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Alcohol-Induced Death in the

SUSAN M. SMITH, PH.D.

Exposure to alcohol during gestation can have profound consequences, but not all cells within the embryo are affected equally. Recent advances in molecular have allowed an exploration of this variation. Much of this research has focused on the embryo’s vulnerability to the facial malformations characteristic of fetal alcohol syndrome. Studies using mice and chicks show that alcohol exposure at specific stages of early embryo development results in significant death among the cells destined to give rise to facial structures (i.e., cranial cells). This type of cell death is through activation of the cell’s own “self-destruct” machinery (i.e., apoptosis). Researchers have advanced several theories to explain how alcohol triggers apoptosis in the neural crest cells. These theories include deficiency in a type of vitamin A compound, retinoic acid; reduced levels of antioxidant compounds (i.e., free radical scavengers) that protect against damage from toxic oxygen molecules (i.e., free radicals); and interference with the cell’s normal internal communication pathways. KEY WORDS: congenital facial anomaly; fetal alcohol syndrome; prenatal alcohol exposure; cytolysis; gestation; teratogens; neural cell; temporal context; retinoic acid; free radicals; oxygen; ; model; literature review

lcohol is capable of directly are unplanned. By the time these preg- Clarren 1996). An excellent correlation inducing abnormalities during nancies are confirmed, major embryonic exists between these structural malfor- A prenatal development that can events already have occurred. (For a mations (i.e., dysmorphologies) and a lead to lifelong and profound disabili- brief overview of normal embryo de- history of prenatal alcohol exposure. ties (i.e., it is a teratogen1). In fact, velopment, see sidebar, p. 296Ð297.) Nevertheless, many children born to prenatal exposure to alcohol—the most Early diagnosis and intervention for alcoholic mothers lack these facial prevalent teratogen in Western society— children affected by prenatal alcohol defects, although the children do exhibit is the most common known cause of exposure can reduce their risk for social alcohol-related neurodevelopmental mental retardation and neurobehavioral difficulties later in life (e.g., problems deficits, such as mental retardation, deficits. By conservative estimates, 12 with employment or trouble with the attention deficits, hyperactivity, im- percent of all patients in long-term law resulting from impulsive behavior pulsiveness, and poor judgmental skills. institutionalized care have fetal alcohol and lack of inhibition) (Streissguth et To help define the risks associated syndrome (FAS) (see Stratton et al. al. 1996). Identifying these children with maternal alcohol use during 1996 for an extensive discussion of can be difficult, however. One useful , researchers are seeking to FAS). Although the simplest prevention FAS screening tool relies on the follow- understand the mechanisms of alcohol’s strategy is for women to avoid alcohol ing trio of distinctive facial features that teratogenic effects on various tissues. consumption when pregnant or planning characterize prenatal alcohol exposure: to conceive, one-half of all pregnancies small eye openings (i.e., short palpebral SUSAN M. SMITH, PH.D., is an associ- fissures), a thin upper lip, and a flattened ate professor in the Department of 1For a definition of this and other technical or absent groove between the upper lip Nutritional Sciences at the University terms used in this article, see glossary, p. 295. and nose (i.e., philtrum) (Astley and of Wisconsin—Madison.

VOL. 21, NO. 4, 1997 287 The use of animal models to reproduce actions and affect only a subset of cells fertilization. For example, alcohol is many of the deficits seen in FAS allows within the embryo. The timing of expo- most likely to cause heart defects investigators to study alcohol’s terato- sure plays a role in the embryo’s outcome during the time when cellular signals genic effects in detail under controlled as well: A given ’s susceptibility are directing the heart’s division into treatment conditions. In addition, recent to disruption peaks during distinct its various chambers and great vessels, findings in molecular embryology have timeframes of development (i.e., critical starting 4 weeks after fertilization. greatly advanced scientific knowledge sensitivity windows). Thus, a close In work that was critical in drawing about the signals and agents that govern examination of the target tissue’s devel- attention to alcohol’s early effects on the normal embryo development. Applying opmental history may reveal clues as embryo, Sulik and colleagues (1981) this knowledge can help elucidate how to the toxicant’s mechanism and the used a mouse model developed by prenatal alcohol exposure results in birth basis for the tissue’s sensitivity. Such Webster and colleagues (1980) to defects. It also can clarify our under- examination also may identify addi- convincingly demonstrate that alcohol standing of why certain physical defects tional, previously unsuspected targets. targets early events in formation. are useful diagnostics for prenatal alco- For example, because the face, limbs, Using an experimental protocol that hol exposure, and it places these deform- urogenital tract, and central nervous involved exposing mouse to ities within the overall criteria that system all use the same set of genes to alcohol during a specific period of define whether a potentially affected direct their growth and development, gestation (corresponding to the third person may qualify for support services. it is not surprising that certain toxicants week of human pregnancy),2 the research- This article first reviews current or genetic mutations will disrupt all of ers produced facial malformations understanding about the embryo’s these tissues to some degree. consistent with the visible characteris- vulnerability to facial malformations The concept of critical sensitivity tics of FAS in people (i.e., a narrow and the alcohol-induced cell death windows is useful in examining how forehead, short palpebral fissures, a responsible for producing such deform- alcohol produces its teratogenic effects. small nose and midface, and a long ities. The article then explores several Certain embryonic tissues, such as those upper lip with a deficient philtrum), as theories on how alcohol triggers cell of the face, heart, and urogenital tract, shown in figure 1. The peak maternal death in embryos and concludes with appear to be particularly sensitive to blood alcohol concentration (BAC) in a discussion of possible future re- alcohol-induced malformations during the mice used in this research ranged search directions. the processes that establish location, from 0.2 to 0.5 percent; the lower growth, and three-dimensional shape as the tissues develop from their primitive 2 ALCOHOL-INDUCED CELL DEATH origins. In humans, many of these early Protocols with alcohol exposure during other specific periods of gestation produced malfor- developmental events occur during a mations in different body systems (e.g., the Many toxic substances (i.e., toxicants), time when a woman may be unaware heart and limbs), as predicted by the critical including alcohol, are specific in their of her pregnancy, 3 to 6 weeks after windows hypothesis.

ABC

Narrow forehead Short palpebral fissures Small nose Small midface Long upper lip with deficient (flat) philtrum

Figure 1 Similarities of facial defects found in (A) humans and (B) mice exposed prenatally to alcohol. Panel C shows a control mouse fetus not exposed to alcohol. (Photograph courtesy of Kathy K. Sulik.)

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BAC levels, in particular, are achiev- able in alcoholics. The fact that alcohol causes similar A. Normal embryo B. Alcohol-exposed embryo facial defects in people and rodents implies that alcohol probably affects Primitive eye embryonic events common to both species. Closer examination of alcohol- Primitive eye exposed mouse embryos revealed Primitive significant cell death within specific groups (i.e., populations) of cells, in- Primitive brain cluding the primitive neural and facial Primitive tissues. In particular, cell death was observed in a group of cells called the neural crest (see figure 2 for similar Heart Primitive ear cell death in chick embryos) (e.g., Sulik et al. 1988). Rats exposed to alcohol during late gestation or soon after birth experienced a similar death of cells within specific brain regions. The restriction of alcohol- induced cell death to selected brain Primitive regions supports the concept that alcohol spinal column affects specific target tissues. Moreover, the causative relationship between Figure 2 Neural crest cell death in the chick embryo. Panel A shows a normal alcohol and cell death is reinforced by embryo after 48 hours of incubation (corresponding to 22 to 25 days of the fact that several alcohol-affected human gestation), when the dark-stained neural crest cells (see arrows) embryonic tissues later result in body migrate from the primitive brain toward regions of facial development. structures that exhibit malformations Panel B shows a 48-hour embryo that was exposed to alcohol at a critical consistent with underdevelopment. In developmental time (i.e., at 18 to 36 hours of incubation). The diffuse, addition to the characteristic facial de- weblike staining (see arrows) in the alcohol-exposed embryo indicates the fects seen in FAS, these malformations presence of fewer neural crest cells compared with the many neural crest can include exencephaly, a condition cells indicated by the denser staining in the control embryo. in which the brain is exposed or extrudes from a skull defect; spina bifida, a congenital defect in which part of the which the cell “programs” its own contain apoptotic machinery but keep is exposed through a gap in destruction. (For a discussion of the it checked with a series of regulatory the backbone; absence of the corpus potential contribution of apoptosis to suppressive proteins. A variety of callosum, a brain structure that helps link alcoholic disease, see the article signals may initiate apoptosis, however, the two brain hemispheres (i.e., failure by Nanji and Hiller-Sturmhöfel, pp. including a loss of con- of the corpus callosum to develop); and 325Ð330.) trols (e.g., during the change from a motor losses in the spinal cord. Necrosis and apoptosis differ in normal to a precancerous cell); altered several crucial ways. Necrosis occurs signaling from cell mediators that Type of Cell Death when cell metabolism ceases, leading stimulate proliferation, differentiation, to membrane disintegration and rupturing or cell survival (i.e., growth factors); Both chronic and acute alcohol expo- of the cell’s contents. In contrast, apop- changes in cell attachment to various sures can lead to cell death in the mature tosis depends on activation of the cell’s tissue surfaces; and activation of spe- liver, brain, and certain white blood own internal cell-death machinery cific proteins that trigger cell death cells (i.e., lymphocytes), as observed (White 1996). Apoptotic cells maintain (i.e., “death domain” proteins). in animal models and cell culture sys- their metabolism and membrane integ- Some apoptosis is a natural part of tems. In turn, such aberrant cell death rity but are characterized by damage to development. In adults, for example, might contribute to characteristic tissue cellular repair systems, DNA fragmen- apoptosis is often a desirable process diseases associated with repeated alcohol tation, and cellular breakdown into used to remove damaged or mutated exposure (e.g., liver cirrhosis or immune membrane-enclosed, metabolically active cells (e.g., cells in tumors) that have suppression). Although some of this cell cell fragments (i.e., “blebs”). These escaped from the normal constraints death involves necrosis, much of it is distinctive characteristics are the basis on their growth. In embryos and fetuses, consistent with the elicitation of another for assays that differentiate apoptotic apoptosis shapes and remodels devel- type of cell death called apoptosis, in from necrotic cell death. Most cells oping tissues by removing unwanted

VOL. 21, NO. 4, 1997 289 cell populations (e.g., the “webbing” the collapse of the entire cell into small, shortly after the cells begin migration. between fingers and toes, which is metabolically active subunits that are Many of the structures arising from the deleted during the sixth through eighth soon engulfed by neighboring cells for cranial neural crest potentially could be weeks of gestation in humans). Research disposal. Special dyes can penetrate affected by alcohol exposure, as a sur- has established that the majority of cell these subunits, and stained subunits vey of the various birth defects reported death during normal embryo develop- show up prominently during alcohol- in the FAS literature would show. ment is apoptotic. At critical times or induced cell death (Sulik et al. 1988). A separate neural crest population locations, however, excessive cell death Further evidence that alcohol-induced (i.e., the trunk neural crest) originates beyond the normal bounds of apoptosis cell death within the neuroepithelium from the primitive spinal cord and could produce malformations within and neural crest is apoptotic comes contributes to other body structures susceptible tissues, despite the em- from a study showing that cell death (see table). Unlike the cranial neural bryo’s tremendous capacity to repair was prevented by compounds that in- crest cells, however, the fate of the damage. The realization that alcohol hibit caspase, an enzyme that destroys trunk neural crest cells is not predeter- and other teratogens can stimulate ex- intracellular repair machinery to ensure mined before their migration. Instead, cessive cell death within the embryo the completion of apoptosis (Cartwright the type of tissue they ultimately form was a conceptual breakthrough. et al. 1998). Taken together, these depends on the environment into which Using neuronal cells from chicks, findings suggest that prenatal alcohol they migrate. Scientific literature sug- investigators first documented embry- exposure can result in the apoptotic gests that tissues formed from the trunk onic cell death resulting from prenatal elimination of embryonic cells. Necrosis neural crest cells are less affected by alcohol exposure in the late 1960’s also can occur under certain circumstan- alcohol than are those made by the (Sandor 1968). More than a decade ces (e.g. nutrient or oxgen starvation), cranial neural crest cells. Such resistance later, similar studies of mouse embryos however, and care must be taken to dis- may reflect the greater adaptability the revealed clusters of dying cells, primari- tinguish between these two mechanisms. trunk neural crest cells have with respect ly in tissues that would later form the to their identity and differentiation, central (i.e., in the even after some cells are deleted. neuroepithelium) (Bannigan and Burke ALCOHOL’S EFFECTS ON NEURAL Researchers have not formally exam- 1982). Apparently, the cell death ob- CREST DEVELOPMENT ined this hypothesis, however. served by the researchers was apoptotic: Although the cells continued to synthe- Realizing that the cells of the neural size DNA (thus indicating that they crest are particularly vulnerable to ALCOHOL AND FACIAL DEFECTS: were still active), they had a rounded alcohol-induced death, researchers TIMING IS EVERYTHING appearance and contained condensed have focused on this cell population. material in their nucleus and surround- Analysis of alcohol-induced facial The mouse studies of Sulik and col- ing cell fluid—features not inconsistent defects revealed certain commonalities leagues (1988) clearly highlighted the with current definitions of apoptosis. among the affected structures. Many similarity between the visible charac- (The older scientific literature refers to of these structures (i.e., the upper and teristics of FAS and the facial malfor- embryonic cell death as necrosis, how- lower jaw, nose, , orbital mations produced following abnormal ever, leading to some confusion.) around the eye, and forehead) are cranial neural crest development. More recent studies have provided derived from the same cellular ances- Widespread cell death within embryo support that apoptosis is indeed the type tors in the embryo (i.e., cell lineage), a regions enriched with neural crest cells of cell death at work in embryos exposed subset of the neural crest population provided strong evidence that alcohol to alcohol. Alcohol-exposed cells within called the cranial neural crest. This directly targeted this cell population. the embryo have several structural and lineage originates within the primitive Nevertheless, a key question remained: biochemical features that are unique (i.e., the neurectoderm) (for What mechanism could be triggering to apoptosis. For example, Sulik and a review, see Noden 1991). Cranial their death? colleagues (1988) found that cells in neural crest cells migrate from the neu- Recent advances in developmental alcohol-exposed embryos often had ral tube and differentiate into a wide biology have made this question ap- nuclear DNA that was fractured and variety of structures (see table). The proachable at the molecular level, condensed into small, dense particles specific body structure that cranial because much is now known about the (i.e., pyknotic fragments) characteristic neural crest cells will generate (i.e., their mechanisms involved in neural crest of apoptosis. Through the use of the positional identity, such as whether the development. Several clues have come appropriate biochemical reagents, these cells will contribute to the upper or from research zeroing in on the role pyknotic fragments can be readily lower jaw, for example) is determined played by the precise timing of alcohol detected within the nervous system and before they leave the neurectoderm. exposure in relation to the embryo’s neural crest of embryos exposed to Similarly, their differentiation fate (e.g., developmental stage. Researchers alcohol (Cartwright et al. 1998). Another whether they develop into nerve or have made progress in identifying the characteristic indicator of apoptosis is bone tissue) is determined before or critical sensitivity window for neural

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crest apoptosis, when and where cell death actually occurs, and how alcohol- Body Components Derived From Neural Crest Cells in the Embryo induced and “natural” (i.e., endogenous) apoptosis interact. Structures Derived From Cranial Neural Crest Cells

Facial and , including those of the nose, upper and lower jaw, and Neural Crest Critical around the eye; also the small bone supporting the (i.e., hyoid bone) Sensitivity Window Skull cartilage and bone (front only) To investigate the mechanism of neural Cartilage of the inner ear crest cell death, some researchers have turned to the chick embryo, which has core (i.e., odontoblasts) long been a model of choice for neural of the and the , salivary, pituitary, and tear- crest studies. (Despite the obvious differ- producing (i.e., lacrimal) glands ences among , mice, and people, Calcitonin-producing cells of the thyroid gland neural crest development in each species is similar; nature does not “reinvent the Transparent fibrous supportive tissue (i.e., stroma) and inner lining of the cornea wheel,” but instead uses the same genes Connective tissue surrounding the eye and optic nerve to solve common problems of embryo Eye muscles that control the size of the pupil and curvature of the (i.e., development across species.) Chicks pupillary and ciliary muscles) offer several advantages to investigators of the face and front part of the neck, including the inner, living layer of skin studying embryo development. Because (i.e., ); ; and fat cells the chick develops in an egg outside the mother’s body, studies can avoid Pigment-producing skin cells (i.e., ) potential complicating factors arising Sensory nerve portions of several nerves arising directly from the brain, specifically from maternal and placental metabolism cranial nerves III (oculomotor), V (trigeminal), VII (facial), IX (glossopharyngeal), and nutrition. Moreover, researchers can and X (vagus) cut a small window in the eggshell and Specialized supportive and nutritive tissue (i.e., glial cells) of all cranial nerve cell directly view and manipulate the em- bundles (i.e., ganglia) bryo. Resealing the window with tape Innermost and middle membrane layers (i.e., the pia mater and arachnoid mater, or wax provides sufficient protection respectively) that enclose the lower rear (i.e., occipital) region of the brain to allow the chick embryo’s develop- ment to continue. Smooth muscle and dividing walls (i.e., septa) of the pulmonary artery and aorta As with mouse embryos, chick (i.e., ) embryos exposed to alcohol levels Type I cells of the small body structure involved in monitoring blood levels of greater than 0.15 percent exhibit sig- oxygen, carbon dioxide, and hydrogen (i.e., the carotid body) nificant cell death within regions enriched with cranial neural crest cells Structures Derived From Trunk Neural Crest Cells and distinct sections of the primitive brain (i.e., the midbrain and hindbrain Motor of the involuntary (i.e., sympathetic and parasympathetic) portion of the neural folds) (Cartwright et al. 1998). nervous system outside of the brain and spinal cord (i.e., the peripheral nervous system) Using specific markers, researchers Intestinal, preaortic, and spinal nerve cell bundles (i.e., ganglia) documented directly that neural crest populations are deleted by alcohol Specialized supportive and nutritive tissue (i.e., glial cells) of the peripheral nervous system exposure, and a laboratory technique (i.e., double-stained overlays) con- Sheath-producing cells in the peripheral nervous system (i.e., Schwann cells) firmed that these losses are attributable Innermost and middle membrane layers (i.e., the pia mater and arachnoid mater, to cell death. In addition, just as in respectively) enclosing the spinal cord mouse embryos, comparatively little Epinephrine- and norepinephrine-releasing cells in the adrenal medulla (i.e., cell death occurs in the heart, eyes, chromaffin cells) and precursors of the vertebral column and (i.e., ) of Pigment-producing skin cells (i.e., melanocytes) the chick embryos. This observation SOURCES: Noden, D.M. craniofacial development: The relation between ontogenetic process and suggests that cell death is not likely morphological outcome. Brain, Behavior and Evolution 38(4Ð5):190Ð225, 1991; Larsen, W.J. Human Embryology. to be a consequence of generalized New York: Churchill Livingstone, 1993; Hall, B.K., and Hörstadius, S. The Neural Crest. London: Oxford Science toxicity, but rather that particular Publications, 1988. embryonic cell populations are

VOL. 21, NO. 4, 1997 291 susceptible (or resistant) to this alcohol- induced effect. Studies using mouse embryos pin- A. Normal embryo B. Alcohol-exposed embryo pointed the critical sensitivity window for facial defects to the short time during the creation of the embryo’s three basic cell layers (i.e., ) and the first organization of the primitive ner- Primitive brain Primitive brain vous system (i.e., and early development) (Webster et al. Primitive ear 1980). Researchers also found the iden- Primitive ear tical critical sensitivity window in chicks (Cartwright and Smith 1995). Heart The ability to precisely identify the developmental stage of chick embryos allowed even more refined investigation of the window’s extent with further Primitive Primitive Heart spinal column studies. These studies uncovered a spinal column curious finding about alcohol-induced cell death in chick embryos: Alcohol exposure caused cell death only if the Figure 3 Apoptosis in neural crest cells and the primitive brain. Panel A shows a normal 48-hour embryo stained with a bright dye (i.e., acridine orange) alcohol was administered before the that becomes concentrated in apoptotic cells. These dead cells appear neural crest cells emigrated from their as tiny dots (see arrows). Panel B shows a 48-hour embryo that was birthplace in the neurectoderm. Once exposed to alcohol at a critical developmental time (i.e., at 18 to 36 the cells began migrating toward the site hours of incubation). Many more dead cells, visible as bright dots (see where the chick’s face would develop, arrows), are seen in the primitive brain and face of the alcohol-exposed the neural crest cells were resistant to embryo compared with the normal embryo. Note that some cell groups alcohol-induced death; moreover, are dying in both embryos, but many more cells are dying in the alcohol- alcohol exposure after the start of neural exposed embryo. This observation supports the premise that normal crest cell migration no longer resulted and alcohol-induced cell death occur simultaneously in the early brain in facial defects. Thus, at least for and neural crest. chicks, the critical sensitivity window for producing facial malformations is extremely narrow, lasting only from time before neural crest cell migration at an early point in embryo develop- gastrulation to cranial neural crest mi- do not actually result in cell death until ment produces maximal cell death in gration, or from 18 to 36 hours of egg later, specifically at 46 to 48 hours of the more rostral neural crest cell popu- incubation after laying. incubation (Cartwright et al. 1998). Thus, lations (e.g., the midbrain portion of Determining the critical sensitivity alcohol exposure during a variety of early the neural tube), whereas later expo- window for facial malformations in stages appears to activate the apoptotic sure shifts the majority of cell death to people is not possible, but because mice pathway, but cells do not carry out the more caudal populations (e.g., the and chicks represent such diverse species “self-destruct” sequence until they reach hindbrain segment of the neural tube) sharing a similarly narrow window, a particular stage of development. (Cartwright and Smith 1995). This these findings strongly suggest that a Closer examination of where apop- finding suggests that neural crest cells comparable window exists for humans. If tosis is occurring in the embryo at a must achieve a particular maturation so, then the appearance of facial defects given timepoint further supports this state before carrying out their alcohol- in FAS may reflect peak blood alcohol theory. Because embryos develop in a induced “suicide” and implies the concentration in early gestation more head-to-tail (i.e., rostrocaudal) sequence, existence of certain cellular events strongly than previously suspected. the neural folds do not meet along that convey susceptibility (or resis- their entire length simultaneously, but tance) to apoptosis. Dependence on are “zipped” together to form the neu- Developmental Stage ral tube. Thus, the neural crest cells Alcohol-Induced and arising from the foremost part of the “Natural” Apoptosis Recent studies demonstrate that the neural tube are created and mature regulation of apoptosis depends on the first, whereas those cells toward the Mouse studies by Sulik and colleagues embryo’s developmental stage. In chick opposite end of the tube are progres- (1988) provided another significant embryos, initial alcohol exposure and sively less mature at any given time- clue regarding apoptosis regulation. peak alcohol concentration achieved any point. Likewise, exposure to alcohol Their research found that teratogen-

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induced cell death often was most vation of apoptosis, each with strong possibly increasing the risk for RA- common in embryonic regions already supporting evidence. The following and alcohol-related birth defects. undergoing some degree of endogenous sections explore these hypotheses and Paradoxically, however, RA itself is cell death. Cartwright and Smith (1995) the evidence to support them. also a potent teratogen, and excessive noted the same observation in the chick RA or vitamin A supplementation model, in which alcohol-induced neural Retinoic Acid Deficiency could endanger the embryo. Scientists crest apoptosis coincided with the need better documentation of potential normal deletion of two subgroups of Retinoic acid (RA), an active form of alcohol-RA links before contemplating cranial neural crest cells by endogenous vitamin A, is essential for neural crest nutritional interventions. apoptosis (see figure 3). survival and is a key regulator of body The coincidence of alcohol-induced form and structure development (i.e., Free Radical Damage and normal neural crest death prompted ). RA affects the posi- the hypothesis that alcohol exposure tional identity of cells according to the An alternative hypothesis on the aberrantly activates the apoptotic structure that they will form within mechanism behind neural crest apop- machinery in cranial neural crest cells. diverse embryonic regions, including tosis is based on alcohol’s known This hypothesis predicts that the preva- the cranial neural crest, face, central ability to cause cell injury during its lence or activity of substances involved nervous system, limbs, and urogenital metabolism. Alcohol exposure can in endogenous apoptosis would increase tract. In addition, RA is a potent medi- increase the levels of highly reactive during concurrent alcohol-induced ator of differentiation in many cell oxygen molecules known as free death. Molecular analysis did not sup- lineages. Alcohol exposure is associ- radicals as well as deplete cells of the port this prediction, however: A study ated with localized RA deficiencies in protective compounds (i.e., antioxi- by Cartwright and colleagues (1998) embryos, however (DeJonge and dants) that normally scavenge these found no increase in the prevalence or Zachman 1995; Deltour et al. 1996). toxic molecules. Under such condi- activity of two substances involved in Because low levels of RA result in the tions (i.e., oxidative stress), vital com- endogenous apoptosis (i.e., a transcrip- apoptosis of neural crest populations ponents of the cell may begin to tion factor named msx2 and a growth and their subsequent elimination from combine with free radicals and result factor named BMP-4) (Cartwright et al. the embryo (Maden et al. 1997), RA in injury. In particular, free radicals 1998) during alcohol-induced death. deficiency is implicated in alcohol- can damage cell membranes through a Thus, although the timing of alcohol- induced apoptosis. process called lipid peroxidation. This induced and endogenous apoptosis Studies of a class IV alcohol dehy- process may interfere with many coincides, their processes must be drogenase enzyme found in embryos important cell regulatory processes, activated through different pathways. further supports this hypothesis. This including control over substances This finding opens the possibility of enzyme is involved in the synthesis of entering and leaving the cell, intercel- discovering therapies that could inhibit retinal, an immediate precursor of RA. lular communication, and protein alcohol’s actions without altering the Deltour and colleagues (1996) demon- synthesis. Damage from such interfer- necessary endogenous cell deletions. strated that alcohol concentrations ence could be fatal to cells. found in alcoholic women inhibit Approaches using whole embryo and this enzyme and are associated with cell cultures, particularly the research WHAT TRIGGERS ALCOHOL- reduced RA levels within the embryonic conducted by Chen and colleagues INDUCED APOPTOSIS? face and nervous system. Additional (Chen and Sulik 1996; Chen et al. 1996) research shows that treatment with have been critical in testing the hypo- Understanding the molecular regula- supplemental RA prevents alcohol- thesis relating free radical damage to tion of apoptosis will help explain induced heart defects in embryos (Twal neural crest apoptosis. Noting that how alcohol initiates the apoptotic and Zile 1997). cultured neural crest cells appear to lack pathway and results in the death of Although frank deficiency of vita- an antioxidant compound (i.e., super- some, but not all, cell populations. In min A compounds (i.e., retinoids) is oxide dismutase) that plays an important pursuit of this goal, current research seldom observed in Western popula- role in removing free radicals, the efforts focus on identifying the mech- tions, a recent study in Iowa (Duitsman researchers cultured alcohol-exposed anism that activates the cell-death et al. 1995) found that between 9 and neural crest cells together with super- pathway following alcohol exposure. 26 percent of a sample of low-income oxide dismutase and other free radical In all likelihood, the trigger for alcohol- pregnant women in their third trimester scavengers. The results showed that induced apoptosis lies within the target had marginal retinoid stores, equivalent the addition of antioxidants modestly cell population itself, because many to levels seen in malnourished, non- reduced alcohol-induced cell death other cell populations do not apoptose, industrialized populations. Inadequate (Chen and Sulik 1996). Because free despite equivalent alcohol exposures. vitamin A intake among alcoholic radical mechanisms also participate in Investigators have presented several women could create marginal retinoid endogenous apoptosis, however, it is hypotheses to explain alcohol’s acti- concentrations within the embryo, unclear whether antioxidants interfere

VOL. 21, NO. 4, 1997 293 with alcohol’s action or with the apop- enzyme protein kinase), depending on New approaches also are available tosis process itself. the target cell (Slater et al. 1993; Sta- to study alcohol’s effects on the cell’s Supplementation with gangliosides, checki et al. 1994). Thus, one can easily normal metabolic processes and com- a type of lipid compound usually found speculate that alcohol could interfere munication. For example, highly sensi- abundantly in nerve cell membranes, also with the signaling of a tive intracellular reagents and probes decreases the death of alcohol-exposed critical to a cell’s survival at particular allow scientists to monitor the various neural crest cells (Chen et al. 1996), and timepoints during development and metabolic pathways that link events in protocols similar to those used with anti- lead to apoptosis. The fact that a wider the cell membrane to changes within oxidant supplementation have protected range of embryonic tissues is not overtly the cell. Combining these techniques nerve cells exposed to alcohol. Thus, affected by alcohol exposure may be with specialized computer-driven detec- researchers hypothesize that ganglio- attributed to the embryo’s tremendous tion equipment, such as laser-driven sides may defend against cell membrane capacity to repair damage. In addition, microscopy (i.e., confocal microscopy), damage from lipid peroxidation. the redundancy of cellular signals may will enable investigators to monitor Further research will prove inform- help compensate for alcohol’s interfer- alcohol’s short-term effects on single ative in understanding free radical dam- ence. The power of such compensation cells as they occur. age and how alcohol-induced oxidative can be seen most clearly in studies that Finally, although the cranial neural stress contributes to alcohol’s injury to delete or inactivate a critical gene, yet crest contributes to many tissues, the embryo. Alcohol exposure may result often result in normal embryos. alcohol-related research on these cells in free radical production from several Again, recent advances in decipher- predominantly focuses on facial bone sources. For example, free radicals ing the molecular signals that govern and cartilage. Examination of other could be formed as a consequence of development will greatly facilitate tissues arising from the cranial neural alcohol’s oxidation itself (e.g., through further exploration of altered cellular crest could uncover less recognized enzymes, such as cytochrome P450’s, communication as a potential mecha- targets of prenatal alcohol exposure. or catalases); its alteration of the cell’s nism of apoptosis. Quite likely, a Approaches similar to those undertaken potential for chemical reactions or the combination of RA deficiency, free by researchers studying facial defects chemical chain of events involved in radical damage, and interference with also will benefit studies of brain, heart, generating cell energy (i.e., mitochon- cellular communication, as well as other urogenital tract, and limb development. drial respiration); or the action of other mechanisms, all contribute to alcohol’s Taken together, recent discoveries agents, such as nitric oxide. The sensitive toxicity and subsequent pathologies. in cellular and molecular embryology intracellular probes that are now avail- will greatly advance our understand- able for detecting free radicals and their ing of alcohol’s impact on prenatal sources will enable researchers to test FUTURE DIRECTIONS development. Moreover, understand- these mechanisms directly. ing the limited circumstances that Advances in molecular and cellular create alcohol-induced facial defects Intracellular Communication biology have opened multiple avenues may bolster the recognition that chil- Interference for exploring alcohol’s impact on pre- dren who demonstrate alcohol-related natal development. Many of alcohol’s neurodevelopmental deficits but lack A third mechanism proposed for alcohol- target tissues, including face, nervous overt facial defects represent a syn- induced apoptosis involves alcohol’s system, limb, and urogenital tract tis- drome that can equal FAS in severity ability to perturb the function of cell sues, employ a common set of genes and long-term prognosis. membrane-associated proteins. These that are responsible for coordinating proteins function as channels or recep- tissue growth (Johnson and Tabin tors that transfer critical chemical 1997). Several alcohol-related birth REFERENCES signals from the cell surface to its defects are consistent with altered interior (i.e., transmembrane or intra- embryo development, and examining ASTLEY, S.J., AND CLARREN, S.K. A case defini- cellular communication). Interference the role that genes play in regulating tion and photographic screening tool for the with this process, perhaps through early development in animal models facial phenotype of fetal alcohol syndrome. direct alcohol-protein interactions, may yield insights into how susceptible Journal of Pediatrics 129(1):33Ð41, 1996. could cause aberrant activation or tissues respond to alcohol exposure. In BANNIGAN, J., AND BURKE, P. Ethanol teratogen- inhibition of the communication path- particular, the use of mice with delib- icity in mice: A light microscopic study. ways vital to cell survival. erate gene mutations inserted in their 26(3):247Ð254, 1982. Studies using various cultured cell genetic material (i.e., transgenic mice) lines have documented alcohol’s ability will assist investigators seeking to CARTWRIGHT, M.M., AND SMITH, S.M. Stage- to alter intracellular communication connect specific gene alterations to dependent effects of ethanol on cranial neural crest cell development: Partial basis for the through a variety of mechanisms (e.g., alcohol’s developmental consequences. phenotypic variations observed in fetal alcohol alterations of cyclic AMP, intracellular (See article by Homanics and Hiller- syndrome. Alcoholism: Clinical and Experimental calcium release, and activity of the Stumhöfel, pp. 298Ð309.) Research 19(6):1454Ð1462, 1995.

294 ALCOHOL HEALTH & RESEARCH WORLD Cell Death in the Embryo

CARTWRIGHT, M.M.; TESSMER, L.L.; AND SMITH, relative dose response (MRDR) test. Nutrition STRATTON, K.; HOWE, C.; AND BATTAGLIA, F., S.M. Ethanol-induced neural crest apoptosis is Research 15:1265Ð1276, 1995. EDS. Fetal Alcohol Syndrome: Diagnosis, coincident with their endogenous death, but is Epidemiology, Prevention, and Treatment. JOHNSON, R.L., AND TABIN, C.J. Molecular mechanistically distinct. Alcoholism: Clinical Washington, DC: National Academy Press, 1996. and Experimental Research 22(1):142Ð149, models for vertebrate limb development. Cell 90:979Ð990, 1997. 1998. STREISSGUTH, A.P.; BARR, H.M.; KOGAN, J.; AND BOOKSTEIN, F.L. Understanding the Occurrence MADEN, M.; GRAHAM, A.; GALE, E.; ROLLINSON, CHEN, S., AND SULIK, K.K. Free radicals and of Secondary Disabilities in Clients With Fetal C.; AND ZILE, M. Positional apoptosis during ethanol-induced cytotoxicity in neural crest vertebrate CNS development in the absence of Alcohol Syndrome (FAS) and Fetal Alcohol cells. Alcoholism: Clinical and Experimental endogenous retinoids. Development 124(14): Effects (FAE): Final Report. Seattle, WA: Fetal Research 20(6):1071Ð1076, 1996. 2799Ð2805, 1997. Alcohol and Drug Unit, 1996. CHEN, S.Y.; YANG, B.; JACOBSON, K.; AND NODEN, D.M. Vertebrate craniofacial develop- SULIK, K.K.; COOK, C.S.; AND WEBSTER, W.S. SULIK, K.K. The membrane disordering effect ment: The relation between ontogenetic process Teratogens and craniofacial malformations: of ethanol on neural crest cells in vitro and the and morphological outcome. Brain, Behavior Relationships to cell death. Development protective role of GM1 ganglioside. Alcohol and Evolution 38:190Ð225, 1991. 103(Suppl.):213Ð231, 1988. 13(6):589Ð595, 1996. SANDOR, S. The influence of aethyl-alcohol on SULIK, K.K.; JOHNSTON, M.C.; AND WEBB, M.A. DEJONGE, M.H., AND ZACHMAN, R.D. The effect the developing chick embryo. II. Revue Roumaine Fetal alcohol syndrome: Embryogenesis in a of maternal ethanol ingestion on fetal rat heart d’Embryologie et de Cytology. Serie de Cytologie mouse model. Science 214(4523):936Ð938, 1981. vitamin A: A model for fetal alcohol syndrome. 5:167Ð172, 1968. Pediatric Research 37(4):418Ð423, 1995. TWAL, W.O., AND ZILE, M.H. Retinoic acid reverses SLATER, S.J.; COX, K.J.A.; LOMBARDI, J.V.; HO, ethanol-induced cardiovascular abnormalities DELTOUR, L.; ANG, H.L.; AND DUESTER, G. Etha- C.; KELLY, M.B.; RUBIN, E.; AND STUBBS, C.D. in quail embryos. Alcoholism: Clinical and nol inhibition of retinoic acid synthesis as a Inhibition of protein kinase C by alcohols and Experimental Research 21(6):1137Ð1143, 1997. potential mechanism for fetal alcohol syndrome. anesthetics. Nature 364(6432):82Ð84, 1993. Federation of American Societies for Experimental WEBSTER, W.S.; WALSH, D.A.; LIPSON, A.H.; Biology (FASEB) Journal 10(9):1050Ð1057, 1996. STACHECKI, J.J.; YELIAN, F.D.; SCHULTZ, F.J.; AND MCEWEN, S.E. Teratogenesis after acute LEACH, R.E.; AND ARMANT, D.R. alcohol exposure in inbred and outbred mice. DUITSMAN, P.K.; COOK, L.R.; TANUMIHARDJO, cavitation is accelerated by ethanol- or Neurobehavior and Toxicology 2:227Ð234, 1980. S.A.; AND OLSON, J.A. Vitamin A inadequacy in ionophore-induced elevation of intracellular socioeconomically disadvantaged pregnant calcium. Biology of Reproduction 50(1):1Ð9, WHITE, E. Life, death, and the pursuit of apop- Iowan women as assessed by the modified 1994. tosis. Genes and Development 10:1Ð15, 1996.

GLOSSARY

Dysmorphology: Abnormal development of tissue form and Morphogenesis: The development of the form and structure structure; a congenital malformation or birth defect. of the body and its parts, including the growth and differ- : The outermost of the three distinct layers of cells in entiation of cells and tissues. the early embryo—enclosing the and — Neural crest: Cells at the junction of the folded tips that from which the nervous system and skin ultimately form. enclose the neural tube. In humans, these cells detach from Endoderm: The innermost of the three distinct layers of the the neural tube during the fourth week of gestation and early embryo. The endoderm produces the lining of the migrate through the embryo to initiate development of a digestive tract and its associated organs (i.e., the , wide range of body structures (see table p. 291). liver, and so forth). Neural tube: The embryological structure from which the brain and spinal cord develop. The neural tube is formed Gastrulation: Extensive cell rearrangements that result in when the mesoderm stimulates the midline of the embryo three distinct cell regions (i.e., germ layers) within the ectoderm to fold up and over, transforming the embryo’s embryo: the ectoderm, mesoderm, and endoderm. All shape from a flattened disk to a primitive vertebrate body. subsequent embryo tissue is derived from one of these three germ layers. In humans, gastrulation begins to take Neurectoderm: The primitive neural tube. place approximately 16 days after fertilization. Somite: Any of the paired segmented divisions of the mesoderm Mesoderm: The middle layer of the three distinct layers of the that develop along the neural tube, forming the vertebral early embryo—lying between the ectoderm and endo- column. The somites differentiate into bones, connective derm—from which several of the body’s organs (i.e., the tissue, voluntary muscle, and the deeper layers of the skin. heart, kidneys, and gonads), bones and musculature, and Teratogen: Any agent capable of causing abnormalities during blood cells ultimately form. prenatal development.

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he union of sperm and egg at fertilization sets blastocyst. Approximately 4 days after fertilization into motion a remarkable series of events des- takes place in the fallopian tube (i.e., oviduct), the blas- Ttined to culminate in a fully developed adult. As tocyst arrives in the uterus.1 Within a few more days, development proceeds toward this goal, cells divide, the blastocyst attaches to the wall of the uterus, where it differentiate into specific types, and organize into body will remain anchored until birth. By the time attachment tissues and systems according to both an ancient general (i.e., implantation) occurs, the rate of cell division has schematic for the species and the new person’s unique slowed somewhat, and the cells of the blastocyst have genetic heritage. rearranged themselves into a hollow ball. A flat disk of The first 8 weeks of this process (i.e., the embryo cells inside the ball will become the embryo proper, stage) are especially busy and are crucial in laying the while the outer cells will develop into placental tissues. foundation for future growth (see figure 1). From its Approximately 2 weeks after fertilization, further cell tiny, one-celled beginning, a fertilized egg undergoes a rearrangement (i.e., gastrulation) begins to establish a process of rapid cell division (i.e., ) and soon multilayered, vertebrate body plan in the embryo. Three becomes a roughly spherical cluster of cells called a distinct “founder regions” of cells (i.e., germ layers)

Fertilization Cleavage Blastocyst Precursor Inner of placenta Sperm cell mass (embryo)

Egg

Day 1 Days 1Ð4 Day 6Ð7 Gastrulation (cross view)

Fluid-filled Ectoderm amniotic cavity Notochordal (embryo and ) plate Mesoderm () Endoderm

Definitive yolk sac

Day 14 Day 18 Embryonic Rearrangement (side view)

Amniotic Fusing neural folds cavity Amniotic cavity

Embryo Neural tube Heart Yolk sac Yolk sac Day 21 Day 22Ð23 Growth and Differentiation

Primitive eye Arm bud

Umbilical Hand cord

Leg bud Heart

5 weeks 6 weeks 8 weeks

Figure 1 Key stages of embryo development. (Note: Structures not drawn to scale.)

296 ALCOHOL HEALTH & RESEARCH WORLD form during gastrulation: the ectoderm, endoderm, and mesoderm. Unfolding genetic codes direct further development of each , transforming it from A Ectoderm C Precursors of Neural crest an initially undifferentiated set of cells to increasingly neural crest Precursor of complex, genetically programmed tissue patterns that neural tube eventually become recognizable body structures. Thus, Ectoderm the skin and nervous system are formed from the outer- most layer of cells (i.e., the ectoderm), while the lining Mesoderm of the digestive tract, respiratory tubes, and their associ- Endoderm ated organs derive from the innermost layer (i.e., the B D Neural endoderm). Between these two layers, the mesoderm Neural tube spreads outward and will generate all the organs be- Neural folds crest cells tween the ectodermal wall and the endodermal tissue, including the cardiovascular system, bones, muscles, and connective tissue. The cells of the three germ layers interact with one another in an intricate sequence to coordinate the devel- opment of all the body structures. For example, a strip of mesoderm cells (i.e., the notochord) lying directly Body cavity beneath the ectoderm along the embryo’s midline causes a portion of the ectoderm to wrinkle and form two ridges (see figure 2). The tops of these ridges (i.e., the neural Figure 2 Formation of the neural tube (cross view). Early folds) curve inward and by the fourth week of gestation, in an embryo’s development, a strip of specialized meet and fuse to enclose a hollow tube (i.e., the neural cells called the notochord (A) induces the cells of the ectoderm directly above it to become the tube) from which the brain and spinal cord will devel- primitive nervous system (i.e., neuroepithelium). op. This developmental milestone helps transform the The neuroepithelium then wrinkles and folds embryo from a flattened disk to a three-dimensional over (B). As the tips of the folds fuse together, primitive body. Soon after the neural folds fuse, cells a hollow tube (i.e., the neural tube) forms (C)— the precursor of the brain and spinal cord. originating at their junction (i.e., neural crest cells) Meanwhile, the ectoderm and endoderm detach and migrate to diverse locations in the embryo continue to curve around and fuse beneath the in order to initiate the development of many vital embryo to create the body cavity, completing body structures. the transformation of the embryo from a flattened disk to a three-dimensional body. Cells originating As the neural crest cells migrate, increasingly com- from the fused tips of the neurectoderm (i.e., plex developmental activity in the embryo results in neural crest cells) migrate to various locations considerable progress toward a recognizable human throughout the embryo, where they will initiate form. Over the course of the fourth week, the heart be- the development of diverse body structures (D). gins to pump; arm and leg buds appear; and the ground- work is laid for the liver, , eyes, , and other body systems. The embryo doubles in length to become pea size. During the next several weeks, rapid progress in 1 development and refinement continues as nerves sprout, Although embryo development is basically the same in all , the timeframes given here correspond specifically to humans. lymphatic and coronary vessels appear, brain structures become visible, the intestinal loop forms, the kidneys begin to ascend, and many other milestones are achieved. Bibliography Hands begin to develop in the fifth week, and finger rays GILBERT, S.F. . 2d ed. Sunderland, MA: appear in the sixth; feet and toe rays follow a week later. Sinauer Associates, 1988. In the seventh week, bone begins to replace the ’s GILBERT, S.F., AND RAUNIO, A.M., EDS. Embryology: Constructing the cartilage foundation, eyelids form, and the first folli- Organism. Sunderland, MA: Sinauer Associates, 1997. cles appear. By the end of the eighth week of gestation, LARSEN, W.J. Essentials of Human Embryology. New York: Churchill the embryo is just over 1 inch long, and all major body Livingstone, 1998. systems are in place. From this point onward, the embryo (now termed “fetus”) primarily undergoes further refine- ment and growth, which continue after birth at a slower rate until adult form and stature are attained. Mary Beth de Ribeaux is a science editor of Alcohol —Mary Beth de Ribeaux Health & Research World.

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