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Teratogen-induced oxidative stress targets glyceraldehyde-3-phosphate dehydrogenase in the organogenesis stage mouse embryo.

Schlisser AE, Yan J, Hales BF - Toxicol. Sci. (2010)

Bottom Line: Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism.The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%.We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

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Intensity mean analysis of nuclear GAPDH, provided by IMARIS software, of embryos treated with saline (control) or HU (600 mg/kg, HU600). Asterisk denotes a significant difference between control and treated group (*p < 0.05).
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fig7: Intensity mean analysis of nuclear GAPDH, provided by IMARIS software, of embryos treated with saline (control) or HU (600 mg/kg, HU600). Asterisk denotes a significant difference between control and treated group (*p < 0.05).

Mentions: In cell cultures, oxidative stress has been reported to induce the nuclear translocation of GAPDH (Nakajima et al., 2007; Oritz-Oritz et al., 2010); thus, we focused on image analysis to localize GAPDH. Initially, confocal microscopy presented discreet homogeneous spots of immunoreactive GAPDH in both vehicle- and HU-treated embryos, and it was challenging to accurately measure nuclear versus cytoplasmic localization in both treatment groups (Fig. 5). Consequently, we quantified the subcellular localization of GAPDH with z-stack imaging coupled with IMARIS, an advanced 3D imaging software. The lumbosacral somites, the caudal area with the most significant malformations, were the focus of this analysis. A 3D surface render of GAPDH revealed the subcellular localization of GAPDH. In both vehicle- and HU-treated groups, GAPDH immunoreactivity was unevenly distributed within the 3D projection of a given cell; although GAPDH was present both in the cytoplasm and nucleus, its density increased toward the nuclear membrane (Figs. 6A and 6B). A masking technique in IMARIS was applied that subtracted the cytoplasmic from the nuclear staining using DAPI as the nuclear border to quantify nuclear GAPDH (Fig. 6D). Interestingly, although GAPDH was present within the nucleus of nontreated groups, in agreement with the literature, a twofold increase in the nuclear localization of GAPDH was observed in embryos exposed to HU600 compared with saline-treated embryos (Fig. 7).


Teratogen-induced oxidative stress targets glyceraldehyde-3-phosphate dehydrogenase in the organogenesis stage mouse embryo.

Schlisser AE, Yan J, Hales BF - Toxicol. Sci. (2010)

Intensity mean analysis of nuclear GAPDH, provided by IMARIS software, of embryos treated with saline (control) or HU (600 mg/kg, HU600). Asterisk denotes a significant difference between control and treated group (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984529&req=5

fig7: Intensity mean analysis of nuclear GAPDH, provided by IMARIS software, of embryos treated with saline (control) or HU (600 mg/kg, HU600). Asterisk denotes a significant difference between control and treated group (*p < 0.05).
Mentions: In cell cultures, oxidative stress has been reported to induce the nuclear translocation of GAPDH (Nakajima et al., 2007; Oritz-Oritz et al., 2010); thus, we focused on image analysis to localize GAPDH. Initially, confocal microscopy presented discreet homogeneous spots of immunoreactive GAPDH in both vehicle- and HU-treated embryos, and it was challenging to accurately measure nuclear versus cytoplasmic localization in both treatment groups (Fig. 5). Consequently, we quantified the subcellular localization of GAPDH with z-stack imaging coupled with IMARIS, an advanced 3D imaging software. The lumbosacral somites, the caudal area with the most significant malformations, were the focus of this analysis. A 3D surface render of GAPDH revealed the subcellular localization of GAPDH. In both vehicle- and HU-treated groups, GAPDH immunoreactivity was unevenly distributed within the 3D projection of a given cell; although GAPDH was present both in the cytoplasm and nucleus, its density increased toward the nuclear membrane (Figs. 6A and 6B). A masking technique in IMARIS was applied that subtracted the cytoplasmic from the nuclear staining using DAPI as the nuclear border to quantify nuclear GAPDH (Fig. 6D). Interestingly, although GAPDH was present within the nucleus of nontreated groups, in agreement with the literature, a twofold increase in the nuclear localization of GAPDH was observed in embryos exposed to HU600 compared with saline-treated embryos (Fig. 7).

Bottom Line: Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism.The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%.We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

Show MeSH
Related in: MedlinePlus