Oxidative damage in chemical teratogenesis.
The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential 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 cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [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 teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, 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 embryopathies, 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. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.
Wells
P G
PG
Faculty of Pharmacy, University of Toronto, Ont., Canada. pg.wells@utoronto.ca
Kim
P M
PM
Laposa
R R
RR
Nicol
C J
CJ
Parman
T
T
Winn
L M
LM
D000014:Abnormalities, Drug-Induced / Q000209:etiology / Q000517:prevention & control; D000818:Animals; D000927:Anticonvulsants / Q000633:toxicity; D000975:Antioxidants / Q000494:pharmacology / Q000627:therapeutic use; D001564:Benzo(a)pyrene / Q000633:toxicity; D001711:Biotransformation; D004249:DNA Damage; D005314:Embryonic and Fetal Development / Q000187:drug effects; D005609:Free Radicals; D051379:Mice; D009602:Nitrosamines / Q000633:toxicity; D018384:Oxidative Stress; D010672:Phenytoin / Q000633:toxicity; D011506:Proteins / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D017382:Reactive Oxygen Species; D015398:Signal Transduction / Q000187:drug effects; D013723:Teratogens / Q000493:pharmacokinetics / Q000494:pharmacology*; D013792:Thalidomide / Q000633:toxicity; D015262:Xenobiotics / Q000493:pharmacokinetics / Q000633:toxicity*
D000927:Anticonvulsants; D000975:Antioxidants; D005609:Free Radicals; D009602:Nitrosamines; D011506:Proteins; D017382:Reactive Oxygen Species; D013723:Teratogens; D015262:Xenobiotics; D001564:Benzo(a)pyrene; D013792:Thalidomide; D010672:Phenytoin; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone