Peters, J.M. , Duncan, J.R., Wiley, L.M. and Keen, C.L.. Influence of antioxidants on cadmium toxicity of mouse preimplantation embryos in vitro. Toxicology (1995) 99:11-18.

Peters, J.M. , Duncan, J.R., Wiley, L.M. and Keen, C.L.. Influence of antioxidants on cadmium toxicity of mouse preimplantation embryos in vitro. Toxicology (1995) 99:11-18.
Toxicology 99 ( 1995) 11- 18
ELSFVIFR
Influence of antioxidants on cadmium toxicity of mouse
preimplantation embryos in vitro *
Jeffrey M. Petersa, John R. Duncanb,
Lynn M. Wiley”, Carl L. Keen*C*d
aDepartments of Obstetrics and Gynecology, Division of Reproductive Biology and Medicine. University of Calijbrnia.
Davis, CA 95616, USA
bDeparrmenr of Biochemistry and Microbiology. Rhodes University, Grahamstown, South Africa
‘Department of Nutrition, University of California. Davis, CA 95616. USA
dDepartment of Internal Medicine, University of California. Davis, CA 95616, USA
Received 30 August 1994; accepted 24 October 1994
Abstract
To test the hypothesis that the developmental
toxicity of cadmium (Cd) is due in part to oxidative damage, embryos
were cultured in medium containing 0.0, 1.O, 3.0, or 6.0 PM Cd with or without various antioxidants for 72 h. Ascorbate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene
(BHT) and glutathione (GSH) were all effective at
ameliorating 1.0 PM Cd-induced embryotoxicity.
For embryos cultured in medium containing either 3.0 or 6.0 PM
Cd, GSH was effective at ameliorating Cd toxicity while the other antioxidants tested were ineffective. Pretreating embryos with antioxidants for 24 h prior to exposing them to Cd and antioxidants did not significantly alter the previously
observed improvement with the exception that pretreatment with GSH virtually eliminated Cd-induced embryotoxicity
between 1.0 and 6.0 PM Cd. A 4-h exposure to GSH prior to culture in Cd markedly improved embryo development
suggesting that GSH taken up during pretreatment
can provide protection against Cd-induced embryotoxicity.
This
work supports the hypothesis that the developmental
toxicity of Cd is in part due to oxidative damage that can be
modulated by select antioxidants.
Keywords:
Cadmium;
Antioxidants;
Oxidative
damage;
1. Introduction
Development;
Glutathione
mouse preimplantation
Cadmium (Cd) exposure is known to inhibit
both cell proliferation
and differentiation
during
* This work was supported by NIH grants HDO1743
(C.L.K.) and ES05409 (L.M.W.).
* Corresponding
author,
Department
of
Nutrition,
Universitv of California.
Davis. CA 95616. USA. Tel.: (916)
~ I
752 6331;- Fax: (916) 75; 8966.
development
with chronic
in vitro
with
Cd exposure
at concentrations
less than 2 PM (Pedersen and
Lin, 1978). While it is clear that Cd induces significant alterations
during early development,
the
mechanism(s)
underlying
this phenomena
is/are
not known. Suggested mechanisms to account for
Cd-induced cellular toxicity include: interference
with metalloenzymes
(Pedersen and Lin, 1978;
these
0300-483X/95/%09.50
0 1995 Elsevier Science Ireland
SSDI 0300-483X(94)02989-8
Embryo;
effects
Ltd. All rights reserved
occurring
12
J.M.
Peters rr ul. /Toxicology
Hussain et al., 1987; Shukla et al., 1987) alterations in thiol proteins (Chan and Cherian, 1992; Li
et al., 1993), inhibition
of energy metabolism
(Muller,
1986), alterations
in DNA structure
(Christie and Costa, 1984; Coogan et al., 1992) altered membrane structure/function
(Muller, 1986;
Shukla et al., 1987) and excessive oxidative
damage (Omaye and Tappel, 1975; Gabor et al.,
1978; Klimczak et al., 1984; Muller, 1986; Ochi et
al., 1987; Shukla et al., 1987; Hussain et al., 1987;
Manta et al., 1991).
Evidence that oxidative damage occurs with Cdtoxicity is provided by observations
in whole animals and in cell culture systems whereby Cd exposure can result in increased
levels of lipid
peroxidation
(Omaye and Tappel, 1975; Gabor et
al., 1978; Klimczak et al., 1984; Muller, 1986; Ochi
et al., 1988; Shukla et al., 1987; Hussain et al.,
1987; Manta et al., 1991 j, and/or inhibition of enzymes involved in preventing
oxidative damage,
such as superoxide dismutase or glutathione
peroxidase (Omaye and Tappel, 1975; Shukla et al.,
1987; Hussain et al., 1987; Manta et al., 1991).
Cellular levels of compounds thought to be involved in preventing
oxidative
damage,
including
metallothionein
and glutathione (GSH), have also
been shown to be affected by in vivo and in vitro
Cd exposure (Ochi et al., 1988; Chan and Cherian,
1992). Furthermore,
it has been shown that some
antioxidants,
including
acid,
(Yascorbic
tocopherol and butylated hydroxytoluene
(BHT),
can modulate Cd toxicity in tissues such as kidney,
liver and testes (Fox and Fry, 1970; Ray et al.,
1981; Sajiki et al., 1982; Onosaka et al., 1987; Ochi
et al., 1988; Shiraishi et al., 1993). Since alterations
in systems
involved
in preventing
oxidative
damage may in part underlie
Cd toxicity, we
evaluated the effect of adding antioxidants
to culture medium as a means of ameliorating
Cdinduced alterations in cellular differentiation
and
proliferation
during preimplantation
embryo development.
2. Materials and methods
2.1. Mouse preimplantation embryo recovery
Swiss ICR female mice were housed in plastic
cages in a temperatureand light-controlled
envi-
99 (1995)
II-IX
ronment (20°C 14 h light/l0 h dark cycle). Preimplantation
embryos were obtained
from superovulated females by flushing oviducts with modified Hank’s Balanced Salt Solution ‘Ll5’, (Goldstein et al., 1975). Embryos were rinsed through
three microdroplets
of L15 and placed in microdroplets of modified bicarbonate
buffered culture
medium ‘T6’ (Wiley et al., 1986) overlaid with
silicon oil in a temperature- and gas-controlled
incubator (37°C 95% O,, 5% COz). Collected embryos from mice were pooled and randomly
assigned to different treatment groups.
2.2. Embryo culture in medium containing cadmium
To evaluate the threshold for Cd toxicity of preimplantation
embryos, we cultured embryos in
varying concentrations
of Cd. Culture medium Cd
concentration
was manipulated
by adding 2 ~1 of
an appropriately
diluted stock CdCl, solution per
1 ml of T6 culture medium to give final concentrations ranging from 1.0 to 30.0 PM Cd (0.1 l-3.3
&ml). Culture medium Cd concentrations
were
verified by measuring samples using flame atomic
absorbance
spectrophotometry
(Clegg et al.,
1981 j. Preimplantation
embryos were cultured for
72 h and the frequency of blastocyst formation
after 72 h of culture was recorded as a measure of
embryonic cell differentiation.
At the end of the 72
h culture period, embryos were fixed and stained
to quantify individual
embryo cell number as a
cell
measure
of
proliferation
(Tarkowski, 1966).
2.3. Embryo culture in medium containing cadmium
and antioxidants
Two-cell preimplantation
embryos were cultured for 72 h as described above in medium containing 0.0, 1.O, 3.0, or 6.0 PM Cd with and
without one of the following antioxidants:
1j 1.0
or 2.0 mM GSH, 2) 0.01, 0.1, or 1.0 mM sodium
ascorbate, 3) 100 PM butylated
hydroxytoluene
(BHT), 4) 100 PM BHA, and 5) 100 PM dl-crtocopherol (AT). Media were prepared by adding
2 ~1 of an appropriately
diluted stock solution per
1 ml of T6 culture medium. The above concentrations of Cd were used because they result in low,
moderately
high, and high levels of toxicity as
assessed in the previous section by cell number and
J.M. Peters er al. / Toxicology 99 (1995)
frequency of blastocyst formation in our culture
system. Concentrations of antioxidants used were
based on unpublished work which determined
ranges of concentrations that were able to go into
solution and that did not interfere with embryonic
differentiation and proliferation. Developmental
progress was assessed and embryo cell number
quantified as described above (Section 2.2.).
2.4. Pretreatment of embryos with antioxidants
To test the idea that pretreating preimplantation
embryos for the first 24 h of culture with antioxidants would allow more time for embryos to accumulate the antioxidants thereby increasing the
probability of observing an amelioration of Cdinduced embryotoxicity,
2-cell embryos were
treated in the following manner. Groups of embryos were cultured for 24 h in medium with and
without one of the following antioxidants: GSH
(1 .O mM), sodium ascorbate (100 FM), BHT (100
@M), BHA (100 PM), AT (100 PM). After this 24
h pretreatment, embryos at the compacted g-cell
stage were transferred to medium containing 1.Oor
6.0 PM Cd with the respective antioxidant. Embryos were assessed for the 48 h culture duration
as described above (Section 2.2.)
2.5. Pretreatment of embryos with GSH
As GSH was shown to significantly enhance embryo viability while cultured in medium containing
Cd and GSH, the following experiment was undertaken to determine whether or not intracellular effects of GSH were in part responsible for the
observed amelioration. Embryos were exposed to
1.0 mM GSH for 4 h and subsequently rinsed
through five microdroplets of equilibrated T6 culture medium to remove any loosely bound GSH.
Pretreated 2-cell embryos were then transferred to
medium containing 0.0, 2.5, 5.0, or 10.0 PM Cd
without GSH. Embryonic differentiation and proliferation were assessed as described above (Section 2.2.) at the end of the 72 h culture.
2.6. Statistical analysis
For all experiments undertaken, there were no
significant interexperimental
variations among
treatment groups as assessed by 2-way ANOVA.
Thus, embryo cell number data were pooled within
13
II-18
groups and l-way ANOVA performed to determine differences between treatment groups. For
the blastocyst formation data, Chi-square analysis
was used to determine differences between treatment groups. Data were pooled when more than
one replicate was done after demonstrating that
there were no differences in observed ratios of
blastocysts to ‘non’-blastocysts using Chi-square
analysis (JMP: Statistical Visualization for the
Macintosh, SAS Institute).
3. Results
3.1. Embryo culture in medium containing cadmium
Blastocyst formation and embryonic cell number after 72 h were significantly lower in preimplantation embryos cultured in medium containing between 1.0 and 2.5 PM Cd in a dosedependent manner compared to controls (Table 1).
Preimplantation embryos cultured in medium with
Cd concentrations >5.0 PM exhibited complete
inhibition of embryonic development at, or prior,
to the morula stage.
Table 1
Cadmium
toxicity
of mouse preimplantation
embryos
in vitro
Treatment
N’
% Blastocysts2
Mean cell
no. 3
% Contro14
Control
1.0 PM Cd
2.5 pM Cd
5.0 pM Cd
10.0 PM Cd
20.0 PM Cd
30.0 ELMCd
30
30
30
30
15
15
15
1OOa
98a
75b
OC
OC
OC
OC
91 l
69 l
46 f
15 f
9*
8 *
5 *
100
76
51
16
10
9
5
3a
9b
6C
Id
1e
le
If
a-fValues within the same column with different superscripts
are significantly different at P 5 0.05.
‘N = the number of embryos.
2The percentage
of embryos
which
developed
into
blastocysts after 72 h of culture. Raw numbers were used to
perform Chi-square analysis.
‘The mean number of cells per embryo. Values represent the
mean f S.E.M..
?h e percent of the mean control embryo cell number.
J. M. Peters EI al. / Toxicology
14
3.2. Embryo culture in medium containing cadmium
and antioxidants
As observed with the previous experiment, embryos cultured in medium containing
1.0 PM Cd
developed into blastocysts at a similar frequency
Table 2
Effect of culture medium antioxidants
on cadmium
mouse preimplantation
embryos in vitro
Treatment’
N2
o/u
Blastocysts7
Control
I gM Cd
143
44
95”
87a
3pMCd
6rMCd
1 pM Cd + 0.01 mM Asc.
3 pM Cd + 0.01 mM Asc.
92
85
42
IO
6 PM Cd + 0.01 mM Asc.
1 gM Cd + 0.1 mM Asc.
3 PM Cd + 0.1 mM Asc.
6 PM Cd + 0.1 mM Asc.
IpMCd+l.OmMAsc.
3 pM Cd + 1.0 mM Asc.
6 PM Cd + 1.0 mM Asc.
1 /.LM Cd + 100 pM BHA
10
41
15
14
43
12
12
42
3pMCd+
IOOBMBHA
6aMCd+lOOpMBHA
l~MCd+lOO~MBHT
3 gM Cd + 100 pM BHT
6pMCd+
100pMBHT
lpMCd+lOQpMAT
3pMCd+
lOOpMAT
6 /LM Cd + 100 /LM AT
lpMCd+
l.OmMGSH
3pMCd+
l.OmMGSH
6pMCd+
l.OmMGSH
1 PM Cd + 2.0 mM GSH
3pMCd+2.0mMGSH
6pMCd+2,0mMGSH
17
17
43
14
13
38
14
16
44
48
43
45
17
13
;h”
87”
Ob
Ob
89a
Ob
Ob
61’
g:
80d
;:
87”
Ob
Ob
88a
;::
91”
68d
33e
96=
1ooa
90”
toxicity
Mean
cell
no.4
‘%
Contro1S
77 f 2a
62 f 5b
ll*lC
6+ld
73 f 5”
6*ld
4*ld
66*5b
4kld
3*ld
60~5~
4*ld
3*ld
78 f 6”
9 f 1c
4*ld
76 + 6”
9 f IC
4*1d
70 f 4b
8+ld
4*1d
75 f 5”
43 f 4e
29*5f
73 f 4”
57*7b
57 zt 8b
100
81
14
8
95
8
5
86
5
4
78
5
4
101
12
5
99
12
5
91
10
5
97
56
38
95
74
74
a-rValues within the same column with different
are significantly different at P I 0.05.
of
superscripts
‘Asc. = sodium ascorbate,
AT = dl-or-tocopherol,
BHA =
butylated
hydroxyanisole,
BHT = butylated
hydroxytoluene,
GSH = glutathione.
‘N = the number of embryos.
‘The percentage of embryos which developed into blastocysts after 72 h of culture. Raw numbers were used to perform
Chi-square analysis.
‘?he mean number of cells per embryo. Values represent the
mean f S.E.M.
‘The percent of the mean control embryo cell number.
99 ( 1995)
I I- 18
as controls, but had -80% of the cell number
compared to controls (Table 2). Embryos that
were cultured in medium containing either 3.0 or
6.0 PM Cd did not develop into blastocysts and
had between S-14% of the mean control embryo
cell number (Table 2). Embryos cultured in medium containing
1.0 PM Cd and supplemented
with
antioxidants
exhibited
a similar frequency
of
blastocyst formation compared to that of embryos
cultured in medium without Cd, as well as to those
cultured solely in 1.0 PM Cd (Table 2). More importantly though, there were no differences observed in the mean embryo cell number in embryos
that were cultured in 1.0 PM Cd supplemented
with ascorbate, BHA, BHT, or GSH compared to
controls embryos cultured in medium without Cd
(Table 2). Supplementing
culture medium containing 1.0 PM Cd with AT did not result in a higher
mean cell number as was observed with the other
antioxidants.
Supplementing
culture
medium
containing
either 3.0 or 6.0 PM Cd with ascorbate,
BHA,
BHT, or AT did not improve embryo development
in terms of blastocyst formation or mean embryo
cell number compared to controls cultured without antioxidants.
However, compared to embryos
cultured in medium containing 3.0 and 6.0 PM Cd,
embryonic cell number was markedly higher in
groups of embryos supplemented
with 1.0 mM
GSH (Table 2). In addition, blastocyst formation
was markedly higher in groups of embryos that
were exposed to either 3.0 or 6.0 PM Cd in the
presence of 1.0 mM GSH compared to embryos
cultured solely in either 3.0 or 6.0 PM Cd. When
culture medium GSH concentration
was increased
to 2.0 mM, Cd-induced
embryotoxicity
was further ameliorated
based on both the incidence of
blastocyst formation, a measure of differentiation,
and embryonic cell number, a measure of cellular
proliferation
(Table 2).
3.3. Pretreatment of embryos with antioxidants
On average, embryos cultured in control medium for 24 h and transferred to medium containing
1.0 PM Cd for the remaining 48 h at the compacted g-cell stage differentiated
into blastocysts
at the same frequency as controls (Table 3). However, the mean embryo cell number was signiti-
J.M. Peters et al. /Toxicology
Table 3
Effect of 24 h pretreatment
with antioxidant
on cadmium
toxicity
99 (1995)
II-18
of mouse preimplantation
15
embryos
in vitro
(Cdl (PM)*
N’
% Blastocysts4
Mean cell no.’
% Control6
No antioxidant
No antioxidant
0
No
No
100
100
antioxidant
antioxidant
gM AT
pM AT
100
100
100
100
pM
gM
PM
PM
3.0
6.0
1.0
3.0
6.0
1.0
3.0
6.0
1.0
3.0
6.0
1.0
95
42
20
28
54
I8
13
50
15
I5
9a=
87a
93=
Ob
91a
87a
Ob
88a
93a
Ob
95a
93a
Ob
88a
94s
Ob
90=
1OOa
1ooa
84 zt
72 f
70 f
l2+
76 zt
68 f
I7 f
75 f
80 f
I2 f
79 f
76 f
I2 f
81 f
67 f
9*
82 f
78 f
73 f
loo
83
80
14
87
78
20
86
92
I4
91
87
14
93
77
II
94
90
84
Treatment
’
AT
BHA
BHA
BHA
100 pM BHT
100 FM BHT
100 PM BHT
100 pM Asc.
100 pM Asc.
100 pM Asc.
1 mM GSH
I mM GSH
1 mM GSH
I.0
3.0
6.0
1.0
3.0
6.0
54
15
13
49
I5
I7
55
20
I6
2a
5b
6b
1c
3b
7b
2d
3b
gaqb
lC
4=-b
5a,b
lC
4a*b
5b
IC
3a
qasb
5b
a-dValues within the same column with different superscripts
are significantly different at P I 0.05.
‘AT = dl-a-tocopherol,
BHA = butylated
hydroxyanisole,
BHT = butylated
hydroxytoluene,
Asc = sodium
ascorbate,
GSH =
glutathione.
2Embryos were pretreated in medium containing respective antioxidant
and no Cd for 24 h, then 8-cell embryos were cultured in
medium containing these concentrations
of Cd with respective antioxidant
for the remaining 48 h culture period.
‘N = the number of embryos.
‘?he percentage of embryos which developed into blastocysts after 72 h of culture. Raw numbers were used to perform Chi-square
analysis.
‘The mean number of cells per embryo. Values represent the mean f S.E.M..
6Th e percent of the control embryo cell number.
cantly lower in this group compared to controls.
Interestingly, although it has been reported that
compacted 8-cell embryos are less sensitive to Cd
toxicity than earlier stages of developing embryos
(Pedersen and Lin, 1978; Yu et al., 1985; Yu and
Chan, 1988), compared to embryos that were exposed to 1.OPM Cd throughout the entire 72 h culture period there were no differences in the
frequency of blastocyst formation or mean embryo
cell number (Tables 2, 3).
Pretreating embryos for 24 h with AT, BHA,
BHT, or ascorbate before transferring compacted
8-cell embryos to medium containing 1.0 PM Cd
resulted in a similar trend to that observed without
pretreating. Embryos that were exposed to AT,
BHA, BHT, or ascorbate prior to simultaneous
Cd/antioxidant exposure had higher mean embryo
cell numbers compared to embryos exposed only
to 1.0 PM Cd, but this trend was not statistically
significant. In contrast, pretreating 2-cell preimplantation embryos for 24 h in medium containing
GSH before exposure to 1.0 PM Cd with 1.0 mM
GSH improved embryonic development in terms
of the mean embryonic cell number compared to
controls (Table 3). The frequency of blastocyst
formation was also similar for this group compared to controls.
Embryos cultured in control medium for 24 h
and transferred at the &cell stage to medium containing only 3.0 PM Cd differentiated into blastocysts at a similar frequency as controls but had
fewer cells per embryo compared to controls
16
J M. Perers et ul. / To.ricologv
(Table 3). This observation is consistent with other
reports suggesting that compacted g-cell embryos
are less sensitive
to Cd than earlier
stages
(Pedersen and Lin, 1978; Yu et al., 1985; Yu and
Chan, 1988). Similarly, compared to embryos that
were exposed to 6.0 PM Cd for the entire 72 h culture period, there were no differences in the frequency of blastocyst formation,
while the mean
embryo cell number was higher in embryos that
were only exposed to 6.0 PM Cd for the last 48 h
of culture (Tables 2, 3).
Pretreating embryos for 24 h in medium containing ascorbate, AT, BHA, or BHT, before exposure to 3.0 or 6.0 PM Cd with respective
antioxidant
did not result in improved embryonic
differentiation
into blastocysts,
nor consistently
significant
higher mean embryo cell numbers,
compared to their respective controls. However,
for embryos exposed to AT before culture in medium with 6.0 ,uM Cd, the mean embryo cell number
was significantly
higher compared to that of embryos exposed solely to 6.0 PM Cd after 24 h of
culture. Pretreating embryos for 24 h with 1.O mM
GSH before transferring
compacted
g-cell embryos to medium containing
either 3.0 or 6.0 FM
Cd resulted in a significantly higher frequency of
blastocyst formation and mean embryo cell num-
Table 4
Influence of 4-h glutathione
(GSH) pretreatment
on cadmium
toxicity of mouse preimplantation
embryos in vrtro
Group
N’
% Blastocysts2
Mean cell
no. 3
% Control4
Control
GSH
58
56
93
98
2.5 pM Cd
5.0 pM Cd
10.0 gM Cd
62
82
57
79
17
4
93
91
77
26
12
100
97
83
28
13
f 4”
*44a
f 4b
f 3c
f Id
a-dValues within the same column with different
scripts are significantly different at P 5 0.05.
super-
‘N = the number of embryos.
‘The
percentage
of embryos
which
developed
into
blastocysts after 72 h of culture. Raw numbers were used to
perform Chi-square analysis.
‘The mean number of cells per embryo. Values represent the
mean * S.E.M.
?h e percent of the mean control embryo cell number.
99 i I995
I I I-IX
bers compared
6.0 PM Cd.
to embryos
exposed
only to 3.0 or
3.4. Pretreatment of embryos with GSH
Embryos that were pretreated
with 1.0 mM
GSH for 4 h prior to culture in medium without
Cd and antioxidants differentiated into blastocysts
and had similar mean embryo cell number compared to untreated controls (Table 4). Interestingly, compared to embryos that were cultured in
equimolar
concentrations
of Cd without GSH
pretreatment (Table l), embryos that were exposed
to GSH for 4 h and subsequently cultured in medium with 2.5-10.0
PM Cd differentiated
into
blastocysts at a greater frequency (Table 4). In addition, the mean embryo cell number was signilicantly higher in embryos pretreated with GSH for
4 h, and then transferred to medium with Cd and
no GSH compared
to non-pretreated
controls
(Tables 1, 4).
4. Discussion
These data show that for 2-cell mouse preimplantation
embryos, culture medium containing
Cd in concentrations
from l-2.5 PM results in a
decrease in both the frequency of blastocyst formation and mean embryo cell number after 72 h of
culture. Culture medium Cd in concentrations
>2.5 PM eliminates blastocyst formation and inhibits embryonic development to less than 2-3 cell
cycles. This observation is consistent with previous
reports that mouse preimplantation
embryos cultured in vitro are sensitive to low concentrations
of
Cd (Pedersen and Lin, 1978).
Our observation that morula stage embryos are
less sensitive to Cd-induced alterations in embryonic differentiation
and proliferation
than earlier
stages of embryos when cultured with 3.0 or 6.0
PM Cd is consistent
with previous
reports
(Pedersen and Lin, 1978; Yu et al., 1985; Yu and
Chan, 1988). However, in contrast to this observation, the inhibitory effect of 1.O FM Cd on embryo
development
was not influenced by delaying embryonic exposure to Cd until the morula stage of
development,
suggesting that at least some of the
effects of Cd-induced
embryotoxicity
are not influenced by the stage of embryogenesis.
J.M. Peters et al. / To.ricology 99 (1995)
Given the relative uniqueness of the effects of
1.OPM Cd exposure on embryo development compared to higher level of Cd exposure, it is of interest that the influence of low-level Cd exposure
on preimplantation embryo development can be
ameliorated by supplementing culture medium
with antioxidants. It is reasonable to suggest that
some of the events associated with Cd-induced embryotoxicity include alterations in oxidative status,
given that it has been shown that Cd exposure can
result in significantly elevated tissue levels of lipid
per-oxidation indices, as well as lower activity of
enzymes involved in preventing cellular oxidative
damage including superoxide dismutase and the
selenoenzyme glutathione peroxidase (Omaye and
Tappel, 1975; Gabor et al,, 1978; Klimczak et al.,
1984; Muller, 1986; Ochi et al., 1988; Shukla et al.,
1987; Hussain et al., 1987; Manta et al., 1991).
However, it has also been suggested that Cdinduced cellular toxicity is due in part to alterations in membrane structure/function
(Muller,
1987; Shukla et al., 1987), alterations in nucleic
acid metabolism (Christie and Costa, 1984;
Coogan et al., 1992), inhibition of energy metabolism (Pedersen and Lin, 1978; Muller, 1987) and/or
interference with metalloenzymes (Pedersen and
Lin, 1978; Hussain et al., 1987; Shukla et al.,
1987). Our observations support the idea that oxidative damage can be a major mediator of Cdinduced embryotoxicity at low Cd concentrations.
However, since an amelioration of Cd-induced
embryotoxicity by culture medium antioxidants
was not observed at high levels of Cd exposure (3.0
or 6.0 PM), with the exception of GSH (and AT to
a much lesser extent), it can be speculated that
there are other mechanisms, in addition to oxidative damage, which contribute to Cd-induced
developmental toxicity when the concentration of
the metal exceeds 1.0 PM and probable Cd uptake
increases.
Our results demonstrate that GSH can ameliorate Cd-induced embryotoxicity which is consistent with others who have shown that intracellular
GSH is involved in the protection of cells from Cd
exposure (Christie and Costa, 1984; Ochi et al.,
1987; Kang et al., 1989; Chan and Cherian, 1992;
Chubatsu et al., 1992; Li et al., 1993). Pretreating
embryos for 24 h with GSH, prior to exposure to
II-I8
17
medium containing both GSH and Cd in concentrations capable of eliminating blastocyst formation and significantly inhibiting cell divisions,
virtually eliminated Cd toxicity. In addition, pretreating embryos with GSH for 4 h followed by
thorough rinsing and subsequent culture in medium with toxic concentrations of Cd, and without
GSH, significantly improved embryo differentiation and proliferation, which suggests that intracellular GSH is involved in preventing Cd-induced
alterations in early development. This concept is
supported by reports that cells with low intracellular GSH, resulting from Cd exposure and/or inhibition of GSH synthesis, exhibit increased
sensitivity to Cd toxicity (Ochi et al., 1988; Kang
et al., 1989; Kang and Enger, 1990; Chan and
Cherian, 1992; Chubatsu et al., 1992). It is not
known exactly how GSH prevents cellular Cd toxicity, although it has been suggested that GSH
could alter the cellular uptake of Cd (Kang et al.,
1989; Chan and Cherian, 1992), affect the cellular
distribution of Cd (Kang et al., 1989; Chan and
Cherian, 1992), or inhibit Cd binding to protein
sulfhydryls thus preventing subsequent alterations
in the Cd-protein/enzyme function(s) (Li et al.,
1993). While our results are consistent with other
reports of antioxidants preventing Cd-induced
toxicity and suggest that GSH could be involved in
preventing oxidative damage, it is possible that
GSH is having other specific effects on Cd metabolism which in turn contribute
to GSHamelioration of Cd-induced embryotoxicity.
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