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Evidence for Ras-dependent signal transduction in phenytoin teratogenicity
1Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S 2S2, Canada.
Abstract:
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.
Insights
Phenytoin causes birth defects by affecting Ras proteins, not by mutating the K-ras gene. Inhibiting Ras activation prevents these phenytoin-induced embryopathies in mice.
Area of Science:
- Developmental toxicology
- Molecular biology
- Teratology
Background:
- Reactive oxygen species (ROS) play dual roles in cell signaling and macromolecular damage.
- K-ras oncogene activation by ROS is implicated in certain cancers.
- Phenytoin, a teratogen, is known to initiate ROS, particularly in sensitive A/J mice.
Purpose of the Study:
- To investigate whether the Ras pathway mediates phenytoin embryopathy at the protein or gene level.
- To determine if Ras pathway activation is responsible for phenytoin-induced developmental abnormalities.
Main Methods:
- Mouse embryo culture using A/J and CD-1 strains.
- Exposure to phenytoin and a farnesyl-protein transferase inhibitor (alpha-hydroxyfarnesylphosphonic acid).
- Assessment of developmental parameters and analysis of K-ras gene mutations and Ras protein activation.
Main Results:
- Both A/J and CD-1 embryos exhibited similar susceptibility to phenytoin, showing reduced anterior neuropore closure, turning, yolk sac diameter, and somite development.
- The farnesyl-protein transferase inhibitor mitigated phenytoin embryotoxicity in A/J embryos (except for yolk sac diameter) and fully blocked it in CD-1 embryos.
- Phenytoin exposure increased GTP-bound Ras levels in both strains without causing mutations in K-ras codon 12.
Conclusions:
- Ras protein activation, rather than K-ras gene mutation, is implicated in phenytoin teratogenicity.
- The Ras pathway is a likely mediator of phenytoin embryopathy.
- These findings suggest a novel mechanism for teratogen-induced developmental toxicity involving posttranslational protein modification.