Abstract
Axial skeletal development is part of the complex, inclusive process of axial or midline development. It involves the interaction of many tissues including the embryonic notochord, neural tube, somite compartments, intersomitic angiopotent cells, and neural crest cells. These tissues give rise to the axial skeleton, intervertebral discs, spinal cord, trunk musculature and dorsal dermis, intervertebral arteries, and spinal ganglia. Development of these tissues occurs in an interdependent and hierarchical manner over an extended period of time. These characteristics may make the axial skeleton disproportionately susceptible to environmental influence, accounting for the high incidence of axial skeletal defects among live and stillbirths. It may also account for the many manifestations of axial skeletal defects observed.
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Introduction
Axial skeletal development is part of the complex, inclusive process of axial or midline development. It involves the interaction of many tissues including the embryonic notochord, neural tube, somite compartments, intersomitic angiopotent cells, and neural crest cells. These tissues give rise to the axial skeleton, intervertebral discs, spinal cord, trunk musculature and dorsal dermis, intervertebral arteries, and spinal ganglia. Development of these tissues occurs in an interdependent and hierarchical manner over an extended period of time. These characteristics may make the axial skeleton disproportionately susceptible to environmental influence, accounting for the high incidence of axial skeletal defects among live and stillbirths. It may also account for the many manifestations of axial skeletal defects observed.
Data show that the axial skeleton is one of several organ systems with a high frequency of abnormality, 1 in 1,000 live births (Brent and Fawcett 2007, Cohen 1997, Dias 2007, Erol et al. 2002, Jaskwhich et al. 2000, O’Rahilly and Müller 1996, Oskouian et al. 2007) and a very low heritable component, estimated to be between 0.5 and 2%. Congenital axial skeletal defects may occur in isolation or as a component of more widespread syndromes or sequences (Cohen 1997, Dias, 2007, Erol et al. 2002, Jaskwhich et al. 2000, Oskouian et al. 2007) (Table 3.1 and Chapter 7). It is estimated that the skeletal defect is accompanied by an intra-spinal neural defect in 40% of cases. In addition, approximately 50–60% cases of congenital scoliosis suffer additional congenital defects in other organ systems including urogenital and cardiovascular systems (approximately 20% and 10–12%, respectively), gastrointestinal and limb defects (2–5%). These combinations of congenital defects and their frequencies are reflective of the degree of concurrent development of the different organ systems.
While dramatic axial skeletal defects do occur in the context of syndromes and other anomalies, the majority of congenital spinal anomalies involve single structural defects of the spine and frequently few obvious coincident malformations or functional deficits (Erol et al. 2002, Jaskwhich et al. 2000, Oskouian et al. 2007), indicating that a time-dependent, tissue-specific insult may be involved. The complexity of axial skeletal development and the variety of axial skeletal defects suggest a variety of loci and mechanisms through which environmental factors may cause axial skeletal dysmorphogenesis.
Faced with the high social costs of resultant morbidity, it is critical to determine the possible impact any environmental factor may have on the embryo. Although many of the known human teratogens can produce axial skeletal defects, the etiology of over half of observed axial skeletal defects is unknown and is assumed to be multi-factorial, a combination of genetic susceptibility and environmental insult (Cohen 1997, Jaskwhich et al. 2000). This fact highlights the need for investigating the role of environmental factors, alone or in combination, in the production of this particular class of defects. Such study requires the convergence of at least two broad fields of study. The first is developmental biology, to understand the details of normal development and identify new markers, loci, and perhaps possible mechanisms of teratogenesis. The second field is teratology, a discipline closely related to reproductive toxicology that involves assessing the impact of environmental factors on the new biological markers, loci, and mechanisms discovered and characterized in developmental biology.
Vertebral Dysmorphogenesis in Human Congenital Scoliosis
Clinically, congenital scoliosis is defined as a spinal curvature of over 10% caused by a structural vertebral defect (Dias 2007, Erol et al. 2002, Oskouian et al. 2007). The abnormal spinal curvature is further defined by its anterior–posterior location and the plane of curvature as coronal for scoliosis and sagittal for kyphosis. The characteristic feature of congenital axial skeletal defects is the malformation of vertebral bodies or processes evident at birth. Broadly, these vertebral defects are clinically classified as failures in formation and morphogenesis represented by hemivertebrae, wedge vertebrae, open vertebral arches, bifid vertebrae, and vertebral agenesis or failures in segmentation represented by unilateral unsegmented bars or block vertebrae bilateral fusions (Fig. 3.1) (Dias 2007, Erol et al. 2002, Jaskwhich et al. 2000, Oskouian et al. 2007). Developmentally, however, all of these defects have their origin in somitogenesis, the initial manifestation of the vertebral column’s metameric segmentation.
Different forms of congenital scoliosis: block vertebrae (a), unilateral bar, (b), wedge vertebrae, (c), multiple hemivertebrae, (d), single, semi-segmented vertebrae, (e), non-segmented hemivertebrae, (f), incarcerated hemivertebrae, (g), defects in segmentation can produce these defects
Normal Development of the Axial Skeleton
As discussed in previous chapters, the axial skeleton is derived from the paraxial mesoderm, a primary germ layer, which undergoes the molecularly timed process of somitogenesis to produce blocks of tissue symmetrically arranged on either side of the midline neural tube and notochord (Table 3.2, Fig. 3.1) (Christ et al. 2000, Stockdale et al. 2000, Gridley 2006). The somite is a transient embryonic structure that plays an important role in the patterning of the axial skeleton (comprised of vertebral bodies, ribs, and intervertebral discs) and its associated tissues: the hypaxial and epaxial muscles of the spine, the dorsal dermis of the trunk, and the intervertebral arteries. The morphogenic description of somitogenesis can be conceptually divided into several phases: patterning, morphogenesis, differentiation, and growth and maturation (Tam and Trainor 1994, Christ et al. 2000, Alexander et al. 2007a). These are helpful classifications when characterizing and studying birth defects and their causes.
Among the tissues of the spine, the axial skeleton and its composite tissues undergo multiple rounds of patterning, differentiation, and growth events, including somitogenesis, resegmentation, and ossification, among other processes. Briefly, the axial skeleton is derived from the sclerotome, the ventromedial quadrant of each somite. Cells of sclerotome are initially part of the epithelial somite. Shortly after expressing the paired-box gene Pax1 (Wallin et al. 1994, Barnes et al. 1996a), the cells de-epithelialize and relocate themselves to surround the notochord. These cells then begin expressing Sox9, a chondrocyte-specific transcription factor, and producing prodigious amounts of cartilage matrix to form the cartilage anlage of the vertebral body (Healy et al. 1999). There is a distinct polarity to the somite as it matures (Tam and Trainor 1994) that is consequential in the course of resegmentation, in which the posterior half of one somite merges with the anterior half of the posterior somite (Christ et al. 2004). Together, these halves combine to form a vertebral body out of phase with the other tissue, characteristic of the vertebral motor unit.
Development of the axial skeleton and the surrounding tissues occurs in an interdependent and hierarchical manner over an extended period of time. This may make the axial skeleton disproportionately susceptible to environmental influence, accounting for the high incidence of axial skeletal defects among live and stillbirths. It may also account for the many manifestations of axial skeletal defects observed. Understanding these processes (the normal development of the spine) and their effects upon the surrounding tissues is important in deciphering the etiology of various forms of congenital scoliosis and the mechanisms by which environmental agents may initiate abnormal development.
Experimental Axial Skeletal Teratology
Given that the majority of axial defects have no known genetic cause (Cohen 1997, Dias 2007, Erol et al. 2002, Jaskwhich et al. 2000, Oskouian et al. 2007), the assumption must be made that there is an environmental component. The principles that helped define environmental teratogical agents were popularized in the wake of the “thalidomide experience” and with some modification remain applicable today (Wilson 1977, Sulik 1997, Sadler and Hunter 1994). In establishing the role of an environmental agent in inducing a congenital axial skeletal defect, we know that it must first affect the development and function of axial tissues and those that influence their differentiation including the notochord, neural tube, paraxial mesoderm, and overlying ectoderm. Second, the exposure must occur somewhere between the 4th and 10th week of human gestation, or organogenesis, during which time gastrulation, neurulation, and somitogenesis occur (Table 3.2) (Nishimura et al. 1974, O’Rahilley and Müller 1996). Third, the target of the teratogen must play a necessary role in the affected developmental process (e.g., somitogenesis) by acting via a specific mechanism. Finally, we must observe the dose-response effect of the environmental agent on embryonic development in both frequency and degree of malformation, including the graded manifestations of abnormal development: death, dysmorphogenesis, inhibition of growth or developmental delay, and functional deficit.
In the etiology of scoliosis, target organs may include the paraxial mesoderm and somites, the neural tube and notochord, and the overlying ectoderm. In particular, the patterning of the somite boundaries and the subsequent boundaries of differentiation defined by integrated signaling pathways under the influence of the surrounding tissues figures prominently. Morphological processes that may be affected include somitogenesis, neurulation, and gastrulation, which involve cell migration, epithelialization, and laminar fusion, as well as proliferation and apoptosis. Finally the differentiation, growth, and maturation of the axial skeletal elements may also play an important role (Table 3.3).
Pathogenesis of Abnormal Axial Development
The identification of the structural defect in congenital scoliosis in the fetus or neonate remains an analysis conducted long after the initial pathogenic events inducing the malformation. Identifying and understanding the initial pathogenic event is a critical step in characterizing the mechanisms of teratogenesis, which can then lead to the development of appropriate interventions. Environmental insults to a developing organism occur at molecular or sub-cellular levels. While the list of possible environmental insults is very large, the insults may be translated into types of cellular responses that result in recognizable patterns of dysfunction of dysmorphogenesis among tissues and organs (Table 3.4) (Wilson 1977, Sulik 1997).
Although teratogens are often discrete in nature (e.g., of known structure/composition and chemical characteristic), the determination of teratogenic mechanism is complicated. The main reasons for this are as follows. First, not all the possible targets of a teratogen have been identified, since many potentially affected targets remain unknown, i.e., normal developmental mechanisms still need identification and characterization. Related to this is the fact that it is highly unlikely that most teratogens act upon a single molecule or even a cellular pathway. Multiple mechanistic pathways may combine to produce a single pathogenic mechanism contributing to the resultant congenital defect. Second, our ability to monitor the effect of the teratogen on the biochemistry of individual cellular targets is limited. Specifically, probes with sufficient sensitivity and specificity are unavailable for many processes and applications. Contributing to these issues is the fact that the amount of tissue available for study is usually very limited. Intertwined within these shortcomings is the difficulty of experimental interpretation, which varies with probe, detection, and particularly the endpoint chosen.
Despite these complications, we can hypothesize several intracellular processes that may be targets of teratogens. The teratogen generates its effects on the embryo often through a mode of molecular mimicry co-opting or undermining normal cellular processes such that they are activated, inactivated, or diverted in a manner inconsistent with developmental timing. Such processes include mitotic interference (mutagenesis and carcinogenesis), epigenetic changes (methylation and acetylation state), altered membrane function (composition or porosity), altered signal transduction, altered/inhibited energy metabolism, inhibition of waste (intermediary) metabolism, changed redox status, specific or general enzyme inhibition, and disturbances in nucleic acid synthesis, among other possibilities. The cellular responses to these insults may be grouped into several common outcomes, including necrosis or apoptosis, reduced biosynthesis, failed cell/cell or cell/matrix interactions, impeded morphogenetic movement, and mechanical disruption of tissues. Ultimately, the final defect may be manifested via loss of cells or tissue or imbalances in growth and differentiation.
While specific mechanisms of many teratogenic insults remain largely unknown, the characterization of cellular responses has been more successful. One particularly well-characterized outcome is the correlation of tissue-specific patterns of cell death and impending malformation (Sadler and Hunter 1994, Sulik et al. 1988, Sulik 1997, Zakeri and Ahuja 1997). This correlation has highlighted several characteristics of teratogenic action including the principles that different cell populations are sensitive to teratogenic insults at different time points, different agents target different tissues, and many teratogens expand areas of normal, developmentally regulated cell death. The observed changes in normal cell death patterns indicate the target tissue often plays a role in the subsequent dysmorphogenesis; however, the apoptotic cells do not participate in subsequent tissue formation – thus the effect of the teratogen on the surviving cells is important and presumed to be related to the cause of cell death. Nonetheless, the increase in cell death serves as an early marker for the teratogenic action. As we learn more about development and toxicological responses on the molecular level, we will create more sensitive cell response markers that will allow greater resolution of the teratological action.
Overview of Agents and Conditions Associated with Axial Skeletal Teratogenesis
As stated above, axial skeletal malformations are often linked to exposure to teratogenic conditions. The following summarizes the types of teratogens and teratogenic conditions associated with spinal malformations (Schardein 2000). Detailed descriptions of some of these factors will be presented in the following sections.
Recreational teratogens: Recreational drugs such as alcohol, cocaine, and cigarettes are known to significantly reduce fetal and post-natal growth, increase infant mortality, and cause congenital malformations of various types and severity.
Pharmaceutical teratogens: Most embryonic organs and the central nervous system are extremely sensitive to the teratogenic affects of pharmaceuticals such as thalidomide, diethylstilbestrol, retinoic acid, valproic acid, warfarin, chemotherapy, lithium, and nicotinic acid.
Industrial and environmental teratogens: Industrial processes required to provide for growing populations worldwide release a substantial amount of waste products into the environment, with the toxicologic and teratogenic effect of many species as yet uncharacterized. Among the chemicals with known teratogenic effects are organic solvents; arsenic, cadmium, and lead anesthetic gases; and organic mercury.
Agricultural teratogens: Insecticides and herbicides are critical to providing nutrition to growing populations. Studies have determined that organochlorine insecticides such as DTT, parathion, and malathion may interfere with fertility and reproduction by mimicking estrogen-like compounds. Among herbicides, the byproduct of Agent Orange, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), is highly teratogenic causing cleft palate and congenital renal abnormalities.
Infectious diseases: Microbial chemicals may act as teratogens. Microbes such as syphilis, cytomegalovirus, rubella, herpes, toxoplasma, and fifth disease affect 1–5% of all live births. These infections may cause a group of associated malformation known as the TORCH complex, as well as isolated structural defects and functional deficits.
Metabolic conditions: Some metabolic disorders, most prominently diabetes and hyperthermia also induce congenital malformations in the embryos. Diabetic pregnancy increases the frequency of a wide variety of congenital defects over background including cardiac defects, eye and ear defects, renal defects, and functional deficits in addition to a high rate of congenital scoliosis, in addition to increased embryonic death and life-long metabolic disorders.
Non-genetically Linked Conditions Characterized by Axial Skeletal Defects: VATER Association
The VATER spectrum is a non-random association characterized by vertebral anomalies (V), anal atresia (A), tracheoesophageal (TE) fistula, and renal (R) anomalies (Botto et al. 1997, Cohen 1997, Martínez-Frías and Frías 1997). This spectrum may also be associated with cardiovascular (C) anomalies and limb (L) anomalies (VACTERL). The incidence of VATER in diabetic mothers is 200× higher than in the general population, which occurs at a rate of 16 per 100,000 births (Pauli 1994, Cohen 1997, Martínez-Frías et al. 1998a). Vertebral defects in this association can involve agenesis, hypoplasia, and hemivertebrae, often afflicting many contiguous vertebral units. As the acronym suggests, many associated tissues are affected. The association of these different mesenchymally derived tissues to the vertebral column and the timing of their development are critical to hypothesizing the origin and mechanism of the defect(s). Analyses of the frequency and co-occurrence of the features of VACTERL and other syndromes suggest that the anomalies can extend to various cranio-caudal levels suggesting a time dependency and critical period through a defect in a common mechanism of dysmorphogenesis (Stewart et al. 1993). The VACTERL sequence can be conceptually included in a group of progressively severe spectrums of which it may be the most severe (Table 3.5). This broad spectrum of malformations has been coined the axial mesodermal dysplasia complex (AMDC) (Stewart et al. 1993). Some confounding features to any hypothesis are the broad range of defects sometimes involving tissues derived from all germ layers, its largely spontaneous occurrence, and low rate of subsequent inheritance.
There are two related, non-exclusive models currently employed to explain the etiology of AMDC suggesting that the collection of defects may arise from a single environmental insult at a time early in post-implantation development. In the first theory, the embryo at the time of early gastrulation is comprised of a single morphogenetic field, the primary developmental field (Optiz et al. 2002, Martinez-Frias et al. 1998b). At this time the embryo responds essentially as a single, homogeneous entity. The primary effect of the insult at this time is to affect growth (proliferation) within the embryo, drastically affecting the existence and position of organizing centers and tissue morphogenesis throughout the embryo as this primary developmental field subdivides into secondary developmental fields that will give rise to the various organs and structures of the embryo including the axial skeleton (Martinez-Frias and Frias 1999). If the insult occurs at this time, there is necessarily a wide range of structures affected (polytopic defects) often of mesenchymal origin, but involving ectodermal and endodermal germ layers as well.
A second variant on the theory holds that the broad spectrum of defects reflects a common mechanistic cause in many tissues of a more heterogeneous entity comprised of multiple secondary developmental fields, such that different tissues of the embryo respond in specific manners to produce the wide spectrum of observed defects (Martínez-Frías and Frías 1997, Bohring et al. 1999). The defect then is thought to lie more in mechanisms of patterning or morphogenesis as the insult or defect occurs slightly later in development. This latter variation on developmental field defects and the etiology of multiple congenital anomalies such as VATER appears to more easily explain the wide spectrum of cranio-caudal positions of the defects and the wide degree of severity observed in several multiple congenital defect associations by allowing for a longer critical period. Both of these theories have been characterized theoretically and statistically to the range of defects observed in infants born to diabetic mothers, one of the most frequently recognized “causative” factors of the VATER spectrum (Martínez-Frías et al. 1998a). The high incidence of the VATER and other AMDC variants in diabetic mothers suggests an etiology that involves a fundamental metabolic imbalance in energy production or a dysfunction in a critical component of the embryonic stress response. Some investigators have suggested that the defects may arise from malformation or dysfunction of the notochord, which is critical to the establishment and maintenance of embryonic axes and the patterning and differentiation of many mesenchymal tissues (Gilbert-Barness et al. 2001). It has been suggested that notochord mutants such as brachyury (T) or sonic hedgehog (SHH) knock-outs could be used as models for VATER and AMDC (Arsic et al. 2002). We discuss the potential for energy metabolism dysregulation as the locus affected resulting in VATER in the context of diabetes-induced congenital scoliosis below.
Environmental Factors That Cause Axial Skeletal Dysmorphogenesis
Valproic Acid
Valproic acid (VPA) is an anti-epileptic drug that is associated with a 20-fold increased incidence of spina bifida, a neural tube defect, in children born to pregnant mothers undergoing VPA treatment (Lammer et al. 1987, Nau et al. 1991). Experimentally, VPA has been shown to be teratogenic in mouse, rat, chick, hamster, rabbit, and rhesus monkeys (Ehlers et al. 1992, Menegola et al. 1996, Vorhees et al. 1987, Barnes et al. 1996b, Basu and Wezeman 2000, Hendrickx et al. 1988). Skeletal abnormalities in these models were most commonly observed, involving vertebrae, ribs, digits, and craniofacial bones. These frequently occur in the context of other cardiovascular, urogenital, and neurological anomalies that together comprise the fetal valproate syndrome. The axial skeletal defects can include presacral vertebrae, cervical and thoracic ribs, indicating possible homeotic transformations. The defects may also include structural vertebral defects, indicating segmentation defects.
In general, the primary locus of teratogens causing spina bifida including VPA is believed to be the neural tube, resulting in failure of neural tube closure (Turner et al. 1990). Subsequently, the neural arches are unable to fuse. However, vertebral defects such as block vertebrae and hemivertebrae sometimes coincide with a neural tube defect have also been observed following VPA exposure (Barnes et al. 1996b). More detailed studies have shown that important patterning genes, such as Pax1 and paraxis (Tcf15), are down-regulated by the administration of VPA in chick embryos (Barnes et al. 1996b, Barnes et al. 1997). The malformations produced by VPA can be mimicked through the administration of anti-sense deoxynucleotides during somitogenesis (Barnes et al. 1996b, Barnes et al. 1997). This type of data confirms that dysregulation of these genes can be teratogenic, but does not indicate a specific mechanism for how this may occur. The down-regulation of these genes may be caused by decreased signaling or reduced or delayed differentiation caused by increased ROS production or altered nucleic acid metabolism (Fantel 1996, Nau et al. 1991), as suggested by studies showing that folic acid administration can significantly reduce the incidence of experimentally induced VPA axial skeletal defects (Green and Copp 2005). More recently, VPA has been shown to also inhibit histone deacetylase activity at therapeutic levels, and that this activity is correlated with axial skeletal defects and exencephaly (Menegola et al. 2006). In a comparison of the teratogenicity and changes in gene expression by VPA and TSA (Trichostatin A), many of the shared genetic effects were specific to skeletal and cardiac muscle, assigning a more specific mechanistic action of VPA to dysregulating epigenetic control which leads to altered gene expression.
Hypoxia
Congenital vertebral anomalies have been produced in newborn animals experimentally by transient hypoxia and transient exposure during the embryonic period (Grabowski and Paar 1958, Ingalls and Philbrook 1958, Rivard 1986, Webster and Abela 2007). In these studies, many gross vertebral and associated skeletal defects have been induced, including hemivertebrae, vertebral fusions, fragmented vertebral bodies, bifid ribs, or junctions of two or more ribs. The nature and extent of skeletal malformations induced have been dependent upon the precise stage of somite formation at the time when maternal stress has been induced. Hypoxia is thought to affect the early embryo through the induction of increased reactive oxygen species (ROS) homotopically (where ROS are already prominent; Fantel 1996) and later through altered vascularization (Grabowski 1961, Danielson et al. 1992, Webster and Abela 2007). Less well defined is the idea that hypoxia itself or its management is important in and of itself for morphogenic process or cell function during embryonic development (Chen et al. 1999, Semenza 1999).
During early organogenesis as the embryonic circulation develops, the embryo is known to undergo a transition from anaerobic respiration to aerobic respiration (Hunter and Sadler 1989, Hunter and Tugman 1995, Mackler et al. 1975, Miki et al. 1988a). Recent studies have confirmed that oxygenation and the cellular response to oxygenation as interpreted through expression patterns of heat shock proteins (protective chaparones) (Edwards et al. 1997, Mirkes 1997), antioxidant (superoxide dismutases) (Wells and Winn 1996, Ornoy 2007, Forsberg et al. 1996, Yon et al. 2008, Zaken et al. 2000), and HIF1alpha expression (Iyer et al. 1998, Maltepe et al. 1997, Jain et al. 1998, Minet et al. 2000) vary between different tissues of the embryo over time. Some of these variations have been correlated to periods of teratogenic susceptibility (Ornoy et al. 1999, Forsberg et al. 1996). In this transition, mitochondrial respiration may be inefficient producing higher-than-usual amounts of ROS at a time when embryonic defenses against ROS damage are not well developed. This combination can lead to excess ROS-induced cell stress and cell death (Dennery 2007, Dumollard et al. 2007, Burton et al. 2003). One hypothesis is that those tissues undergoing energetically demanding process such as morphogenesis are most susceptible to the oxygenation transition, a hypothesis furthered in diabetic embryopathy. The neural tube and somatic mesoderm have been shown to have a higher metabolic activity (Raddatz and Kucera 1983, Miki et al. 1988a, b, Mackler et al. 1971, Mackler et al. 1975) than surrounding tissues during early organogenesis, the time of greatest susceptibility to environmentally induced axial skeletal defects.
Carbon Monoxide
Early work studying the effects of hypoxia utilized carbon monoxide as a chemical hypoxic agent. Carbon monoxide (CO) is an odorless, colorless, non-irritating gas produced by the incomplete combustion of carbon containing materials. There have been no epidemiological studies of the direct effect of CO on human pregnancies (Schardein 2000). However, there are a number of case reports and anecdotes suggesting that CO may be a teratogen in humans (Robkin 1997, Longo 1977). Anecdotal accounts were given in Brander (Robkin 1997) and reported congenital malformations, such as microcephaly, micrognathia, and limb defects including hip dysplasia, tetraplegia, equinovarus, and limb reduction. Indirect epidemiological information can be obtained from the observations of pregnancy outcomes among women who smoke. Maternal smoking is associated with various adverse outcomes including low birth weight, decreases in successful births (Fichtner et al. 1990), and various behavioral defects that can be mimicked by CO alone in animal models (Bnait and Seller 1995).
There are a limited number of studies linking CO to congenital malformations. Early studies in chick, rabbit, and rat showed a causative relationship (Baker and Tumasonis 1972, Murray et al. 1979); however, later studies failed to confirm this connection (Astrup et al. 1975). More recent studies exploring threshold levels and critical periods related to CO-induced effects upon the embryo have documented CO-induced dysmorphogenesis (Bailey et al. 1994, Daughtrey et al. 1983, Loder et al. 2000, Alexander and Tuan 2003). CO exposures during early organogenesis, the critical period, resulted in vertebral anomalies, microphthalmia, and a phenotype similar to caudal dysgenesis syndrome. Such malformations have been reported with CO exposures administered during organogenesis in the context of other teratogens at sub-teratogenic levels (Singh et al. 1993, Singh 2006). This may be a significant problem worldwide since acute carbon monoxide exposures may be higher and more frequent than often reported (Fichtner et al. 1990, Ralston and Hampson 2000).
CO does impair oxygen delivery to and into cells by binding hemoglobin, myoglobin, and other porphyrins; however, it may also function as a signaling molecule in the context of nitric oxide (NO) signaling (Maines 1997). When administered after the vascular system is developed, the axial defects caused by CO are attributed to vascular leakage and subsequent mechanical disruption of developing tissue (Baker and Tumasonis 1972). However, during early organogenesis, axial defects involve the reduction of important segmentation genes including Pax1 and paraxis (Tcf15) (Alexander and Tuan 2003), resulting possibly in the impaired inductive interaction of the neural tube with the paraxial mesoderm with CO acting as a signaling molecule. Nitric oxide is known to regulate neurulation and other early embryonic processes (Lee and Juchau, 1994), and CO can alter the production of NO in axial tissues (Alexander et al. 2007a). The impaired interaction is likely due to a loss of cell function characterized or indicated by increased neural tube apoptosis and loss of neural tube-derived somite epithelialization signals.
Diabetes
Maternal diabetes is known to have many teratogenic effects (Finnell and Dansky 1991, Aberg et al. 2001). Malformations including neural tube defects, caudal dysgenesis, vertebral defects, congenital heart defects, femoral hypoplasia, renal, and craniofacial anomalies are described in infants of diabetic mothers. Caudal regression syndrome is a severe condition characterized by agenesis, regression, and or disorganization of the posterior (sacral–lumbar) vertebrae and the malformation of the soft tissue at that level and below (Bohring et al. 1999, Martinez-Frias et al. 1998b). It occurs 200 times more frequently in diabetic than in nondiabetic pregnancies. Other major malformations of the midline are also much more frequent including VATER, OAV, and other major malformations. Together, these can be placed in a related and progressive spectrum of syndromes and non-random associations belonging to the ADMC.
Mouse models utilizing “diabetic environments” or hyperglycemia report various anomalies encompassing the full spectrum of embryonic embryopathy (Akazawa 1995, Ornoy et al. 1999). These models together reveal that hyperglycemia is sufficient to cause most of the defects observed in diabetic embryopathy including neural tube defects, axial skeletal defects, heart and craniofacial abnormalities, rib and renal defects – although no individual model phenocopies the condition completely. At physiological levels of hyperglycemia or ketosis, the most consistent outcome is a failure of anterior and posterior neuropore closure (Sadler et al. 1988, Sadler and Horton 1983, Horton and Sadler 1983, Ornoy et al. 1986, Sadler et al. 1989). Researchers have determined that the diabetic environment increases ROS production in these regions of the neural tube and in the primordia of the organs listed above including craniofacial region, otic, and optic cups, Hensen’s node, and the notochord, caused by the diabetic environment. Coincident with the high ROS is an increase in cell death and a decrease in Pax3, a factor critical in neural tube closure (Fine et al. 1999, Loeken 2005). Application of folic acid and other antioxidants greatly reduced the incidence of ROS production (Ornoy 2007), insipient cell death and the reduction of Pax3 expression.
The caudal agenesis/dysgenesis syndrome can be phenocopied by prolonged exposure to hyperglycemia, hyperketonemia, and streptozotocin. The collection of defects in these severely affected animals indicates an early patterning event is disturbed. The notochord is laid down during gastrulation and is responsible for dorsoventral and mediolateral patterning as well as survival of the mesoderm during axis elongation. High levels of cell death in the notochord are observed in severely affected animals, suggesting the notochord function is likely to be compromised, and mutations in the T-box gene brachyury (Rashbass et al. 1994) and disruption of Shh function (Kim et al. 2001) have been presented as possible models of caudal dysgenesis and other manifestations of ADMC.
The incidence and severity of malformations in diabetic pregnancies are correlated with poor glycemic control in the first trimester and can be reduced by instituting tight glycemic control prior to conception, and the evidence presented above of various antioxidants and insulin provides hope that a cocktail can be developed and delivered harmlessly to prevent the initiation of the diabetic embryopathic condition. While prevention of the condition appears at hand, the initial biochemical imbalance presents us with an interesting pattern. The condition of hyperglycemia provides a “free” energy source that is readily available to the mitochondrion for ATP production, a condition opposite to hypoxia, in which ATP production in greatly decreased. A reasonable hypothesis incorporating these two opposite conditions is that molecular regulation of any developmental process can be disturbed by abnormal maternal fuel metabolism, and the timing of specific episodes of poor glycemic control determines which organ systems are affected.
Retinoic Acid
Retinoic acid (RA) is an analog of vitamin A commonly used to treat acne and other skin conditions. In humans, prenatal exposure results in a characteristic pattern of defects including abnormalities in the ears, mandibles, palates, aortic arch, and central nervous system. In animal models, many similar defects are observed (Gudas 1994). At higher doses delivered during organogenesis, RA can induce axial skeletal defects as well as including homeotic transformations (Kessel and Gruss 1991, Rubin and LaMantia 1999, Kawanishi et al. 2003), and at higher doses axial skeletal truncations (Padmanabhan 1998).
Aside from being a well-characterized teratogen, retinoic is also a naturally occurring chemical involved in many aspects of embryonic patterning, including the patterning of the somites. The teratogenic effects of retinoic acid above are consistent with the in situ expression of RA receptors (Maden 1994, Cui et al. 2003, Iulianelle et al. 1999) and metabolic-transforming enzymes (Swindell et al. 1999, Niederreither et al. 2002, Reijntjes et al. 2004, Cammas et al. 2007) as well as the effect of knocking down these molecules in murine models. RA, its receptors, and CYP26 are expressed in the paraxial mesoderm and act as critical regulators in the coordination of the somitogenesis clock and HOX gene expression (Duester 2007, reviewed in Sewell and Kusumi 2007). At higher levels, it is hypothesized that RA interrupts tissue morphogenesis, neural crest migration, and at highest doses causes cell death in morphogenically critical tissues, such as the neural tube, notochord, and paraxial mesoderm, resulting in a phenotype similar to caudal dysgenesis syndrome (Iulianelle et al. 1999).
Hyperthermia
Exposure of the human fetus to high temperatures (for example, 2°C over normal), as in the case of high fever or prolonged hot tub usage, is associated with neural tube defects, heart defects, microphthalmia, and functional deficits (Graham et al. 1998, Edwards et al. 1997). There is no epidemiological evidence suggesting heat shock causes axial skeletal defects. In studying the mechanisms of heat shock teratogenesis in animal models, vertebral defects were observed in many species including mice, rats, and chicks (Breen et al. 1999, Mirkes and Cornel 1992, Primmet et al. 1988). The severity of these defects is correlated to the time and duration of exposure. Experimental studies in chick embryos revealed that at moderate levels and exposure times (42°C for 20 minutes), one or two adjacent segments were fused into a single large somite. This effect was repeated every 7–8 somites separated by normal somitogenesis (Primmet et al. 1988). This result suggested a cell cycle-dependent mechanism to the defect and to somitogenesis itself, prompting the proposal of a clock and wave-front model for the patterning process of somitogenesis (Primmet et al. 1989).
The response of the embryo to hyperthermia is very dependent upon the degree of temperature increase, its duration, and the stage at which the heat shock is experienced (Graham et al. 1998). There is a steep threshold for embryonic survival and resorption, which suggests the general outcome of hyperthermia is embryonic resorption. At levels of hyperthermia inducing embryonic survival and malformation, tissue-specific cell death is observed. Investigators identified the induction of heat shock proteins (HSP) as a prominent feature of the embryonic response. These molecular chaperones play important roles in regulating protein folding during normal cell function, but they also serve to protect cells from environmental insult. In the process, the HSP-bound proteins are not able to perform their function (Buckiova et al. 1998, Walsh et al. 1999). During teratological doses of hyperthermia, the cell cycle is slowed, suggesting a mechanism of the vertebral anomalies observed. Recently the mechanism of somitogenesis was shown to involve the tightly controlled, cyclic expression of a variety of proteins many belonging to the Notch/Delta signaling system (Shifley and Cole 2007). During heat shock, some of these proteins or their targets may be bound by HSP, and we can hypothesize that this would disrupt the somitogenic clock resulting in disrupted pattern and ultimately vertebral defects. An important feature of the protective heat shock response then and its relation to teratogenesis is that their activation and function may reduce or delay tissue development or morphogenic actions. In fact, many teratogenic insults induce HSP activity, and as such HSP activation may be an underlying commonality in teratogenic mechanisms along with ROS production and apoptosis.
Arsenic
Arsenic, a metal pollutant, is found naturally in groundwater and unnaturally in mine waste sites, industrial byproducts, and in agricultural runoff. It is toxic to humans and is known to cause birth defects including spina bifida, craniofacial defects, developmental retardation and to decrease birth weight and increase incidences of fetal mortality, miscarriage, and still birth (Willhite and Ferm 1984, DeSesso et al. 1998). In experimental in vitro models, arsenic is teratogenic in mice, rats, and chicks (Hood and Bishop 1972, Chaineau et al. 1994, Beaudoin 1974, Lindgren et al. 1984, Peterková and Puzanová 1976, DeSesso et al. 1998), with neural tube defects being common among all of them (Shalat et al. 1996, Takeuchi 1979). Its toxicity is greatly dependent on its redox state: arsenate vs. arsenite. The structure of arsenate can mimic that of phosphate groups, imparting arsenate with the ability to disrupt various cell processes including nucleic acid metabolism, lipid metabolism, and electron transport. Inefficient electron transport can lead to high production of ROS, which have documented cell destructive activities and teratogenic capacity (Hunter 2000, Kitchin and Ahmad 2003, Bernstam and Nriagu 2000). In addition, arsenate can reduce to arsenite. The effects of arsenite on disruption of cell cycle and cytoskeletal structure have been attributed to its reaction to sulfhydryl groups (Levinson et al. 1980), which may account for its strong induction of the heat shock response (German et al. 1986, Mirkes and Cornel 1992, Bernstam and Nriagu 2000). In addition, arsenite can disrupt the citric acid cycle and electron transport via binding to thiol group enzymatic active sites (DeSesso et al. 1998). A disruption in the energy status of different tissues of the developing embryo is attributed to teratogenicity of arsenic causing similar malformations to those observed in hypoxic or hyperglycemic environment; however, arsenic has other distinctive effects on the embryo (DeSesso et al. 1998). Arsenic and other metal compounds are very effective inducers of the heat shock response (Mirkes and Cornel 1992, Bernstam and Nriagu 2000), which may protect cell from molecular damage, but induce birth defects in its own right via disruption of the cell cycle and other cyclic and time-dependent morphogenetic processes (Wlodarczyk et al. 1996). In addition, cells surviving the initial arsenic insult, may pass on genetic damage that contribute to subsequent carcinogenic transformation later in ontogeny (Bernstam and Nriagu 2000). These multiple, interacting mechanisms may account for the wide range of malformations observed following acute arsenic exposure.
Ethanol
Ethanol, widely consumed as a recreational drug, has long been strongly associated with teratogenesis as fetal alcohol syndrome (FAS). FAS is present in one in three children of alcoholic mothers, with an estimated 40,000 children born every year in the United States (Schardein 2000, Thackray and Tift 2001). FAS manifestations include growth deficiency, central nervous system problems, characteristic facial features, and organ malformations. Features of FAS have been observed in animal models exposed to ethanol in utero or in vitro, including mice, rat, chick, and others (Sulik et al. 1981, Becker et al. 1996, Fernandez et al. 1983, Sanders and Cheung 1990, Yelin et al. 2007, Schardein 2000, Chaudhuri 2000, Padmanabhan and Muawad 1985).
The mouse model has been a particularly effective model in elucidating the etiology of ethanol-induced birth defects. One mechanism, of ethanol-induced teratogenesis is through ethanol impaired placental blood flow to the fetus by constricting blood vessels and inducing embryonic/fetal hypoxia and malnutrition (Shibley et al. 1999). Since ethanol rapidly crosses the placenta into the fetus, there are other direct embryonic and fetal targets of ethanol. The mouse model has been a particularly effective model in elucidating the etiology of ethanol-induced birth defects. Ethanol has been shown to increase cell death in critical cell populations including anterior neural folds and neural crest cells (Sulik et al. 1988, Rovasio and Battiato 1995, Dunty et al. 2001), which play a critical role in the morphogenesis of the face. Neural crest cells are particularly vulnerable to ethanol, inducing delayed/altered migration and cell death (Rovasio and Battiato 1995). Correlations have been made to increased ROS production within the neural crest population (Kotch et al. 1995), mitochondrial dysfunction and cell death in the etiology of ethanol and other teratogens (Ornoy 2007). The anterior neural tube and cranial neural crest have been the subject of intense scrutiny in the teratogenic mechanisms of ethanol, however, other tissues are also affected, including the eye, ear, heart, renal system, and axial skeleton (Kennedy and Elliott 1986, Parnell et al. 2006, Sulik 2005, Webster and Abela 1984, Assadi and Zajak 1992, Sanders and Cheung 1990, Carvan et al. 2004). With respect to the axial skeleton, investigators observed a misalignment or segmentation defect in ethanol-exposed embryos. Despite the substantial morphological difference with heat shock- treated embryos, the investigators suggested that the mechanism may be similar to heat shock (Carvan et al. 2004), involving the induction of the stress response by increased ROS production.
Methanol
Methanol is an alcohol encountered frequently during industrial processes. When the effects of inhaled ethanol and methanol were compared, the highest doses of methanol significantly increased the incidence of various defects including skeletal malformations (Nelson et al. 1985). Skeletal malformations were the most prevalent congenital defects observed and included vertebral abnormalities and an increased incidence of cervical ribs. Other skeletal abnormalities caused by methanol have been observed including holoprosencephaly, facial dysmorphogenesis, basicranial malformation, duplications of the atlas and axis and cervical vertebral abnormalities, and abnormal number of presacral vertebrae (Connelly and Rogers 1997, Rogers et al. 2004). Initial cellular responses appear similar to ethanol at the level of tissue-specific cell death (Abbott et al. 1995). In contrast to ethanol, many of the axial skeletal defects indicate homeotic shifts in segment identity.
Conclusion
Advances in cell and molecular biology with respect to normal development and somitogenesis and the pathogenesis and mechanisms of teratogenesis are occurring at a tremendous rate. This allows teratologists and developmental toxicologists the opportunity to revisit old problems with new tools. Despite the large number of cellular processes that may be disturbed by a teratogen, there are only a limited number of cellular and morphological outcomes. This has led investigators to strive for the identification of very defined critical periods and doses in a variety of model systems to aid in the identification of the initial targets of a teratogen and the true, hypothetically singular target molecule or process, as proposed by Wilson in 1956. Applying genomic and proteomic technologies to the problem of teratogenesis should begin to reveal the full spectrum of cellular processes affected, and elucidate links between variations in genotype and the effect of the environment on the phenotype that produce birth defects such as congenital scoliosis. The identification, at least in part, of this “holy grail” will aid in the development of new preventative treatments to a variety of teratogenic insults.
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Supported in by the Intramural Research Program of the National Institute of Arthritis, and Musculoskeletal and Skin Diseases, NIH (ZO1 AR41131).
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Alexander, P.G., Tuan, R.S. (2010). Environmental Factors and Axial Skeletal Dysmorphogenesis. In: Kusumi, K., Dunwoodie, S. (eds) The Genetics and Development of Scoliosis. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-1406-4_3
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