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Protection against cadmium-induced teratogenicity in vitro by glycine
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Toxicology in Vitro 22 (2008) 75–79 www.elsevier.com/locate/toxinvit
Protection against cadmium-induced teratogenicity in vitro by glycine Norma Paniagua-Castro a, Gerardo Escalona-Cardoso a, Eduardo Madrigal-Bujaidar b, Elizdath Martı´nez-Galero c, Germa´n Chamorro-Cevallos c,* a
Department of Physiology, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico b Department of Morphology, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico c Department of Pharmacy, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico Received 20 May 2007; accepted 13 August 2007 Available online 23 August 2007
Abstract Cadmium (Cd) has an embryotoxic effect on laboratory animals expressed by growth retardation and induced craniofacial and skeletal malformations. Some of the mechanisms suggested to account for this reproduction damage include oxidative stress and lipoperoxidation. It has been shown that due to its antioxidant activity, glycine protects embryos from in vivo cadmium-induced teratogenicity. However, it is not known whether such protection may also be found in embryo cultures and what its possible mechanism of action might be. The purpose of this study was to determine whether the effect of glycine (1 mM) against the damage of CdCl2 (1 lM) on the embryo, was direct or indirect. The amino acid was found to have significantly counteracted the effects of Cd by reducing the growth retardation and preventing the opening of the neural tube. Such protective effect seems to be partly due to decreased lipoperoxidation levels in embryos exposed to the metal, which would make it a direct effect. 2007 Elsevier Ltd. All rights reserved. Keywords: Cadmium teratogenicity; Glycine protection
1. Introduction
Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CAT, catalase; Cd, cadmium; CdCl2, cadmium chloride; CO2, carbon dioxide; DNA, deoxyribonucleic acid; GD, gestational day; Gly, glycine; GPx, glutathione peroxidase; GSH, glutathione; GSSG/GSG, oxidized glutathione/reduced glutathione; HCl, hydrochloric acid; MDA, malondialdehyde; N2, nitrogen; O2, oxygen; ROS, oxygen reactive species; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; TNF, tumor necrosis factor. * Corresponding author. Address: IPN, Agencia de Correos 220, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico. Tel.: +52 55 5396 8929; fax: +52 55 5729 6000x46211. E-mail address: [email protected] (G. Chamorro-Cevallos). 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.08.005
Cadmium (Cd) is considered to be one of the most toxic substances occurring in the environment, due to the kind of deleterious effects it has and its long half life in the body (Nordberg and Nordberg, 2000; Domingo, 1994). Chronic exposure to Cd in humans causes mainly bone, kidney and pulmonary defects (Berglund et al., 2002) and Cd has been classified as a carcinogen in humans (IARC, 1993). Cd has also been reported as having an effect on laboratory animal reproduction (RCHAS, 1996). Teratogenic Cd effects vary depending on the dose, period of administration, route used to expose, and animal species (Hovland et al., 1999; Mahalik et al., 1995). In embryos of rodents exposed to Cd prior to neurulation, the most common malformation seen is in open neural tube
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defects. On the other hand, when exposure occurs after the neural tube is closed, bone alterations occur (Fernandez, 2005). Cd embryotoxicity is partly due to oxidative DNA changes associated to increased production of oxygen reactive species (ROS), such as superoxide ion, hydroxyl radicals, and hydrogen peroxide (Stohs et al., 2000) decreased antioxidant enzyme levels, and the interaction of this metal with the enzymes that repair damaged DNA (Asmuss et al., 2000; Ochi et al., 1987). Cd has also been found to activate several transcription factors involved in apoptosis processes (Fernandez et al., 2003). It has been demonstrated that some teratogenic effects of Cd are counteracted by pretreatment with bismuth nitrate in mice (Naruse and Hayashi, 1989) and by administration of caffeine (Lutz and Beck, 2000). In the same way, it has been reported that the developmental toxicity by cadmium in mouse preimplantation embryos in vitro was modulated by select antioxidants, such as ascorbate, butylated hydroxyanisole, butylated hydroxytoluene and glutathione (Peters et al., 1995). Glycine is the simplest and a non-essential amino acid in the body which protects the cells from Cd toxicity (Shaikh and Tang, 1999) and from other toxic substances (Tariq and Moutaery, 1997; Zhong et al., 1998; Zhong et al., 2003). It has been proven that the protection caused by this amino acid against these toxicities is due to its antioxidant effects (Matilla et al., 2002: Senthilkumar et al., 2004a,b). In vivo studies demonstrated that glycine decreases the mouse embryo malformations induced by Cd (PaniaguaCastro et al., 2007), those caused by hyperglycemia (Martı´nez et al., 2003), as well as those provoked by cholinomimetic drugs in chicken embryos (Landauer, 1975). However, in vitro studies have not been carried out in order to determine the possible mechanism of protection of glycine. The advantage of the studies of whole culture embryos is their capacity to reveal whether the protective effect is direct or indirect. Accordingly, the purpose of this research was to determine whether glycine can protect embryos directly from the effect of cadmium, and whether this effect is due to the antioxidant activity. 2. Materials and methods Experiments in animals were approved by the Laboratory Animal Care Committee of our Institution and were conducted in compliance with the Mexican Official Standard (NOM – 062-200-1999) regarding technical specifications for production, care and use of laboratory animals. 2.1. Chemicals and substances CdCl2 and glycine were of analytical grade and were obtained from commercial sources. CdCl2 and glycine were dissolved in water.
2.2. Embryo culture Virgin Swiss Webster female mice were maintained in a room at a constant temperature (24 ± 2 C) and humidity (50%) and under a controlled 12-h light cycle. Males were placed with females for 3 h between 6:00 a.m. and 9:00 a.m. during the last hours of the dark cycle. The presence of a vaginal plug at the end of this period indicated that mating had occurred and was rated as day 0 of pregnancy. After eight days of gestation, dams were killed by cervical dislocation and the embryos removed from the uterus. The decidual tissue, trophoblast, Reicherts’ membrane and parietal yolk sac were carefully removed, leaving the ectoplacental cone intact. Four embryos with three to five somites were placed in a culture tube with a solution of 3.5 ml of culture medium and 0.5 ml of 0.9% saline or 0.5 ml of treatment substances. Embryos were initially gassed in the 5% O2/5% CO2/90% N2 saturated medium. After 12 h they were re-gassed with 20% O2/5% CO2/ 75% N2, at 24 h with 40% O2/5% CO2/55% N2 and at 36 h with 95% O2/2.5% CO2/2.5% N2, and remained in incubation for another 12 h. All cultures were spun at 40 rpm at 37 C. Embryos were randomized in four groups receiving (1) 0.5 ml of 0.9% saline (control), (2) 0.5 ml of glycine, (3) 0.5 ml of CdCl2, or (4) 0.25 ml of glycine plus 0.25 ml of CdCl2. Both glycine and cadmium were added to the medium to a final concentration of 1 mM or 1 lM, respectively. These concentrations were chosen based on previous experiments in our laboratory that determined the maximum concentration of glycine that was not teratogenic, and the maximum teratogenic concentration of cadmium that was not embryolethal (results not shown). The culture medium was prepared from blood of male Wistar rats by aseptic dorsal aortic puncture with the animals under ether anesthesia. Blood was centrifuged at 3500 rpm for 5 min at 5 C in a cryocentrifuge. Sera were removed and heat inactivated at 56 C for 30 min and then stored at 70 C. At the end of the culture period all embryos were removed from the culture vessel and examined microscopically. The explants were evaluated for growth and differentiation using Brown and Fabro (1981) criteria, which included increased yolk sac diameter and circulation, crown-rump length and number of somites. In order to assess the morphology, an examination was made of embryonic flexion, heart, neural tube, brain development, otic, optic and olfactory systems, branchial bars, mandibular and maxillary processes, and fore and hind limb-buds. Morphogenesis was scored as normal or abnormal compared to untreated controls. Two people, independently each other, blindly scored, the embryos for morphogenesis and the average of both results was considered. Some embryos were stored at 20 C for later DNA quantification using Hoescht colorant (Labarca and Paigen, 1980).
N. Paniagua-Castro et al. / Toxicology in Vitro 22 (2008) 75–79
2.3. Lipid peroxidation Lipid peroxidation was assessed by measuring the malondialdehyde (MDA) content according to Buege and Aust (1978), as modified by Kotch et al. (1995) in controls, glycine and CdCl2-exposed embryos, and CdCl2 exposed embryos co-treated with glycine. Embryo homogenates, prepared by pooling the tissue of three controls or three treated embryos, were mixed thoroughly with trichloroacetic acid [(TCA)-thiobarbituric acid (TBA) – HCl [15% (w/v) TCA – 0.375% (w/v) TBA – 0.25 N HCl]. The solution was heated for 60 min in a boiling water bath. After cooling, the flocculent was removed by centrifugation at 1000 g for 10 min. The absorbance of the sample was determined at 535 nm against an only reagent blank. The MDA content in the sample was calculated as a 1.56 · 10 5 M 1 cm 1 extinction coefficient. Altogether 12 embryos for each treatment group and 12 control embryos were used for this analysis. The protein content of these embryos was determined by the coomassie blue method (Bradford, 1976) using bovine serum albumin (BSA) as a standard.
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length were also reduced. Furthermore, the percentage of embryos in which the anterior neural tube remained open was significantly increased. Table 1 also shows that the embryos exposed to 1 mM glycine for 48 h had no significant changes in the variables mentioned. Moreover, in none of the embryos was an open neural tube observed. Adding glycine (1 mM) and CdCl2 (1 lM) to the culture medium significantly increased the morphology score and the number of somites, the embryo DNA, the yolk sac diameter, and the cephalic length compared to CdCl2 group. In addition, there was a significant decrease in the number of embryos with a neural tube opening compared to the CdCl2 group. The MDA embryo content is shown in Fig. 1. Lipoperoxidation levels are seen to be significantly higher than in CdCl2 exposed embryos, while embryo cultures in a glycine supplemented medium showed a lower MDA content. Adding glycine (1 mM) to the CdCl2 culture medium (1 lM) significantly decreased embryo lipoperoxidation compared to the CdCl2 group.
2.4. Statistical analysis 120
*
100
nM MDA/mg protein
Differences between experimental and control groups were statistically analyzed as follows. Somite numbers were analyzed using the ANOVA method and Duncan’s test for multiple comparisons. Embryonic rotations were analyzed using a ranked ANOVA and Newman-Keuls test for multiple comparisons. The incidence of neural tube and craniofacial defects was analyzed using a Fisher’s exact probability test between experimental and control groups. The MDA content in treated and control groups was analyzed using a one-way ANOVA test and then the Turkey test (post hoc test). A p < 0.05 value was considered to be significant.
80
*
*a
60 40 20 0
3. Results
Control
Table 1 shows that 48 h embryo cultures in a CdCl2 medium at a 1 lM concentration presented a significantly decreased morphology score. The number of somites, the embryo DNA content, the yolk sac diameter, and the brain
Gly
CdCl2
Gly+CdCl2
Fig. 1. MDA content in embryos cultivated in medium with different treatment: Gly: glycine 1 mM: CdCl2: cadmium chloride 1 lM; Gly + CdCl2: glycine 1 mM plus CdCl2 1 lM. (*) denotes significant difference compared to the control group; (a) difference compared to CdCl2 1 lM.
Table 1 Comparison of growth and developmental parameters between control and treated embryos 48 h post-treatment initiation
Control Glycine 1 mM CdCl2 1 lM Glycine 1 mM + CdCl2 1 lM a 1 *
N
Morphological score
Somite No.
Embryonic DNA (lg)
Yolk-sac diameter (mm)
Crown-rump length (mm)
Cephalic length (mm)
Embryos with ONT1 (%)
20 20 18 20
53.4 ± 0.76 52.2 ± 0.41 43.2 ± 2.1* 50.1 ± 1.46a
33.7 ± 0.9 32.8 ± 1.0 31.4 ± 1.27* 36.4 ± 1.07a
30.5 ± 1.18 32.0 ± 1.5 28.3 ± 0.9* 30.2 ± 0.9a
8.7 ± 0.27 8.5 ± 0.22 7.6 ± 0.24* 8.0 ± 0.24a
7.5 ± 0.25 7.1 ± 0.27 7.0 ± 0.28 7.2 ± 0.14
3.7 ± 0.13 3.5 ± 0.14 3.4 ± 0.13* 3.95 ± 0.10a
5 0 88.9* 5a
p < 0.05 vs CdCl2-treated group. Open neural tube (anterior). p < 0.05 vs control group.
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4. Discussion Cadmium given to dams in early gestation stages results in fetus malformations such as excencephaly, anophthalmia, cleft palate, phocomelia, and other skeletal deficiencies, in addition to growth retardation (Domingo, 1994). These alterations have been associated with multiple metal effects both on the maternal and embryo bodies, including placenta necrosis and poor nutrient supply to the embryo (Levin and Miller, 1980), deficiency of iron (Akesson et al., 2002; Piasek et al., 2004), magnesium (Semczuk and Semczek-Sikora, 2001); zinc (Brzo´ska and Moniuszko-Jakoniuk, 2001; Warner et al., 1984), or calcium (Otha et al., 2002). While the maternal and placental factors play a major role in Cd toxicity, the direct action on the embryo sac and the embryo itself may not be obvious in in vivo studies. In this study, as with many embryo culture models, the maternal factor was ruled out and CdCl2 was analyzed for its direct effect on embryos. The results showed that Cd increased the number of embryos with an open anterior neuropore expressed during the fetal stage which is known as excencephaly, which is consistent with the reports by other authors (Domingo, 1994; Hovland et al., 1999). It also resulted in growth and embryo differentiation retardation. However, even though the results suggest that Cd produces free radicals, the mechanism whereby they are generated is still unknown. Hypotheses submitted by other authors suggest a direct rupture of DNA chains and the resulting generation of free radicals such as 8-hydroxideoxiguanosine (Fernandez et al., 2003) and oxidant species such as superoxide anions and nitrogen oxide (Shaikh et al., 1999) in several cell types. The combination of these actions may be the cause for the cadmium embryotoxic effect. In general, antioxidant agents prevent the toxic effects of metals on liver and kidney cells in rats (Shaikh et al., 1999); in mouse embryo cells (Warren et al., 2000), and human HeLa (Dally and Hartwig, 1997; Szuster-Ciesielska et al., 2000). In this sense, glycine has shown its cell protective effect in several studies. In pre-implanted embryos, it acts as an osmotic regulating agent that increases cell viability (Dawson and Baltz, 1997; Dawson et al., 1998). It prevents hepatotoxicity and nephrotoxicity resulting from ischemic shock and reperfusion thus decreasing the production of oxygen reactive species (Matilla et al., 2002; Weinberg et al., 1990). The glycine antioxidant mechanism has been associated to macrophage inactivation and inhibition of TNFa. It also prevents decreased levels of SOD, GPx, and CAT (Mauriz et al., 2001; Wheeler et al., 2000), enzymes which are inhibited by cadmium (Morales et al., 2006; Fernandez, 2005; Koyoturk et al., 2006; Hoissain and Bhattacharya, 2006). At the same time, it has been observed that glycine decreases the parameters of oxidative stress such as TBARS and the GSSG/GSG relation (Matilla et al., 2002).
Martı´nez et al. (2003) showed that glycine prevents fetus dismorphogenesis, which is due to maternal diabetes. The authors suggest that such protection results from decreased protein glycation and from the potential antioxidant action. In a previous in vivo study, Paniagua-Castro et al. (2007) reported that 4 mg/kg of CdCl2 given to dams subcutaneously on days 8 through 10 of gestation mainly produce fetus excencephaly associated with increased embryo lipoperoxidation levels, which were significantly reduced by giving the dams 2% glycine. According to the results of this study, adding glycine (1 mM) to the CdCl2 contaminated culture medium significantly decreased the incidence of neural tube opening in embryos and prevented growth retardation, by means of a direct action, while increasing the DNA content, the number of somites and the morphology score. Such protective effect seems to be partly due to decreased lipoperoxidation levels in embryos exposed to the metal, possibly preventing the production of Cd generated ROS as well as the decrease of antioxidant enzymes. Conflict of interest statements There is no conflict of interest involved in this study. Acknowledgements The authors thank Dr. Francois Spezia and Dr. Michael Collins for their teaching and advising on whole embryo culture. A special acknowledgement is due to Dr. Antonio Pen˜a for the facilities provided for DNA determinations. References Akesson, A., Berglund, M., Schutz, A., Bjellerup, P., Bremme, K., Vahter, M., 2002. Cadmium exposure in pregnancy and lactation in relation to iron status. American Journal of Public Health 92, 284–287. Asmuss, M., Mullenders, L.H.F., Eker, A., Hartwig, A., 2000. Differential effects of toxic metal compounds on the activities of Fpg and XPA, two zinc finger proteins involved in DNA repair. Carcinogenesis 21, 2097–2104. Berglund, M., Akesson, A., Bjellerup, P., Vahter, M., 2002. Metal-bone interactions. Toxicology Letters, 219–225. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. In: Fleischer, S., Packer, L. (Eds.), . In: Biomembranes: Part C: biological oxidations, microsomal, cytochrome P-450, and other hemoprotein systems. Academic Press, San Diego, pp. 302–310. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry 72, 248–254. Brown, N.A., Fabro, S., 1981. Quantitation of rat embryonic development in vitro: a morphological scoring system. Teratology 24, 65–78. Brzo´ska, M.M., Moniuszko-Jakoniuk, J., 2001. Interactions between cadmium and zinc in the organism. Food and Chemical Toxicology 39, 967–980. Dally, K., Hartwig, A., 1997. Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 5, 1021–1026. Dawson, K.M., Baltz, J.M., 1997. Organic osmolytes and embryos: substrates of the Gly and b transport systems protect mouse zygotes
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Toxicology in Vitro 22 (2008) 75–79 www.elsevier.com/locate/toxinvit
Protection against cadmium-induced teratogenicity in vitro by glycine Norma Paniagua-Castro a, Gerardo Escalona-Cardoso a, Eduardo Madrigal-Bujaidar b, Elizdath Martı´nez-Galero c, Germa´n Chamorro-Cevallos c,* a
Department of Physiology, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico b Department of Morphology, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico c Department of Pharmacy, National School of Biological Sciences, National Polytechnic Institute, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico Received 20 May 2007; accepted 13 August 2007 Available online 23 August 2007
Abstract Cadmium (Cd) has an embryotoxic effect on laboratory animals expressed by growth retardation and induced craniofacial and skeletal malformations. Some of the mechanisms suggested to account for this reproduction damage include oxidative stress and lipoperoxidation. It has been shown that due to its antioxidant activity, glycine protects embryos from in vivo cadmium-induced teratogenicity. However, it is not known whether such protection may also be found in embryo cultures and what its possible mechanism of action might be. The purpose of this study was to determine whether the effect of glycine (1 mM) against the damage of CdCl2 (1 lM) on the embryo, was direct or indirect. The amino acid was found to have significantly counteracted the effects of Cd by reducing the growth retardation and preventing the opening of the neural tube. Such protective effect seems to be partly due to decreased lipoperoxidation levels in embryos exposed to the metal, which would make it a direct effect. 2007 Elsevier Ltd. All rights reserved. Keywords: Cadmium teratogenicity; Glycine protection
1. Introduction
Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; CAT, catalase; Cd, cadmium; CdCl2, cadmium chloride; CO2, carbon dioxide; DNA, deoxyribonucleic acid; GD, gestational day; Gly, glycine; GPx, glutathione peroxidase; GSH, glutathione; GSSG/GSG, oxidized glutathione/reduced glutathione; HCl, hydrochloric acid; MDA, malondialdehyde; N2, nitrogen; O2, oxygen; ROS, oxygen reactive species; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; TNF, tumor necrosis factor. * Corresponding author. Address: IPN, Agencia de Correos 220, Prolongacio´n Carpio y Plan de Ayala s/n, Colonia Santo Toma´s, 11340 Me´xico D.F., Mexico. Tel.: +52 55 5396 8929; fax: +52 55 5729 6000x46211. E-mail address: [email protected] (G. Chamorro-Cevallos). 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.08.005
Cadmium (Cd) is considered to be one of the most toxic substances occurring in the environment, due to the kind of deleterious effects it has and its long half life in the body (Nordberg and Nordberg, 2000; Domingo, 1994). Chronic exposure to Cd in humans causes mainly bone, kidney and pulmonary defects (Berglund et al., 2002) and Cd has been classified as a carcinogen in humans (IARC, 1993). Cd has also been reported as having an effect on laboratory animal reproduction (RCHAS, 1996). Teratogenic Cd effects vary depending on the dose, period of administration, route used to expose, and animal species (Hovland et al., 1999; Mahalik et al., 1995). In embryos of rodents exposed to Cd prior to neurulation, the most common malformation seen is in open neural tube
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defects. On the other hand, when exposure occurs after the neural tube is closed, bone alterations occur (Fernandez, 2005). Cd embryotoxicity is partly due to oxidative DNA changes associated to increased production of oxygen reactive species (ROS), such as superoxide ion, hydroxyl radicals, and hydrogen peroxide (Stohs et al., 2000) decreased antioxidant enzyme levels, and the interaction of this metal with the enzymes that repair damaged DNA (Asmuss et al., 2000; Ochi et al., 1987). Cd has also been found to activate several transcription factors involved in apoptosis processes (Fernandez et al., 2003). It has been demonstrated that some teratogenic effects of Cd are counteracted by pretreatment with bismuth nitrate in mice (Naruse and Hayashi, 1989) and by administration of caffeine (Lutz and Beck, 2000). In the same way, it has been reported that the developmental toxicity by cadmium in mouse preimplantation embryos in vitro was modulated by select antioxidants, such as ascorbate, butylated hydroxyanisole, butylated hydroxytoluene and glutathione (Peters et al., 1995). Glycine is the simplest and a non-essential amino acid in the body which protects the cells from Cd toxicity (Shaikh and Tang, 1999) and from other toxic substances (Tariq and Moutaery, 1997; Zhong et al., 1998; Zhong et al., 2003). It has been proven that the protection caused by this amino acid against these toxicities is due to its antioxidant effects (Matilla et al., 2002: Senthilkumar et al., 2004a,b). In vivo studies demonstrated that glycine decreases the mouse embryo malformations induced by Cd (PaniaguaCastro et al., 2007), those caused by hyperglycemia (Martı´nez et al., 2003), as well as those provoked by cholinomimetic drugs in chicken embryos (Landauer, 1975). However, in vitro studies have not been carried out in order to determine the possible mechanism of protection of glycine. The advantage of the studies of whole culture embryos is their capacity to reveal whether the protective effect is direct or indirect. Accordingly, the purpose of this research was to determine whether glycine can protect embryos directly from the effect of cadmium, and whether this effect is due to the antioxidant activity. 2. Materials and methods Experiments in animals were approved by the Laboratory Animal Care Committee of our Institution and were conducted in compliance with the Mexican Official Standard (NOM – 062-200-1999) regarding technical specifications for production, care and use of laboratory animals. 2.1. Chemicals and substances CdCl2 and glycine were of analytical grade and were obtained from commercial sources. CdCl2 and glycine were dissolved in water.
2.2. Embryo culture Virgin Swiss Webster female mice were maintained in a room at a constant temperature (24 ± 2 C) and humidity (50%) and under a controlled 12-h light cycle. Males were placed with females for 3 h between 6:00 a.m. and 9:00 a.m. during the last hours of the dark cycle. The presence of a vaginal plug at the end of this period indicated that mating had occurred and was rated as day 0 of pregnancy. After eight days of gestation, dams were killed by cervical dislocation and the embryos removed from the uterus. The decidual tissue, trophoblast, Reicherts’ membrane and parietal yolk sac were carefully removed, leaving the ectoplacental cone intact. Four embryos with three to five somites were placed in a culture tube with a solution of 3.5 ml of culture medium and 0.5 ml of 0.9% saline or 0.5 ml of treatment substances. Embryos were initially gassed in the 5% O2/5% CO2/90% N2 saturated medium. After 12 h they were re-gassed with 20% O2/5% CO2/ 75% N2, at 24 h with 40% O2/5% CO2/55% N2 and at 36 h with 95% O2/2.5% CO2/2.5% N2, and remained in incubation for another 12 h. All cultures were spun at 40 rpm at 37 C. Embryos were randomized in four groups receiving (1) 0.5 ml of 0.9% saline (control), (2) 0.5 ml of glycine, (3) 0.5 ml of CdCl2, or (4) 0.25 ml of glycine plus 0.25 ml of CdCl2. Both glycine and cadmium were added to the medium to a final concentration of 1 mM or 1 lM, respectively. These concentrations were chosen based on previous experiments in our laboratory that determined the maximum concentration of glycine that was not teratogenic, and the maximum teratogenic concentration of cadmium that was not embryolethal (results not shown). The culture medium was prepared from blood of male Wistar rats by aseptic dorsal aortic puncture with the animals under ether anesthesia. Blood was centrifuged at 3500 rpm for 5 min at 5 C in a cryocentrifuge. Sera were removed and heat inactivated at 56 C for 30 min and then stored at 70 C. At the end of the culture period all embryos were removed from the culture vessel and examined microscopically. The explants were evaluated for growth and differentiation using Brown and Fabro (1981) criteria, which included increased yolk sac diameter and circulation, crown-rump length and number of somites. In order to assess the morphology, an examination was made of embryonic flexion, heart, neural tube, brain development, otic, optic and olfactory systems, branchial bars, mandibular and maxillary processes, and fore and hind limb-buds. Morphogenesis was scored as normal or abnormal compared to untreated controls. Two people, independently each other, blindly scored, the embryos for morphogenesis and the average of both results was considered. Some embryos were stored at 20 C for later DNA quantification using Hoescht colorant (Labarca and Paigen, 1980).
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2.3. Lipid peroxidation Lipid peroxidation was assessed by measuring the malondialdehyde (MDA) content according to Buege and Aust (1978), as modified by Kotch et al. (1995) in controls, glycine and CdCl2-exposed embryos, and CdCl2 exposed embryos co-treated with glycine. Embryo homogenates, prepared by pooling the tissue of three controls or three treated embryos, were mixed thoroughly with trichloroacetic acid [(TCA)-thiobarbituric acid (TBA) – HCl [15% (w/v) TCA – 0.375% (w/v) TBA – 0.25 N HCl]. The solution was heated for 60 min in a boiling water bath. After cooling, the flocculent was removed by centrifugation at 1000 g for 10 min. The absorbance of the sample was determined at 535 nm against an only reagent blank. The MDA content in the sample was calculated as a 1.56 · 10 5 M 1 cm 1 extinction coefficient. Altogether 12 embryos for each treatment group and 12 control embryos were used for this analysis. The protein content of these embryos was determined by the coomassie blue method (Bradford, 1976) using bovine serum albumin (BSA) as a standard.
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length were also reduced. Furthermore, the percentage of embryos in which the anterior neural tube remained open was significantly increased. Table 1 also shows that the embryos exposed to 1 mM glycine for 48 h had no significant changes in the variables mentioned. Moreover, in none of the embryos was an open neural tube observed. Adding glycine (1 mM) and CdCl2 (1 lM) to the culture medium significantly increased the morphology score and the number of somites, the embryo DNA, the yolk sac diameter, and the cephalic length compared to CdCl2 group. In addition, there was a significant decrease in the number of embryos with a neural tube opening compared to the CdCl2 group. The MDA embryo content is shown in Fig. 1. Lipoperoxidation levels are seen to be significantly higher than in CdCl2 exposed embryos, while embryo cultures in a glycine supplemented medium showed a lower MDA content. Adding glycine (1 mM) to the CdCl2 culture medium (1 lM) significantly decreased embryo lipoperoxidation compared to the CdCl2 group.
2.4. Statistical analysis 120
*
100
nM MDA/mg protein
Differences between experimental and control groups were statistically analyzed as follows. Somite numbers were analyzed using the ANOVA method and Duncan’s test for multiple comparisons. Embryonic rotations were analyzed using a ranked ANOVA and Newman-Keuls test for multiple comparisons. The incidence of neural tube and craniofacial defects was analyzed using a Fisher’s exact probability test between experimental and control groups. The MDA content in treated and control groups was analyzed using a one-way ANOVA test and then the Turkey test (post hoc test). A p < 0.05 value was considered to be significant.
80
*
*a
60 40 20 0
3. Results
Control
Table 1 shows that 48 h embryo cultures in a CdCl2 medium at a 1 lM concentration presented a significantly decreased morphology score. The number of somites, the embryo DNA content, the yolk sac diameter, and the brain
Gly
CdCl2
Gly+CdCl2
Fig. 1. MDA content in embryos cultivated in medium with different treatment: Gly: glycine 1 mM: CdCl2: cadmium chloride 1 lM; Gly + CdCl2: glycine 1 mM plus CdCl2 1 lM. (*) denotes significant difference compared to the control group; (a) difference compared to CdCl2 1 lM.
Table 1 Comparison of growth and developmental parameters between control and treated embryos 48 h post-treatment initiation
Control Glycine 1 mM CdCl2 1 lM Glycine 1 mM + CdCl2 1 lM a 1 *
N
Morphological score
Somite No.
Embryonic DNA (lg)
Yolk-sac diameter (mm)
Crown-rump length (mm)
Cephalic length (mm)
Embryos with ONT1 (%)
20 20 18 20
53.4 ± 0.76 52.2 ± 0.41 43.2 ± 2.1* 50.1 ± 1.46a
33.7 ± 0.9 32.8 ± 1.0 31.4 ± 1.27* 36.4 ± 1.07a
30.5 ± 1.18 32.0 ± 1.5 28.3 ± 0.9* 30.2 ± 0.9a
8.7 ± 0.27 8.5 ± 0.22 7.6 ± 0.24* 8.0 ± 0.24a
7.5 ± 0.25 7.1 ± 0.27 7.0 ± 0.28 7.2 ± 0.14
3.7 ± 0.13 3.5 ± 0.14 3.4 ± 0.13* 3.95 ± 0.10a
5 0 88.9* 5a
p < 0.05 vs CdCl2-treated group. Open neural tube (anterior). p < 0.05 vs control group.
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4. Discussion Cadmium given to dams in early gestation stages results in fetus malformations such as excencephaly, anophthalmia, cleft palate, phocomelia, and other skeletal deficiencies, in addition to growth retardation (Domingo, 1994). These alterations have been associated with multiple metal effects both on the maternal and embryo bodies, including placenta necrosis and poor nutrient supply to the embryo (Levin and Miller, 1980), deficiency of iron (Akesson et al., 2002; Piasek et al., 2004), magnesium (Semczuk and Semczek-Sikora, 2001); zinc (Brzo´ska and Moniuszko-Jakoniuk, 2001; Warner et al., 1984), or calcium (Otha et al., 2002). While the maternal and placental factors play a major role in Cd toxicity, the direct action on the embryo sac and the embryo itself may not be obvious in in vivo studies. In this study, as with many embryo culture models, the maternal factor was ruled out and CdCl2 was analyzed for its direct effect on embryos. The results showed that Cd increased the number of embryos with an open anterior neuropore expressed during the fetal stage which is known as excencephaly, which is consistent with the reports by other authors (Domingo, 1994; Hovland et al., 1999). It also resulted in growth and embryo differentiation retardation. However, even though the results suggest that Cd produces free radicals, the mechanism whereby they are generated is still unknown. Hypotheses submitted by other authors suggest a direct rupture of DNA chains and the resulting generation of free radicals such as 8-hydroxideoxiguanosine (Fernandez et al., 2003) and oxidant species such as superoxide anions and nitrogen oxide (Shaikh et al., 1999) in several cell types. The combination of these actions may be the cause for the cadmium embryotoxic effect. In general, antioxidant agents prevent the toxic effects of metals on liver and kidney cells in rats (Shaikh et al., 1999); in mouse embryo cells (Warren et al., 2000), and human HeLa (Dally and Hartwig, 1997; Szuster-Ciesielska et al., 2000). In this sense, glycine has shown its cell protective effect in several studies. In pre-implanted embryos, it acts as an osmotic regulating agent that increases cell viability (Dawson and Baltz, 1997; Dawson et al., 1998). It prevents hepatotoxicity and nephrotoxicity resulting from ischemic shock and reperfusion thus decreasing the production of oxygen reactive species (Matilla et al., 2002; Weinberg et al., 1990). The glycine antioxidant mechanism has been associated to macrophage inactivation and inhibition of TNFa. It also prevents decreased levels of SOD, GPx, and CAT (Mauriz et al., 2001; Wheeler et al., 2000), enzymes which are inhibited by cadmium (Morales et al., 2006; Fernandez, 2005; Koyoturk et al., 2006; Hoissain and Bhattacharya, 2006). At the same time, it has been observed that glycine decreases the parameters of oxidative stress such as TBARS and the GSSG/GSG relation (Matilla et al., 2002).
Martı´nez et al. (2003) showed that glycine prevents fetus dismorphogenesis, which is due to maternal diabetes. The authors suggest that such protection results from decreased protein glycation and from the potential antioxidant action. In a previous in vivo study, Paniagua-Castro et al. (2007) reported that 4 mg/kg of CdCl2 given to dams subcutaneously on days 8 through 10 of gestation mainly produce fetus excencephaly associated with increased embryo lipoperoxidation levels, which were significantly reduced by giving the dams 2% glycine. According to the results of this study, adding glycine (1 mM) to the CdCl2 contaminated culture medium significantly decreased the incidence of neural tube opening in embryos and prevented growth retardation, by means of a direct action, while increasing the DNA content, the number of somites and the morphology score. Such protective effect seems to be partly due to decreased lipoperoxidation levels in embryos exposed to the metal, possibly preventing the production of Cd generated ROS as well as the decrease of antioxidant enzymes. Conflict of interest statements There is no conflict of interest involved in this study. Acknowledgements The authors thank Dr. Francois Spezia and Dr. Michael Collins for their teaching and advising on whole embryo culture. A special acknowledgement is due to Dr. Antonio Pen˜a for the facilities provided for DNA determinations. References Akesson, A., Berglund, M., Schutz, A., Bjellerup, P., Bremme, K., Vahter, M., 2002. Cadmium exposure in pregnancy and lactation in relation to iron status. American Journal of Public Health 92, 284–287. Asmuss, M., Mullenders, L.H.F., Eker, A., Hartwig, A., 2000. Differential effects of toxic metal compounds on the activities of Fpg and XPA, two zinc finger proteins involved in DNA repair. Carcinogenesis 21, 2097–2104. Berglund, M., Akesson, A., Bjellerup, P., Vahter, M., 2002. Metal-bone interactions. Toxicology Letters, 219–225. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. In: Fleischer, S., Packer, L. (Eds.), . In: Biomembranes: Part C: biological oxidations, microsomal, cytochrome P-450, and other hemoprotein systems. Academic Press, San Diego, pp. 302–310. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry 72, 248–254. Brown, N.A., Fabro, S., 1981. Quantitation of rat embryonic development in vitro: a morphological scoring system. Teratology 24, 65–78. Brzo´ska, M.M., Moniuszko-Jakoniuk, J., 2001. Interactions between cadmium and zinc in the organism. Food and Chemical Toxicology 39, 967–980. Dally, K., Hartwig, A., 1997. Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 5, 1021–1026. Dawson, K.M., Baltz, J.M., 1997. Organic osmolytes and embryos: substrates of the Gly and b transport systems protect mouse zygotes
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