OXIDATIVE DAMAGE IN CHEMICAL TERATOGENESIS

The teratogenicity of many xenobiotics is thought to depend at least i n part upon their bioactivation by embryonic cytochromes P450, prostag landin ii synthase (PHS) and lipoxygenases (LPOs) to electrophilic and /or free radical reactive intermediates that covalently bind to or oxi dize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carci nogens benzo[ a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl) -1-butanone (NNK), the anticonvulsant drug phenytoin, structurally rel ated anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimet hadione) and the sedative drug thalidomide, we have examined the poten tial teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cu ltured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, character ized by electron spin resonance spectrometry, that initiated ROS forma tion, including hydroxyl radicals, which were characterized by salicyl ate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyg uanosine formation), protein (carbonyl formation), glutathione (GSH) a nd lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-( p-hydroxyphenyl)-5-phenylhydanto in (HPPH), thalidomide, B[a]P and NNK in vivo and/or in embryo culture , the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicit y of phenytoin and thalidomide were reduced by PI-IS inhibitors. Oxida tive macromolecular lesions and teratogenicity also were reduced by th e free radical trapping agent phenylbutylnitrone (PEN), and the antiox idants caffeic acid and vitamin E. In embryo culture, addition of supe roxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity i nitiated by phenytoin and B[a]P, suggesting a major contribution of RO S, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopat hies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Ox idation of DNA may be particularly critical, since transgenic mice wit h +/- or -/- deficiencies in the p53 tumor suppressor gene, which faci litates DNA repair, are more susceptible to phenytoin and B[a]P terato genicity. Even p53-deficient mice treated only with normal saline show ed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These r esults suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a r eactive intermediate, as well in the mechanism of embryopathies occurr ing in the absence of xenobiotic exposure, (C) 1997 Elsevier Science B .V.