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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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Illustration of the separation of the mouse embryo. Head part (h), from the cranial end (top) of the embryo to the caudal end of the first branchial arch; body part (b), the region between the head and the tail part; tail part (t), from the cranial border of the third somites (counted from the caudal end) to the caudal end of the embryo. (A) Western blot analysis of 4-HNE-protein adducts in the three parts of embryos exposed to vehicle (saline) or HU (HU400, 400 mg/kg or HU600, 600 mg/kg). All 4-HNE-protein adducts were quantified by scan densitometric analysis, as indicated in (B). Each bar (mean ± SEM) represents three litters. “Dagger” denotes a significant difference between different parts of the embryo within the same treatment group (†p < 0.05).
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fig1: Illustration of the separation of the mouse embryo. Head part (h), from the cranial end (top) of the embryo to the caudal end of the first branchial arch; body part (b), the region between the head and the tail part; tail part (t), from the cranial border of the third somites (counted from the caudal end) to the caudal end of the embryo. (A) Western blot analysis of 4-HNE-protein adducts in the three parts of embryos exposed to vehicle (saline) or HU (HU400, 400 mg/kg or HU600, 600 mg/kg). All 4-HNE-protein adducts were quantified by scan densitometric analysis, as indicated in (B). Each bar (mean ± SEM) represents three litters. “Dagger” denotes a significant difference between different parts of the embryo within the same treatment group (†p < 0.05).

Mentions: Timed-pregnant CD1 mice were purchased from Charles River Canada Ltd (St Constant, Quebec, Canada) and housed in the McIntyre Animal Resource Centre (McGill University, Montreal, Quebec, Canada). Animal protocols were conducted in accordance with the guidelines outlined in the Guide to the Care and Use of Experimental Animals. CD1 mice mated between 8:00 and 10:00 A.M. (GD 0) were treated with saline (control) or HU (Aldrich Chemical Co., Madison, WI) at 400 or 600 mg/kg by ip injection at 9:00 a.M. on GD 9. Female mice were euthanized by CO2 and cervical dislocation on GD 9 at 3 h after treatment; each treatment group consisted of 7–12 litters. On GD 9, the uteri were removed and embryos were explanted to Hanks’ balanced salt solution (Invitrogen Canada, Inc., Ontario, Canada). Embryos were cut into head, body, and tail sections (Fig. 1). Protein extracts were obtained immediately for assessment of 4-HNE-protein adducts by Western blotting or the analysis of GAPDH enzymatic activity. For two-dimensional (2D) gel electrophoresis, the tail parts from four litters of embryos exposed to vehicle or HU600 were pooled and subjected to protein extraction immediately. Embryos were left whole for lactate assays and immunofluorescence.


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

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

Illustration of the separation of the mouse embryo. Head part (h), from the cranial end (top) of the embryo to the caudal end of the first branchial arch; body part (b), the region between the head and the tail part; tail part (t), from the cranial border of the third somites (counted from the caudal end) to the caudal end of the embryo. (A) Western blot analysis of 4-HNE-protein adducts in the three parts of embryos exposed to vehicle (saline) or HU (HU400, 400 mg/kg or HU600, 600 mg/kg). All 4-HNE-protein adducts were quantified by scan densitometric analysis, as indicated in (B). Each bar (mean ± SEM) represents three litters. “Dagger” denotes a significant difference between different parts of the embryo within the same treatment group (†p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Illustration of the separation of the mouse embryo. Head part (h), from the cranial end (top) of the embryo to the caudal end of the first branchial arch; body part (b), the region between the head and the tail part; tail part (t), from the cranial border of the third somites (counted from the caudal end) to the caudal end of the embryo. (A) Western blot analysis of 4-HNE-protein adducts in the three parts of embryos exposed to vehicle (saline) or HU (HU400, 400 mg/kg or HU600, 600 mg/kg). All 4-HNE-protein adducts were quantified by scan densitometric analysis, as indicated in (B). Each bar (mean ± SEM) represents three litters. “Dagger” denotes a significant difference between different parts of the embryo within the same treatment group (†p < 0.05).
Mentions: Timed-pregnant CD1 mice were purchased from Charles River Canada Ltd (St Constant, Quebec, Canada) and housed in the McIntyre Animal Resource Centre (McGill University, Montreal, Quebec, Canada). Animal protocols were conducted in accordance with the guidelines outlined in the Guide to the Care and Use of Experimental Animals. CD1 mice mated between 8:00 and 10:00 A.M. (GD 0) were treated with saline (control) or HU (Aldrich Chemical Co., Madison, WI) at 400 or 600 mg/kg by ip injection at 9:00 a.M. on GD 9. Female mice were euthanized by CO2 and cervical dislocation on GD 9 at 3 h after treatment; each treatment group consisted of 7–12 litters. On GD 9, the uteri were removed and embryos were explanted to Hanks’ balanced salt solution (Invitrogen Canada, Inc., Ontario, Canada). Embryos were cut into head, body, and tail sections (Fig. 1). Protein extracts were obtained immediately for assessment of 4-HNE-protein adducts by Western blotting or the analysis of GAPDH enzymatic activity. For two-dimensional (2D) gel electrophoresis, the tail parts from four litters of embryos exposed to vehicle or HU600 were pooled and subjected to protein extraction immediately. Embryos were left whole for lactate assays and immunofluorescence.

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