Department of Pharmacology and Therapeutics, McGill University, Montreal, QC, Canada
1 To whom correspondence should be addressed at Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. Fax (514) 398-7120. E-mail: barbara.hales{at}mcgill.ca.
Received December 12, 2004; accepted March 3, 2005
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ABSTRACT |
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Key Words: c-Fos; c-Jun; teratogen; oxidative stress; glutathione; redox-sensitive transcription factor.
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INTRODUCTION |
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Oxidative damage to embryonic macromolecules has been observed after maternal exposure to various embryotoxic chemicals (Wells et al., 1997). Oxidative stress disturbs the cellular redox status, inducing oxidative damage to cellular macromolecules (DNA, lipid, and protein) and altering gene expression, possibly primarily by post-transcriptional modification of redox-sensitive transcription factors. Activator Protein-1 (AP-1) is a redox-sensitive early response transcription factor composed of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra2) families of nuclear proteins. The AP-1 members form heterodimers (Fos-Jun) or homodimers (Jun-Jun) that recognize the DNA consensus sequence 5'-TGAG/CTCA-3' (Angel and Karin, 1991
). Oxidative stress regulates the activation of AP-1 through a variety of mechanisms, including the phosphorylation of c-Fos or c-Jun by mitogen-activated protein kinase (MAPK), or oxidative/reductive modification of the cysteine residues present in the DNA binding sites of both c-Fos and c-Jun (Abate et al., 1990
; Hirota et al., 1997
). Increased AP-1 DNA binding activity in response to oxidative stress regulates the transcription of genes associated with antioxidant defense, cell cycle control, and apoptosis, all processes that are important in protecting embryos from oxidative damage or disrupting normal development. Previous in vitro studies with organogenesis stage whole embryos reported that culture induced an increase in the ratio of oxidized to reduced glutathione (GSSG:GSH) and AP-1 DNA binding activity (Ozolins and Hales, 1997
, 1999
); depletion of GSH with L-buthionine-S,R-sulfoximine induced embryotoxicity and prolonged AP-1 binding activity (Ozolins et al., 2002
).
NF-B, also a redox-sensitive transcription factor, is involved in development, positional signaling, and programmed cell death. NF-
B is a ubiquitous, pleiotropic, multisubunit transcription factor, made up of five subunits (p50, p52, p65 or RelA, c-Rel, and Rel-B) that form homodimers and heterodimers (Verma et al., 1995
). The predominant forms, p50 and p65 (RelA), are sequestered in the cytoplasm by association with I-
B; dissociation of the NF-
B:I-
B complexes, followed by translocation of the released NF-
B into the nucleus, activates NF-
B post-translationally. Exposures that induce oxidative stress, such as H2O2, tumor necrosis factor, phorbol esters, glutathione depletion, and ultraviolet (UV) or ionizing radiation, induce NF-
B DNA binding activity (Gius et al., 1999
; Haddad et al., 2000
; Li and Karin, 1998
, 1999
; Marshall et al., 2000
). The regulation of NF-
B is particularly important during development. NF-
B may act in the developing limb to disturb fibroblast growth factor (FGF) signals between the apical ectodermal ridge and the underlying mesenchyme (Bushdid et al., 1998
; Kanegae et al., 1998
). Inhibition of NF-
B/Rel activity results in a dysmorphic apical ectodermal ridge, loss of digit formation, and reversal of the direction of limb outgrowth. Interestingly, there is evidence that NF-
B may be involved in the teratogenesis of thalidomide (Hansen et al., 2002
) and phenytoin (Kennedy et al., 2004
).
Glutathione, the major intracellular nonprotein thiol, exists mainly in the reduced form (GSH). Upon oxidative stress, GSH is oxidized to GSSG, protecting cellular macromolecules; GSSG can be reduced to GSH in the presence of glutathione reductase and NADPH. The GSSG:GSH ratio is tightly regulated, maintaining cellular redox status; this ratio has been used as a marker of oxidative stress (Bajt et al., 2004; Lauterburg et al., 1984
; Ozolins and Hales, 1997
; Suliman et al., 2004
).
The goal of this study was to elucidate the impact of an exposure to hydroxyurea that is teratogenic during early organogenesis on oxidative stress and the activation of redox-sensitive transcription factors in the conceptus. Pregnant mice were treated with hydroxyurea on gestational day 9, during early organogenesis, the period of development most susceptible to oxidative stress and teratogenic insult.
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MATERIALS AND METHODS |
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Double staining for fetal skeletal analysis.
Ethanol-fixed fetuses were immersed in a water bath (70°C) for 7 s. The fetuses were skinned, eviscerated, and placed in 95% ethanol overnight. The ethanol was decanted and replaced with alcian blue solution (15 mg alcian blue; 80 ml 95% ethanol; 20 ml glacial acetic acid) for 24 h. The solution was then replaced with 95% ethanol. After 24 h, the ethanol was substituted with alizarin red S solution (25 mg/l alizarin red S in 1% potassium hydroxide) for 48 h. The dye was drained and replaced with 0.5% potassium hydroxide for 24 h. The skeletons were placed in a 2:2:1 solution (2 parts 70% ethanol: 2 parts glycerin: 1 part benzyl alcohol). After 24 h, stained skeletons were placed in 1:1 solution (1 part 70% ethanol:1 part glycerin) for subsequent evaluation and storage.
Glutathione determinations.
At the time of collection, the embryos and yolk sacs from each litter were placed in 60 µl of RIPA buffer (150 mM NaCl; 1% NP-40; 0.5% deoxycholate; 0.1 % SDS; 50 mM Tris, pH 7.5) containing 10 µl/ml protease inhibitor cocktail (Active Motif, Carlsbad, CA). The samples were homogenized with an ultrasonicator (Sonics & Materials Inc., Newtown, CT) and centrifuged at 10,000 x g for 10 min at 4°C. From each sample, 30 µl of supernatant was removed, added to 90 µl of 5% 5-sulfosalicylic acid, and centrifuged at 10,000 x g for 10 min at 4°C. Total glutathione (GSSG + GSH) and GSSG were measured spectrophotometrically, using an enzymatic recycling assay, as previously described (Ozolins and Hales, 1997). The remaining supernatant from each sample (prior to the addition of 5-sulfosalicylic acid) was aliquoted, flash frozen in liquid nitrogen, and stored at 80°C for protein assays (Bradford, 1976
) (Bio-Rad Laboratories, ON, Canada) and the ELISA tests.
Electrophoretic mobility shift assays (EMSA).
To prepare nuclear extracts, tissues were placed in 3 ml/g ice-cold complete hypotonic buffer (20 mM Hepes, pH 7.5; 5 mM NaF; 10 µM Na2MoO4; 0.1 mM EDTA; 1 µl/ml 1M DTT; 1 µl/ml detergent) and homogenized on ice with a pellet pestle (Kontes, Vineland, NJ). After 15 min incubation on ice, the samples were centrifuged at 4°C for 10 min at 850 x g. The pellet was resuspended in 100 µl hypotonic buffer and incubated on ice for 15 min. After the addition of 5 µl of detergent, the samples were vortexed for 10 s at the highest setting. After centrifugation at 14,000 x g for 30 s, 5 µl complete lysis buffer was added to the pellet, which was then sonicated for a few seconds and rocked on ice for 15 min. The supernatant was obtained by centrifugation at 14,000 x g for 10 min. After determination of the protein content (Bradford, 1976; Bio-Rad Laboratories, ON, Canada), the extracts were adjusted to the same protein concentration by the addition of complete lysis buffer, flash frozen in liquid nitrogen, and stored at 80°C. Hypotonic buffer, lysis buffer, dithiothreitol, protease inhibitor cocktail, and detergent were supplied in a Nuclear Extract Kit (Active Motif).
DNA fragments containing the AP-1 DNA binding site (5'-CGCTTGATGAGTCAGCCGGAA-3') were endlabeled with 32P-ATP by T4 polynucleotide kinase (Promega Corporation, Madison, WI) and purified by chromatography on a MicroSpin G-25 column (Amersham Biosciences UK Limited, Little Chalfont, UK). Nuclear extracts (9 µg protein) were incubated for 20 min at 4°C with 4 µl binding buffer supplied in a GelShift kit (Active Motif). Samples were then reacted with 1 µl labeled AP-1 probe (about 200,000 cpm) for 20 min at 4°C and electrophoresed on 5% polyacrylamide gels in 1x TGE buffer at 200 V. Gels were dried, and radioactivity was detected by autoradiography at 80°C. To confirm the specificity of binding, a 50-fold excess of the unlabeled oligonucleotide probes or mutated AP-1 oligonucleotides (5'-CGCTTGAGGAGTCGGCCGGAA-3') was preincubated with the sample for 20 min at 4°C.
Enzyme-linked immunosorbent assays (ELISA).
Embryo and yolk sac extracts were prepared as described above for glutathione determinations. The DNA binding activity of the c-Fos heterodimer complex or the c-Jun homo/heterodimers was detected using ELISA transcription factor assay kits (Active Motif). Briefly, a 96-well plate was coated with oligonucleotide containing the AP-1 consensus site, 5'-TGAGTCA 3'. Complete binding buffer (30 µl) (10 mM Hepes, pH 7.5; 8 mM NaCl; 12% glycerol; 0.2 mM ethylene diamine tetraacetic acid [EDTA]; 0.1% bovine serum albumin [BSA]; 0.88 mM dithiothreitol [DTT]; 0.17 mg/µl poly [d(1-C)]) was added to each well. To confirm the specificity of binding, 20 pmol AP-1 wild-type oligonucleotide or 20 pmol mutated oligonucleotide was added to two wells, respectively. As a positive standard, K562 cell nuclear extract (2.5 µg) in 20 µl complete lysis buffer (20 mM Hepes, pH 7.5; 400 mM NaCl; 20% glycerol; 0.1 mM EDTA; 10 mM NaF; 10 µM Na2MoO4; 1 mM NaVO3; 10 mM PNPP; 10 mM ß-glycerophosphate; 0.89 mM DTT; 0.01% protease inhibitor cocktail) was added to two wells containing complete binding buffer. Embryo or yolk sac extracts were diluted with complete lysis buffer to 20 µl and then added to the remaining wells; embryo or yolk sac extracts containing 20 µg protein were used for the examination of c-Jun dimer binding activity and 40 µg protein for the c-Fos dimer assays. The plates were incubated for 1 h and washed with washing buffer (10 mM phosphate buffer, pH 7.5; 50 mM NaCl; 0.1% Tween 20; 2.7 mM KCl). One hour after the addition of the phospho-c-Jun or c-Fos antibody, the plates were washed with washing buffer. Horseradish proxidase (HRP)-conjugated secondary antibody was added and incubated for 1 h. After washing, the plates were developed with developing solution (TMB substrate solution in 1% DMSO) for 510 min; the reaction was terminated by the addition of the stop solution (0.5 M H2SO4). The plates were read on a spectrophotometer (SPECTRAmax Plus 384, Molecular Devices Corporation, Sunnyvale, CA) at 450 nm with correction at 655 nm. The binding activity of samples was normalized by the relative absorbance of the positive standard and then divided by the protein content in the samples. Protein content was determined in triplicate (Bradford, 1976) (Bio-Rad Laboratories, ON, Canada)
The NF-B p65 or p50 dimer DNA binding activities were detected separately using the p65 or p50 transcription factor assay kits (Active Motif). The experiments were done as described above for AP-1 assays, except that the oligonucleotide containing the NF-
B consensus site 5'-GGGACTTTC-3' was immobilized on the 96-well plate, poly [d(1-C)] was replaced by the herring sperm DNA in the complete binding buffer, DTT was increased to 2 mM in the complete binding buffer and 5 mM in the complete lysis buffer, and Jurkat nuclear extract was used as a positive control for NF-
B p65 or p50 dimer binding activities. Embryo or yolk sac extracts containing 10 µg protein were used for the examination of p65 dimer binding activity and 40 µg protein for the p50 dimer.
Immunohistochemistry.
Embryos on GD 9 were fixed for 3 h at 4°C in Bouin's fluid. After fixation, the embryos were dehydrated in ethanol, embedded in paraffin, and serially sectioned. The tissue serial sections (5 µm) were dried, deparaffinized, and hydrated. The c-Fos immunohistochemistry staining was done using a rabbit ABC kit (Vector Laboratories, Inc., Burlingame, CA) as follows. The sections were incubated for 0.5 h in 0.3% H2O2 in PBS, followed by two rinses for 3 min each with phosphate buffered saline (PBS). The sections were then incubated in blocking serum (1.5% goat normal serum in PBS) for 0.5 h. The excess diluent solution was removed from the slides and the sections were incubated at 4°C overnight with a rabbit polyclonal anti-c-Fos IgG (sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:100 in blocking serum. After two rinses for 5 min each with PBS, the biotinylated anti-rabbit IgG diluted in blocking serum was applied to the sections for 30 min. After they were washed for 5 min in PBS, the sections were stained with a 3,3'-diaminobenzidine solution, and counterstained with methyl blue. As a negative control for c-Fos staining, the primary antibody was omitted.
Statistical analysis.
Statistical analyses were done by two-way analysis of variance (ANOVA), one-way ANOVA, or one-way ANOVA on rank's, as appropriate, using the SigmaStat computer program, followed by a post hoc Holm Sidak TEST. The a priori level of significance was p < 0.05.
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RESULTS |
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Double-stained skeletons of representative fetuses from each treatment group are shown in Figure 1D. The cartilage is stained blue and the bone is red. No skeletal abnormalities were apparent in the control and 400 mg/kg hydroxyurea treatment groups. In contrast, one third of the surviving fetuses in the 500 mg/kg treatment group had skeletal abnormalities; the rate of skeletal malformations reached 90.2% after exposure to 600 mg/kg hydroxyurea (Fig. 1C). The skeletal malformations observed included limb malformations (missing digits, hemimelia, amelia), curly tails, and short ribs (Fig. 1D).
The Effects of Hydroxyurea Exposure on AP-1 DNA Binding Activity
Electrophoretic mobility shift assays (EMSA) were done to test the hypothesis that teratogenic exposures of hydroxyurea induce AP-1 DNA binding activity (Fig. 2). In the absence of nuclear extract (free probe), the migration of the oligonucleotide was not impeded; addition of nuclear extract retarded the migration of the probe, indicated as the AP-1 DNA binding band. The addition of excess unlabeled wild-type oligonucleotide inhibited this binding, but excess mutated sequence did not influence the binding (Fig. 2A). An increase in AP-1 DNA binding activity (relative to control) was detected in the nuclear extracts prepared from the hydroxyurea-treated embryos and yolk sacs collected 3 h after treatment on GD9 (Fig. 2B and 2C). DNA binding activity increased in a dose-dependent manner with hydroxyurea dose (Fig. 2B).
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DISCUSSION |
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As the major cellular thiol-disulfide redox buffer, GSH is important in maintaining the cellular redox status. Changes in the intracellular thiol/disulfide status trigger signal transduction pathways which increase antioxidant defenses to maintain redox homeostasis, but at same time, influence normal cellular function, such as cell proliferation, differentiation, adhesion, or death. In the embryo, the ratio of GSSG/GSH was about twofold lower than in the yolk sac or maternal liver, suggesting that redox homeostasis in the embryo may be different from that in the maternal liver or yolk sac. Interestingly, after exposure of whole embryos in culture to diamide, more extensive S-thiolation of protein sulfhydryls by GSH was observed in the yolk sac compared to the embryo proper; thus, protein sulfhydryls in the yolk sac were more sensitive or accessible to oxidation (Hiranruengchok and Harris, 1995).
Exposure to teratogenic doses of hydroxyurea did not significantly induce oxidative stress in the maternal liver, embryo, or yolk sac, as assessed by the ratio of GSSG/GSH. Previous studies reported that antioxidants or a free radical scavenger delayed the cell death and partially rescued the developmental toxicity induced by hydroxyurea (DeSesso, 1981; DeSesso and Goeringer, 1990
; DeSesso et al., 1994
), suggesting that oxidative stress was involved in the teratogenesis of hydroxyurea. Although hydroxyurea exposure did not alter the ratio of oxidized to reduced glutathione in the whole conceptus, it remains possible that hydroxyurea may induce a localized oxidative stress in specific regions of the embryo. The antioxidant defense system is not equally distributed throughout the embryo; interestingly, limbs and the neural tube may have lower antioxidant defenses than do other regions (Mackler et al., 1998
).
Significantly, AP-1 DNA binding activity was dramatically increased, even after exposure to a dose of hydroxyurea (400 mg/kg), which did not induce developmental toxicity. Higher doses of hydroxyurea (500 and 600 mg/kg), which were teratogenic, further elevated AP-1 DNA binding activity. If, as seems likely, there are a number of components that contribute to hydroxyurea teratogenicity, the risk of having a malformation may be a continuous variable, with a threshold value (Fraser, 1976). Thus, it is possible that exposure to 400 mg/kg hydroxyurea induces AP-1 activity but is below the "threshold" for malformations. AP-1 regulates the transcription of genes that are important in cell differentiation, proliferation, and apoptosis, all basic processes during embryo development. AP-1 activity may be induced by various noxious stimuli, such as heat shock (Andrews et al., 1987
), heavy metals (Jin and Ringertz, 1990
; Ramesh et al., 1999
), and alkylating chemicals (Futscher and Erickson, 1990
), all of which can be teratogenic. Interestingly, c-Fos expression was elevated in the deciduas of embryos 6 h after exposure to ethanol on GD 8 (Poggi et al., 2003
). Stable overexpression of Bcl-2, a gene that protects against cell death by apoptosis, enhanced AP-1 DNA binding activity in cell lines (Feng et al., 2004
). In the presence of different stimuli and in various cell types, the activation of AP-1 may have different consequences, from protecting the cell to triggering cell death. It is not clear whether hydroxyurea induced activation of AP-1 indicates a role for AP-1 in protecting the embryo from the potential teratogenic insult or in disrupting normal embryonic development.
We have shown that c-Fos heterodimers contribute to the AP-1 DNA binding activity increase induced by hydroxyurea and that c-Jun homo-/hetero dimers do not. Different AP-1 members display multiple roles in regulating embryonic development. The targeting of individual AP-1 members in transgenic null mouse experiments has demonstrated that c-Jun, JunB, or Fra-1 are essential for embryo development; however, mice lacking c-Fos, JunD, and FosB are viable but have a variety of birth defects. C-Jun null mice died during the fetal period because of cardiac defects (Eferl et al., 1999); the knockout of Fra-I or JunB induced embryolethality as a consequence of defects in the extra-embryonic tissues (Schorpp-Kistner et al., 1999
; Schreiber et al., 2000
). Mice lacking FosB or JunD had a nurturing defect, or male sterility, respectively (Gruda et al., 1996
; Thepot et al., 2000
). Knockout c-Fos mice were viable and fertile but lacked osteoclasts, and they produced progeny with osteopetrosis (Johnson et al., 1992
; Wang et al., 1992
). Overexpression of c-Fos induced the transformation of osteoblasts leading to osteosarcomas (Wang et al., 1995
).
The developmental toxicity of hydroxyurea was characterized by skeletal malformations, including curly tails, abnormal limbs, and short ribs. The evidence that c-Fos is involved in osteogenesis (Closs et al., 1990; Sakano et al., 1997
) leads us to suggest that the effects of hydroxyurea on skeletal morphogenesis may be a consequence of disturbances in the regulation of c-Fos expression and activity. The relationship between where increased c-Fos immunostaining was found in the embryos exposed to hydroxyurea and the subsequent malformations is interesting. Increased c-Fos immunostaining was found in the somites of the hydroxyurea-exposed embryos, many of which are likely to develop shortened ribs. After exposure to 600 mg/kg hydroxyurea, c-Fos expression increased dramatically in the caudal region of the neural tube; neural tube defects at the caudal region of the tail result in curly tail malformations. Although we did not observe craniofacial defects in this study, hydroxyurea has been reported to induce craniofacial defects and cardiovascular anomalies in rats (Aliverti et al., 1980
). In utero exposure of rabbits to hydroxyurea altered the embryonic cardiovascular system as early as 2 min post-treatment, followed by petechial hemorrhages and hematomas in the forebrain, postocular region, and the mandibular and nasal processes, as well as apparent collapse of the vasculature in the forelimb bud (Millicovsky and DeSesso, 1980
). Our data show that exposure to doses of hydroxyurea that are teratogenic dramatically increased the immunoreactivity of c-Fos in the brain regions and in areas around the blood vessels. It is possible that the absence of cardiac and craniofacial defects among the fetuses in this study may be due to the protective effects of c-Fos in these regions or, alternatively, that the insult in these regions leads to an increase in embryolethality that is manifested in embryo resorptions. Elucidation of the different combinations of the c-Fos heterodimers activated by hydroxyurea in various regions of the embryo may help to explain specificity of the response to insult.
In contrast to the effect on AP-1 DNA binding activity, NF-B DNA binding activity was not influenced by hydroxyurea treatment, although both NF-
B and AP-1 are redox-sensitive transcription factors, and NF-
B is important in limb development. This may be due to a difference in the sensitivity of these two transcription factors to insult. These data indicate that different mechanisms are involved in regulating the response of embryonic AP-1 and NF-
B DNA binding activity to stress.
AP-1 DNA binding activity may be triggered by genotoxicity or by oxidative stress. Hydroxyurea inhibits ribonucleotide reductase, resulting in inhibition of DNA synthesis, affecting the cell cycle at G0 to S phase. AP-1 family members are involved in promoting cell cycle progression; specifically, AP-1 has been linked to the transcriptional regulation of cyclin D1. Stress induces a complex program of c-Fos and Fra-1 chromatin trafficking that is required for cyclin D1 expression during cell cycle re-entry (Burch et al., 2004). The induction of AP-1 DNA binding activity in the conceptus by hydroxyurea may disturb cell cycle recovery in the conceptus. Whether increased AP-1 DNA binding activity mediates the response of the conceptus to insult or represents a failed attempt at self-protection, it is evident that it represents a sensitive and early indicator of insult.
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ACKNOWLEDGMENTS |
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