Original ContributionEvidence for embryonic prostaglandin H synthase-catalyzed bioactivation and reactive oxygen species-mediated oxidation of cellular macromolecules in phenytoin and benzo[a]pyrene teratogenesis1
Introduction
The anticonvulsant drug phenytoin (diphenylhydantoin; Dilantin®), which is commonly used for the treatment of epilepsy, has been shown to be teratogenic in mice,1, 2rats,[3]frogs,[4]chickens,[5]rabbits,[6]and humans.7, 8, 9, 10While numerous reports suggest that phenytoin-initiated teratogenesis results from the bioactivation of phenytoin to a reactive arene oxide intermediate,11, 12, 13, 14there are several discrepancies in this hypothesis that have led to the investigation of other enzymatic systems known to bioactivate xenobiotics, including peroxidase systems such as prostaglandin H synthase (PHS) and related enzymes such as lipoxygenases (LPOs).13, 14
In vivo and in vitro studies from our laboratory indicate that phenytoin-initiated teratogenesis at least in part may involve peroxidase-catalyzed bioactivation of phenytoin to a reactive free radical intermediate, which if not detoxified may initiate oxidative stress leading to the oxidation of embryonic lipids, proteins, and DNA13, 14(Fig. 1). Alternatively, peroxyl radicals generated during eicosanoid biosynthesis may be involved in the epoxidation of xenobiotics, although the teratological contribution of this pathway for phenytoin and benzo[a]pyrene is speculative. We hypothesize that highly reactive oxygen species, such as hydroxyl radicals (016/OH), could be generated by the phenytoin free radical. Potentially, 016/OH could be generated indirectly by the phenytoin free radical via the Fenton reaction.[16]In vivo catalytic iron may be found loosely bound to membrane lipids, DNA, and phosphate complexes such as ADP.17, 18While 016/OH is generally thought to be a primary intermediate of in vivo damage,[16]there is some controversy over which reactive oxygen species is ultimately responsible for cellular damage.[19]However, it is known that if these reactive oxygen species are not detoxified by cytoprotective enzymes such as superoxide dismutase (SOD) and catalase, they can cause lipid peroxidation and protein oxidation and degradation.20, 21, 22, 23Evidence for embryotoxic oxidative stress initiated by a phenytoin free radical intermediate includes: (a) characterization of a carbon-centered phenytoin free radical intermediate by electron spin resonance spectrometry;[24](b) in vivo phenytoin-initiated hydroxyl radical formation measured by the 3-hydroxylation of salicylate;[25](c) phenytoin-initiated oxidation of protein, lipid,26, 27and DNA;28, 29and (d) complete inhibition of phenytoin-initiated embryonic DNA oxidation and embryotoxicity in embryo culture by the antioxidative enzymes SOD and catalase.[29]
In vivo and in vitro evidence from our laboratory indicates that peroxidase-catalyzed embryonic protein oxidation may constitute an important molecular mechanism mediating the teratogenicity of phenytoin.26, 27Oxidative modification of protein structure can be directly linked with increased proteolytic susceptibility.[30]Many changes occur in proteins as a result of exposure to reactive oxygen species, including decreases in native fluorescence, shifts in isoelectric point, and both increases and decreases in molecular weight due to either covalent interactions or peptide cleavage.30, 31, 32Oxidative damage to proteins has been shown to result in increased protein turnover and decreased enzymatic function, and has been associated with a number of pathological processes including emphysema, atherosclerosis, and neurological diseases.[33]
Glutathione (GSH) is the most abundant nonprotein thiol in the cell and is an important component of the cell's antioxidant defense. GSH can play a critical role in the detoxification of reactive oxygen species by serving as a substrate for glutathione peroxidase in the removal of hydrogen peroxide, in which case GSH is oxidized to glutathione disulfide (GSSG). GSH also can be protective by its direct interaction with reactive oxygen species, again leading to the formation of GSSG.[34]Finally, reduced glutathione is essential for several critical cellular functions, such as macromolecular synthesis (protein and DNA), microtubule assembly, maintenance of cellular structure and integrity, and modulation of protein conformation and enzyme activities.[35]In vitro, GSH can reduce phenytoin covalent binding to rat and mouse liver microsomal protein.36, 37, 38In vivo and in embryo culture, depletion of GSH or inhibition of its synthesis has been shown to increase phenytoin teratogenicity.39, 40, 41These results together suggest that thiols, and specifically GSH, provide important cytoprotective activity against phenytoin teratogenesis.
Benzo[a]pyrene is a polycyclic aromatic hydrocarbon found widely in the environment. Benzo[a]pyrene is a classic DNA-damaging carcinogen[42]and teratogen.43, 44Benzo[a]pyrene itself is relatively nontoxic; however, it can be bioactivated to a toxic reactive intermediate by cytochromes P450[45]and peroxidases46, 47(Fig. 1). If the reactive intermediate is not detoxified, it can both oxidize and arylate DNA and protein.47, 48, 49, 50, 51Although DNA arylation by benzo[a]pyrene has been postulated to play a role in teratologic initiation,[43]the teratological relevance of oxidation has not been investigated. DNA oxidation may constitute an important molecular mechanism mediating phenytoin teratogenicity,28, 29and transgenic p53-deficient mice that have low DNA reparative capabilities are more susceptible to the teratogenicity of both benzo[a]pyrene[44]and phenytoin.52, 53Therefore, we hypothesize that embryonic peroxidase-catalyzed bioactivation of benzo[a]pyrene may lead to the oxidation of DNA and protein, which may initiate teratogenesis.
To determine the potential for embryonic PHS-catalyzed bioactivation and oxidation of protein and thiols as potential molecular targets mediating phenytoin teratogenicity, a mouse embryo culture model was used to measure embryonic PHS content and to assess protein and thiol oxidation resulting from exposure to a concentration of phenytoin (20 μl/ml, 80 μM) that is within the therapeutic range in human and mouse[54]maternal plasma. Similarly, the potential for DNA and protein oxidation as molecular mechanisms of benzo[a]pyrene (10 μM) teratogenicity was evaluated. Following a characterization of benzo[a]pyrene embryopathy in embryo culture, addition to the culture medium of the antioxidative enzyme SOD, which completely blocks phenytoin embryopathy,[29]was evaluated for its ability to increase embryonic SOD activity and inhibit benzo[a]pyrene embryopathy. The method for assessment of oxidized proteins was first validated by using a known hydroxyl radical-generating system consisting of vanadyl sulfate and H2O2, with bovine serum albumin (BSA) or embryonic protein as the protein targets. To determine the potential involvement of free radical-initiated reactive oxygen species in protein oxidation and the molecular mechanism of phenytoin teratogenicity, the potential cytoprotective effects of the antioxidative enzymes SOD and catalase, and the reducing agent dithiothreitol (DTT), were evaluated.
These studies provide direct evidence that, in the absence of maternal and placental processes, embryonic PHS-catalyzed bioactivation and reactive oxygen species-dependent oxidative stress may play a critical role in phenytoin and benzo[a]pyrene teratogenesis. The results also suggest that embryonic proteins, thiols, and DNA may constitute teratologically important molecular targets.
Section snippets
Animals
Virgin female CD-1 mice (Charles River Canada Ltd., St. Constant, Quebec, Canada) were housed in plastic cages with ground corn cob bedding (Beta Chip, Northeastern Products Corp., Warrensburg, NY, USA) and maintained in a temperature-controlled room with a 12-h light–dark cycle. Food (Laboratory Rodent Chow 5001, PMI Feeds Inc., St. Louis, MO, USA) and tap water were provided ad lib. Three females were housed with one male from 1700 to 0900 h. The presence of a vaginal plug in a female mouse
Detection of Prostaglandin H Synthase
Immunochemical detection was used to determine whether PHS was present in day 9.5 embryos. In contrast to low or negligible P450 levels, substantial PHS content was detectable in mouse embryos during organogenesis (Fig. 2).
BSA Control Experiments
The preliminary protein oxidation studies using BSA as a control protein showed that BSA was oxidized in a concentration-dependent manner by the vanadyl sulfate–H2O2 hydroxyl radical-generating system (Fig. 3Fig. 4).
Vanadyl Sulfate–H2O2-initiated Embryonic Protein Oxidation
Having established the method for BSA, further control
Discussion
The results from these embryo culture studies support the hypothesis of embryonic peroxidase-catalyzed bioactivation of phenytoin and benzo[a]pyrene to reactive free radical intermediates that generate reactive oxygen species, which can oxidize embryonic proteins, thiols, and DNA, potentially initiating teratogenesis. It has been shown in vivo and in vitro that peroxidases such as prostaglandin H synthase and related enzymes such as lipoxygenases are capable of bioactivating phenytoin.37, 41, 65
Acknowledgements
We thank Dr. J. S. Leeder at the Toronto Hospital for Sick Children for his valuable help with the protein oxidation studies. This research was supported by a grant from the Medical Research Council of Canada.
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Preliminary reports of this research were presented at the 33rd, 34th, and 35th Annual Meetings of the Society of Toxicology, Dallas, Texas, March 1994 (Toxicologist 14:164; 1994); Baltimore, Maryland, March 1995 (Toxicologist 15:276; 1995); and Anaheim, California, March 1996 (Fundam. Appl. Toxicol. 30(Suppl. No. 1, Part 2: The Toxicologist):244; 1996); and at the 27th Annual Symposium of the Society of Toxicology of Canada, Montreal, December 1994 (Proceedings, p. 77; 1994).