Embryopathic effects of thalidomide and its hydrolysis products in rabbit embryo culture: evidence for a prostaglandin H synthase (PHS)-dependent, reactive oxygen species (ROS)-mediated mechanism.

Thalidomide (TD) causes birth defects in humans and rabbits via several potential mechanisms, including bioactivation by embryonic prostaglandin H synthase (PHS) enzymes to a reactive intermediate that enhances reactive oxygen species (ROS) formation. We show herein that TD in rabbit embryo culture produces relevant embryopathies, including decreases in head/brain development by 28% and limb bud growth by 71% (P<0.05). Two TD hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaric acid (PGA), were similarly embryopathic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment with the PHS inhibitors eicosatetraynoic acid (ETYA) or acetylsalicylic acid (ASA), or the free-radical spin trap phenylbutylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopathies initiated by TD, PGMA, and PGA. This is the first demonstration of limb bud embryopathies initiated by TD, as well as its hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating that all likely contribute to TD teratogenicity in vivo, in part through PHS-dependent, ROS-mediated mechanisms.

Receptor- and reactive intermediate-mediated mechanisms of teratogenesis.

Drugs and environmental chemicals can adversely alter the development of the fetus at critical periods during pregnancy, resulting in death, or in structural and functional birth defects in the surviving offspring. This process of teratogenesis may not be evident until a decade or more after birth. Postnatal functional abnormalities include deficits in brain function, a variety of metabolic diseases, and cancer. Due to the high degree of fetal cellular division and differentiation, and to differences from the adult in many biochemical pathways, the fetus is highly susceptible to teratogens, typically at low exposure levels that do not harm the mother. Insights into the mechanisms of teratogenesis come primarily from animal models and in vitro systems, and involve either receptor-mediated or reactive intermediate-mediated processes. Receptor-mediated mechanisms involving the reversible binding of xenobiotic substrates to a specific receptor are exemplified herein by the interaction of the environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or "dioxin") with the cytosolic aryl hydrocarbon receptor (AHR), which translocates to the nucleus and, in association with other proteins, binds to AH-responsive elements (AHREs) in numerous genes, initiating changes in gene transcription that can perturb development. Alternatively, many xenobiotics are bioactivated by fetal enzymes like the cytochromes P450 (CYPs) and prostaglandin H synthases (PHSs) to highly unstable electrophilic or free radical reactive intermediates. Electrophilic reactive intermediates can covalently (irreversibly) bind to and alter the function of essential cellular macromolecules (proteins, DNA), causing developmental anomalies. Free radical reactive intermediates can enhance the formation of reactive oxygen species (ROS), resulting in oxidative damage to cellular macromolecules and/or altered signal transduction. The teratogenicity of reactive intermediates is determined to a large extent by the balance among embryonic and fetal pathways of xenobiotic bioactivation, detoxification of the xenobiotic reactive intermediate, detoxification of ROS, and repair of oxidative macromolecular damage.

Resistance of CD-1 and ogg1 DNA repair-deficient mice to thalidomide and hydrolysis product embryopathies in embryo culture.

Thalidomide (TD) displays remarkable species specificity, causing birth defects (teratogenesis) in humans and rabbits, but not rats or mice; yet, few determinants of species susceptibility have been identified. Also, certain mouse strains are susceptible to the embryopathic effects of some teratogens in embryo culture despite their resistance in vivo. Herein we show that CD-1 mouse embryos in culture are resistant to limb embryopathies caused by TD and two of its hydrolysis products, 2-phthalimidoglutaramic acid and 2-phthalimidoglutaric acid, although all three compounds cause these embryopathies in rabbit embryo culture. These results show that the resistance of CD-1 mice to TD teratogenesis is inherent to the embryo and is not dependent upon maternal factors, including differential in vivo exposure to the many hydrolysis products of TD. In utero TD exposure of rabbit but not mouse embryos elevates levels of the teratogenic oxidative DNA lesion 8-oxoguanine, which is repaired by oxoguanine glycosylase 1 (OGG1). However, DNA repair-deficient ogg1 knockout mice proved resistant to TD-initiated embryopathies in culture and teratogenesis in vivo, indicating that the resistance of mice is not due to a higher level of DNA repair.

Fluorothalidomide: a characterization of maternal and developmental toxicity in rabbits and mice.

The expanding therapeutic uses of thalidomide (TD) are limited by its teratogenic side effects. Although the therapeutic and teratogenic effects may be stereoselectively separable, rapid in vivo racemization of the TD isomers confounds the corroboration of this distinction. Herein we evaluated the potential of fluorothalidomide (FTD), the closest structural analog of TD with stable, nonracemizing isomers, as a model compound for studying stereoselectivity in TD teratogenesis. In contrast to TD, FTD was a potent maternal and fetal toxicant in both rabbits and mice in vivo. Furthermore, FTD rapidly degraded in vivo, presumably via hydrolysis, which in vitro was up to 22-fold faster for FTD than TD. Most critically, in an established rabbit embryo culture model for TD teratogenesis, FTD did not initiate the limb bud embryopathies observed with TD. The chemical instability and strikingly different maternal and developmental toxicological profiles of FTD and TD make FTD an unsuitable compound for studying stereoselective mechanisms of TD teratogenesis.

Oxidative stress in developmental origins of disease: teratogenesis, neurodevelopmental deficits, and cancer.

In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.