
and biochemical responses (Bandiera et al, 1982; Safe,
1984, 1994; Ahlborg et al, 1994). Since environmental matrices invariably contain complex mixtures of PCDDs,
Polychlorinated dibenzo-/>-dioxins (PCDDs) and dibenzoPCDFs, and PCBs, the TEF approach is being considered
furans (PCDFs) are industrial and combustion by-products
for hazard and risk assessment of TCDD-like PCB congeners
which have been detected as complex mixtures in diverse
(Ahlborg et al, 1994; Safe, 1994).
environmental matrices including fish, wildlife, various
The TEF approach for hazard and risk assessment of
foods, and human tissues (Ballschmiter et al, 1989). The
PCBs, PCDDs, and PCDFs assumes that contributions from
major concerns regarding emissions and exposure to PCDDs
individual congeners are essentially additive. However, the
and PCDFs are due to the relatively high toxicity of 2,3,7,8results obtained for complex and simple (binary) mixtures
tetrachlorodibenzo-/?-dioxin (TCDD) and several structurof PCBs indicate that there are species- and response-specific
ally related 2,3,7,8-substituted congeners (Poland and Knutadditive (Eadon et al, 1986; Van der Kolk et al, 1992; De
son, 1982; Safe, 1990). Hazard and risk assessment of
Jongh et al, 1992, 1993a,b; Silkworth et al, 1993) and
PCDDs and PCDFs utilizes a mechanism-based toxic equivnonadditive (primarily antagonistic) (Bannister et al, 1987;
alency factor (TEF) approach (Bellin and Barnes, 1989; Safe,
Haake et al, 1987; Biegel et al., 1989; Davis and Safe,
1990; Ahlborg et al, 1992) in which the TCDD or toxic
1989, 1990; Morrissey et al, 1992; Harper et al, 1995)
equivalents (TEQ) of a mixture is equal to summation of
interactions. For example, the induction of CYPlAl-depenthe concentration of individual (/) congeners times their cordent hepatic microsomal enzyme activities by commercial
responding TEF,.
Aroclor mixtures in the rat were essentially additive contributions from the individual Ah receptor agonists in the mix1
ture (Harris et al, 1993). In contrast, the immunotoxicity of
To whom correspondence should be addressed. Fax: (409) 862-4929.
0272-0590/97 $25.00
Copyright © 1997 by the Society of Toxicology
All rights of reproduction in any form reserved.
ZHAO ET AL.
these same Aroclor mixtures in the mouse was significantly
lower than expected due to nonadditive (antagonistic) interactions with other PCB congeners in the mixtures (Harper
et ai, 1995). There are numerous reports which demonstrate
that commercial PCB mixtures and 2,2',4,4',5,5'-hexaCB, a
compound which is not an Ah receptor agonist, inhibit both
2,3,7,8-TCDD-induced toxicity (immunotoxicity and fetal
cleft palate) and 3,3',4,4',5-pentaCB-induced immunotoxicity in mice (Haake et ai, 1987; Biegel et ai, 1989; Davis
and Safe, 1989, 1990; Morrissey et ai, 1992; Harper et ai,
1995). Additionally, nonadditive species-specific interactions have also been observed in cell culture for induction
of CYP1A1-dependent activity (Schalk et ai, 1993; Bosveld
et ai, 1995).
At present, in vivo nonadditive (antagonistic) interactions
between Ah receptor agonists and PCBs such as 2,2',
4,4',5,5'-hexaCB have reported only been for immunotoxicity and fetal cleft palate in the mouse. The chick embryo
has previously been shown to be highly sensitive to biochemical and toxic responses (such as enzyme induction, embryolethality, malformations, edema, and liver lesions) induced
by TCDD and related compounds, and the chicken embryo
also expresses the Ah receptor (Denison et ai, 1986;
Goldstein et ai, 1976; McKinney et ai, 1976; Poland and
Glover, 1977; Schrankel et ai, 1982; Brunstrbm and Danerud, 1983; Rifkind et ai, 1984; Brunstrbm, 1986, 1988;
Brunstrom and Andersson, 1988).
Quantitative structure-activity relationships (QSARs) for
embryo mortality by PCBs have been established, and the
rank order of potency is 3,3',4,4',5-pentaCB > 3,3',4,4'tetraCB > 3,3',4,4',5,5'-hexaCB > 2,3,3',4,4'-pentaCB >
2,3,4,4',5-pentaCB, with 2,2',4,5'-tetraCB, 2,2',4,4',5,5'hexaCB, and 2,2',3,3',6,6'-hexaCB being inactive. This order is similar to the typical Ah receptor-mediated response
in the chicken embryo, i.e., cytochrome P4501A1 induction
(Brunstrom and Andersson, 1988; Brunstrbm, 1988). The
rank order for EROD induction by PCBs is 3,3',4,4',5-pentaCB > 3,3',4,4'-tetraCB > 3,3',4,4',5,5'-hexaCB (Brunstrom and Andersson, 1988).
Previous studies have investigated the interactive effects
of a potent Ah receptor agonist, typified by TCDD and
3,3',4,4',5-pentaCB, with mixtures or compounds such as
the commercial Aroclors or 2,2',4,4',5,5'-hexaCB (Haake et
ai, 1987; Biegel et ai, 1989; Davis and Safe, 1989, 1990;
Morrissey et ai, 1992; Harper et ai, 1995). The results
indicate that antagonistic interactions are observed for some
toxic responses including immunotoxicity and teratogenicity
(i.e., fetal cleft palate) in mice. Other studies have reported
that the chicken embryo is highly sensitive to the toxic effects of TCDD, coplanar PCB congeners, and related HAHs
(Poland and Glover, 1977; Rifkind et ai, 1984; Brunstrom
and Anderson, 1988).
3,3',4,4',5-PentaCB is the most potent Ah receptor agonist
among the PCBs and is a major contributor to the TEQ value
for several commercial mixtures and environmental mixtures
of PCBs (Harper et ai, 1995). 2,2',4,4',5,5'-HexaCB exhibits minimal Ah receptor agonist activity and is a major component in commercial PCB mixtures and environmental extracts (Ahlborg et ai, 1992; Safe, 1990). Since the Ah receptor has been detected in 5-day-old chicken embryos, the
CHEST bioassay was utilized in the following studies as a
rapid and simple method to investigate the potential interactions between the two model PCB congeners, i.e.,
3,3',4,4',5-pentaCB and 2,2',4,4',5,5'-hexaCB. These findings were further compared with previous studies in vivo
and in vitro.
MATERIALS AND METHODS
Chemicals. 3,3',4,4',5-PentaCB and 2,2',4,4',5,5'-hexaCB were synthesized as previously described (Mullin et ai, 1984) and were >98% pure
as determined by gas chromatography. All other chemicals and biochemicals
were the highest quality available from commercial sources.
Chicken embryo studies. Fertilized hen's eggs from the same flock
were purchased from Hy-Line International (Bryan, TX). The eggs were
left at room temperature for a period of 24 hr and then incubated at 37.5°C
and 80% relative humidity at the USDA/ARS Poultry Toxicology Laboratory (College Station, TX). Eggs were turned every 6 hr, and after 4 days
of incubation all eggs were candled. Those that were infertile or contained
dead embryos were discarded. The remaining eggs were weighed and
cleaned at the blunt end with 70% ethanol, and a small hole was made in
the shell outside the air sac using a dental drill. 3,3',4,4',5-PentaCB or
2,2',4,4',5,5'-hexaCB was dissolved in corn oil, and corn oil, pentaCB (at
doses of 0.5, 1.0, 2.0, 3.0, 4.0, 8.0, or 12.0 Mg/kg), 2,2',4,4',5,5'-hexaCB
(at doses of 10, 25 or 50 mg/kg), or 3,3',4,4',5-pentaCB (2.0 ^g/kg) plus
2,2',4,4',5,5'-hexaCB (10, 25 or 50 mg/kg) was injected into the egg yolk.
The volume of corn oil used was 1 ml/kg egg weight. The hole was sealed
with paraffin wax after each injection. Some eggs were left untreated to
serve as controls. The eggs were candled every other day postinjection, and
dead embryos were recorded and discarded. The experiment was terminated
on Day 18. Embryonic lethality was determined, and surviving embryos
were evaluated for malformations (including head, eye, beak, body wall,
and limb), pericardia! and subcutaneous edema, and gross liver lesions.
Representative samples of liver were fixed in 10% buffered formalin and
embedded in paraffin. Sections (5-jim-thick) were stained with hematoxylin
and eosin for routine histologic examination.
Statistics. All data were subjected to analysis of variance using the
General Linear Models Procedure of the Statistical Analysis System (SAS
Institute). The significance of the differences among treatment groups with
variable means was determined by Waller-Duncan k ratio / test. All statements of significance were at a probability level of p =s 0.05.
RESULTS
Chicken embryos were treated as described with corn oil,
different doses of 3,3',4,4',5-pentaCB (0.5 to 12 /xg/kg),
2,2',4,4',5,5'-hexaCB (10 to 50 mg/kg), and binary mixtures
of 3,3',4,4',5-pentaCB (2.0 Mg/kg) plus 2,2',4,4',5,5'-hexaCB (10, 25, or 50 mg/kg). Exposure of chicken embryos
to 3,3',4,4',5-pentaCB at doses from 0.5 to 12 fig/kg resulted
in a dose-dependent increase in embryolethality, malformations, edema, and liver lesions. At the high doses (8.0 and 12
INHIBITION OF 3.3'.4.4',5-PENTACB-lNDUCED EMBRYOTOX1CITY BY 2.2'.4.4',5,5'-HEXACB
/Ltg/kg), embryolethality varied from 80 to 100%; significant
toxic responses were observed at doses of 1.0 and 2.0 fig/
kg (Table 1). Some of the malformations in the 3,3',4,4',5pentaCB-treated chicken embryos included eye defects (anophthalmia and microphthalmia), beak deformities, exencephaly, microcephaly, absence of wings, and club foot.
3,3',4,4',5-PentaCB (2.0 Mg/kg) caused 42% embryolethality, 28% embryonic deformations, 69% edema, and 70%
liver lesions. The results in Fig. 1 illustrate the histopathology of the 3,3',4,4',5-pentaCB-induced liver lesions observed at the 2.0 Mg/kg dose. Histological examination of
livers revealed subcapsular zones of coagulative necrosis
often with mineralization, and occasionally associated with
vacuolization of hepatocytes. Mild to minimal bile duct proliferation with heterophilic infiltration also was observed in
the livers.
In contrast, treatment with 10 to 50 mg/kg 2,2',4,4',5,5'hexaCB did not significantly induce embryolethality, malformations, edema, or liver lesions in the chicken embryos
(Table 1). The chicken embryos were also cotreated with a
toxic dose of 3,3',4,4',5-pentaCB (2.0 Mg/kg) and different
doses of 2,2',4,4',5,5'-hexaCB (10, 25, or 50 mg/kg). The
results (Table 2 and Fig. 2) demonstrate that all doses of
2,2',4,4',5,5'-hexaCB protected the embryos from 3,3',
4,4',5-pentaCB-induced malformations, edema, and liver lesions, but no inhibition of 3,3',4,4',5-pentaCB-induced mortality was observed.
DISCUSSION
Several studies have reported that the chicken embryo is
highly sensitive to the toxic effects of TCDD, coplanar PCB
congeners, and related HAHs (Poland and Glover, 1977;
Rifkind et al, 1984; Brunstrom and Andersson, 1988). The
Ah receptor can be detected in 5-day-old embryos, and although receptor levels are variable in the embryo and neonate (Denison et al, 1986), induction of CYP1A1-dependent
activity is observed throughout embryonic development and
in the neonate. The embryotoxicity of coplanar PCBs has
been characterized extensively (Brunstrom and Darnerud,
1983; Brunstrom and Lund, 1988; Brunstrom and Andersson, 1988; Brunstrom, 1988; Denison et al, 1986; Nikolaidis
et al, 1988a,b, 1990; Rifkind et al., 1984, 1985) and the
results are consistent with those summarized in Table 1 and
Fig. 1. ED50 values for most of the responses (including
lethality) were observed in the dose range of 1.0-3.0 A*g/kg,
and this corresponds to the LD50 of 3.1 //g/kg for chicken
embryolethality previously reported by Brunstrom and coworkers (1990). Brunstrom and Andersson (1988) reported
that at doses up to 50 mg/kg, 2,2',4,4',5,5'-hexaCB did not
cause a significant increase in embryonic mortality or abnormalities, and these observations were consistent with the
results obtained in this study for this congener. Rifkind and
co-workers (1984) reported some hepatic effects of
2.2',4.4',5.5'-hexaCB; however, these responses were observed only at a high dose (i.e., 50.000 nmol/egg or 18.1
mg/egg) which was approximately 6 to 30 times higher than
the doses used in the present study.
Previous studies in this laboratory and others have investigated the interactive effects of potent Ah receptor agonists,
typified by TCDD and 3,3',4,4',5-pentaCB, with compounds
or mixtures such as the commercial Aroclors or 2,2',
4,4',5,5'-hexaCB (Haake et al, 1987; Biegel et al, 1989;
Davis and Safe, 1989, 1990;Morrissey et al, 1992; Harper et
al, 1995). The results indicate that nonadditive antagonistic
interactions are observed for some toxic responses including
immunotoxicity in adult mice and fetal cleft palate in mice.
The present study has shown that interaction of 2,2'.
4,4',5,5'-hexaCB (10 to 50 mg/kg) with a toxic dose of
3,3',4,4',5-pentaCB (2.0 /xg/kg) resulted in a significant decrease in malformations, edema, and liver lesions in the
chicken embryo. In contrast, no protection was observed
from 3,3',4,4',5-pentaCB-induced embryolethality in eggs
cotreated with both PCB congeners. Mortality occurred in
different frequencies during the incubation days between 6
and 18 in embryos treated with 3,3',4,4',5-pentaCB alone
or in combination with 2,2',4,4',5,5'-hexaCB. The highest
percentage of embryolethality occurred before Day 12. More
research is warranted to ascertain the mechanism of PCBinduced mortality. However, previous research has demonstrated that many of the toxic responses elicited by the PCBs
are mediated through the Ah receptor. Thus, it is possible
that the malformations, edema, and liver lesions in chick
embryos exposed to 3,3',4,4',5-pentaCB are Ah receptormediated.
The mechanism leading to hepatic necrosis is unknown.
The pattern suggests that there should be a vascular lesion.
Vessel compromise would cause focally extensive areas of
coagulative necrosis similar to those observed. Unfortunately, neither thrombi nor vessel degeneration were seen in
samples. Dystrophic mineralization of areas of acute necrosis
is not surprising in embryos; however, the mineral deposition
is nonspecific and does not indicate any particular underlying
mechanism related to the initial cause of the necrosis. Necrosis was not seen in untreated controls and in embryos treated
with hexaCB.
Our results have demonstrated the response-specific antagonistic effects of 2,2',4,4',5,5'-hexaCB in the chicken
embryo treated with a toxic dose of 3,3',4,4',5-pentaCB and
extended the number of examples of nonadditive interactions
between Ah receptor agonists and other HAHs. The nonadditive (antagonistic) interactions were also observed in chick
embryo hepatocytes cotreated with TCDD and 2,2',4,4',5,5'hexaCB in which the EC50 value for induction of CYP1 Aldependent activity was significantly decreased in the cotreated cells (Bosveld et al, 1995). In their study, the dose
ZHAO ET AL.
FIG. 1. Histopathology of chicken embryo livers. Livers from 18-day-old chicken embryos treated with 3,3',4,4',5-pentaCB alone (2.0 ^g/kg)
exhibited large subcapsular zones of necrosis and mineralization (top). In contrast, necrosis was not observed in livers from 18-day-old chicken embryos
cotreated with 3,3',4,4',5-pentaCB (2.0 fig/kg) plus 2,2',4,4',5,5'-hexaCB (25 mg/kg) (bottom) and the results were similar to those observed in untreated
embryos (hematoxylin and eosin stain, original magnification X100).
INHIBITION OF 3,3',4,4',5-PENTACB-INDUCED EMBRYOTOXICITY BY 2,2',4,4',5,5'-HEXACB
TABLE 1
Toxicity of 3,3',4,4',5-PentaCB, 2,2',4,4',5,5'-HexaCB, and Their Combination in the Chicken Embryo"
Treatment
Control
Corn oil
PentaCB (0.5 /ug/kg)
PentaCB (1.0/ig/kg)
PentaCB (2.0 j*g/kg)
PentaCB (3.0 /ig/kg)
PentaCB (4.0 Mg/kg)
PentaCB (8.0 /xg/kg)
PentaCB (12.0 /xg/kg)
HexaCB (10 mg/kg)
HexaCB (25 mg/kg)
HexaCB (50 mg/kg)
PentaCB (2.0 Mg/kg)
+ hexaCB (10 mg/kg)
PentaCB (2.0 /xg/kg)
+ hexaCB (25 mg/kg)
PentaCB (2.0 /xg/kg)
+ hexaCB (50 mg/kg)
No. of
eggs
143
104
19
20
55
19
25
10
18
76
71
71
Malformation
(%)
Mortality (%)
0.1
12.9
5.0
33.3
41.5
55.1
63.1
80.0
100.0
20.1
14.6
14.3
±
±
±
±
±
±
±
±
±
±
±
±
0.3
12.8
5.0
8.3*
23.4*
17.6*
9.6*
0.0*
0.0*
8.6
10.6
6.6
Liver lesions
(%)
Edema (%)
0.1
2.8
0.0
16.7
28.3
56.7
61.1
100.0
± 0.3
± 4.3
± 0.0
± 16.7*
± 32.8*
± 23 3*
± 28.3*
± 0.0*
—
9.8 ± 1.8
1.2 ± 0.2
3.5 ± 2.6
0.0
0.0
0.0
0.0
68.9
83.3
100.0
100.0
0.0
0.0
11.1
0.0
69.7
73.3
100.0
100.0
± 0.0
± 0.0
± 0.0
± 0.0
±31.8*
± 16.7*
± 0.0*
± 0.0*
—
—
0.0 ±
0.0 ±
0.0 ±
± 0.0
± 0.0
± 11.1*
± 0.0
±35.1*
± 6.7*
± 0.0*
± 0.0*
0.0 ± 0.0
1.6 ± 2.7
1.6 ± 2.2
0.0
0.0
0.0
43
39.2 ± 17.0*
2.4 ±
2.4t
11.9 ±
28
32.6 ±
0.8*
o.o ± o.ot
0.0 ±
0.0t
3.3 ±
3.3t
27
33.0 ±
0.0*
o.o ± o.ot
0.0 ±
0.0t
6.0 ±
0.0t
2.4*-t
26.2 ±
2.4*-t
" Chicken eggs were treated with 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB and their combination and on Day 18, embryos were examined as described
under Materials and Methods. The data are expressed as means ± SE for at least two different experiments for the treatment groups [except for the
groups treated with 8.0 and 12.0 /xg/kg of 3,3',4,4',5-pentaCB and 3,3'4,4',5-pentaCB (2.0 /xg/kg) plus 2,2',4,4',5,5'-hexaCB (50 mg/kg)].
* Values are significantly different from the solvent control (p =s 0.05). t Values are significantly different from 3,3',4,4',5-pentaCB (2.0 ^tg/kg) (p
=s 0.05).
TABLE 2
Incidence of Malformations in Chicken Embryos Treated with 3,3',4,4',5-PentaCB, 2,2',4,4',5,5'-HexaCB, and Their Combination
Incidence of malformations (%)°
Treatment
Control
Corn oil
PentaCB (0.5 /ig/kg)
PentaCB (1.0 /ig/kg)
PentaCB (2.0 /ig/kg)
PentaCB (3.0 //g/kg)
PentaCB (4.0 /tg/kg)
PentaCB (8.0 /ig/kg)
PentaCB (12.0/xg/kg)
HexaCB (10 mg/kg)
HexaCB (25 mg/kg)
HexaCB (50 mg/kg)
PentaCB (2.0 /ig/kg)
+ hexaCB (10 mg/kg)
PentaCB (2.0 jtg/kg)
+ hexaCB (25 mg/kg)
PentaCB (2.0 /ig/kg)
+ hexaCB (50 mg/kg)
No. of
eggs
No. of live
embryos
143
104
19
20
55
19
25
10
18
76
71
71
142
92
18
13
32
8
9
2
0
61
58
Head
Eye
Beak
Body
wall
Limbs
1
0
8
13
50
44
50
0.7
2
0
15
13
50
33
100
0.7
2
0
0
13
13
22
100
0.7
1
0
0
0
0
0
0
0
0
0
0
0
0
33
100
0
0
0
0
0
0
0
0
4
0
2
60
43
0
0
28
0
28
3
0
0
0
0
19
0
0
0
° Types of malformation included exencephaly, microcephaly, anophthalmia, microphthalmia, beak deformity, coelosmia, absence of wings, and club
foot. Percentages of malformation were calculated based on the number of surviving embryos.
0
27
18
0
ZHAO ET AL
• C o r n oil
(ffllHexaCB 50 mg/kg
EaPentaCB 2.0 ng/kg
^PentaCB 2.0 ug/kg + HexaCB 50 mg/kg
80
70
60
a
1
1
50
40
30
20
10
0
r—fTFPi
Malformations
Edema
Liver lesions
FIG. 2. Antagonist effects of 2,2',4,4',5,5'-hexaCB on 3,3',4.4',5-pentaCB-induced malformations, edema, and liver lesions in chicken embryos.
The percentages of malformations, edema, and liver lesions were calculated
on the total number of live embryos.
ratio for TCDD to 2,2',4,4',5,5'-hexaCB was 1:10,000. In
this study, 3,3',4,4',5-pentaCB was used instead of TCDD
and the dose ratio for 3,3',4,4',5-pentaCB to 2,2',4,4',5,5'hexaCB was within the same range (i.e., 5000-25,000). A
recent paper also reported that 2,2',5,5'-tetraCB and
2,2',3,3',4,4'-hexaCB inhibited TCDD or 3,3',4,4'-tetraCBinduced CYP1A1-dependent responses in mouse hepatoma
Hepa Ic.lc7 cells (Aart et al., 1995).
Possible nonadditive (antagonistic) interactions between
coplanar PCBs and other PCB fractions have also been reported in a study of the embryotoxicity of Lake Ontario fish
extracts in Japanese Medaka (Harris etal., 1994). In contrast,
Van Birgelen and co-workers (1996) reported a dramatic
synergistic interaction between 2,2',4,4',5,5'-hexaCB and
TCDD in the formation of hepatic porphyrins in the rat.
These data suggest that antagonistic or synergistic interactions between halogenated aromatic compounds are species-,
cell type-, and response-specific. It has been reported previously that interactions between 2,2',4,4',5,5'-hexaCB and
TCDD can result in modified pharmacokinetics of the latter
compound; this may contribute to some of the observed in
vivo nonadditive interactions (De Jongh et al., 1992,
1993a,b, 1995). However, pharmacokinetic considerations
would not explain nonadditive interactions observed in cell
culture (Aarts et al., 1995; Bosveld et al., 1995). The response-specific inhibition of 3,3',4,4',5-pentaCB-induced
toxicity by 2,2',4,4',5,5'-hexaCB in the chicken embryo suggests that the inhibitory mechanisms may be complex and
cannot be explained by simple competition for binding to the
Ah receptor. The end-point and species-specific nonadditive
interactions of Ah receptor agonists and PCBs such as
2,2',4,4',5,5'-hexaCB represent a problem or limitation for
utilizing the TEF approach for hazard assessment of HAHs
which assumes additivity for individual compounds in mixtures.
The interactive effects of PCBs are complex and the mechanism^) of the nonadditive (antagonistic) interactions are
unknown. Previous studies demonstrate that unlike Aroclor
1254, a complete displacement of [3H]TCDD from the Ah
receptor was not observed in 2,2',4,4',5,5'-hexaCB competitive binding assays; moreover, [l25I2]2,2',5,5'-tetrachloro4,4'-diiodobiphenyl did not bind directly to the Ah receptor
(Davis and Safe, 1990). These results suggest that the antagonism observed for 2,2',4,4',5,5'-hexaCB may occur by
mechanisms other than through the Ah receptor (Davis and
Safe, 1990). A recent study by De Jongh et al. (1995) has
shown that the concentration of TCDD in the liver (% dose/
g tissue) was increased in the animals cotreated with TCDD
and 2,2',4,4',5,5'-hexaCB compared to the animals treated
with TCDD alone. The increase in the hepatic retention and
subsequent decrease in available TCDD reaching the palate
may contribute to the antagonistic action of 2,2',4,4',5,5'hexaCB. This was supported by a study (Biegel et al., 1989)
demonstrating that cotreatment of animals with TCDD and
2,2',4,4',5,5'-hexaCB significantly decreased the level of the
TCDD reaching the palate. Earlier studies have reported the
antagonistic interactions between Ah receptor agonists and
PCBs (such as 2,2',4,4',5,5'-hexaCB) for immunotoxicity
and fetal cleft palate in mice. Although the toxic potencies
of individual PCBs have been characterized earlier in the
chicken embryo, this is the first report of interactive effects.
More research on simple and complex mixtures of HAHs
should be carried out to further define toxic responses which
are nonadditive (i.e., antagonistic or synergistic) and to determine the mechanisms of these interactions.
ACKNOWLEDGMENTS
The National Institutes of Health (P42-ES04917) and the Texas Agricultural Experiment Station (H62I5) are gratefully acknowledged. S.S. is a
Sid Kyle Professor of Toxicology.
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