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

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.

FIG. 1.

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).

FIG. 2.

2D gel electrophoresis of the tail samples obtained from embryos treated with saline (controls, A) or HU (600 mg/kg, B) and the corresponding 2D Western blots illustrating immunoreactive 4-HNE-protein adducts (C, control; D, HU 600 mg/kg treated). Spots 1–8 were analyzed by MS (see Table 1); (n = 2).

FIG. 3.

2D gel electrophoresis of the tail samples obtained from embryos treated with saline (control, A) or HU (600 mg/kg, HU600) (B) and the corresponding 2D Western blots illustrating GAPDH immunoreactive protein spots (control, C; HU600, D); (n = 2).

FIG. 4.

(A) Spectrophotometric analysis of NADH as a measure of GAPDH activity in whole embryo samples after treatment with saline (control), low-dose HU (400 mg/kg, HU400), or high-dose HU (600 mg/kg, HU600); (n = 7). (B) Lactate measurements of whole embryo samples treated with saline (control), low-dose HU (400 mg/kg, HU400), or high-dose HU (600 mg/kg, HU600); (n = 3). Asterisk denotes a significant difference between control and treated group (*p < 0.05).

FIG. 5.

Confocal microscopy images of saline (control, A) or HU (600 mg/kg, HU600, B)-treated embryos. Green fluorescence represents GAPDH immunoreactivity; (n = 5).

FIG. 6.

IMARIS 3D-rendered surface of lumbosacral somites (control sample) to show technique. (top left) Raw data, blue: DAPI, green: GAPDH; (top right) 3D-rendered surface of GAPDH and DAPI combined; (bottom left) 3D-rendered surface of DAPI; (bottom right) 3D surface of DAPI removed leaving nuclear GAPDH; (control n = 6, HU600 n = 7).

FIG. 7.

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).