A considerable number of teratogens, including the anticonvulsant drug phenytoin and structurally related drugs and environmental chemicals, may be bioactivated by peroxidases, such as prostaglandin H synthase (PHS) and lipoxygenases (LPOs), to a reactive free radical intermediate that initiates birth defects. However, the molecular targets of the reactive free radical intermediates mediating chemical teratogenesis, and hence the fundamental determinants of susceptibility, are poorly understood. In these studies, a teratogenic dose of phenytoin (65 mg/kg), when injected into pregnant CD-1 mice during organogenesis on gestational day 12, initiated the oxidation of DNA in maternal hepatic and embryonic nuclei, forming 8-hydroxy-2'-deoxyguanosine. Significant maternal and embryonic DNA oxidation occurred at 6 and 3 h, respectively, suggesting relative embryonic deficiencies in free radical-related cytoprotective enzymes, although the rates appeared similar. Maximal DNA oxidation in both maternal and embryonic tissues occurred at 6 h, presumably reflecting the balance of DNA oxidation and repair, the latter of which appeared similar in both tissues. Inhibition of phenytoin-initiated embryonic DNA oxidation by the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (41.5 mg/kg), and by acetylsalicylic acid (10 mg/kg), an inhibitor of the cyclooxygenase component of PHS, was consistent with the previously reported reduction by these inhibitors of phenytoin-initiated murine birth defects. In vitro studies using a horseradish peroxidase (0.5 mg/ml)-H2O2 (5.45 micrograms/ml) bioactivating system for drug-initiated oxidation of 2'-deoxyguanosine (3.74 mM), indicated that the potency of xenobiotic-initiated formation of 8-hydroxy-2'-deoxyguanosine for the structurally related drugs and metabolites phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, trimethadione, dimethadione, l-mephenytoin, l-nirvanol, d-nirvanol (80 microM each), or thalidomide (64 microM), reflected their murine teratogenic potency. Given the relatively low activities of cytochromes P450, compared to PHS and LPOs, in human and rodent embryonic tissues, these data support the potential teratological importance of peroxidase-catalysed bioactivation of xenobiotics with structural similarities to phenytoin. These studies provide the first evidence that peroxidase-catalysed embryonic DNA oxidation may constitute a critical molecular mechanism mediating the teratogenicity of phenytoin and related drugs and environmental chemicals, and suggest the potential teratological importance of additional embryonic processes, such as DNA repair and tumor suppressor genes, as determinants of susceptibility.