Misregulation of gene expression in the redox‐sensitive NF‐κb‐dependent limb outgrowth pathway by thalidomide

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Abstract
Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide‐induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox‐sensitive processes, such as transcription factor activation and/or binding. Nuclear factor‐kappa B (NF‐κB), a redox‐sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide‐induced redox shifts. Thalidomide‐resistant Sprague‐Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide‐sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF‐κB and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf‐10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF‐κB binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N‐acetylcysteine and α‐N‐t‐phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide‐treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf‐8, and Fgf‐10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf‐8, and Fgf‐10 expression. These findings show that NF‐κB binding is diminished due to selective thalidomide‐induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth.