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). 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