Current Pharmaceutical Design - Volume 10, Issue 22, 2004

Developmental toxicology is primarily concerned with adverse effects of environmental and therapeutic exposures to the unborn child. This is the second issue of Current Pharmaceutical Design to focus on this important and controversial area. In line with the previous issue, the emphasis of this set of articles is to discuss emerging aspects in the field. Vitamin A metabolites, and particularly retinoic acid, are essential for normal embryonic development and alteration of retinoid signalling can result in severe dysmorphogenesis of almost all organ systems. In their paper Lee, Kochhar and Collins [1] discuss the phenotypic features, pathogenesis and molecular bases of retinoid-induced limb teratogenesis. The article also provides the reader with a description of normal limb development. Among birth defects, cleft palate is one of the most common. Greene and Pisano [2] offer an up-to-date discussion on the role of growth factors in normal and abnormal orofacial development. Major consideration is given to the transforming growth factor gene family. Folate metabolism represents a major area of research in developmental toxicology. The article by Brauer and Tierney [3] covers the recent developments regarding the impact of elevated homocysteine on morphogenesis of neural tube and neural crest cell-derived structures. There is an intensive search for alternative tests to in vivo animal models in developmental toxicity testing. In the article authored by Bremer and Hartung [4], the reader is provided with an overview of the current status of in vitro test development. The use, limitations and further needs of embryonic stem cell assays, one of the most promising models, are illustrated. Phytoestrogens are plant-derived isoflavonoids and lignans with estrogen-like activities. The article presented by You [5] reviews recent findings on the developmental effects of genistein, one of the most important phytoestrogens. The article also describes the potential of genistein to alter the toxicological response to the pesticide methoxychlor. The last manuscript, from Clementini and myself [6], focuses on the recently evolving topic of the teratological consequences of nitric oxide synthesis inhibition. I would like to express my sincere appreciation to the contributors.

The developing limb has been studied extensively and is a useful model to study morphogenesis. During embryogenesis, limb formation is initiated as a budding off from the embryonic lateral body wall. Limb pattern is specified by a series of epithelial-mesenchymal interactions, directing proximodistal, dorsoventral and anteroposterior axes. Vitamin A metabolites, especially retinoic acid, are known to play an important role in limb development, and the effects of retinoic acid may be mediated through the retinoid receptor signaling pathways. Accumulated evidence has shown that inadequate levels (excess or deficiency) of retinoic acid cause a wide range of limb malformations. Some species have the capacity to regenerate amputated limbs, and retinoids certainly affect this process, but there is debate regarding the extent that regeneration recapitulates development. In this review, phenotypic features, pathogenesis and the molecular basis of retinoid-induced limb malformations are discussed with a description of normal limb development and endogenous retinoid pathways.

Despite the fact that orofacial clefts represent one of the most common birth defects, the molecular mechanisms by which the embryonic primordia of the midface grow and differentiate are not completely understood. A multiplicity of genes must be expressed and their protein products must interact in a highly orchestrated fashion to affect normal orofacial development. Several families of growth factors have emerged as key contributors to the choreography of cellular and tissue interactions contributing to morphogenesis of the orofacial region. This overview focuses on those growth factors that are generally accepted as playing a pivotal role in normal as well as abnormal development of first branchial arch-derived structures. Particular emphasis is given transforming growth factor-ß (TGFß) and TGFα family members, as these have been the most extensively investigated with regard to their role in development of orofacial structures. Consideration is also given to evidence implicating developmental contributions from members of the bone morphogenetic protein and fibroblast growth factor families.

Elevated maternal homocysteine (Hcys) is a well-established risk factor for embryonic toxicity and the development of congenital defects, particularly neural tube closure defects and neurocristopathies. The mechanisms responsible are unclear but early work has focused on the role of folate metabolism because these defects are greatly reduced by folate supplementation. As a consequence, elevated Hcys is often looked upon as being an indirect consequence of faulty folate metabolism, although more recent studies show Hcys may act directly as a teratogen. Because Hcys is at the crossroads of protein and DNA metabolism, has a propensity to chemically modify proteins directly, can generate free radicals, and even perturb ligand binding to certain receptors, the developmental processes Hcys can potentially disturb are enumerable. But in recent years, investigators have begun identifying cellular and molecular targets for the direct action of Hcys. While elevating Hcys can alter a myriad of basic cellular activities needed for normal development, our current understanding as to the specific etiological mechanisms responsible for congenital defects is very speculative. Here we provide an overview of what is currently known regarding the toxicity and teratogenicity of elevated Hcys during embryonic development, paying particular attention to neural tube and neural crest cell morphogenesis.

The future chemical policy of the European Union as well as the 7th amendment of the cosmetic directive is calling for the development of alternative tests to animal experimentation for toxicological safety testing. In the field of embryotoxicity one of the most promising in vitro models are based on embryonic stem cells. The embryonic stem cell test has already been validated in comparison to in vivo results in an international blind collaboration study. The presented review is discussing the use, limitations and further needs for the test in order to be fully suitable for regulatory acceptance. In this context, it is summarized which requirements for an in vitro embryotoxicity test have to be fulfilled for regulatory toxicity testing. In addition, an overview about the current status of test development of other embryonic stem cell tests is presented. Several workshops have reviewed the progress of in vitro tests for developmental toxicity testing. A general consensus of these workshops has been that one single test will not be sufficient to cover all manifestations of developmental toxicity. The establishment of a test battery for developmental toxicity is required. This will be even more challenging since the principles of a composing test strategy and its validation have not been defined yet. Finally, the unique possibility of combining the human embryonic stem cell technology and the microarray techniques might lead to a deeper understanding of the toxicological mechanisms of human developmental toxicants.

Phytoestrogens are plant-derived compounds with estrogen-like activities. Certain foods such as soyderived products are known to have high levels of phytoestrogens, and about 25% of commercial infant formulas used in the United States are soy-based. One of the most important phytoestrogens is the isoflavone genistein. Human exposures to genistein occur through normal dietary intake and through the use of genistein or other isoflavone extracts as nutritional supplements. Among the issues raising concerns about human exposure to phytoestrogens is how such exposure may affect responsiveness and sensitivity of the exposed subjects to other xenobiotics, particularly drugs and environmental chemicals with estrogenic or other endocrine activities. This article describes our recent studies on the developmental effects of dietary genistein in rats and its potential to interact with the toxicology of the endocrine-active pesticide methoxychlor. Data from our studies demonstrated that genistein is capable of altering the toxicological behaviors of methoxychlor and likely other endocrine active compounds as well. The complexities of such interactions are difficult to predict based on their in vitro steroid receptor reactivities.

Nitric oxide (NO) is generated by a family of NO synthase (NOS) enzymes, including endothelial (eNOS), inducible (iNOS) and neuronal (nNOS). NO is an important bioregulator of a wide variety of physiological processes. Recent experimental evidence indicates that inhibition of NO synthesis can lead to teratogenesis. The current review focuses on this aspect of NOS. Exposure of pregnant rodents to non-selective NOS inhibitors, such as NG-nitro- Larginine- methyl ester (L-NAME) and NG-nitro-L-arginine (L -NNA), has been linked to limb reduction defects. The teratogenic phenotype, characterized by hemorrhage and transverse terminal tissue destruction, has been regarded to be compatible with a vascular origin. The critical time for teratogenic response was traced to advanced stages of gestation. Similar limb reduction defects have been described in mice deficient in eNOS, but not in other NOS isoforms. Several observations have led to the proposal that hypoxia and possible consequential generation of reactive oxygen species are involved in the causation of NOS inhibitors induced limb defects.

Proteinase-activated Receptor 2 (PAR2) is a potential target for the design of drug treatments for vascular diseases. Its unique mechanism of activation by serine proteinases, questions regarding the identities of endogenous agonists and its apparent multiple activities in the vasculature contribute to complex pharmacology. The progress of the pursuit to understand the function of PAR2 relies on the design of short specific peptides as selective agonists for PAR2 in receptor-selective cultured cell expression systems and is limited by the lack of any PAR2 antagonists. Fortunately, the utilization of transgenic PAR2-deficient mice enables the identification of the actions of selective PAR2-derived activating peptides attributed to activation solely of PAR2 in more physiologically complex systems. Of multiple pharmacological responses, PAR2-derived peptide agonists reduce vascular tone, and therefore increase blood flow, via nitric oxide-dependent and -independent paracrine actions of the endothelium upon the underlying vascular smooth muscle cells of blood vessels. PAR2-mediated endothelial-dependent relaxation and hyperpolarization of vascular smooth muscle in select arterial vascular beds via a nitric oxide / cyclooxygenases-independent mechanism suggests a strategy for correction of endothelium-based vascular dysfunction. Vascular tissues respond to progression of vascular diseases such as atherosclerosis or to injury with variable changes of PAR2 expression. With further research and drug development, PAR2 agonists and antagonists may become a basis for a new class of therapeutic agents for treatment of vascular diseases.

Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily and form heterodimers with retinoid X receptor. To date, three PPARs isoforms have been isolated and termed α, β (or δ), and γ. Although PPARγ is expressed predominantly in adipose tissue and associated with adipocyte differentiation and glucose homeostasis, it has been recently demonstrated that PPARγ is present in a variety of cell types. Synthetic antidiabetic thiazolidinediones (TZDs) and natural prostaglandin D2 (PGD2) metabolite, 15-deoxy-Δ12, 14- prostaglandin J2 (15d-PGJ2), are well-known as ligands for PPARγ. After it has been reported that activation of PPARg suppresses production of proinflammatory cytokines in activated macrophages, medical interest in PPARg have grown and a huge research effort has been concentrated. PPARg, is currently known to be implicated in various human chronic diseases such as diabetes mellitus, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, and Alzheimer's disease. Moreover, PPARγ ligands have potent tumor modulatory effects against colorectal, prostate, and breast cancers. Recent studies suggest that TZDs not only ameliorate insulin sensitivity but also have pleiotropic effects on many tissues and cell types. Although activation of PPARγ seems to have beneficial effects on atherosclerosis and heart failure, the mechanisms by which PPARγ ligands prevent the development of cardiovascular diseases are not fully understood. This review will focus on the latest developments in the PPARγ field and the roles of PPARg-dependent pathway in cardiovascular diseases.