Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.
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-kappaB), 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-kappaB 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-kappaB 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 alpha-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-kappaB 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.
Hansen
Jason M
JM
Toxicology Program, Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109, USA.
Gong
Siew-Ging
SG
Philbert
Martin
M
Harris
Craig
C
D000818:Animals; D005121:Extremities / Q000196:embryology*; D018507:Gene Expression Regulation, Developmental; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008164:Luminescent Proteins / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010663:Phenylmercury Compounds / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013723:Teratogens; D013792:Thalidomide; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D016328:NF-kappa B; D010663:Phenylmercury Compounds; D013723:Teratogens; D049452:Green Fluorescent Proteins; D013792:Thalidomide; D005978:Glutathione; C031504:mercury orange