Evidence for Ras-dependent signal transduction in phenytoin teratogenicity.
Reactive oxygen species (ROS) can transduce intracellular signals or damage macromolecules, including mutational activation of the K-ras oncogene, particularly in A/J mice, which are reported to be highly sensitive to the ROS-initiating teratogen phenytoin. Here, we determined in embryo culture whether the Ras pathway mediated phenytoin embryopathy at the protein or gene level. Embryos from pregnant inbred A/J dams and outbred CD-1 dams were cultured with a therapeutic concentration of phenytoin, with or without alpha-hydroxyfarnesylphosphonic acid, an inhibitor of the enzyme farnesyl-protein transferase, which is required for posttranslational Ras activation. A/J and CD-1 embryos were similarly susceptible to phenytoin embryopathies, with reductions in anterior neuropore closure, turning, yolk sac diameter, and somite development (p < 0.05). The farnesyl-protein transferase inhibitor blocked phenytoin embryotoxicity in A/J embryos for all parameters except yolk sac diameter (p < 0.05) and completely blocked embryotoxicity in CD-1 embryos (p < 0.05). Embryonic DNA did not show phenytoin-initiated mutations in codon 12 of the K-ras gene in either A/J or CD-1 embryos, but phenytoin substantially increased the levels of GTP-bound Ras in both CD-1 and A/J embryos. These results provide the first direct evidence that Ras proteins may be involved in the teratogenicity of phenytoin, likely via a mechanism other than mutational activation.
Winn
Louise M
LM
Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S 2S2, Canada.
Wells
Peter G
PG
D000014:Abnormalities, Drug-Induced / Q000235:genetics*; D000818:Animals; D000927:Anticonvulsants / Q000633:toxicity*; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005204:Farnesol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D011905:Genes, ras; D051379:Mice; D008805:Mice, Inbred A; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D009924:Organ Culture Techniques; D063065:Organophosphonates / Q000494:pharmacology; D010672:Phenytoin / Q000633:toxicity*; D011247:Pregnancy; D015398:Signal Transduction; D013045:Species Specificity; D013723:Teratogens
C080570:(alpha-hydroxyfarnesyl)phosphonic acid; D000927:Anticonvulsants; D063065:Organophosphonates; D013723:Teratogens; D005204:Farnesol; D010672:Phenytoin; D004247:DNA; D015689:Oncogene Protein p21(ras)