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2J mice
Camp. B&hem. Physiol. Vol. 113C, No. 3, pp. 393-398, Copyright 0 1996 Elsevier Science Inc.
ISSN SO742-8413/96/$15.00 PII SO742-8413(96)00011-2
1996
ELSEVIER
Comparative Teratological Studies on TCDD, Endrin and Lindane in C57BL/6J and DBA/ZJ Mice E. A. Hussoun and S. _7.Stoh SCHOOLOF PHARMACYAND ALLIEDHEALTH PROFESSIONS, CREIGHTONUNIVERSITY,OMAHA, NE 68178 USA
ABSTRACT.
The teratogenic
by TCDD
effects
of endrin
of C57BL/6]
and
lindane
have
been
determined
and
compared
to those
and DBA/ZJ mice after the administration
of single oral doses to pregnant mice on day 12 of gestation. TCDD produced dose-dependent decreases in fetal weight, fetal thymic weight and placental weight, and dose-dependent increases in fetolethality, cleft palate formation and hydronephrosis at doses of lo-30 and 30-60 pgg/kg body weight in C57BL/6J and DBA/ZJ mice, respectively. No maternal death was observed at the given doses in both strains of mice. Endrin (4.5 and 6 mg/kg body weight) and lindane (30 and 45 mg/kg body weight) produced significant decreases in fetal weight and placental weight in C57BL/6] and DBA/ZJ mice, and dose-dependent decreases in fetal thymic weight in C57BL/6J mice but not DBA/ZJ mice. Endrin and lindane caused O-25 and 14-25% maternal deaths, respectively, at the above mentioned doses. Neither cleft plate nor hydronephrosis were induced by endrin or lindane in the two strains of mice. The results support the hypothesis that TCDD-induced cleft plate and hydronephrosis involve mechanisms that are Ah (aryl hydrocarbon) receptor mediated. However, other fetotoxic effects induced by TCDD, and the fetotoxic effects induced by endrin and lindane may involve additional unknown mechanisms that are not related to the Ah-receptor. COMP BIOCHEMPHYSIOL113C, 393-398, 1996. induced
in the fetuses
KEY WORDS. TCDD, endrin, lindane, teratology, C57BL/6J mice, DBA/ZJ mice, fetotoxicity
Endrin is a highly toxic chlorinated
INTRODUCTION (PCH)
The polyhalogenated
cyclic hydrocarbons
variety
such as endrin and lindane, and con-
of pesticides,
taminants p-dioxin
include
a
of pesticides such as 2,3,7,&tetrachlorodibenzo(TCDD).
Although
lindane,
endrin and TCDD
are structurally dissimilar, they share the properties of being highly stable, halogenated, TCDD
cyclic and lipophilic
(7,8,13).
is the most toxic compound in this group, inducing a
variety of toxic responses in laboratory animals (33,34,35). Endrin and lindane are known to induce toxic responses in laboratory animals that are similar in some aspects to those induced by TCDD
which include hepatotoxicity
17), induction of drug metabolizing enzymes (5,37), hypoplasia (3), weight loss and eventually Lindane has been a common component agricultural fertilizers, household insecticide parasitic (38).
medications
Although
and animal
the production
(2,3,16, thymic
death (22). in a variety of sprays, human
parasiticidal
solutions
of lindane was banned in
the United States in 1977, it is still present in commercial products and found in significant quantities in the environment, and is thus a potential hazard to human health (38).
Address reprint requests to: S. 1. Stohs, School of Pharmacy and Allied Health Professions, Creighton University, 2500 California Plaza, Omaha, NE 68178 USA. Tel: 402-280-2950. Received 1 June 1995; revised 19 December 1995; accepted 19 Decemher 1995.
which has been used for domestic,
hydrocarbon
pesticide
agricultural and public
health purposes. The mechanisms of toxicity of these compounds are not known, although various possible mechanisms have been proposed (2,3,5,16,18,27,36,39). In addition to their toxic effects, these PCH are teratogenie and fetolethal 24,32,34,37).
TCDD
in laboratory
animals
(4,9,14,15,23,
is the most extensively
studied of
these compounds and is considered the most potent experimental teratogen duced by TCDD dronephrosis,
(28,34).
The most prominent
effects in-
in the fetuses of mice are cleft palate, hy-
thymic hypoplasia, reduced fetal weight and
fetal death (9,11,21,28,34). Lindane when given to mice during early pregnancy
at
a dose which is 50% of the LDSO caused total absence of any implantation
sites (37). When the same dose was given
at mid-pregnancy, lindane caused total resorption of fetuses. At late pregnancy, lindane resulted in low birth weight pups which subsequently died 12-15 Neither
days after parturition
this study nor others examined
(37).
the effects of lin-
dane when given to pregnant mice at doses below those causing 100% fetolethality with observation of fetal malformation. Studies with endrin have shown marked species differences in teratogenic sensitivity, with hamsters being the most affected species (15,23,32). produced conflicting
Studies in CD-l
mice have
results, with one study failing to show
394
E. A. Hassoun and S. J. Stohs
any teratogenic
or fetolethal
given
at doses that
other
study,
however,
when
given
to pregnant
effects
caused
even when
maternal
cndrin
lethality
was shown
mice
endrin (24).
was
In an-
to be teratogenic
at a single
oral dose which
was 50% of the LDsa and given on day 9 of gestation. At this dose, endrin caused a high incidence of congenital anomalies
without
or growth
impairment
fects of endrin
a concurrent
been
(14). Therefore of teratogenicity
are not known, suggested
21,34). appear
to TCDD
and have
(9,10,11,
in teratogenic which locus
receptor
of the genetic which
and its congeners This
has
AH
been
as well as a number
receptor
for mechanisms
has
locus
shown
is the
to bind
of polycyclic
greatly
stimulated
and
teratogenicity
of toxicity
received
TCDD
ceived
of
were checked
at 30, 45 or 60 pg/kg.
the vehicle
taining
used to dissolve
10% acetone).
Other
mice received
or lindane vehicle
at doses
which
All chemicals at 7500 @/kg TCDD
volume
body weight.
formations when The
the maximal
to pregnant
animals
gestation
C57BL/6J
carbon
and sacrificed
dioxide
resorbed
or live fetuses.
Live fetuses
and live fetuses
were checked
is considered
external
malformations
to the stimu-
collected
to the AH receptor
to be the first step in a series of events lation
of the polysubstrate
other
enzyme
systems
not related
of TCDD (1,4,27). Since endrin and lindane fects with TCDD, are similar
fore, the fetotoxic investigated
induced
C57BL/6J
fetotoxic
toxic efeffects
that
if they are adminis-
to mice as TCDD. and lindane
with
and also
conversion
some common
by TCDD
effects of endrin
and compared
in AH responsive DBA/ZJ
share
the same conditions
system
to metabolic
they may produce
to those
tered under
leading
monooxygenasc
those
There-
were further
induced
by TCDD
mice and in AH nonresponsive
then
number
and cleft
in Bouin’s
the presence
of hydroncphrotic
L1ata for each of variance
(TCDD) was Resources Program,
tional
MD).
Institute
drin were purchased All chemicals
(Bethesda, from Supelco,
were of the highest
Lindane
Inc. (Bellefonte,
ohNa-
and enPA).
grade that is commercially
available.
Animals Male
Thymuses The
of were
fetuses
were
for later examination
for
were subjected
Scheffe’s
S method
post hoc test. The data are expressed dard error of the mean (SEM). The statistical
unit for comparisons, as an average
as the mean + standam was used as the
and the fetotoxic
of each effect among
different
to analysis
was used as the
effects are
the fetuses of
superscripts
are consid-
(P < 0.05).
RESULTS As can be seen in Table decreases weight cleft
1, TCDD
in fetal weight,
as well as dose-dependent palate
formation
produced
fetal thymic and
dose-dependent
weight
increases
hydronephrosis
and placental in fetolethality, in C57BL/6J.
The same effects were also produced by TCDD in the DBA/ 2J mice (Table 2). The ED, values for TCDD for the production of cleft palate and hydronephrosis in C57BL/6J mice were 25 and 18 pg/kg, respectively, while these values
and Treatment
and female
mice
of C57BL/6J
and
(Jackson Laboratories, Bar Harbor, ME), weeks old, were maintained at 25°C with cycle, and were given a commercial pellet dent Lab Chow #SOOl) and tap water ad Males
were
for the presence
METHODS
2,3,7,8-Tetrachlorodibenzo-p-dioxin tained from the Chemical Research Cancer
and placentae
palates.
that dam. Values with non-identical AND
uteri dead,
kidneys.
group of animals
(ANOVA).
ered significantly MATERIALS Chemicals
the
of implantations,
and weighed.
solution
and
Data Analysis
calculated
mice.
for the
from the fetuses
placed
doses on
on day 18 of
anesthesia,
weighed
( 12). Binding
and resorption
at teratogenic
embryos
sive embryos
investigated
studies,
rate of mal-
(10,19,21).
were weighed
using
on
was kept
Based on our previous
were
strains of mice but not in nonrespon-
intubation
of the vehicles
produce
oil
and lindane.
with the lowest rate of fetal death
given
and
or the corn
endrin
were given orally by gastric
and its congeners
re-
oil con-
at doses of 4.5 and 6 mg/kg
of 30 and 45 mg/kg,
The
animals
(corn
groups of both C57BL/6J
endrin
TCDD and related compounds (6,.30,35). AH receptors are found in several embryonic tissues of midgestational murine of responsive
for vaginal
Control
TCDD
was used to dissolve
day 12 of gestation
the
mice
lowing treatment groups. Groups of C57BL/6J mice were given either TCLX) at 10, 20, or 30 pg/kg. DBA/ZJ mice
day 12 of gestation.
occurred,
A product
hydrocarbons.
of TCDD
and its bioisosteres
( 11,19,20,34). AH
endrin
mechanisms
differences
day 0) the female
plugs. Before treatment, pregnancy was confirmed by abdominal palpation. The animals were assigned into the fol-
DBA/ZJ
with the AH (aryl hydrocarbon)
cytosolic
search
several
strain
cf-
clarification.
of TCLX),
although
marked
to segregate
TCDD
the teratogenic
for the teratogenicity
In mice,
sensitivity
in fetal mortality
in mice may need further
The mechanisms lindane
increase
day (=
and females
were mated
DBA/2J
strains
approximately 8 a 12 h light/dark diet (Purina Rohum.
overnight
and the next
in DBA/ZJ TCDD
mice were
55 and 50 pg/kg,
had to be given to DBA/ZJ
respectively.
Thus,
mice at doses that were
2-3 times higher than those given to the C57BL/6J mice (Table 1). No maternal deaths were induced by these doses in either of the two strains used for this study. A significant reduction in maternal weight was induced by TCDD at a
Comparative
Teratological
Studies on TCDD,
TABLE 1. Administration
Endrin and Lindane
395
of 2,3,7,8-tetrachlorodibenzoqwiioxin
(TCDD)
to pregnant C57BL16J mice on day 12 of gestation
% of Mean of
number/dam f S.E.M. (total number)
fetuses/dam dead or resorbed f S.E.M. (No. of late/early)
6.7 + 0.6” (40) 6.8 + 0.5” (41) 6.2 + 0.5” (37) 8.2 ? 0.2,’ (41)
7.9 2 4.6* (O/2) 8.6 + 3.5” (O/2) 8.1 2 3.3” (l/2) 14.4 2 1.71 (4/2)
implantation Treatment
Number of dams
ChIltd
6
TCDD (10 e/kg) TCDD (20 /e&g) TCDD (30 &/kg)
6 6 5
Values with nonidentical
Mean of weight (&/dam f S.E.M.
Mean of fetal thymic weight (mg)/dam f S.E.M.
% of fetuses/dam having cleft palate f S.E.M.
% of fetus/ dam having hydronephrosis f S.E.M.
0.98 k 0.023
0.14 + 0.01”
1.21 2 0.05”
0”
0’
30.1 2 0.5,’
0.95 + 0.03’
0.14 + 0.01”
1.08 + 0.04h
6.3 I’ 3.8h
13.2 + 2.7h
29.5 + 0.4”
0.79 + O.Olh
0.12 + O.Olh
1.02 k 0.02,
17.1 -t 4.2‘
43.3 2 6.4’
28.8 2 0.5’
0.59 + 0.01’
0.11 i- 0.01’
0.09 + 0.01”
83.6 :? 6.2”
88.3 5 2.7’
Mean of maternal weight(g) f S.E.M.
Mean of fetal weight (gwm f S.E.M.
30.3 + 0.4’
superscripts within a column are significantly
different (P < 0.05)
dose of 30 ,ug/kg body weight given to the C57BL/6J mice (Table 1) and at doses of 45 ,ug/kg body weight or higher given to the DBA/2J mice (Table 2). Table 3 shows that endrin, when given at doses of 4.5 and 6 mg/kg body weight, produced significant decreases in fetal weight, fetal thymic weight and placental weight in C57BL/6J mice. When endrin was given to the DBA/ZJ mice (Table 4), it caused significant reduction in fetal weight at doses of 4.5 and 6 mg/kg body weight, and placental weight at a dose of 6 mg/kg body weight. At this latter dose, endrin caused 25% maternal death in both C57BL/ 6J and DBA/2J mice. No cleft palate or significant hydronephrosis were observed following endrin administration to either strain of mice. The effects of lindane on pregnancy outcome in C57BL/ 6J and DBA/2J mice are shown in Tables 5 and 6, respectively. As with endrin, lindane when given at doses of 30 and 45 mg/kg body weight produced significant decreases in fetal weight, fetal thymic weight and placental weight in C57BL/6] mice (Table 5). When given to the DBA/2J mice, lindane caused significant reduction in fetal and placental weight at a dose of 45 mg/kg body weight (Table 6). Lindane caused 14 and 25% maternal deaths when given at doses of 30 and 45 mg/kg, respectively, in both strains of mice. However, as with endrin, lindane produced no significant reduction in maternal weight at either of the two
TABLE 2. Administration
placetWdl
doses given to the two strains of mice (Tables 5 and 6). Neither cleft palate nor hydronephrosis were induced by lindane in the fetuses of C57BL/6J and DBA/ZJ mice (Tables 5 and 6) as compared with TCDD (Tables 1 and 2). Doses of lindane lower than 30 mg/kg body weight produced no maternal toxicity or death, and no significant teratogenic effects were observed in the fetuses of C57BL/6] and DBA/ZJ mice (results not shown). DISCUSSION The results clearly demonstrate that TCDD, endrin and lindane are fetotoxic. TCDD induced malformations, fetal and placental weight retardation and fetolethality in TCDD (Ah) responsive C57BL/6J and nonresponsive DBA/ZJ mice (Tables 1 and 2). These results are in agreement with previous studies (20,31). However, TCDD had to be given at doses that are approximately 2-3 times higher to DBA/ 2J mice as compared to the doses given to the C57BL/6J mice (Table 2). Ah receptors are present in non-responsive mouse strains as DBA/2J. However, the affinity with which TCDD binds to receptors in DBA/ZJ mice is about ten-fold weaker than in C57BL/6J mice (28). These receptors may be occupied and activated in the tissues of the DBA/2J mice only upon exposure to high doses of TCDD. TCDD induced approximately 70-80% malformations
of 2,3,7&btetrachlorodibenzo-p-dioxin
(TCDD)
to pregnant DBA12J mice on day 12 of gestation
96of
Treatment
Number of dams
Control
5
TCDD (30 pgkg) TCDD (45 pgkg) TCDD (60 pgs/W
6 6 5
Values with nonidentical
Mean of implantation number/dam f S~~~L($tal
7.4 i 0.2” (37) 8.5 k 0.2h (51) 6.3 2 0.2’ (38) 6.5 + 0.8’ (36)
fetuses/dam dead or resorbed * S.E.M. (No. of late/early) 7.9 + 2.7* (O/3) 9.7 + 1.8” (312) 21.2 ?z 4.1h (O/10) 50.0 I 6.7‘ (O/W
Mean of Mean of maternal weight (g) f S.E.M.
Mean of fetal weight (g)/dam f S.E.M.
30.3 k 0.7”
0.90 t 0.01*
30.4 2 0.6’
0.88 -t O.Olh
(mgs)zo . . . =
% of fetoseskiam having cleft palate 2 S.E.M.
% of fetus/ dam having hydronephrosis 2 S.E.M.
0.15 ? O.Ol^
1.10 2 0.05,’
0”
2.9 k 2.3,’
0.15 2 0.01”
1.10 2 0.05’
0”
1.9 2 1.7”
plX&d
weight (g)ldam * S.E.M.
Mean of fetal thymic weight
26.7 2 0.6b
0.71 ? 0.01’
0.15 k 0.01”
1.10 + 0.04”
27.6 + 0.4’
0.62 2 0.01’
0.12 c 0.01”
0.08 ? O.Olh
superscripts within a column are significantly
different (P < 0.05)
7.6 t- 3.0h 66.7 + 5.4‘
13.1 + 2.4h 83.3 T 5.4’
E. A. Hassoun
3%
and S. J. Stohs
TABLE 3. Administration of endrin to pregnant C57BL16J mice on day 12 of gestation
Treatment
NUlDber of dame surviving
Gnrr0l
6
Endrm (4.5 mg/kg) Endrm (6 w/kg)
5 6
Mean of im&wion number/dam f S.E.M. (total tWIlber)
% of fetuses/dam dead or nxwiled* S.E.M. (No. of late/early)
6.7 + 3.5’ (40) 5.4 i 0.6’ (37) 5.i Z 0.6 (42)
5.i t 1.3’ (C/2) 10.2 ? 3.61 I’ (2/l) 28.‘) + 3.2b (416)
TABLE 4. Adminhation
imphnbltiOll
Nllttlber of dams survivimr
number/dam * S.E.M. (total tllUI&r)
( :onml
5
Endrm (4.5 mg/kK) Endrm (6 w/kg)
5
5.8 z C.7 (17) 6.C z 0.5’ (30) 6.7 - 1.3’ (43)
6
Mean of fetal weight (g)lb * S.E.M.
Mean of pltW?tltnl weight (gVdam f S.E.M.
Mean of fetal thymic weight (me)/~ f S.E.M.
% of fetuses/dam having cleft *=* S.E.M.
30.1 A 0.4’
0.98 ? 0.02’
0 14 . 0.31’
I.21 z 0.05’
0’
C’
33. I
+ C.5
il.%?‘- O.CL’.
0.12
I 05
L7.
C
‘9.5
? 0.6
11.71 .. 3.01
0.11 ? WI”
+ C.CI
I 0.31’
1.01 z 0.32”
C
% of fetus/ dam having hydronephrai SAM.
i.6
f
T 5.P
of endrin to pregnant DBA12J mice on day 12 of gestation
Mean of
Treatment
Mean of maternal weight (g) t S.E.M.
% of fetwesldam dend or redorbed* S.E.M. (No. of late/early) 12.4 2 5.1’ (O/3) 13.3 A 2.3~ (C/2) 134 i 7.1,
Mean of
% of fetuseeldam having cleft
Mean of maternal weight(g) zt S.E.M.
Mean of fetal weight (gVd_ * SAM.
w-t (gVd=l * S.E.M.
Mean of fetal thymic weight (meV~ * S.E.M.
10.3 L 37
3.YC * O.CI
0 15 ? C.01’
I IO I 0.35
C,’
2.9 ? 2.4’
3CY + 3.7
L785 f C.01’
3.15 z &Cl’
l.IC
0’
0’
2Y.I
0 78 3 L?.Cl
0.12 f 3.01.
I.117 ? o.C44’
3’
2.R 2 2.5.
I L77-
phC&d
2 0.05’
% of fetus/ dam h&ng hydronephmeis S.EM.
*
(012)
TABLE 5. Adminiatmtion of hndnne to pregnant C57BL/6J mice on day 12 of gestation Mean of iI+llItMiOfl
Treatment
Number ofdams swvivhu
number/dam f S.E.M. (total IUlIlber)
% of fetuses/dam dead or lworbed* S.E.M. (No. of late/edY)
TABLE 6. Administration of hdane
Treatment
Number ofduns surviving
Mean of implantation number/dam f SAM. (total Wltlber)
Mean of maternal weight(g) ?z S.E.M.
Mean of fetal weight (g)ldun f SAM.
Mean of plscetlbll weight (gIldam * S.E.M.
Mean of fetal thymic weight (meVd=n f S.EM.
% of fehlnesldam having cl&i pPL@f S.E.M.
% of fetus/ dam having hydmnephmsis S.E.M.
2
to pregnant DBA/2J mice on day 12 of gestation
% of fetuses/dam dead 01 lrwxbed* S.E.M. (No. of Intelearly)
Mean of maternal weight (g) f S.E.M.
Mean of fetal weight (gVdom * S.E.M.
Mean of pLcelltpl weight (g)ldam * S.E.M.
Mean of fetal thymic weight (mgV~ * S.E.M.
% of fetunes/dMl having cleft
% of fetls/ dam having hydmnephrosb S.E.M.
*
Comparative
Teratological
Studies on TCDD,
such as cleft palate and hydronephrosis
Endrin and Lindane
in the C57BL/6J
and DBA/2J mice at doses of 30 and 60 pg/kg body weight, respectively.
However,
50% fetolethality
these same doses produced 14 and
in the C57BL/6J
and DBA/2J
mice, re-
397
ever, it has not been clearly demonstrated on the involvement
increases in the induction
of these latter effects of TCDD,
and lindane may contribute ative mechanisms
spectively. Significant
that these effects
segregate with the fetal Ah receptor. Further investigations to our understanding
endrin
of the rel-
involved in their fetotoxicities.
of malformations
and other fetotoxic effects such as decreases in fetal, placental and fetal thymic weights were observed when TCDD
These studies were supportedby a grant (#ES06818) from the National Institutes of Health. The authors thank Ms. LuAnn Schweryfur
was given to DBA/ZJ mice at doses that caused maternal
technical assistance.
toxicity
as indicated
by significant
reduction
in maternal
weight. However, these effects are significantly induced by TCDD in the C57BL/6J mice at doses that did not affect maternal TCDD
weight. Therefore,
maternal
might be a contributing
fetotoxicities
induced by
factor in TCDD-induced
including fetolethalities
in the DBA/ZJ mice.
Endrin and lindane induce fetotoxic lar to those produced by TCDD
toxicity
effects that are simi-
(Tables 3-6).
These effects
include fetal growth retardation, and decreases in fetal thymic and placental weights. However, unlike TCDD, endrin and lindane produced no cleft palate or hydronephrotic
kid-
neys in the fetuses of the two strains of mice used in this study, even when they were given at doses that caused maternal toxicity
as indicated
by the deaths of some litters.
Although endrin and lindane share the properties with TCDD of being highly stable, halogenated, cyclic and lipophilic (7,13),
these compounds may have very low affinity
towards the fetal AH receptor which explains their inabilities to induce malformations TCDD. tion
With the exception
which
C57BL/6J
was induced
similar to those induced by of fetal thymic weight reduc-
by endrin
and lindane
in the
but not in the DBA/2J mice, endrin and lindane
induced fetal growth retardation
and decreased
placental
weight in both strains of mice. These observations the involvement
of additional mechanisms
suggest
in the fetotoxi-
cities of these compounds. As with TCDD-induced
fetotox-
icity in the DBA/2J mice, maternal toxicity as a factor in endrin- and lindane-induced fetotoxic effects should not be excluded, since the two compounds were given at doses that produced
some maternal
deaths
in both
C57BL/6J
and
DBA/ZJ mice. The results of this study support the hypothesis that malformations
such as cleft palate and hydronephrosis
by TCDD
may involve direct interactions
AH receptor (12).
as a first step in the mechanism
However,
placental lindane
fetal growth retardation
weight induced by TCDD may be initiated
known mechanisms
through
induced
with the fetal of toxicity
and decreases in
as well as endrin and additional,
though un-
that may or may not be closely related
to those involved in the induction of malformations. Oxidative stress induced lipid peroxidation and DNA damage, and the enhancement in response to TCDD and contribute
of the excretion of lipid metabolites may be mediated by the AH receptor
to the fetotoxicity
(17). These mechanisms
have also been shown to be involved in TCDD-, endrinand lindane-induced toxicity in animals (2,18,25). How-
References 1. Abbott, B.D.; Perdew, G.H.; Bimbaum, L.S. Ah-receptor in embryonic mouse palate and effects of TCDD on receptor expression. Toxicol. Appl. Pharmacol. 126:16-25;1994. 2. Bagchi, M.; Hassoun, E.; Akubue, P.; Bagchi, D.; Stohs, S.J. Comparative effects of endrin on hepatic lipid peroxidation, DNA damage and nitric oxide production by peritoneal macrophages from C57BL/6J and DBA/ZJ mice. Comp. Biochem. Physiol. 105C:525-529;1993. 3. Bagchi, M.; Hassoun, E.; Bagchi, D.; Stohs, S.J. Endrininduced increases in hepatic lipid peroxidation, membrane microviscosity and DNA damage in rats. Arch. Environ. Contam. Toxicol. 23:1-5;1992. 4. Bimbaum, L.S. Developmental toxicity of TCDD and related compounds: Species sensitive and differences. In: Gallo et al. (eds). Biological basis for risk assessment of dioxins and related compounds, Banbury Report 35, Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1991:51-67. 5. Campbell, M.A.; Gyorkas, J.; Leece, B.; Homonko, K.; Safe, S. The effects of twenty-two organochlorine pesticides as inducers of hepatic drug-metabolizing enzymes. Gen. Pharmacol. 14:445-454;1983. J.M.B.; Elfstrom, J.G.; Snockowski, M.; 6. Carlstedt-Duke, Hogberg, B.; Gustafsson, J.A. Detection of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver by isoelectric focusing in hepatic cytosol. J. Biol. Chem. 251:49364946;1976. 7. Chadwick, R.W.; Copeland, M.F.; Froelich, R.; Cooke, N. Chlorobenzene, lindane or chlorobenzene plus lindane. J. Toxicol. Environ. Hlth. 12:599-610;1983. 8. Courtney, K.D.; Moore, J.A. Teratology studies with 2,4,5trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzop-dioxin. Toxicol. Appl. Pharmacol. 20:396-403;1971. 9. Couture, L.A.; Abbot, B.D.; Bimbaum, L.S. A critical review of the developmental toxicity and teratogenicity of 2,3,7,8tetrachlorodibenzo-p-dioxin: recent advances toward understanding the mechanism. Teratology 42:619-627;1990. 10. d’Argy, R.; Hassoun, E.; Dencker, L. Teratogenicity of TCDD and the congener 3,3’,4,4’-tetrachloroazoxybenzene in sensitive and non-sensitive mouse strains after reciprocal blastocyst transfer. Toxicol. Lett. 21:197-202;1984. 11. Dencker, L.; Hassoun, E.; d’Argy, R.; Alm, G. Fetal thymus organ culture as an in vitro model for the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin and its congeners. Mol. Pharmacol. 27:130-140;1985. 12. Dencker, L.; Pratt, R.M. Association between the presence of the Ah-receptor in embryonic murine tissues and sensitivity to TCDD-induced cleft palate. Teratogens, Carcinogens, Mutagens 1:399-406;1981. 13. Ecobicon, D.J. Toxic effects of pesticides. In: Klaassen et al. (eds). Casarett and Doull’s Toxicology: The basic science of poisons, 4th ed. New York: Maxwell McMillan Pergamon; 1991:573-580.
398
14. Good, E.E.; Ware, G.W. Effects of insecticides on reproduction in the laboratory mouse. IV. Endrin and dieldrin. Taxicol. Appl. Pharmacol. 14:201-203;1969. 15. Gray, J.A.; Gray, L.E. Jr.; Sovocool, G.W. Perinatal toxictty of endrin in rodents. 1. Fetotoxic effects of prenatal exposure in hamsters. Toxicology 13:155-165;1979. 16. Hassan, M.Q.; Numan, I.T.; Nasiri, N.A.; Stohs, S.J. Endrininduced histopathological changes and lipid peroxidation in livers and kidneys of rats, mice, guinea pigs and hamsters. Toxicol. Pathol. 19:108-l 14;1991. 17. Hassoun, E.; Bagchi, I).; Stohs, S.J. Evidence of TCDDinduced tissue damage in fetal and placental tissues and changes in amniotic fluid lipid metaholites of pregnant CFl mice. Toxicol. Lett. 76:245-250;1995. 18. Hassoun, E.; Bagchi, M.; Bagcht, LX; Stohs, S.J. Comparattvc studies on lipid peroxidation and L)NA-single strand breaks induced by lindane, DDT, chlordane, and endrin m nits. Comp. Biochem. Physiol. 104C:427-431;1993. 19. Hassoun, E.; d’Argy, R.; Denckcr, L.; Sundstrom, G. Tcratological studies on the TCDL) congener 3,3’,4,4’-tetrachloroazoxybenzene in sensitive and non-sensitive mouse strains. Evidence for direct effect on embryonic tissues. Arch. Toxicol. 55:20-26;1984. 20. Hassoun, E.; d’Argy, R.; Denckrr, L.; Lundin, L.C.; Borwell, P. Teratogenicity of 2,3,7,8-tetrachlorodihenzofuran in rccombinant inbred strains. Toxicol. Lett. 23:37-42;1984. 2 1. Hassoun, E.; Dencker, L. TCDD embryotoxicity in the mouse may be enhanced by /Xnaphthoflavone, another ligand of the Ah-receptor. Toxicol. Lett. 12:191-198;1982. 22. Hayes, W.J. Jr. Clinical handbook on economic poisons. US Dept Health, Education and Welfare, Public Health Service, Communicable Disease Center, Toxicology Section, Atlanta, GA.; 1963. 23. Kavlock, R.J.; Chemoff, N.; Ham&h, R.C.; Gray, J.; Rogers, E.; Gray, L.E. Jr. Perinatal toxicity of cndrin in rodents. II. Fetotoxic effects of prenatal exposure in rats and mice. Toxicology 21:141-150;1981. 24. Kavlock, R.J.; Chemoff, N.; Rogers, E.H. The effect of acute maternal toxicity on fetal development in the mouse. Temtogen. Carcinogen. and Mutagen. 5:3- 13;1985. 25. Mohammadpour, H.; Murray, W.J.; Stohs, S.J. 2,3,7,8-Tetrachlorodibenzo-p-dioxm.induccd lipid peroxidation in genetically responsive and non-responsive mice. Arch. Environ. Contam. Toxicol. 17:645-650;1988. 26. Murphy, R.; Harvy, C. Residues and metabolites of selected persistent halogenated hydrocarbons in blood specimens from a general population survey. Environ. Hlth. Persp. 60:115120;1981.
E. A. Hassoun
and S. J. Stohs
27. Nebert, L).W.; Etsen, H.J.; Niyishi, M.; Lang, M.A.; Hjelmeland, H.J.; Okey, A.B. Genetic mechanisms controlling the induction of polysubstrate monooxygenasc (P-450) activities. Ann. Rev. Pharmacol. Toxicol. 21:431-462;1981. 28. Neubert, D.; Zenz, P.; Rothen Wallmer, A.; Merker, H.J. A survey of the emhryotoxic effects of TCDD in mammalian species. Environ. Hlth. Persp. 5:63-75;1973. 29. Neubert, D.; Dillmann, 1. Emhryotoxic effects in mice treated with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Nauyn Schmiedeberge Arch. Pharmacol. 272:243-264;1972. 30. Okey, A.B.; Bandy, G.P.; Mason, M.E.; Kahl, G.F.; Eisen, 1t.].; Guenthner, T.M.; Nebert, D.W. Regulatory gene product of the Ah-receptor: Characterization of the cytosolic induccr-receptor complex and evidence for its nuclear translocanon. J. Biol. Chem. 254:11636-l 1648;1979. 3 I. Gkey, A.B.; Riddick, D.S.; Harper, P.A. The Ah receptor: Mcdiator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TODD) and related compounds. Toxicol. Lett. 70: l-22;1994. 32. Ottolcnghi, A.D.; Haseman, J.K.; Suggs, F. Teratogenic effects of aldrin, dieldrin and endrin in hamsters and mice. Teratology 9:11-16;1973. 33. Pohjanverta, R.; Tuomisto, J. Short-term toxicity of 2,3,7,8tetrachlorodihenzo-p-dioxin in laboratory animals: Effects, mechanisms and animal models. Pharmacol. Rev. 46:483549; 1994. 34. Poland, A.; Glovet, E. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: Segregation wtih the Ah-locus. Mol. Pharmacol. 17:86-94; 1980. 35. Poland, A.; Clover, E.; Kende, A.S. Stcreospcitic high affintry binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by polyacrylamide gels. Toxicol. Lett. 2:365-373;1978. 36. Poland, A.; Knutson, J.C. 2,7,7,8-Tetrachlorodibenzo-pdioxin and related halogenated aromatic hydrocarbones: Exammation of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 22:517-554;1982. 37. Sircar, S.; Lahir, P. Lindane ( FHCH) causes reproductive failure and fetotoxtcity in mice. Toxicology 59:171-177;1989. 38. U.S. Public Health Service. Toxicological profiles for alpha-, hem-, and gamma-, and delta-hexachlorocyclohexane. In: Agency for toxic substances and disease registry. Clement Associates, Preparer. Washington: U.S. Government Printing CXce; 1989. 39. Viviani, A.; Lutz, W.K.; Schlatter, C. Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes hy phenobarbital, 3-methyl cholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dieldrin and other inducers. Bitrhem. Pharmacol. 27:2 103%2108;1978.
ISSN SO742-8413/96/$15.00 PII SO742-8413(96)00011-2
1996
ELSEVIER
Comparative Teratological Studies on TCDD, Endrin and Lindane in C57BL/6J and DBA/ZJ Mice E. A. Hussoun and S. _7.Stoh SCHOOLOF PHARMACYAND ALLIEDHEALTH PROFESSIONS, CREIGHTONUNIVERSITY,OMAHA, NE 68178 USA
ABSTRACT.
The teratogenic
by TCDD
effects
of endrin
of C57BL/6]
and
lindane
have
been
determined
and
compared
to those
and DBA/ZJ mice after the administration
of single oral doses to pregnant mice on day 12 of gestation. TCDD produced dose-dependent decreases in fetal weight, fetal thymic weight and placental weight, and dose-dependent increases in fetolethality, cleft palate formation and hydronephrosis at doses of lo-30 and 30-60 pgg/kg body weight in C57BL/6J and DBA/ZJ mice, respectively. No maternal death was observed at the given doses in both strains of mice. Endrin (4.5 and 6 mg/kg body weight) and lindane (30 and 45 mg/kg body weight) produced significant decreases in fetal weight and placental weight in C57BL/6] and DBA/ZJ mice, and dose-dependent decreases in fetal thymic weight in C57BL/6J mice but not DBA/ZJ mice. Endrin and lindane caused O-25 and 14-25% maternal deaths, respectively, at the above mentioned doses. Neither cleft plate nor hydronephrosis were induced by endrin or lindane in the two strains of mice. The results support the hypothesis that TCDD-induced cleft plate and hydronephrosis involve mechanisms that are Ah (aryl hydrocarbon) receptor mediated. However, other fetotoxic effects induced by TCDD, and the fetotoxic effects induced by endrin and lindane may involve additional unknown mechanisms that are not related to the Ah-receptor. COMP BIOCHEMPHYSIOL113C, 393-398, 1996. induced
in the fetuses
KEY WORDS. TCDD, endrin, lindane, teratology, C57BL/6J mice, DBA/ZJ mice, fetotoxicity
Endrin is a highly toxic chlorinated
INTRODUCTION (PCH)
The polyhalogenated
cyclic hydrocarbons
variety
such as endrin and lindane, and con-
of pesticides,
taminants p-dioxin
include
a
of pesticides such as 2,3,7,&tetrachlorodibenzo(TCDD).
Although
lindane,
endrin and TCDD
are structurally dissimilar, they share the properties of being highly stable, halogenated, TCDD
cyclic and lipophilic
(7,8,13).
is the most toxic compound in this group, inducing a
variety of toxic responses in laboratory animals (33,34,35). Endrin and lindane are known to induce toxic responses in laboratory animals that are similar in some aspects to those induced by TCDD
which include hepatotoxicity
17), induction of drug metabolizing enzymes (5,37), hypoplasia (3), weight loss and eventually Lindane has been a common component agricultural fertilizers, household insecticide parasitic (38).
medications
Although
and animal
the production
(2,3,16, thymic
death (22). in a variety of sprays, human
parasiticidal
solutions
of lindane was banned in
the United States in 1977, it is still present in commercial products and found in significant quantities in the environment, and is thus a potential hazard to human health (38).
Address reprint requests to: S. 1. Stohs, School of Pharmacy and Allied Health Professions, Creighton University, 2500 California Plaza, Omaha, NE 68178 USA. Tel: 402-280-2950. Received 1 June 1995; revised 19 December 1995; accepted 19 Decemher 1995.
which has been used for domestic,
hydrocarbon
pesticide
agricultural and public
health purposes. The mechanisms of toxicity of these compounds are not known, although various possible mechanisms have been proposed (2,3,5,16,18,27,36,39). In addition to their toxic effects, these PCH are teratogenie and fetolethal 24,32,34,37).
TCDD
in laboratory
animals
(4,9,14,15,23,
is the most extensively
studied of
these compounds and is considered the most potent experimental teratogen duced by TCDD dronephrosis,
(28,34).
The most prominent
effects in-
in the fetuses of mice are cleft palate, hy-
thymic hypoplasia, reduced fetal weight and
fetal death (9,11,21,28,34). Lindane when given to mice during early pregnancy
at
a dose which is 50% of the LDSO caused total absence of any implantation
sites (37). When the same dose was given
at mid-pregnancy, lindane caused total resorption of fetuses. At late pregnancy, lindane resulted in low birth weight pups which subsequently died 12-15 Neither
days after parturition
this study nor others examined
(37).
the effects of lin-
dane when given to pregnant mice at doses below those causing 100% fetolethality with observation of fetal malformation. Studies with endrin have shown marked species differences in teratogenic sensitivity, with hamsters being the most affected species (15,23,32). produced conflicting
Studies in CD-l
mice have
results, with one study failing to show
394
E. A. Hassoun and S. J. Stohs
any teratogenic
or fetolethal
given
at doses that
other
study,
however,
when
given
to pregnant
effects
caused
even when
maternal
cndrin
lethality
was shown
mice
endrin (24).
was
In an-
to be teratogenic
at a single
oral dose which
was 50% of the LDsa and given on day 9 of gestation. At this dose, endrin caused a high incidence of congenital anomalies
without
or growth
impairment
fects of endrin
a concurrent
been
(14). Therefore of teratogenicity
are not known, suggested
21,34). appear
to TCDD
and have
(9,10,11,
in teratogenic which locus
receptor
of the genetic which
and its congeners This
has
AH
been
as well as a number
receptor
for mechanisms
has
locus
shown
is the
to bind
of polycyclic
greatly
stimulated
and
teratogenicity
of toxicity
received
TCDD
ceived
of
were checked
at 30, 45 or 60 pg/kg.
the vehicle
taining
used to dissolve
10% acetone).
Other
mice received
or lindane vehicle
at doses
which
All chemicals at 7500 @/kg TCDD
volume
body weight.
formations when The
the maximal
to pregnant
animals
gestation
C57BL/6J
carbon
and sacrificed
dioxide
resorbed
or live fetuses.
Live fetuses
and live fetuses
were checked
is considered
external
malformations
to the stimu-
collected
to the AH receptor
to be the first step in a series of events lation
of the polysubstrate
other
enzyme
systems
not related
of TCDD (1,4,27). Since endrin and lindane fects with TCDD, are similar
fore, the fetotoxic investigated
induced
C57BL/6J
fetotoxic
toxic efeffects
that
if they are adminis-
to mice as TCDD. and lindane
with
and also
conversion
some common
by TCDD
effects of endrin
and compared
in AH responsive DBA/ZJ
share
the same conditions
system
to metabolic
they may produce
to those
tered under
leading
monooxygenasc
those
There-
were further
induced
by TCDD
mice and in AH nonresponsive
then
number
and cleft
in Bouin’s
the presence
of hydroncphrotic
L1ata for each of variance
(TCDD) was Resources Program,
tional
MD).
Institute
drin were purchased All chemicals
(Bethesda, from Supelco,
were of the highest
Lindane
Inc. (Bellefonte,
ohNa-
and enPA).
grade that is commercially
available.
Animals Male
Thymuses The
of were
fetuses
were
for later examination
for
were subjected
Scheffe’s
S method
post hoc test. The data are expressed dard error of the mean (SEM). The statistical
unit for comparisons, as an average
as the mean + standam was used as the
and the fetotoxic
of each effect among
different
to analysis
was used as the
effects are
the fetuses of
superscripts
are consid-
(P < 0.05).
RESULTS As can be seen in Table decreases weight cleft
1, TCDD
in fetal weight,
as well as dose-dependent palate
formation
produced
fetal thymic and
dose-dependent
weight
increases
hydronephrosis
and placental in fetolethality, in C57BL/6J.
The same effects were also produced by TCDD in the DBA/ 2J mice (Table 2). The ED, values for TCDD for the production of cleft palate and hydronephrosis in C57BL/6J mice were 25 and 18 pg/kg, respectively, while these values
and Treatment
and female
mice
of C57BL/6J
and
(Jackson Laboratories, Bar Harbor, ME), weeks old, were maintained at 25°C with cycle, and were given a commercial pellet dent Lab Chow #SOOl) and tap water ad Males
were
for the presence
METHODS
2,3,7,8-Tetrachlorodibenzo-p-dioxin tained from the Chemical Research Cancer
and placentae
palates.
that dam. Values with non-identical AND
uteri dead,
kidneys.
group of animals
(ANOVA).
ered significantly MATERIALS Chemicals
the
of implantations,
and weighed.
solution
and
Data Analysis
calculated
mice.
for the
from the fetuses
placed
doses on
on day 18 of
anesthesia,
weighed
( 12). Binding
and resorption
at teratogenic
embryos
sive embryos
investigated
studies,
rate of mal-
(10,19,21).
were weighed
using
on
was kept
Based on our previous
were
strains of mice but not in nonrespon-
intubation
of the vehicles
produce
oil
and lindane.
with the lowest rate of fetal death
given
and
or the corn
endrin
were given orally by gastric
and its congeners
re-
oil con-
at doses of 4.5 and 6 mg/kg
of 30 and 45 mg/kg,
The
animals
(corn
groups of both C57BL/6J
endrin
TCDD and related compounds (6,.30,35). AH receptors are found in several embryonic tissues of midgestational murine of responsive
for vaginal
Control
TCDD
was used to dissolve
day 12 of gestation
the
mice
lowing treatment groups. Groups of C57BL/6J mice were given either TCLX) at 10, 20, or 30 pg/kg. DBA/ZJ mice
day 12 of gestation.
occurred,
A product
hydrocarbons.
of TCDD
and its bioisosteres
( 11,19,20,34). AH
endrin
mechanisms
differences
day 0) the female
plugs. Before treatment, pregnancy was confirmed by abdominal palpation. The animals were assigned into the fol-
DBA/ZJ
with the AH (aryl hydrocarbon)
cytosolic
search
several
strain
cf-
clarification.
of TCLX),
although
marked
to segregate
TCDD
the teratogenic
for the teratogenicity
In mice,
sensitivity
in fetal mortality
in mice may need further
The mechanisms lindane
increase
day (=
and females
were mated
DBA/2J
strains
approximately 8 a 12 h light/dark diet (Purina Rohum.
overnight
and the next
in DBA/ZJ TCDD
mice were
55 and 50 pg/kg,
had to be given to DBA/ZJ
respectively.
Thus,
mice at doses that were
2-3 times higher than those given to the C57BL/6J mice (Table 1). No maternal deaths were induced by these doses in either of the two strains used for this study. A significant reduction in maternal weight was induced by TCDD at a
Comparative
Teratological
Studies on TCDD,
TABLE 1. Administration
Endrin and Lindane
395
of 2,3,7,8-tetrachlorodibenzoqwiioxin
(TCDD)
to pregnant C57BL16J mice on day 12 of gestation
% of Mean of
number/dam f S.E.M. (total number)
fetuses/dam dead or resorbed f S.E.M. (No. of late/early)
6.7 + 0.6” (40) 6.8 + 0.5” (41) 6.2 + 0.5” (37) 8.2 ? 0.2,’ (41)
7.9 2 4.6* (O/2) 8.6 + 3.5” (O/2) 8.1 2 3.3” (l/2) 14.4 2 1.71 (4/2)
implantation Treatment
Number of dams
ChIltd
6
TCDD (10 e/kg) TCDD (20 /e&g) TCDD (30 &/kg)
6 6 5
Values with nonidentical
Mean of weight (&/dam f S.E.M.
Mean of fetal thymic weight (mg)/dam f S.E.M.
% of fetuses/dam having cleft palate f S.E.M.
% of fetus/ dam having hydronephrosis f S.E.M.
0.98 k 0.023
0.14 + 0.01”
1.21 2 0.05”
0”
0’
30.1 2 0.5,’
0.95 + 0.03’
0.14 + 0.01”
1.08 + 0.04h
6.3 I’ 3.8h
13.2 + 2.7h
29.5 + 0.4”
0.79 + O.Olh
0.12 + O.Olh
1.02 k 0.02,
17.1 -t 4.2‘
43.3 2 6.4’
28.8 2 0.5’
0.59 + 0.01’
0.11 i- 0.01’
0.09 + 0.01”
83.6 :? 6.2”
88.3 5 2.7’
Mean of maternal weight(g) f S.E.M.
Mean of fetal weight (gwm f S.E.M.
30.3 + 0.4’
superscripts within a column are significantly
different (P < 0.05)
dose of 30 ,ug/kg body weight given to the C57BL/6J mice (Table 1) and at doses of 45 ,ug/kg body weight or higher given to the DBA/2J mice (Table 2). Table 3 shows that endrin, when given at doses of 4.5 and 6 mg/kg body weight, produced significant decreases in fetal weight, fetal thymic weight and placental weight in C57BL/6J mice. When endrin was given to the DBA/ZJ mice (Table 4), it caused significant reduction in fetal weight at doses of 4.5 and 6 mg/kg body weight, and placental weight at a dose of 6 mg/kg body weight. At this latter dose, endrin caused 25% maternal death in both C57BL/ 6J and DBA/2J mice. No cleft palate or significant hydronephrosis were observed following endrin administration to either strain of mice. The effects of lindane on pregnancy outcome in C57BL/ 6J and DBA/2J mice are shown in Tables 5 and 6, respectively. As with endrin, lindane when given at doses of 30 and 45 mg/kg body weight produced significant decreases in fetal weight, fetal thymic weight and placental weight in C57BL/6] mice (Table 5). When given to the DBA/2J mice, lindane caused significant reduction in fetal and placental weight at a dose of 45 mg/kg body weight (Table 6). Lindane caused 14 and 25% maternal deaths when given at doses of 30 and 45 mg/kg, respectively, in both strains of mice. However, as with endrin, lindane produced no significant reduction in maternal weight at either of the two
TABLE 2. Administration
placetWdl
doses given to the two strains of mice (Tables 5 and 6). Neither cleft palate nor hydronephrosis were induced by lindane in the fetuses of C57BL/6J and DBA/ZJ mice (Tables 5 and 6) as compared with TCDD (Tables 1 and 2). Doses of lindane lower than 30 mg/kg body weight produced no maternal toxicity or death, and no significant teratogenic effects were observed in the fetuses of C57BL/6] and DBA/ZJ mice (results not shown). DISCUSSION The results clearly demonstrate that TCDD, endrin and lindane are fetotoxic. TCDD induced malformations, fetal and placental weight retardation and fetolethality in TCDD (Ah) responsive C57BL/6J and nonresponsive DBA/ZJ mice (Tables 1 and 2). These results are in agreement with previous studies (20,31). However, TCDD had to be given at doses that are approximately 2-3 times higher to DBA/ 2J mice as compared to the doses given to the C57BL/6J mice (Table 2). Ah receptors are present in non-responsive mouse strains as DBA/2J. However, the affinity with which TCDD binds to receptors in DBA/ZJ mice is about ten-fold weaker than in C57BL/6J mice (28). These receptors may be occupied and activated in the tissues of the DBA/2J mice only upon exposure to high doses of TCDD. TCDD induced approximately 70-80% malformations
of 2,3,7&btetrachlorodibenzo-p-dioxin
(TCDD)
to pregnant DBA12J mice on day 12 of gestation
96of
Treatment
Number of dams
Control
5
TCDD (30 pgkg) TCDD (45 pgkg) TCDD (60 pgs/W
6 6 5
Values with nonidentical
Mean of implantation number/dam f S~~~L($tal
7.4 i 0.2” (37) 8.5 k 0.2h (51) 6.3 2 0.2’ (38) 6.5 + 0.8’ (36)
fetuses/dam dead or resorbed * S.E.M. (No. of late/early) 7.9 + 2.7* (O/3) 9.7 + 1.8” (312) 21.2 ?z 4.1h (O/10) 50.0 I 6.7‘ (O/W
Mean of Mean of maternal weight (g) f S.E.M.
Mean of fetal weight (g)/dam f S.E.M.
30.3 k 0.7”
0.90 t 0.01*
30.4 2 0.6’
0.88 -t O.Olh
(mgs)zo . . . =
% of fetoseskiam having cleft palate 2 S.E.M.
% of fetus/ dam having hydronephrosis 2 S.E.M.
0.15 ? O.Ol^
1.10 2 0.05,’
0”
2.9 k 2.3,’
0.15 2 0.01”
1.10 2 0.05’
0”
1.9 2 1.7”
plX&d
weight (g)ldam * S.E.M.
Mean of fetal thymic weight
26.7 2 0.6b
0.71 ? 0.01’
0.15 k 0.01”
1.10 + 0.04”
27.6 + 0.4’
0.62 2 0.01’
0.12 c 0.01”
0.08 ? O.Olh
superscripts within a column are significantly
different (P < 0.05)
7.6 t- 3.0h 66.7 + 5.4‘
13.1 + 2.4h 83.3 T 5.4’
E. A. Hassoun
3%
and S. J. Stohs
TABLE 3. Administration of endrin to pregnant C57BL16J mice on day 12 of gestation
Treatment
NUlDber of dame surviving
Gnrr0l
6
Endrm (4.5 mg/kg) Endrm (6 w/kg)
5 6
Mean of im&wion number/dam f S.E.M. (total tWIlber)
% of fetuses/dam dead or nxwiled* S.E.M. (No. of late/early)
6.7 + 3.5’ (40) 5.4 i 0.6’ (37) 5.i Z 0.6 (42)
5.i t 1.3’ (C/2) 10.2 ? 3.61 I’ (2/l) 28.‘) + 3.2b (416)
TABLE 4. Adminhation
imphnbltiOll
Nllttlber of dams survivimr
number/dam * S.E.M. (total tllUI&r)
( :onml
5
Endrm (4.5 mg/kK) Endrm (6 w/kg)
5
5.8 z C.7 (17) 6.C z 0.5’ (30) 6.7 - 1.3’ (43)
6
Mean of fetal weight (g)lb * S.E.M.
Mean of pltW?tltnl weight (gVdam f S.E.M.
Mean of fetal thymic weight (me)/~ f S.E.M.
% of fetuses/dam having cleft *=* S.E.M.
30.1 A 0.4’
0.98 ? 0.02’
0 14 . 0.31’
I.21 z 0.05’
0’
C’
33. I
+ C.5
il.%?‘- O.CL’.
0.12
I 05
L7.
C
‘9.5
? 0.6
11.71 .. 3.01
0.11 ? WI”
+ C.CI
I 0.31’
1.01 z 0.32”
C
% of fetus/ dam having hydronephrai SAM.
i.6
f
T 5.P
of endrin to pregnant DBA12J mice on day 12 of gestation
Mean of
Treatment
Mean of maternal weight (g) t S.E.M.
% of fetwesldam dend or redorbed* S.E.M. (No. of late/early) 12.4 2 5.1’ (O/3) 13.3 A 2.3~ (C/2) 134 i 7.1,
Mean of
% of fetuseeldam having cleft
Mean of maternal weight(g) zt S.E.M.
Mean of fetal weight (gVd_ * SAM.
w-t (gVd=l * S.E.M.
Mean of fetal thymic weight (meV~ * S.E.M.
10.3 L 37
3.YC * O.CI
0 15 ? C.01’
I IO I 0.35
C,’
2.9 ? 2.4’
3CY + 3.7
L785 f C.01’
3.15 z &Cl’
l.IC
0’
0’
2Y.I
0 78 3 L?.Cl
0.12 f 3.01.
I.117 ? o.C44’
3’
2.R 2 2.5.
I L77-
phC&d
2 0.05’
% of fetus/ dam h&ng hydronephmeis S.EM.
*
(012)
TABLE 5. Adminiatmtion of hndnne to pregnant C57BL/6J mice on day 12 of gestation Mean of iI+llItMiOfl
Treatment
Number ofdams swvivhu
number/dam f S.E.M. (total IUlIlber)
% of fetuses/dam dead or lworbed* S.E.M. (No. of late/edY)
TABLE 6. Administration of hdane
Treatment
Number ofduns surviving
Mean of implantation number/dam f SAM. (total Wltlber)
Mean of maternal weight(g) ?z S.E.M.
Mean of fetal weight (g)ldun f SAM.
Mean of plscetlbll weight (gIldam * S.E.M.
Mean of fetal thymic weight (meVd=n f S.EM.
% of fehlnesldam having cl&i pPL@f S.E.M.
% of fetus/ dam having hydmnephmsis S.E.M.
2
to pregnant DBA/2J mice on day 12 of gestation
% of fetuses/dam dead 01 lrwxbed* S.E.M. (No. of Intelearly)
Mean of maternal weight (g) f S.E.M.
Mean of fetal weight (gVdom * S.E.M.
Mean of pLcelltpl weight (g)ldam * S.E.M.
Mean of fetal thymic weight (mgV~ * S.E.M.
% of fetunes/dMl having cleft
% of fetls/ dam having hydmnephrosb S.E.M.
*
Comparative
Teratological
Studies on TCDD,
such as cleft palate and hydronephrosis
Endrin and Lindane
in the C57BL/6J
and DBA/2J mice at doses of 30 and 60 pg/kg body weight, respectively.
However,
50% fetolethality
these same doses produced 14 and
in the C57BL/6J
and DBA/2J
mice, re-
397
ever, it has not been clearly demonstrated on the involvement
increases in the induction
of these latter effects of TCDD,
and lindane may contribute ative mechanisms
spectively. Significant
that these effects
segregate with the fetal Ah receptor. Further investigations to our understanding
endrin
of the rel-
involved in their fetotoxicities.
of malformations
and other fetotoxic effects such as decreases in fetal, placental and fetal thymic weights were observed when TCDD
These studies were supportedby a grant (#ES06818) from the National Institutes of Health. The authors thank Ms. LuAnn Schweryfur
was given to DBA/ZJ mice at doses that caused maternal
technical assistance.
toxicity
as indicated
by significant
reduction
in maternal
weight. However, these effects are significantly induced by TCDD in the C57BL/6J mice at doses that did not affect maternal TCDD
weight. Therefore,
maternal
might be a contributing
fetotoxicities
induced by
factor in TCDD-induced
including fetolethalities
in the DBA/ZJ mice.
Endrin and lindane induce fetotoxic lar to those produced by TCDD
toxicity
effects that are simi-
(Tables 3-6).
These effects
include fetal growth retardation, and decreases in fetal thymic and placental weights. However, unlike TCDD, endrin and lindane produced no cleft palate or hydronephrotic
kid-
neys in the fetuses of the two strains of mice used in this study, even when they were given at doses that caused maternal toxicity
as indicated
by the deaths of some litters.
Although endrin and lindane share the properties with TCDD of being highly stable, halogenated, cyclic and lipophilic (7,13),
these compounds may have very low affinity
towards the fetal AH receptor which explains their inabilities to induce malformations TCDD. tion
With the exception
which
C57BL/6J
was induced
similar to those induced by of fetal thymic weight reduc-
by endrin
and lindane
in the
but not in the DBA/2J mice, endrin and lindane
induced fetal growth retardation
and decreased
placental
weight in both strains of mice. These observations the involvement
of additional mechanisms
suggest
in the fetotoxi-
cities of these compounds. As with TCDD-induced
fetotox-
icity in the DBA/2J mice, maternal toxicity as a factor in endrin- and lindane-induced fetotoxic effects should not be excluded, since the two compounds were given at doses that produced
some maternal
deaths
in both
C57BL/6J
and
DBA/ZJ mice. The results of this study support the hypothesis that malformations
such as cleft palate and hydronephrosis
by TCDD
may involve direct interactions
AH receptor (12).
as a first step in the mechanism
However,
placental lindane
fetal growth retardation
weight induced by TCDD may be initiated
known mechanisms
through
induced
with the fetal of toxicity
and decreases in
as well as endrin and additional,
though un-
that may or may not be closely related
to those involved in the induction of malformations. Oxidative stress induced lipid peroxidation and DNA damage, and the enhancement in response to TCDD and contribute
of the excretion of lipid metabolites may be mediated by the AH receptor
to the fetotoxicity
(17). These mechanisms
have also been shown to be involved in TCDD-, endrinand lindane-induced toxicity in animals (2,18,25). How-
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