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2
Issues in Research on Fetal Drug Effects

Alcohol is one of a number of chemically diverse compounds currently recognized to be toxic to the developing central nervous system (CNS) of humans. The neurotoxic properties of these compounds have generally been confirmed in animal studies. Based on their effects, these agents can be divided into two classes of neurotoxicants: some are teratogens in that they produce CNS malformations with associated neurobehavioral dysfunction (e.g., alcohol, methylmercury), whereas others produce neurobehavioral dysfunction in the absence of CNS malformations (e.g., lead, polychlorinated biphenyls). The agents in Table 2-1 are presented here in order to put alcohol in the context of other developmental toxic agents and the way these agents can injure the developing CNS, both pre- and postnatally.

Alcohol is a member of one broad category of teratogenic chemicals; this category also includes other substances of abuse (nicotine), pharmacologic agents (phenytoin or retinoic acid), and environmental toxicants (lead, mercury). Other teratogens can be categorized as either physical agents, such as ionizing radiation or hyperthermia; infectious agents, such as rubella virus; and factors related to maternal health and nutrition, such as malnutrition or maternal hyperglycemia secondary to diabetes. Although the effects of exposure to many of these compounds are well described, the mechanisms of action are not. Teratogens differ in their periods of susceptibility, the duration of exposure required to cause adverse fetal outcome, and the manifestations of the insult. A few examples of well-known teratogens are provided below, but this discussion is not exhaustive. This



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Page 33 2 Issues in Research on Fetal Drug Effects Alcohol is one of a number of chemically diverse compounds currently recognized to be toxic to the developing central nervous system (CNS) of humans. The neurotoxic properties of these compounds have generally been confirmed in animal studies. Based on their effects, these agents can be divided into two classes of neurotoxicants: some are teratogens in that they produce CNS malformations with associated neurobehavioral dysfunction (e.g., alcohol, methylmercury), whereas others produce neurobehavioral dysfunction in the absence of CNS malformations (e.g., lead, polychlorinated biphenyls). The agents in Table 2-1 are presented here in order to put alcohol in the context of other developmental toxic agents and the way these agents can injure the developing CNS, both pre- and postnatally. Alcohol is a member of one broad category of teratogenic chemicals; this category also includes other substances of abuse (nicotine), pharmacologic agents (phenytoin or retinoic acid), and environmental toxicants (lead, mercury). Other teratogens can be categorized as either physical agents, such as ionizing radiation or hyperthermia; infectious agents, such as rubella virus; and factors related to maternal health and nutrition, such as malnutrition or maternal hyperglycemia secondary to diabetes. Although the effects of exposure to many of these compounds are well described, the mechanisms of action are not. Teratogens differ in their periods of susceptibility, the duration of exposure required to cause adverse fetal outcome, and the manifestations of the insult. A few examples of well-known teratogens are provided below, but this discussion is not exhaustive. This

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Page 34 TABLE 2-1  Neurobehavioral Outcome of Prenatal Exposure in Humans or Animals       Alcohol Methylmercury Ionizing Radiation Phenytoin PCBs Lead Opioids Marijuana Tobacco Gross neuropathology Pos., NDR Pos., DR Pos., DR — — NE NE NE NE Mental retardation Pos., DR Pos., DR Pos., DR NE ? NE NE NE NE Reduced IQ scores Pos., DR Pos., DR Pos., DR Pos., NDR Pos., NDR Pos., DR NE ? Pos., DR Hyperactivity Pos., DR — — — Pos., NDR ? NE NE Pos., DR Attention deficit Pos., DR — — — — ? ? Pos., NDR Pos., NDR Developmental delays Pos., DR Pos., DR — Pos., NDR Pos., DR — ? Pos., NDR Pos., DR Gait abnormality Pos., DR Pos., DR — — NE NE NE NE NE Fine/gross coordination Pos., DR — — — Pos., DR ? ? NE NE Sensory deficits Pos., DR Pos., DR — — NE Pos., NDR NE NE Pos., Dr Neonatal withdrawal Pos., NDR — — — — — Pos., DR ? Pos., DR NOTE: Pos., DR = Positive findings, dose-related; Pos., NDR = positive findings, non-dose-related; ? = suspected, some reports; NE = no effects; and—= not tested, unknown.    

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Page 35 chapter is intended to highlight issues of concern in FAS; the interested reviewer is referred to the published literature for more detail on the other teratogens. Alcohol is a recognized human teratogen that produces fetal alcohol syndrome (FAS) and a variety of other alcohol-related effects in children exposed during prenatal life. Of all the substances of abuse, including heroin, cocaine, and marijuana, alcohol produces by far the most serious neurobehavioral effects in the fetus. The teratogenicity of X-irradiation has been known since the early part of this century, and it is well-established that the developing CNS is particularly sensitive to irradiation-induced injury. Population-based studies of the effects of in utero exposure in Hiroshima and Nagasaki have indicated a risk of mental retardation based on gestational age at the time of exposure. What would become known as "fetal Minamata disease" results from maternal ingestion of methyl-mercury-contaminated food. Mercury pollutants contained in wastewater and discharged from a chemical plant in Minamata, Japan, accumulated in fish and shellfish, an important staple of the local inhabitants' diet. Even though most of the exposed mothers did not show symptoms of mercury poisoning, exposure of the fetus during gestation months 6-8 produced widespread neuropathological effects in the cerebrum and cerebellum (Burbacher et al., 1990). Of all the neurotoxicants, lead has been the most extensively studied and is viewed as a serious pediatric public health problem. Pregnant women and children living in major urban centers where soil and dust become contaminated with lead-based paint are at risk for undue exposure. Increases in blood levels of lead are known to occur in children as soon as they begin to crawl and to place contaminated hands and toys in their mouth. Neurobehavioral processes of cognition, learning, and behavior are all known to be adversely affected in exposed children (Davis et al., 1990). The polychlorinated biphenyls (PCBs), first introduced in the 1930s, are a group of synthetic hydrocarbons that had widespread commercial use as electrical insulating fluid, in heat exchangers, plastics, transformers, capacitors, and carbonless copy paper. As a result of their largely uncontrolled disposal and exceptional persistence, they became a major environmental pollutant in the air, water, and soil of industrialized countries. Children prenatally exposed to PCBs show neurobehavioral effects on cognition, learning, and behavior (Tilson et al., 1990). Diphenylhydantoin (DPH, phenytoin) is one of several prescription medications, used often in combination, to treat seizures associated with epilepsy. DPH has been implicated as a human teratogen. Although only a relatively small number of children prenatally exposed to DPH have been assessed for neurobehavioral effects, results tentatively suggest a reduction in IQ scores (Adams et al., 1990). The opiates heroin and methadone are not teratogenic, and effects on the developing CNS appear to be mediated by the opioid receptors. Neonates show acute but transitory symptoms of neonatal withdrawal, a physiological state characterized

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Page 36 by CNS irritability. The symptoms, though life-threatening when severe, are timelimited and resolve in several weeks to months (Hutchings, 1990). Although there have been reports of mild neonatal withdrawal following maternal use of marijuana, neurobehavioral deficits are not evident between 6 months and 2 years of age. Beginning at 3 years of age, however, there have been reports of reduced IQ scores (Day et al., 1994), attention deficit (Fried et al., 1992), and developmental delay (Fried, 1993). Cigarette smoking during pregnancy is associated with dose-related effects on withdrawal (i.e., increased tremors), reduced IQ scores, developmental delay, and poor auditory responsiveness; several reports have linked maternal smoking with hyperactivity and impaired attention (Fried, 1994). Prenatal exposure to tobacco is clearly linked to low birth weight. Although reports from the mid 1980s suggested that the use of cocaine during pregnancy increased the risk of genitourinary tract malformations, abruptio placenta, intrauterine growth retardation, sudden infant death syndrome as well as a number of postnatal neurobehavioral deficits, subsequent reports largely failed to replicate these initial observations (see Hutchings, 1993 and accompanying commentaries). A major problem in interpreting the results of virtually all of the studies that reported these adverse effects is the extent to which the outcomes attributed to cocaine resulted from concurrent abuse of alcohol, cigarettes, and other illicit substances. Ongoing prospective studies that control for these and other confounding variables are expected to yield more interpretable results. As noted above, alcohol is a potent teratogen; as with most teratogens, there is considerable variation in the extent and severity of prenatal effects in exposed offspring, and that some but not all deficits appear to be attenuated as the offspring matures. For example, a case series of adolescents with FAS show less striking facial dysmorphia and normal weight in some, particularly adolescent females (Streissguth et al., 1985). There are no data suggesting that cognitive deficits ameliorate. Identification of risk factors to explain differences in susceptibility to and expression of alcohol teratogenesis has therefore become an important area of investigation for both basic and clinical researchers. The following sections discuss the contribution of basic research to our understanding of teratology and the potential risk factors that modulate outcomes in fetal alcohol-exposed offspring. This chapter focuses on data from animal models that are relevant to difficult or unanswered issues in clinical FAS research. Chapters 4 and 8 contain discussion relevant to the clinical teratology of alcohol exposure. PRINCIPLES OF TERATOLOGY AND DEVELOPMENTAL TOXICOLOGY Wilson (1973) set forth several organizing principles that specify the relationship of environmental agents to adverse outcome: (1) developmental stage; (2) species susceptibility and genotype; and (3) dose-response. Schardein (1985) has succinctly characterized these factors as they relate to teratology by paraphrasing

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Page 37 a well-known axiom of Karnofsky (1965); a version modified to include the broader domain of developmental toxicology would be as follows: "A toxic response in developing organisms depends upon the administration of a specific treatment or a particular dose to a genetically susceptible species when the offspring are in a susceptible stage of development." In this section, we extend these basic principles of teratology originally set forth by Wilson (1973) and elaborated by Schardein (1985) to the domain of prenatal alcohol exposure and include the variable of postnatal environment or experience, which is now known to influence developmental outcome. SUSCEPTIBLE STAGES OF DEVELOPMENT It is convenient to divide prenatal development into three periods: the predifferentiation period, the period of the embryo, and the period of the fetus. The distinction, however, is only conceptual. The conceptus, throughout gestation, is in a continual state of orderly biochemical and structural transition during which new constituents are being formed and spatially rearranged. At any time in the total span of development, these ongoing processes can be subtly deflected, severely perturbed, or abruptly halted, resulting in death or abnormal development. Furthermore, although the effects of exposure during specific stages or "critical periods" of development are probably best documented for the anatomical or dysmorphogenic effects of various teratogenic agents, data on stage-specific effects on growth and functional deficits are increasing, particularly in relation to prenatal alcohol exposure. The interval between fertilization of the oocyte and its implantation in the endometrium is approximately six days in both rats and humans, and is referred to as the predifferentiation period. During this time, the ovum, while remaining relatively undifferentiated, undergoes a series of mitotic divisions, changing from a unicellular zygote to a multicellular blastocyst. Agents that produce malformations later in development are generally thought to be without teratogenic effects during this early period. It appears either that chemicals are toxic to the entire blastocyst, resulting in its death (i.e., spontaneous abortion in humans or resorption in rodents) or that if they are toxic to a limited number of cells, regulative mechanisms result in repair with no apparent damage. Relatively few studies using animal models have examined the effects of alcohol during the predifferentiation period. An early study reported no adverse effects of alcohol given on gestation day 6 on pregnancy outcome in mice (Lochry et al., 1982). However, in another study (Padmanabhan and Hameed, 1988) it was found that alcohol exposure on gestation days 1-6 did not influence litter size (implantation), but markedly increased prenatal mortality (resorptions) and resulted in increased placental weights, decreased umbilical cord length, and decreased fetal weight at gestation day 15 in surviving litters. Interestingly, malformations (craniofacial, eye, urogenital, limb) were also noted in 80-100 percent of

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Page 38 viable fetuses. These somewhat surprising results suggest that very early alcohol exposure, at least in certain mouse strains, may in fact be teratogenic as well as lethal to the embryo. The period between early germ layer differentiation and the completion of major organ formation (i.e., organogenesis) is referred to as the period of the embryo. This phase of development, characterized by the formation of complex, multicellular tissues and organs of diverse origins and functions, begins soon after implantation and, in humans, continues through approximately the eighth week of gestation. During organogenesis, the embryo is maximally susceptible to gross structural malformation if exposed to teratogenic agents. The nature of the defects will depend both on the effects of the agent on embryonic cells and the gestational age at the time of exposure. In contrast to the predifferentiation period, the effects of acute alcohol exposure on specific days during the embryonic period have been studied fairly extensively. Various mouse strains have been used in these studies, and it is important to note that the results may differ among strains (see section below on "Susceptible Species and Genotype" for discussion of this issue). Teratogenic doses of alcohol induce excessive cell death. Most cell types appear to be vulnerable to alcohol-induced cell death, but neuronal cell populations may be particularly vulnerable. Exposure on gestation days 7 and 8 in mice was shown to result in craniofacial defects similar to those seen in FAS (e.g., micrognathia, low-set ears, short philtrum, cleft palate, cleft lip) (Sulik et al., 1981), as well as brain anomalies (e.g., microcephaly, exencephaly, deficiencies in cerebral hemispheres, striatum, olfactory bulbs, limbic structures, corpus callosum, lateral ventricles) (Sulik et al., 1984; Webster et al., 1980, 1983). In addition, ocular defects (anophthalmia, microphthalmia, corneal and lens anomalies) were associated with acute alcohol exposure on gestation day 7 (Cook et al., 1987; Webster et al., 1983), whereas cardiac (e.g., reduced size of the cardiac tube, abnormalities of the A-V canals, ventricular-septal defects, anomalies of great vessels) (Daft et al., 1986; Webster et al., 1984) and skeletal anomalies (involving vertebrae, sternum, ribs) (Blakely and Scott, 1984; Ciociola and Gautieri, 1988; Padmanabhan and Muawad, 1985; Stuckey and Berry, 1984) were associated with acute exposure on gestation day 8. It was concluded (Kotch and Sulik, 1992) that alcohol teratogenesis and the patterns of malformations induced at times corresponding to late in the third and early in the fourth week postfertilization in the human can be directly correlated to the selective cytotoxic effects of alcohol coupled with the selective vulnerability of the cells at the margin of the anterior neural folds. In contrast, acute alcohol exposure on gestation days 9 and 10 was found to produce urogenital anomalies (hydronephrosis, hydroureter) (Boggan et al., 1989; Gage and Sulik, 1991; Gilliam and Irtenkauf, 1990; Randall et al., 1989) and limb anomalies involving forelimbs (including ectrodactyly, polydactyly, syndactyly) (Gilliam and Irtenkauf, 1990; Kotch et al., 1992; Randall et al., 1989; Webster et

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Page 39 TABLE 2-2    Periods of Susceptibility to Developmental Injuries to the Nervous System Developmental injuries to the nervous system have a protracted period of susceptibility that extends beyond organogenesis. For the central nervous system these include the following events. 1. Neurogenesis 2. Neuronal differentiation and migration 3. Arborization and synaptogenesis 4. Functional synaptic organization 5. Myelination 6. Gliogenesis, glial migration, and glial differentiation SOURCE: Adapted from Vorhees (1989). al., 1980, 1983). A much lower incidence of malformations was observed when alcohol exposure was restricted to gestation days 12-14. The interval from the end of organogenesis until parturition (approximately weeks 9-40 of human pregnancy, corresponding in rats to approximately gestation day 15 to birth) is referred to as the period of the fetus. Toxic exposure during the fetal period generally does not produce gross structural malformations. Such exposure can, however, produce histologic changes in tissues, inhibit growth, and produce subtle damage in the developing CNS (often manifested as neurobehavioral effects) and other organ systems by interfering with histogenesis, synaptogenesis, the formation of myelin, and other biochemical processes (Table 2-2). Such effects are characterized for alcohol by changes in function or functional organization and include a number of well-documented clinical entities such as microcolon, islet cell hyperplasia, and ventricular septal hypertrophy. These processes continue well into the postnatal period and, for the human CNS, into the second year of life. In mouse models, prenatal alcohol exposure on gestation day 15 or 18 was shown to result in dose-dependent decreases in fetal body weight, decreased brain DNA (deoxyribonucleic acid) synthesis, and delayed neonatal reflexive behaviors (Ciociola and Gautieri, 1988). Gestation days 15-21 in the rat also appear to be a critical period of vulnerability to alcohol-induced reduction in hippocampal n-methyl-D-aspartate (NMDA) receptor binding (Savage et al., 1992). However, in terms of CNS effects in rodents, the early postnatal period may be the most vulnerable to alcohol-induced damage, as it is equivalent to the third trimester of gestation in humans. For example, it was shown that alcohol exposure during either the first half or the last half of gestation had no detectable effect on the development of specific neurons (mossy fibers) within the hippocampus of rats. In contrast, similar alcohol exposure during the first 10 days postpartum dramatically altered hippocampal mossy fiber organization (West

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Page 40 and Hamre, 1985). Furthermore, with respect to the cerebellum, it was shown that both the extent and the location of neuron loss (specifically, Purkinje and granule cells) were extremely sensitive to the timing of alcohol exposure during the brain growth spurt (Hamre and West, 1993). Other studies using rat models have shown that late (days 12-17) but not early (days 5-10) gestation is a sensitive period for growth retardation and for behavioral effects of prenatal alcohol exposure. Mice exposed to relatively low alcohol doses (17 percent alcohol-derived calories) had normal birth weights but showed markedly attenuated growth between 19 and 28 days of age, resulting in a weight reduction for at least 35 days (Middaugh and Boggan, 1991). Mice exposed to 25 percent alcohol-derived calories (which led to peak BAC of approximately 100 mg/dl) did not have normal birth weights. It was suggested that fetal alcohol exposure may compromise the organism's ability to handle the increased demands associated with weaning and accelerated growth. Behaviorally, it was reported that fetal alcohol-exposed mice were slower to respond on high fixed ratio schedules than controls (Middaugh and Gentry, 1992). This compromised function may reflect an adverse effect of fetal alcohol exposure on the development of neuronal systems underlying reward which develop during the latter part of gestation. SUSCEPTIBLE SPECIES AND GENOTYPE Different species show a differential susceptibility to developmental toxicants, and within species, susceptibility can vary with genotype. For teratological effects, Kalter (1968) pointed out that both inter- and intraspecies variability may be manifested in several ways: (1) an agent that is teratogenic in some species may have little or no teratogenic effect in another; (2) a teratogen may produce similar defects in various species, but these defects will vary in frequency; and (3) a teratogen may induce certain abnormalities in one species that are entirely different from those induced in another. Other critical factors associated with genotype and species relate to differences in rates of metabolism; qualitative differences in metabolic pathways; and for prenatal studies, differences in placental structure and maternal-fetal pharmacokinetics (e.g., see Nau, 1986). The majority of studies using rodent models have focused on the period of the embryo, which is the primary period of organogenesis. These studies have shown that some mouse strains appear to be particularly sensitive to the teratogenic effects of both acute and chronic alcohol exposure, whereas other strains appear to be less sensitive to alcohol's teratogenic effects (Cassells et al., 1987; Chernoff, 1977). These differences in susceptibility may occur in relation to the teratogenic effects of alcohol on brain morphology as well as to alcohol's effects on the dysmorphology of a number of organ systems. Furthermore, strains may differ in their sensitivity or resistance to alcohol's teratogenic effects depending

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Page 41 on the day of exposure, in the sensitivity of particular organ systems that may occur within a strain, or the rate of metabolism of alcohol. Several recent studies suggest that differences in alcohol sensitivity among organisms with different genotypes may influence the severity of alcohol's effects on the fetus. Studies in mouse lines selectively bred for differences in the hypnotic effects of acute alcohol administration (long sleep [LS] and short sleep [SS]) reported line differences in fetal growth, dysmorphology, behavioral deficits, and mortality that cannot be explained by differences in maternal weight gain, duration of alcohol exposure, or blood alcohol levels (Gilliam et al., 1987; 1989a,b). Offspring of alcohol-sensitive LS mice were shown to exhibit greater prenatal and postnatal growth retardation, increased skeletal abnormalities, deficits in passive avoidance performance, and increased mortality compared to offspring of alcohol-insensitive SS mice. Differences in the pattern of skeletal abnormalities were also observed, with rib anomalies more common in SS fetuses and anomalies of the sternum more common in LS fetuses. Interestingly, it was shown that alcohol-induced deficits in fetal weight were determined primarily by maternal genetic factors, whereas both maternal and fetal genotype appeared to play a role in determining susceptibility to morphological abnormalities (Gilliam and Irtenkauf, 1990). An important source of information on genetic influences on the expression of teratologic effects comes from studies of twins. Alcohol seems to be similar to many teratogens, such as thalidomide, diethylstilbestrol (DES), and diphenylhydantoin, in that monozygotic twins are more concordant for outcome of prenatal alcohol exposure than dizygotic twins (Streissguth and Dehaene, 1993). This review of 16 pairs of alcohol-exposed twins reports that all five of the monozygotic twin pairs were concordant for diagnosis and that 7 of the 11 dizygotic twins were concordant for diagnosis. In animal studies, not all of the newborns are equally affected. These data suggest that effects of prenatal exposure to alcohol, like many teratogens, can be modulated by genetic influences. Together these data suggest that genetic factors may, in fact, represent important determinants in susceptibility to teratogenesis, conferring special sensitivity or resistance to the teratogenic effects of alcohol at particular periods of development. However, no specific genes have been identified to date. Clearly, these data have implications for the variability of defects observed in children exposed to high doses of alcohol prenatally. DOSE-RESPONSE EFFECTS Dose-response relationships are among the most critical issues in developmental toxicology, yet they are too often misunderstood, oversimplified, or simply neglected. Because prenatal drug studies involve two mutually interacting biological systems, the maternal and fetoplacental dose-response relationships are exquisitely complex and involve interactive pharmacological and toxic effects

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Page 42 in the mother and the offspring. An appreciation of the problem may be developed with a few examples. Both thalidomide and isotretinoin are examples of compounds that produce gross structural malformations in the offspring at levels that are pharmacologically active in the mother (lowest observable effect level, LOEL) but are below the dose that produces maternal toxicity. Among human teratogens, such potent teratological effects are relatively rare. Both vitamin A and salicylate are examples of teratogens that produce functional effects at a lower dose range and dysmorphology at a higher dose range (for review, see Hutchings, 1983). Below a dose level that is lethal to the embryo, there may be two embryotoxic responses, each with its respective threshold. As dose increases above the ''no observable effect level" (NOEL), the first embryotoxic response is an impairment in function (lowest observable adverse effect level, LOAEL), followed by a second threshold after which gross structural malformations are produced. THC (Delta-9-tetrahydrocannabinol), the psychoactive ingredient in marijuana, is an example of a compound that produces fetal lethality or growth retardation in the offspring but only at doses that are highly toxic to the mother (Hutchings et al., 1989). Within the pharmacological range of the compound, and at levels that are not toxic to the mother, there is no embryotoxic response. Embryotoxicity is seen only at levels that produce maternal toxicity. This is one of the most common types of profiles observed in animal studies. Figure 2-1 presents hypothetical dose-response effects for alcohol in the maternal-fetoplacental unit. These relationships are conceptual and not intended to depict empirically established correlations. The general scheme depicts a maternal dose-response function in the lower bar and an embryonic-fetal dose-response function in the upper bar. Overall, as the maternal dose or intake of alcohol increases from no observable effects to progressively greater levels of intoxication, there is a corresponding increase in adverse effects in the fetus. For both the mother and the embryo/fetus, there may be levels of alcohol exposure that produce pharmacological effects (LOEL) but are below the threshold dose for adverse effects (LOAEL). However, the relationship of maternal to fetal dose response effects is incompletely understood. Although the data strongly support a relationship of chronic high levels of maternal alcohol intake to the full FAS, what remains unclear is whether there is a continuum of dose-response effects ranging from anatomic and behavioral changes at low to moderate maternal doses to full-blown FAS at high maternal doses, or if there are two or more thresholds resulting in degrees of impairment in function and structural malformation. As yet undefined is whether there is a LOEL distinct from the LOAEL for alcohol exposure in the fetus. Dose-response effects of prenatal alcohol exposure have been demonstrated in numerous animal studies. Definitions of what constitutes "high," "moderate," and "low" doses of alcohol in the animal literature vary. Given the differences in alcohol metabolism between humans and rodents (most commonly used in animal

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Page 43 FIGURE 2-1 Maternal/fetal dose-response effects (modified after Hutchings, 1985). *Historically referred to as fetal alcohol effects or alcohol-related birth defects (see Chapter 4). NOEL, no observable effect level; LOEL, lowest observable effect level; LOAEL, lowest observable adverse effect level. models), as well as among species and strains of rodents, levels of alcohol exposure are probably best judged by blood alcohol levels reached rather than quantity or dose of alcohol level administered. Blood alcohol concentrations (BAC) in humans above about 100 mg/dl (0.10 percent) can be considered high; levels of about 50-100 mg/dl can be considered moderate; and levels below about 50 mg/dl can be considered low (U.S. Department of Health and Human Services, 1993). A BAC of 100 mg/dl is considered legal intoxication in most states. A man weighing 150 pounds who consumed two drinks in one hour would have a BAC around 40 mg/dl. Women generally achieve a higher BAC than men at the same level of consumption, even if matched for weight. Peak BAC has been shown in animal models to be more relevant to fetal outcome than the dose administered; this is consonant with clinical experience that binge drinking is risky to the fetus. BACs that are associated with adverse fetal outcomes in rodent models can range from below 50 mg/dl over a period of days to over 400 mg/dl on one day, depending on the outcome examined (Middaugh and Bogan, 1991; Savage et al., 1992; Sulik et al., 1981; West, 1987; West et al., 1990). In two early studies using mouse models, dose-response effects were observed for malformations induced by prenatal alcohol exposure. Chernoff (1977) found that prenatal alcohol exposure resulted in embryolethality at high doses; cardiac and eyelid dysmorphology at moderate doses; and deficient occiput ossification, neural anomalies, and low fetal weight at low doses. Randall et al. (1977) found dose-response increases for percentage of malformed offspring (anomalies including skeletal, cardiovascular, ophthalmic, abdominal, and urogenital). Similarly, Abel and Dintcheff (1978) reported that postnatal growth deficits in rats increased with increasing alcohol dose. Dose-response effects for behavioral deficits induced by prenatal alcohol exposure have also been reported. For example, linear dose-response functions for spontaneous alternation, reversal

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Page 44 learning, and conditioned taste aversion learning tasks were observed for rats exposed to alcohol prenatally. Performance in a passive avoidance task was also found to be directly related to the amount of alcohol consumed by pregnant females (Lochry and Riley, 1980; Riley et al., 1979, 1986). Alcohol exposure during the early postnatal period (i.e., the third trimester equivalent, reveals similar dose-response effects on both physical and behavioral development. Effects on brain growth during this period of rodent brain growth spurt were particularly marked. Data from West's laboratory (Bonthius and West, 1988; Bonthius et al., 1988) demonstrated that the pattern of alcohol exposure influences the pattern of blood alcohol levels and the severity of brain growth restriction. A particular dose of alcohol administered in multiple feedings over 24 hours has significantly fewer adverse effects than the same dose condensed and administered over a shorter period of time. That is, the more concentrated the pattern of alcohol administration, the higher are the maximum blood alcohol levels achieved and the more severe is the interference with brain growth. Thus, the pattern of alcohol exposure is important because of the resultant blood alcohol concentrations. Increasing doses of alcohol resulted in effects ranging from no significant microcephaly, to decreases in both total brain weight and cerebellar weight, to significant restriction of brain stem weight, to death. It was also shown that male offspring exhibited greater reduction in brain weight and higher blood alcohol levels than female offspring at a given dose. Thus, males appear more susceptible than females to the adverse effects of alcohol on brain growth (Pierce and West, 1986). Sensorimotor development also appears to be delayed or disrupted with condensed rather than uniform alcohol exposure regimens (Kelly et al., 1987). In addition, deficits in both acquisition and retention of a passive avoidance task were dose related. Together, these data indicate that "bingeing" results in more severe deficits in brain growth, morphology, neuron cell death, sensorimotor development, and behavior than does continuous exposure to the same overall dose of alcohol. Work from Savage's laboratory suggests that in rats exposure to low to moderate doses of alcohol during the prenatal period may alter certain specific aspects of brain function without inducing gross abnormalities. Prenatal exposure (through a 3.5 percent alcohol liquid diet for varying periods during gestation) resulting in peak maternal blood alcohol levels as low as 40 mg/dl resulted in alterations in hippocampal NMDA receptor binding and in long-term potentiation (Queen et al., 1993; Savage et al., 1992). This level of exposure is significantly less than that required for most alcohol-induced teratogenic effects. In general, the hippocampus, which has an integral role in memory, is particularly sensitive to a number of neurotoxicants, including alcohol. The mechanisms are unclear and may be mediated by alcohol effects on other systems. Changes such as NMDA receptor binding could be one biologic mechanism underlying some of the functional deficits associated with prenatal alcohol exposure. Data from Clarren and colleagues (Clarren et al., 1988), using a nonhuman

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Page 45 primate model, examined the effects of weekly exposure to varying doses of alcohol on specific dysfunctional outcomes. In addition to dose effects, these investigators also examined effects of full gestational exposure (from the first week of gestation) compared to those of delayed gestational exposure (from the fifth week of gestation). It was found that no animal showed all the features of human FAS, although facial dysmorphia, growth deficiency, and CNS dysfunction were found in 16 of the 28 alcohol-exposed animals. Importantly, the data demonstrated that full gestational exposure at doses resulting in blood alcohol levels greater than 140 mg/dl led to significant developmental delays, whereas delayed gestational exposure (exposure to much higher levels of alcohol after gestation week 5) led to animals who were more cognitively intact at 6 months of age. This latter observation was confirmed in follow-up studies in which animals exposed to peak maternal blood alcohol levels of › 140 mg/dl once per week in the first six weeks of gestation had the same degree of developmental delay as animals exposed to the same dosages nearly throughout gestation. Thus, measurable teratogenic effects from weekly exposures to alcohol occurred only at intoxicating doses. However, early gestational exposure was more damaging to cognitive function than later—and considerably greater—alcohol exposure. These data are counter-intuitive to current thoughts about nervous system teratology. Although the sample size was small, the data were replicated within the same laboratory. Thus, possible interactive effects of dose of alcohol consumed and susceptible periods of exposure must thus be considered in examining the teratogenic consequences of prenatal alcohol exposure. Interestingly, these investigators have recently shown that modern imaging techniques may be useful in elucidating mechanisms of alcohol teratogenicity. They demonstrated that the choline: creatine ratio in the brain, detected by proton magnetic resonance imaging (MRI), increased significantly with increasing duration of in utero alcohol exposure. These signal alterations occurred in the absence of gross structural brain anomalies (detected by MRI) and were significantly correlated with alcohol-related cognitive and behavioral dysfunction (Astley et al., in press). INTERVENTION AND PREVENTION The area of intervention and treatment of children with FAS is still in a relatively young stage. Animal models designed to investigate mechanisms of alcohol teratogenesis and the effects of postnatal and postweaning experiences on developmental outcome may provide insights useful for developing treatment strategies for children with FAS and other alcohol-related birth defects. A series of studies by Randall and colleagues demonstrated that prostaglandins (PGs) may play a role in the etiology of alcohol-related birth defects. Acute alcohol administration on a single day of pregnancy in mice resulted in decreased fetal weight and increased prenatal mortality and birth defects, particularly kidney and limb defects. Pretreatment with aspirin, which affects prostaglandin

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Page 46 metabolism, was found to reduce prenatal mortality and decrease the incidence of birth defects in a dose-dependent manner. Importantly, aspirin dose-dependently inhibited PG levels in uterine and embryonic tissue, and the magnitude of inhibition was positively correlated with the extent to which aspirin reduced the incidence of alcohol-related birth defects (Randall and Anton, 1984; Randall et al., 1991a). Furthermore, the protective effects of aspirin were not related to an effect on maternal blood alcohol levels. Other nonsteroidal anti-inflammatory (NSAI) agents such as ibuprofen and indomethacin were also effective in attenuating alcohol's teratogenic effects but to a lesser extent than aspirin. For example, ibuprofen antagonized the effects of alcohol on fetal growth retardation and the frequency of birth defects, but did not affect prenatal mortality or the number of implantation sites (Randall et al., 1991b). Indomethacin, which does not cross the placenta as readily as aspirin, was found to reduce the number of fetuses with birth defects and appeared to antagonize prenatal mortality, but only at the highest doses (Randall et al., 1987). Clearly, further investigation is needed on the role of prostaglandin in mediating the teratogenic effects of alcohol and on the possible use of PG inhibitors in attenuating alcohol-related birth defects. A series of studies by Wainwright and colleagues investigated the possibility that adverse effects of fetal alcohol exposure on brain development might be mediated in part by an alcohol-induced reduction of available long-chain polyunsaturated fatty acids to the developing brain (Wainwright et al., 1990a,b). Interestingly, these studies reported that supplementation of the maternal diet with a source of long-chain fatty acids increased maternal weight gain, improved perinatal survival of offspring, increased offspring body weights, and enhanced neurobehavioral development. Furthermore, the fatty acid composition of the maternal diet was found to modulate the effects of prenatal alcohol exposure on the membrane phospholipid composition of the developing brain. Although the magnitude of the effects appears to be small, these data nevertheless suggest the intriguing possibility that alcohol-nutrition interactions may be important in mediating certain critical effects of prenatal alcohol on development. Both the postnatal and the postweaning rearing environment may also have a critical impact on the developmental outcome of fetal alcohol-exposed offspring. Data from Weinberg et al. (1995) demonstrated that a simple noninvasive manipulation, early postnatal handling, can alter or attenuate some but not all deficits resulting from prenatal alcohol exposure. Handling eliminated deficits in preweaning growth of offspring and performance in a step-down avoidance task, and attenuated the hypothermic response to alcohol challenge as well as the increased adrenocortical response to restraint stress. However, handling had no effect on the corticosterone response to alcohol challenge and did not reduce the more prolonged corticoid elevation during restraint stress observed in alcohol-exposed compared to pair-fed and control animals. Hannigan and coworkers (1993) demonstrated that fetal alcohol-exposed offspring placed in an enriched postweaning environment show an attenuation of gait ataxia and improved performance in a Morris water maze compared with offspring reared in isolation.

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Page 47 Together, the data from these studies clearly have important clinical implications. Data from clinical studies suggest that postnatal environment and experience may significantly influence outcome in terms of both behavioral and cognitive development (Brown et al., 1991; Smith and Coles, 1991). Although one cannot directly extrapolate from findings in animals to the clinical setting, the present data certainly indicate one possible direction for future research on the treatment of children exposed to alcohol prenatally. A MULTIFACTORIAL MODEL The data presented above clearly indicate that the teratogenic effects of prenatal alcohol exposure can be influenced by numerous factors, both biological and environmental. The complex nature of these multifactorial influences is illustrated in Figure 2-2. This figure attempts to illustrate the point that the FIGURE 2-2 Theoretical influences on the expression of prenatal alcohol exposure.

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