Cadmium-Induced Changes in Apoptotic Gene Expression Levels and DNA Damage in Mouse Embryos Are Blocked by Zinc

Estíbaliz L. Fernández, Anne-Lee Gustafson1, Maria Andersson, Bjorn Hellman and Lennart Dencker2

Department of Pharmaceutical Biosciences, Division of Toxicology, Biomedical Center, Uppsala University, 75124 Uppsala, Sweden

Received June 12, 2003; accepted July 22, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium is a potent teratogen in laboratory animals, causing exencephaly when administered at early stages of development. Due to its heterogenicity with respect to molecular targets, the mechanisms behind cadmium toxicity are not well understood. In the present study, C57BL/6 pregnant mice were treated with saline, cadmium, or zinc plus cadmium at 8 days post-coitus and studied 24 h after exposure. Cadmium induced significant DNA damage in the embryonic cells. Cadmium also induced embryonic growth retardation, as well as a significant upregulation of p53, p21, and Bax transcription levels. At the same time, there was a downregulation of Bcl-2, shifting the equilibrium Bcl-2/Bax toward the apoptotic pathway. There was an increase in apoptotically stained cells in the cadmium-treated embryos, and pro-caspase-3 was significantly activated. Zinc pretreatment maintained DNA damage at the control levels. It also prevented cadmium-induced effects on the expression levels of p53 and p21. The cadmium-induced decrease in Bcl-2 was inhibited, whereas the Bax levels were maintained closer to the control values. The Bad transcripts did not change at any experimental condition. Morphologically, zinc could maintain the embryological development, where apoptotic areas were as in the controls, and decrease por-caspase-3 activation. In summary, cadmium administered to pregnant mice increased primary DNA damage and activated the apoptotic pathway. These effects could be ameliorated by zinc pretreatment, and, because of that, it is possible that the mechanisms of cadmium-induced teratogenicity are related to zinc metabolism.

Key Words: cadmium; zinc; apoptosis; DNA damage.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium, a transition metal with an extremely long biological half-life, has become a ubiquitous environmental pollutant during the past several decades due to its extensive and continued use in industry and agriculture (Goering et al., 1994Go). Chronic exposure to cadmium in humans is associated with bone, lung, and renal damage (Fells, 1999Go), and there is sufficient evidence in humans to classify cadmium and cadmium compounds as carcinogenic substances (IARC, 1993Go). So far, there are no studies available demonstrating that cadmium is a human teratogen, but maternal exposure to environmental cadmium, higher placental concentration (Loiacono et al., 1992Go), and/or fetal cadmium exposure (Frery et al., 1993Go) have been associated with lower birth weights in humans. Moreover, several studies on laboratory animals (mice, rats, and hamsters) have provided clear evidence that cadmium at higher doses is a potent developmental toxicant (Barr et al., 1973Go, Gale et al., 1973Go, Yu et al., 1985Go). Administered during gestation to laboratory mice, cadmium induces a large range of fetal malformations, varying depending on the strain of mice, dose, and time of administration (Hovland et al., 1999Go). If the embryos are exposed to cadmium before neurulation, the most dramatic malformation observed is an opening in the anterior neural pore (exencephaly). If the exposure occurs after the closure of the neural tube, there is a shift to rib and upper limb defects (Ferm, 1971Go; Nakashima et al., 1988Go).

Cadmium produces oxidative modifications of DNA, such as the formation of 8-hydroxydeoxyguanosine, and the generation of strand breaks in different cell types, for example,. liver and kidney cells (Forrester et al., 2000Go; Littlefield and Hass, 1995Go). Oxidative DNA damage produced by cadmium has been associated with an increased production of reactive oxygen species (ROS) (Ochi et al., 1987Go), and interactions between this metal and DNA repair enzymes (Assmus et al., 2000; Waalkes, 2000Go). Interestingly, there is evidence suggesting that Cd2+ binds covalently to N7 centers of adenine and guanine, and that it can form intrastrand bifunctional adenine-thymine (AT) adducts, suggesting a direct attack on the DNA molecule (Hossain and Huq, 2002Go).

At the cellular level, cadmium also induces different biochemical changes, which are typically associated with apoptosis (Robertson and Orrenius, 2000Go). Apoptosis is a widespread and morphologically distinct process of cell death that plays an important role during normal embryonic development, for example, in modeling structures; regulating cell number; and eliminating abnormal, misplaced, nonfunctional, or harmful cells (Manova et al., 1998Go). This process is particularly important in the development of the central nervous system, where neurons compete for a limited amount of survival factors (neurotrophic factors) and for the development and maintenance of the immune system (Collins et al., 1993; D’Sa-Eipper et al., 2000Go). In human lymphoma cells, cadmium has been shown to cause apoptosis by two independent pathways: the Ca2+–calpain and the caspase–mitochondria pathways (Li et al., 2000Go), indicating that apoptosis could play an important role in acute and chronic toxicity from this metal.

Zinc supplementation prior to cadmium administration prevents several of the effects observed when cadmium is added alone (Dreosti, 2001Go; Ferm and Carpenter, 1967). Thus it has been shown that zinc inhibits the apoptotic protease caspase-3 (Truong-Tran et al., 2001Go), stabilizes the structure of p53 and DNA repair proteins (Chai et al., 1999Go), acts as an antioxidant by decreasing ROS production in cell cultures (Dally and Hartwig, 1997Go; Szuster-Ciesielska et al., 2000Go), and prevents the gross teratogenic effects of cadmium by restoring normal development (Warner et al., 1984Go).

One approach to investigating complex developmental processes and coordinating them with genetic regulation has been to disrupt morphogenesis with specific teratogens and study their consequences at the molecular and morphological levels. In this paper we have studied primary DNA damage by performing the alkaline version of the comet assay, a cadmium-induced alteration in the expression levels of genes by using the reverse transcription-polymerase chain reaction (RT-PCR), and pro-caspse-3 activation by western blot. The genes under study (p53, p21, Bcl-2, Bax, and Bad) were selected according to their implication in cell-cycle regulation and in the apoptotic pathway. In addition, the protective effects of zinc on cadmium-induced teratogenicity were studied in an experimental group of dams, which received a zinc pretreatment injection before cadmium exposure.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment.
C57BL/6 strain of mice was used for these experiments due to their high teratogenic susceptibility to cadmium treatment (Hovland et al., 1999Go). A dose of 4 mg (22 µmol Cd2+)/kg body weight (bw) of CdCl2 was selected because of the great rate of survival embryos with exencephalic endpoint (Nakashima et al., 1988Go). C57BL/6 original breeding pairs were purchased from B&K Universal (Solna, Sweden). At our own facilities, the mice were maintained at constant temperature (24–26°C) and humidity (30–40%) on a 12:12 h light:dark cycle and given food and water ad libitum. Male and female mice were mated from 8:00 A.M. to 10:00 A.M., and the females were checked for the presence of a vaginal plug. This day was considered day 0 post-coitus (dpc). At 10:00 A.M. on the morning of 8 dpc, the dams were randomly separated into four different groups according to their treatment. All of the substances were dissolved and administered in sterile saline and given as an intraperitoneal (ip) injection (4.0 ml/kg bw). The control group received a saline injection, the second group received a single dose of 4.0 mg/kg bw of CdCl2, the third group got an ip injection of 8.0 mg (59.1 µmol Zn2+)/kg bw of ZnCl2 2 h before cadmium treatment, and the last group received a single dose of ZnCl2 (8.0 mg/kg bw). The selected zinc dose and time of administration were based on previous studies done by others (Ferm and Carpenter, 1967; Ferm et al., 1968Go). The dams were killed by cervical dislocation, and the embryos were dissected out 24 h (9 dpc) and 72 h (11 dpc) after treatment, respectively. Twenty-four h after cadmium treatment, at 8 dpc, was the time point selected for studying the cell death pathway because of the high increase in apoptotic cells observed by us and others at this time point (unpublished data; Mirkes and Little, 2000Go). The parameters used to assess embryonic growth and development were crown-rump length (CR length), somite number, and status of the neural tube (NT) (closed NT vs. open NT). Chemicals were obtained from Sigma, St. Louis, MO.

The animal experiments performed in this study were ethically approved by the Uppsala Djurförsökssetiska Nämnd, no. C 196/0 (as of September 29, 2000).

Cell viability.
Before the comet assay, cell viability was always evaluated, using the trypan blue exclusion assay. In brief, a 50-µl aliquot of single-cell suspension was collected from each treatment group and mixed with 50 µl of 0.25% trypan blue (Sigma St. Loius, MO). From this mixture a total volume of 10 µl was visualized in a Burker chamber under a microscope. Trypan blue-stained cells were considered nonviable.

Alkaline comet assay.
For the comet assay (Singh et al., 1988Go), 10 embryos from each treatment group were placed into phosphate buffered saline (PBS) and passed through a 0.4-µm diameter sieve in order to get a single-cell suspension. In the same way, one group of embryos was placed in freshly prepared 100-µM hydrogen peroxide at 37°C for 5 min, as a positive control. After centrifugation at 1100 rpm for 5 min, the cells were resuspended in 200 µl of PBS. Ten-µl cell suspension was added to 70 µl of 0.6% low-melting-point agarose in PBS, and 60 µl of this mixture was layered on top of a microscope slide precoated with 0.8% low-melting-point agarose in water. After the agarose had set, the slides were placed in a lysing solution (2.5-M NaCl, 100-mM Na2-ethylenediaminetetraacetic acid [Na2-EDTA], 10-mM Trizma base, 1% sodium lauryl sarcosinate, pH adjusted to 10 with NaOH, with 1% Triton X-100 and 10% dimethyl sulfoxide added before use) for 1 h at 4°C. From here on, all of the steps were performed in a cold room and under yellow light. The slides were then drained and placed in an electrophoresis unit (Sigma horizontal dual mode) containing an electrophoresis buffer (1-mM Na2-EDTA and 300-mM NaOH, pH > 13) for 40 min, to allow DNA unwinding. Thereafter, electrophoresis was performed for 5 min, using a field strength of 0.7 V/cm (300 mA, 25 V). After electrophoresis, the slides were neutralized with 0.4 M Trizma buffer (pH 7.5) for 15 min, dried at room temperature avoiding dust and particles, and kept in a sealed container until the day of image analysis (Vaghef et al., 1996Go).

The slides were stained with ethidium bromide (20 µg/ml, 35 µl/slide) and examined at x400 magnification, using a fluorescence microscope (excitation filter 515–560 nm, barrier filter 590 nm) attached to a black and white video camera connected to a computer-based image analysis system. Fifty comets per slide (12 to 16 slides per group of treatment) were randomly captured at a constant depth of the gel. Care was taken to avoid debris, comets without an identifiable nucleus, comets that were superimposed, and comets at the edges of the slides (Hellman et al., 1995Go). The image analysis program (Aequitas 1A, version 1.22, DDL Ltd, Cambridge, UK) with its special application for the comet assay (Autocell, version 2.0/9E, Reppalon AB, Sweden) was used for automatic analysis of the digitized images. Two different parameters were used as indicators of DNA damage: the tail moment and the percentage of DNA in the tail (Wiklund and Agurell, 2003Go). All calculations were based on absolute intensities, and all slides were coded before examination.

Reverse transcription (RT).
The embryonic part anterior from the otic vesicle, without including branchial arches and developing heart, was immediately homogenized in TRIzol reagent (Invitrogen, Groningen, The Netherlands), and the total RNA was isolated according to the manufacturer’s instructions. One µg of total RNA isolated from the control and cadmium-treated embryos with and without zinc pretreatment were treated with 1-U DNase I Amp grade (Life Technologies, Taby, Sweden) according to the manufacturer’s instructions and subjected to RT to produce cDNA. The RT was performed in a total volume of 40 µl/sample containing 0.1-µM oligo(dT) primer (Amersham Pharmacia Biotech, Uppsala, Sweden), 1 µl of 5 x RT buffer (Promega, Madison, WI), 1-mM dNTP, and 200-U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The reactions were performed at 37°C for 60 min and stopped by heating at 75°C for 10 min.

Polymerase chain reaction assay (PCR).
Five µl of the cDNA samples were amplified by PCR in a total volume of 50 µl containing 0.25-µM 5' and 3' primers, 10 x PCR buffer (50-mM KCl, 10 -M Tris–HCl, 0.1% Triton X-100, and 2.5 mM MgCl2), 0.2-µM dNTPs, and 1 µl of AdvanTaqTM DNA polymerase (Clontech, Palo Alto, CA). The samples were incubated at 94°C for 4 min, amplified (94°C for 15 s, 58°C for 30 s, and 68°C for 30 s), and ended with a final extension at 68°C for 7 min. PCR primers and the number of cycles shown in Table 1Go were used to detect transcripts from p53, p21, Bcl-2, Bax, Bad, and Cyclophillin (Cyc).


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TABLE 1 Oligonucleotide Primers Used for Gene Expression Level Analysis by Reverse-Transcription PCR Assay
 
After amplification, 15 µl of each PCR sample was run on a 1.5% agarose gel containing 0.4-µg ethidium bromide (EtBr)/ml. Images were captured in a gel documentation system (GDS 5000 from Ultra Violet Products Ltd, Cambridge, UK), and the bands were quantified using the public domain NIH Image program (developed at the U. S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image).

Western analysis.
For detection of caspase-3 protein activation, a total protein extract was made from neural tubes and craniofacial structures anterior to the otic vesicle of the three samples under study (control, cadmium, and zinc plus cadmium treatment) in the presence of a lysis buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1-mM Pefabloc, 10-µg/ml aprotinin, and 1-mM sodium orthovanadate). After centrifugation at 10,000g for 10 min, the protein concentration in each supernatant was determined according to standard methods (bicinchoninic acid protein assay; Pierce, Rockford, IL). The protein extracts (50 µg/lane) were resolved by SDS–polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride membrane (Hybond P; Amersham Pharmacia Biotech, Uppsala, Sweden). The membrane was blocked in 10% nonfat dry milk in Tris-buffered saline (TBS)/0.1% Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (ab) diluted in 5% nonfat dry milk in TBST. The primary ab used were goat polyclonal anti-caspase-3 at 1:2500 (Sigma, St. Louis, MO) and monoclonal anti-actin at 1:5000 (Sigma, St. Louis, MO). HRP-linked anti-mouse or anti-goat secondary antibodies (Amersham Pharmacia Biotech, Uppsala, Sweden) were used at 1:5000 for 1 h, and the membranes were washed twice with TBST and three times with TBS. Antigen–antibody complexes were visualized by development with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Uppsala, Sweden) according to manufacturer’s instructions. Bands were quantified as described for the PCR gels.

Detection of programmed cell death (PCD).
The embryos dissected out from the control and treated animals were fixed in 4% paraformaldehyde (PF) in PBT (0.1% Tween-20 in PBS) overnight, processed into 100% methanol, and stored at -20°C until use. PCD detection was performed with minor changes following the manufacturer’s instructions using the in situ Cell Death Detection Kit, AP (Roche Diagnostics, Bromma, Sweden). Briefly, the embryos were permeabilized for 10 min in 10 µg/ml of proteinase K followed by 4% PF for 10 min before inactivation of the endogenous peroxidase with H2O2. Incubation of the embryos with 50 µl of TUNEL reaction mixture for 1 h at 37°C performed the labeling of the 3'-OH DNA ends. For the colorimetric detection of the apoptotic cells, an AP converter was used, and the embryos were "developed" at room temperature with NTB/BCIP until the desired intensity was achieved.

Statistics.
A chi-square test was used in Table 2Go for comparing two proportions. A one-way ANOVA (analysis of variance) test was used to evaluate the differences between the control, cadmium, and zinc plus cadmium treatment, which were done in at least triplicate, and all contained a minimum of three experiments. Results were considered significant at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).


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TABLE 2 Developmental Status of Mouse Embryos from Each Group of Treatment at Day 8 Post-Coitus, after 24 h (Day 9) and 72 h (Day 11) of Exposure
 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Morphological Effects after Treatment
It is clear from Table 2Go that zinc could almost completely abolish the anterior neural tube defects (NTDs) caused by cadmium administration. At 9 dpc, 95 % of the control embryos had closed anterior NTs (100 % at 11 dpc) (Fig. 1AGo). In contrast, only 13 % of the embryos exposed to a single dose of cadmium had closed anterior NTs at 9 dpc, and this figure remained low at 11 dpc, indicating that most of these embryos had a permanent open anterior neural pore (Fig. 1BGo). The corresponding figures for embryos that were given zinc before cadmium was 44 % (9 dpc) (Fig. 1CGo) and 92 % (11 dpc), respectively. This demonstrates that, although the closure of the anterior neural pore was delayed at 9 dpc, this process could be completed in most of the embryos at 11 dpc.



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FIG. 1. Detection of apoptotic cells by TUNEL in control (A), cadmium (B), and zinc plus cadmium (C) treated embryos at 8 dpc + 24 h. Control and zinc pretreated embryos (A and C) had closed neural tubes, while in the cadmium-treated embryos the neural tube was open at the mid-hindbrain levels (B; arrow). Observe the apoptotic cells (black spots) in the midline of the fused neural tube (A and C), as compared to the increased cell death in the open mid-hindbrain neural tube and in the frontonasal area of the cadmium-treated embryos (B). OV, otic vesicle; HT, heart; FB, forebrain; MB, midbrain; HB, hindbrain; PNP, posterior neuropore.

 
Embryonic development of the cadmium-treated embryos was significantly delayed as demonstrated by CR length and somite number (Table 2Go). These parameters were maintained as in the control embryos when zinc was included as a pretreatment before cadmium as well as when it was given as a single exposure. These figures were true for both developmental stages studied, 9 and 11 dpc

DNA Damage in Mouse Embryos
Cell viability, expressed as the number of cells not stained with trypan blue divided by the total number of cells counted, was constantly found to be >85% in all treatment groups (Fig. 2Go). Tail moment and content of DNA in the tail were used as indicators of DNA damage. Tail moment was defined as the distance between the center of mass of the tail and the center of mass of the head, in microns, multiplied by the percentage of DNA in the tail. It is, as compared to the tail length parameter, better to use since it takes into account both tail length and intensity (Bowden et al., 2003Go).



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FIG. 2. Viability of the cells in suspension used for the comet assay, detected by the trypan blue exclusion assay. Control (saline), cadmium (4 mg/kg bw), and zinc + cadmium (8 mg/kg bw 2 h before cadmium injection + 4 mg/kg bw). H2O2 (100 µM) 5 min at 37°C as a positive control. *Significantly different at p <= 0.05.

 
To check the validity of the alkaline comet procedure, hydrogen peroxide was used as a positive control. As is shown in Figure 3Go, this compound clearly damages DNA. Embryos whose females were treated with cadmium showed an increased level of DNA damage in terms of single-strand breaks. When zinc was injected before cadmium, the tail moment and DNA content of the tail were only slightly, but not statistically, significantly affected when compared to the control, remaining close to control values.



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FIG. 3. Level of primary DNA damage as evaluated by the comet assay 24 h after cadmium administration at 8 day post-coitus. Control (saline), cadmium (4 mg/kg bw), and zinc + cadmium (8 mg/kg bw 2 h before cadmium injection + 4 mg/kg bw). H2O2 (100 µM) 5 min at 37°C as a positive control. **, ***Significantly different at p <= 0.01 and p <= 0.001, respectively.

 
Gene Expression Levels
Gene expression levels of the transcription factors p53, p21, Bcl-2, Bax, and Bad were determined in the control, cadmium, and zinc plus cadmium treated embryos 24 h after administration (Fig. 4Go). The tumor suppressor gene p53, which is involved in arresting the cell cycle after DNA damage, was significantly upregulated after cadmium exposure. In line with this, p21, a Cdk kinase inhibitor and a downstream target of p53, was also significantly upregulated. Bcl-2 and Bax produce mitochondrial-related proteins with antagonistic effects, the former having an anti-apoptotic activity and the latter a pro-apoptotic activity. Bcl-2 was significantly downregulated, more than twofold, as opposed to Bax, the expression levels of which were significantly upregulated. In contrast, another pro-apoptotic gene studied, Bad, did not change in expression but stayed at the basal level (Fig. 4Go).



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FIG. 4. Gene expression levels as evaluated by reverse transcription polymerase chain reaction 24 h after exposure. Treatment was performed on 8 days post-coitus embryos; control (saline), cadmium (4 mg/kg bw), and zinc + cadmium (8 mg/kg bw 2 h before cadmium injection + 4 mg/kg bw). The housekeeping gene Cyc normalized all gene expression values. *, **, ***Significantly different at p <= 0.05, p <= 0.01, and p <= 0.001, respectively.

 
In contrast to embryos given cadmium alone, pretreatment with zinc did not affect the expression levels of p53 and p21 genes, which remained at a control level. As is shown in Figure 4Go, both Bcl-2 and Bax expression levels were significantly upregulated in the embryos given zinc before cadmium. Bad expression was unaffected in all groups of treatment.

Activation of Caspase-3
Caspase-3 exists in all cells as an inactive pro-enzyme that requires cleavage at specific aspartate cleavage sites to yield active subunits of 17 and 12 kilodaltons (kD). The antibody used is directed against the N terminus of caspase-3, thus recognizing the intact pro-caspase-3 of 32 kD and the 17-kD cleaved fragment of caspase-3 by western blot. The levels of endogenous pro-caspase-3 were very similar between the treatments under study (Fig. 5Go). On the contrary, the 17-kD subunit was significantly increased, as compared to the controls in extracts prepared from the heads of cadmium-treated and in zinc plus cadmium–treated embryos. Zinc pretreatment seemingly could significantly decrease the activation of pro-caspase-3 caused by cadmium.



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FIG. 5. (A) Representative western blot picture of pro-caspase-3 activation 24 h after treatment in control (C), cadmium (Cd), and zinc-supplemented (Zn + Cd) embryos. Caspase activation (A) was determined by the presence of the cleaved subunit p17. Subunit p32 corresponded to the pro-enzyme form of caspase-3. Actin levels were also assessed to verify equivalent protein loading. (B) Quantified representation of pro-caspase-3 activation. The protein levels of actin were used to normalized the data. **Significantly different at p <= 0.01, as compared to the control and cadmium, respectively.

 
Programmed Cell Death
Control embryos showed apoptotic cells in the midline of the fused neural tube from the forebrain to the hindbrain, the ventral part of the otic vesicle, the dorsal part of the somites, and at the posterior neural pore (Fig. 1AGo). At the same stage and 24 h after cadmium treatment, the embryos showed an increased apoptotic signal in areas previously seen to contain apoptotic cells in the control embryos. A remarkable difference between the cadmium-treated and control embryos was, however, seen in the head region. Apoptotic cells were highly present in the neuroepithelium of the open neural folds at the mid- and hindbrain levels and in the frontonasal area (Fig. 1BGo). In zinc-pretreated embryos with normal morphology, the apoptotic cell number was as in the control embryos (Fig. 1CGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanisms by which cadmium causes teratogenicity are complex and may implicate a number of known and less known target molecules. Cadmium could, for example, disturb the function of DNA repair enzymes by zinc substitution (Waalkes, 2000Go); produce DNA adducts by direct interaction with adenine or guanine (Hossain and Huq, 2002Go); alter the expression levels of transcription factors implicated in apoptosis pathways (Robertson and Orrenius, 2000Go); form complexes with ascorbate and glutathione, inhibiting their scavenger capacity and therefore increase ROS burden that could attack the DNA molecule (Assmus et al., 2000); and/or inhibit carbonic anhydrase, altering yolk sac blood circulation (Alsen et al., 1976Go). In this paper we have shown that cadmium alters normal anterior embryonic development, causes primary DNA damage, and activates genes implicated in the apoptotic pathway when it is administered to mouse embryos during organogenesis. Most of these cadmium-induced effects could be prevented if the embryos were exposed to zinc prior to cadmium.

The most prominent malformation induced by cadmium in mammalian embryos is a failure of the anterior neural pore to close, leading to exencephalic embryos (Christley and Webster, 1983Go; Ferm, 1971Go; Webster and Messerle, 1980Go). In our study, 80 % of the embryos had an open neural tube 24 or 72 h after an injection of 4 mg/kg bw of cadmium. The anterior defects preferentially compromised the mid- and hindbrain structures, as was previously seen by Nakashima et al.(1988)Go. Embryos were also growth-retarded when compared to the controls, as measured by CR length and somite number. Pretreatment with zinc (molar excess 2.7) before cadmium administration has been shown to have protective effects on cadmium teratogenicity (Ferm and Carpenter, 1968Go). On the other hand, 8 mg/kg bw of zinc by itself did not have any obvious deleterious effects on embryonic development.

Programmed cell death (apoptosis) involves diverse input signaling pathways originating from the plasma membrane, mitochondria, the nucleus, or the cytoskeleton, and all these signals are critical for the involvement of apoptosis in normal brain morphogenesis (D’Sa-Eipper et al., 2001; Ferri and Kroemer, 2001Go). Twenty-four h after cadmium exposure, an increased apoptosis was observed in the mouse embryo (this paper and other, unpublished data; Christley and Webster, 1983Go; Mirkes and Little, 2000Go). Interestingly, several teratogens causing neural tube defects (NTDs) increased apoptosis in the tip of the neural folds (Mirkes and Little, 2000Go). These are areas where physiological apoptosis also takes place (Sah et al., 1995Go), being necessary for neural tube closure (Nonn et al., 2003Go). There are some controversies as to whether this increased apoptosis is part of the mechanism by which neural folds do not elevate and fuse, or if it is just a parallel phenomenon for a number of teratogens (Weil et al., 1997Go). In this paper we showed an increased apoptotic cells, detected by TUNEL, in cadmium-treated embryos as compared to the controls. On the contrary, these apoptotic areas were similar between control and zinc-pretreated embryos exhibiting normal embryogenesis.

As indicated in the present study, cadmium induced an upregulation in p53 expression levels, expected to stop cell cycle and allowing DNA repair mechanisms to act. p53 has been implicated in the regulation of both normal embryonic development and in the prevention of developmental defects after teratogenic exposure (Polyak et al., 1997Go; Sah et al., 1995Go). Developmental abnormalities occur in mice with loss of p53 as well as with overexpression of p53, suggesting that p53 levels are critical for normal cellular processes (Lozano and Liu, 1998Go). Specifically, p53- mice display defects in neural tube closure, resulting in an overgrowth of neural tissue in the region of the midbrain (exencephaly) (Sah et al., 1995Go). In parallel, we also showed an increased primary DNA damage in cells that were exposed to cadmium. What still remains to be shown is the order of these events. (i) If cadmium, through a process of apoptosis, leads to the activation of endonucleases, the inactivation of DNA repair enzymes, and, as a consequence, the production of DNA strand breaks; or (ii) if the primary effect of cadmium is at the DNA level, arresting the cell cycle to allow repair of the DNA damage and secondarily activating apoptotic pathways due to the severe damage. Recent studies demonstrated that cadmium can produce DNA adducts interacting directly with adenine or guanine (Hossain and Huq, 2002Go). Zinc administered 2 h before cadmium exposure was sufficient to maintain p53 expression levels and DNA damage as in the control embryos. Zinc has been shown to inhibit cadmium-induced apoptosis and the production of ROS in cell cultures (Szuster-Ciesielska et al., 2000Go). It is also well known that zinc is an essential metal involved in zinc-finger proteins that are factors controlling cell proliferation, differentiation, and apoptosis through the regulation of gene expression (Urrutia, 1997Go). The specific DNA-binding domain of p53 has a complex tertiary structure that is stabilized by zinc (Dreosti, 2001Go). Cadmium competes and displaces zinc from its normal localization, thus altering zinc homeostasis and its physiological function (Hartwig, 2001Go).

p53 directly interacts with the transcription of p21 that will in turn inactivate cyclin–Cdk complexes inhibiting the elongation step in DNA replication (Bunz et al., 1998Go). Here we show that p21 expression levels were also significally upregulated by cadmium. Besides, p53 regulates other apoptotic effector proteins interacting with members of the Bcl-2 family of proteins, including anti-apoptotic Bcl-2 and Bcl-XL, and pro-apoptotic Bax and Bad proteins (Sionov and Haupt, 1999Go). The activity of some of these proteins can be regulated by phosphorylation and by the ratio of inhibitors to activators, since various family members can dimerize with one another, antagonizing or enhancing the function of the other (Gross et al., 1999Go). Interactions between Bcl-2 and Bax regulate cytochrome c release from mitochondria and establish baseline sensitivity to apoptotic stimuli. In this study we observed a significant downregulation of Bcl-2 and a concurrent upregulation of Bax at the transcription level. It is thus likely that a decreased Bcl-2/Bax ratio promotes apoptosis signaling to be activated (Oltvai et al., 1993Go). By western blot we also showed activation of pro-caspase-3, the executioner caspase, after cadmium administration.

The injection of zinc before cadmium treatment can keep the expression levels of the genes under study at basal levels. Zinc supplementation thus maintained the expression levels of p53 and p21, as well as the Bcl-2/Bax ratio. Pro-caspase-3 activation could not be kept at basal levels, but its activation was not as dramatic as was observed in the cadmium-treated embryos. Anyway, there was a significant decrease in pro-caspase-3 activation between zinc-pretreated and cadmium-treated embryos. It should be said that several different pathways leading to the activation of pro-caspse-3 could be involved, and only a few were taken into consideration in this study.

Protective effects by zinc may be related to metallothionein (MT) activation. It is well known that zinc administration induced MT expression levels in different tissues (liver, kidney, etc.) (Shimoda et al., 2003Go). A role of MT is to detoxify heavy metals, such as cadmium and mercury, and MT induction can prevent apoptosis induced by them (Klaassen et al., 1999Go). In our study, MT induction by zinc prior to cadmium administration could account for the ameliorative effects observed.

In the present study we have clearly shown that cadmium can be a potent inducer of primary DNA damage in embryonic cells, and that it can activate several transcription factors implicated in the apoptotic pathway, such as p53, Bcl-2, and Bax. We have also shown that treatment with zinc could ameliorate the effects induced by cadmium, supporting previous data implicating that the mechanisms of cadmium teratogenicity are in some way related to zinc homeostasis.


    ACKNOWLEDGMENTS
 
The authors wish to acknowledge Raili Engdahl and Lena Norgren for their excellent technical assistance during this study. This work was supported by research grants from the Basque Government (BFI 98.83), up to 2002 by the Swedish funds MISTRA (98003), "A new strategy for the risk management of chemicals," and from 2003 by CFN (02/04-36) "A toxicogenomics approach to developmental toxicology," and by the Swedish Medical Research Council (MFR 99Pu-12723).


    NOTES
 
1 Current address: AstraZeneca Safety Assessment, AstraZeneca R & D, 15185 Södertälje, Sweden. Back

2 To whom correspondance should be addressed at Department of Pharmaceutical Biosciences, Division of Toxicology, Biomedical Center, Uppsala University, P.O. Box 594, SE-751 24 Uppsala, Sweden. Fax: +46-18-471-4253. E-mail: Lennart.Dencker{at}farmbio.uu.se. Back


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 MATERIALS AND METHODS
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 DISCUSSION
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