A mechanism for thalidomide teratogenicity: Redox misregulation of nuclear factor kappa B in limb development

Thalidomide is a potent human and animal teratogen that has recently been introduced in the United States for medicinal purposes. Previous studies have not sufficiently identified the mechanism of thalidomide-induced limb reduction defects. We hypothesize that thalidomide selectively oxidizes rabbit but not rat embryonic limb buds, changing the intracellular redox environment and causing the misregulation of nuclear factor kappa B (NF-κB), a redox-sensitive transcription factor necessary for limb outgrowth.; Utilizing limb bud cell (LBC) cultures from thalidomide-resistant rat and thalidomide-sensitive rabbit embryos, thalidomide was found to deplete glutathione, shift redox potential and produce oxidative stress preferentially in rabbit but not rat LBC cultures. Species selectivity was consistent with thalidomide sensitivity. Rabbit LBCs showed preferential production of oxidative stress in the nucleus as compared to the cytosol, suggesting a greater redox shift in the nucleus that could modulate transcription factor binding.; Following transfection of LBCs with a green fluorescent protein reporter (pNF-κB-d2EGFP), thalidomide (100 μM) decreased NF-κB binding in rabbit LBCs but did not affect rat LBC NF-κB binding. The addition of N-acetyl cysteine restored NF-κB binding. Expression of NF-κB-dependent genes, Twist and Fgf-10, was determined by in situ hybridization. Fgf-8 expression was also measured as it is involved in a limb bud outgrowth feedback loop with FGF-10. Thalidomide treatment in utero (400 mg/kg/day) did not affect the expression of outgrowth genes in the rat embryonic limb, but significantly decreased expression of all outgrowth genes measured in the rabbit embryonic limb on gestation days 10, 11 and 12. Altered gene expression was prevented in rabbit with the pre-treatment of alpha-phenyl bultylnitrone (PBN), a free radical trapping agent. Transfection and in situ hybridization experiments demonstrate that thalidomide induced modulation of NF-κB/DNA binding both in vitro and in vivo under conditions of oxidative stress.; This dissertation describes a mechanism for thalidomide-induced limb reduction defects. Findings are consistent in sensitive and resistant species and show that changes in redox status modulates transcription factor signaling. Redox misregulation of transcription factors may serve as a model for other teratogens that cause specific malformations and functional abnormalities.