Medical University of South Carolina, Department of Psychiatry & Behavioral Sciences, Charleston, South Carolina, 29401.
In its broadest sense, teratology can be viewed as the study of monstrosities, and a teratogen as an agent that causes abnormal development. Interest in malformations in both man and animals has been evident since the earliest days of recorded history, but detailed scientific studies of the phenomenon, especially with regard to ethanol, were not introduced until the early 1900's. These pioneer investigators were not interested in ethanol or alcoholism, per se, but rather were embryologists or environmental teratologists concerned with the study of developmental abnormalities. In an attempt to identify possible teratogens, a number of agents were used. As might be expected, ethanol was among them.
Initially, non-mammalian species were commonly employed subjects. Since fertilization and subsequent development occurs externally in such species, rather than internally as in mammals, experimental observation and intervention was convenient. Embryos bathed in ethanol solutions demonstrated Abnormal cleavage, stunted growth, and central nervous system disturbances, often following atypical and predictable pattern (1). However, effective concentrations were usually five to forty times higher than the highest levels reported in man and are, therefore, of questionable significance.
Since alcohol is a small, readily diffusable uncharged molecule that is equally distributed in the body water, it is not surprising that it easily crosses the placenta and reaches the fetus. Unlike many other drugs, alcohol distribution is not influenced by degree of lipid solubility. Nicloux (2,3) was the first investigator to demonstrate that alcohol ingested by a gravid woman, dog, or guinea pig passed to the fetus in concentrations paralleling those circulating in maternal blood.
These pioneer studies by Nicloux have been confirmed and extended using a higher degree of sophistication than was available at the time. Gas chromatographic analysis demonstrated a rapid equilibration between the mother and fetus in the pregnant ewe (4), while similar findings have been reported using autoradiographic techniques in humans, as well as monkeys, hamsters, and mice (5,6,7). Autoradiographic examination further revealed a pattern of distribution involving most organs, with the highest concentration in the liver, myocardium and bone marrow (7).
It is evident from these studies that the placenta, even late in pregnancy, does not constitute a barrier to ethanol as it does to many other drugs. On the other hand, acetaldehyde, ethanol's major metabolite, does not cross the placenta in appreciable amounts, at least in the near-term rat (8). Rather, it has been suggested that the placenta acts as a metabolic barrier by metabolizing significant amounts of acetaldehyde (9), which may be potentially toxic to the developing fetus.
FETAL AND NEWBORN METABOLIC CAPACITY
Since the placenta does not block ethanol penetration to the fetus, it is concluded that the fetus is exposed to blood alcohol levels similar to maternal levels. If results from the rat can be generalized to the human, the in vivo rate of ethanol metabolism is not affected by pregnancy (10).
Prompted by the popular clinical use of ethanol to prevent premature labor through inhibition of oxytocin, Seppala (11) and Idanpann-Heikkela (12) studied the elimination of ethanol in the pregnant female, fetus and newborn. A gas liquid chromatographic method of analysis indicated the equilibrium between maternal and fetal concentrations was reached sixty minutes after the initial infusion.
At birth and up until thirty minutes post-delivery, the blood alcohol levels in the mother and fetus remained elevated; by four hours post-partum the maternal elimination rate was twice as high as the newborn's. Premature infants infused with 8% w/v ethanol for up to forty minutes post-partum eliminated alcohol in a linear function (13). Higher, but decreasing blood alcohol levels in the fetus and newborn suggest that the near-term fetus and newborn are capable of metabolizing ethanol in the typical zero-order function, but not at adult capacities.
Although the activity of liver alcohol metabolizing enzymes is not necessarily a good predictor, or sufficient explanation, of metabolic capability for ethanol elimination, interesting reports are worthy of mention. In the rat, alcohol dehydrogenase (ADH) activity was minimally detectable at eighteen days gestation and reached adult levels by eighteen days of age (14). In aborted human fetuses, 3-4% of the adult liver ADH activity was evident in eight week old livers and adult levels were reached by five years of age (15). Structural differences between adult and fetal liver ADH seem to exist (16-18). The few studies concerned with liver aldehyde dehydrogenase activity report considerably lower enzyme activity in the human and rat fetal liver than the adult (9, 14). Interestingly, higher circulating levels of acetaldehyde have been reported in peripheral blood from pregnant rats than non-pregnant rats (19), but no detectable acetaldehyde was measured in near-term fetuses (8) whose mothers were given alcohol acutely by injection.
It can be suggested from the literature cited that the near-term fetus and newborn are capable of metabolizing ethanol and acetaldehyde, but at a significantly slower rate than the adult. Younger fetuses, however, do not have the capacity to metabolize drugs and are dependent upon the mother for drug elimination. The significance of exposure to high circulating alcohol and acetaldehyde levels in utero on subsequent development is not understood, but deleterious effects on both physical and mental status, as well as more subtle biochemical effects are clearly suggested. The remainder of this review will cite evidence to support the notion that alcohol is a teratogenic agent.
A dramatic, but often overlooked, measure of teratogenesis is prenatal/postnatal mortality. A high incidence of miscarriages and stillbirths has been ascribed to alcoholic parents (20-23). Two separate issues arise, depending upon which parent was the alcoholic. Often, however, only one parent is considered while drinking habits in the other are ignored (23).
Miscarriages can thus arise from inherited, prenatal, or environmental factors, or any such combination. Until these variables can be partitioned adequately, alcohol, per se, cannot be implicated as responsible for miscarriages or stillbirths. A careful statistical evaluation of the incidence of miscarriages and stillbirths in the general population, as well as in the offspring of non-alcoholic women matched for age, parity, physical condition, and socio-economic standing, is needed before any definitive conclusions can be drawn. However, a potential problem in gathering accurate statistics on this issue in alcoholic women is the absence of perinatal care in the lower socio-economic groups. Miscarriages may often go undetected.
This issue could be more readily resolved with adequate animal models, for species such as the rodent tend to resorb, rather than to abort, fetuses, and implantation sites can readily be quantified and compared to controls. Unfortunately, few experimenters have chosen this approach to the issue. A plethora of reports describe an increased number of prenatal deaths, stillbirths, and decreased litter size and weight calculated after birth, in fetuses exposed prenatally to ethanol (24-28).
Rodents often cannibalize their young, especially if visceral organs are exposed. Parity as well as environmental conditions also influence pup-killing (29). It is, therefore, possible that defective pups were killed before counted. Further, early resorptions are not reflected in post-partum evaluation. Even palpations and subsequent verification at birth (30) can be complicated by repeated handling of the mother and cannot accurately be detected during early stages of gestation.
Another key issue is the fact that alcoholics tend to be undernourished and protein deficient, since ethanol serves as a caloric source. Nutritional variables, then, complicate the picture. Few studies, either in humans or animals have attempted to control for this crucial variable.
The liquid diet technique originally proposed by Freund (31) circumvents this criticism by allowing for a pair-fed control group that receives the same total number of calories per day as the ethanol group, with ethanol-derived calories replaced by sucrose. Since all other variables (e.g. handling and stress) are assumed to be similar for the two groups, differences between them can be attributed to the presence of ethanol. Chernoff (32) utilized the liquid diet procedure of alcohol introduction in CBA and C3H mice and reported dose-related strain differences in ethanol's embryotoxic effect. Although not specified, it is assumed that he utilized the pair-feeding paradigm in his controls.
Our own work with this technique has demonstrated an increased number of resorptions in ethanol-treated primiparous C57BL mice, as compared to lab chow and sucrose pair-fed controls (33). The total number of resorptions increased proportionally to the increased amount of ethanol ingestion; with diets containing 35% of the total calories as ethanol, few viable litters were recovered (34).
From these recent animal models controlling for nutritional variables, ethanol's embryotoxic potential is clearly implicated. It is possible that different periods of exposure, dose, and parity, as well as species or strain differences, may influence the number of resorptions. The significance of these reports remain, however. In the presence of controlled nutrition, ethanol-treated females resorb significantly more fetuses than pair-fed sucrose or lab chow controls.
The most blatant expression of embryopathic drug action is abnormal physical or mental development of the progeny. Since the time of the Greeks and Romans, parental ethanol abuse at the time of conception has been held responsible for skeletal and mental defects in children. Hence, ethanol was prohibited on the wedding night (35).
This point of view was pervasive in the literature, with reports constantly emphasizing examples of such defective children and implying an overall degeneration of the human race (36). The basic premise was that alcohol acted in some deleterious way to alter the egg or sperm. Chromosomal aberrations, or basic inheritance of birth defects is a separate issue, not necessarily similar to in utero exposure to ethanol. Further, even if the fetus were exposed in utero, from a perusal of these studies, two things become obvious. First, the reports are generally clinical observations. Accurate history of pattern of maternal drinking habits, socio-economic status, dietary regimen, perinatal care, marital status, drug use, or parity are frequently not included in the records. Second, the paternal history is omitted. It thus becomes impossible to assess the relative overall contribution of ethanol, per se, to the resultant defective Children. Animal models should have afforded better control of these variables, but unfortunately were plagued by other confounding variables.
Combemale (37) published one of the earliest reports of in utero exposure to alcohol in mammals. Two normal dogs were mated; the bitch was exposed to ethanol for the first twenty-three days of gestation. Three of the six pups were stillborn; the remaining three were of "weak intelligence" and when mated to "normal intelligence" studs, produced defective young. This early report was followed by others suggesting deleterious effects of ethanol on immature organisms (38-40).
Stockard, however, reported the most extensive research in this area (41-45). He devised a specific apparatus for administering alcohol to pregnant guinea pigs without handling them. Briefly, the animal was prodded into a chamber filled with vapors from 95% ethanol and left until "intoxicated" for six days/week. "Defective" young were often found, affected particularly in the hind extremities. Eye defects were also very common, such as opaque cornea, opaque lens, and anopthalmia.
Pictet (46) and Durham and Woods (47) criticized Stockard's work on a number of grounds and set out to re-examine the issue. They argued that similar defects are characteristic of certain lines of guinea pigs and cannot validly be attributed to maternal ethanol treatment. Their different initial stock, method of intense inbreeding and lack of age and parity controls make these findings also of little significance.
This issue continued to foster empirical studies, extended to the mouse (48), chicken (49) and rat (50,51). Similar methods of intoxication were utilized (i.e. inhalation), and when inconsistent results were obtained, differences were attributed to species variation in sensitivity to ethanol.
Interest eventually waned in this area, especially when reports demonstrating "superior" offspring from ethanol-treated parents appeared. Ethanol was proposed as a selective agent allowing only the fittest of the species to survive (52,53).
Only recently has general interest been renewed in the distinct possibility that ethanol itself may result in abnormal physical and/or mental development of the young. LeMoine and his colleagues in France, described a common syndrome in children of alcoholic mothers (54). Of 127 children born to alcoholic parents twenty-five were malformed; growth deficiencies were evident from birth and psychomotor retardation was apparent. I.Q. scores averaged about seventy; EEG recordings were atypical.
Ulleland was one of the first to describe a similar clinical observation in children of alcoholic females in the United States (55). She observed six small-for-gestational age infants, all of whom had alcoholic mothers. Prompted by this coincidence, Ulleland examined the records of deliveries at Harborview Medical Center (Washington) for an eighteen month period. Forty-seven underweight children were born to a total of 1,582 mothers; ten of the twelve mothers were over thirty-five. Since this hospital services poor or underprivileged clientele, inadequate nutrition could have been responsible for the low birth weight in the offspring as well as maternal medical problems, drug abuse, or environmental variables. Eight of eleven mothers when examined post-partum had mild-severe protein or caloric deficiency, presumably also present during pregnancy.
Jones et al. (56) extended these observations. Initially eight unrelated children born to alcoholic mothers were observed. Six of the eight children were ascertained because of similar physical abnormalities and the remaining two subjects because their mother was an alcoholic; three children were American Indians, three Negroes, and two Caucasians, ranging in ages from eleven weeks to four years. The duration of alcoholism in the mothers was two to twenty-three years, with and average of 9.4 years.
All patients demonstrated impaired pre- and post-natal growth. Common craniofacial and limb defects were observed, such as short palpebral fissures, protrusion of one or both jaws, epicanthal folds, limitation of joint movement, altered palmar crease patterns, and cardiac abnormalities. Assessment of mental, motor and social development revealed low performance, better correlated with mental age than chronological age.
A subsequent report (57) identified three more children with similar malformations, all of American Indian descent. Some new abnormalities reported were cleft palate, respiratory difficulty, hypoglycemia, hypocalcemia, and hyperbilirubinemia. The death of one subject allowed a necropsy. The brain was incompletely developed, the cerebral cortex in particular, and lacked a corpus callosum. Neuronal migration was reported to be disoriented. Subsequent reports by others corroborated these initial findings (58-61). This pattern of altered growth and morphogenesis is now commonly referred to as the "fetal alcohol syndrome".
A follow-up study of twenty-three offspring of alcoholic mothers (62) resulted in some profound implications. The subjects were drawn from a population of 55,000 women studied by the National Institute of Neurologic Disease and Stroke. The charts of the mother and child were examined with no direct patient interview. The population represented eleven negroes, eleven caucasians, and one American Indian, ranging in age from twenty-one to forty, and of lower socio-economic class.
Seventeen percent of the twenty-three offspring included in the study died before one week of age. Of the remaining nineteen subjects, only thirteen were available for evaluation at seven years of age. Intellectual impairment was the most common occurrence with 12% of the cases three standard deviations below the mean and 63% between one and two standard deviations below the mean. Half of the subjects remained with their mother while the other half lived with relatives or in a foster home. Those remaining with their biological mother tended to have lower I.Q. scores. Six subjects were diagnosed on the basis of physical abnormalities to have the "fetal alcohol syndrome". The authors suggested that since 43% of the subjects were adversely affected (four dead and six with the fetal alcohol syndrome) the consideration should be given to termination of pregnancy in chronic alcoholic women. Some investigators have questioned the validity of this suggestion (63). A recent up-date now includes a total of sixty cases of the fetal alcohol syndrome (64).
These sensational reports of a common syndrome of dysmorphology in children of alcoholic women suggest the distinct possibility that ethanol is teratogenic to the human fetus. As cautioned by Green (65), additional variables may complicate the picture. Economic status, nutritional status, drug use parity, and prenatal care are extremely difficult to control in a clinical situation. Thus, it becomes inherently difficult to ascertain the relative contribution of ethanol, per se, to the observed deleterious effects.
The animal experiments initially designed to assess the effect of maternal alcohol consumption on fetal development are, unfortunately, plagued by a common criticism of the clinical reports, that is, no nutritional controls (66-71), and will not be reviewed in detail. It can generally be concluded from these reports that maternal ethanol consumption results in decreased birth weight and/or number, retarded development, and impaired learning ability. Data from the open-field apparatus yields conflicting results (69,72).
More recently, Chernoff (32) reported a mouse model of the fetal alcohol syndrome attempting to circumvent the nutritional issue by feeding ethanol to mice in a nutritionally balanced liquid diet representing from 0-35% of the total calories as ethanol for at least thirty days prior to and throughout gestation. Strain differences existed. Alcohol-treated CBA progeny demonstrated open eyes and deficient ossification at a concentration supplying 25% of the total calories as ethanol; there was an increasing incidence of cardiac and neural anomalies with higher concentrations evident in both CBA and C3H mice. The author implicates strain differences in alcohol dehydrogenase activity as a possible explanation for the observed teratogenic and embryo-toxic effect, although metabolic rates and/or acetaldehyde levels were not reported.
Kronick (73) also administered ethanol to pregnant mice and observed a high incidence of anomalous development in the progeny. B6D2F1/J mice were injected intraperitoneally with approximately 5.71 g/kg ethanol on gestation days 8, 9, 10 and 11, or singly on one of the gestation days 7 through 12. External malformations were evident in 27% of the females treated on days 8 and 9 and 57% of the females treated on days 10 and 11. Single injections on day 9 and 10 produced the highest incidence of abnormal fetuses (41/68 and 26/51 respectively). Anomalies included coloboma of the iris, extrodactyly of the forepaws, hydronephrosis, hypoplastic atria, and exencephaly.
Our own work is also concerned with an animal model approximating the fetal alcohol syndrome in mice (C57BL/J6). We hypothesized that if alcohol acted similarly to other teratogens (74), its effects should be demonstrable during a finite period of critical development. For this reason we chose to introduce the ethanol on gestation day 5 (immediately following implantation) and remove it on gestation-day 11. Our method of drug exposure was via a nutritionally balanced liquid diet containing either 17, 25 or 30% of the total calories as ethanol. We pair-fed controls a similar diet isocalorically substituted with sucrose instead of ethanol.
Fetuses were removed on gestation day 19 by caesarian section and examined for external and internal anomalies (75). We found a dose-related increase in anomalous fetuses as the proportion of ethanol-derived calories increased to 30%. Nearly total resorptions were evident at higher concentrations.
The pattern of anomalies was similar across doses, but the incidence varied. We observed frequent limb anomalies, including adactyly, syndactyly, and ectrodactyly of the forelimbs, cardiovascular anomalies including abnormalities of both the major branches of the aorta and vena caval system and intercardiac anomalies, such as atresia of the mitral valve and interventricular septal defects. Hydronephrosis and hydroureter of varying degrees was also commonly observed, as were micropthalmia. Other anomalies included gastroschisis, exencephaly, hydrocephalus, and anopthalmia (34).
Our results, and those of Chernoff (32) and Kronick (73) clearly demonstrate the teratogenicity and embryotoxicity of ethanol in the mouse. Our results in particular further demonstrate this effect in the presence of adequate nutrition, a common criticism of the clinical reports. The anomalies observed by us and others in animal models are strikingly similar to those reported in children of alcoholic mothers (76).
There is no reason to believe that all teratological effects should be obvious at birth. In addition to gross structural and physical anomalies, more subtle alterations in body chemistry and function probably exist, but either are latent or not manifest until challenged and, therefore, are not immediately recognizable post-delivery. Since the identification and characterization of the fetal alcohol syndrome is relatively recent, a paucity of data exist on possible latent effects (62). In fact, many of the children identified to date have not yet reached puberty (76).
Animal models have yielded little information on longterm pathogenesis or functional anomalies except for reproductive capacity (30). Changes in behavioral variables (69, 72,77), EEG (78,79), neurochemistry (80,81), acid-base balance (82), seizure susceptibility (72), liver enzyme activity (83) and alcohol preference (69) have all been demonstrable shortly following birth or weaning. Whether these effects are permanent, or reversible with age is unknown.
POSSIBLE MECHANISMS OF ACTION
It is strongly suggested, from both clinical and empirical reports, that ethanol is a teratogenic agent capable of producing a variety of developmental anomalies. Since the first report by Jones et al. (56) describing the fetal alcohol syndrome, an additional fifty-two children have been identified as having the syndrome. Clinicians have been amply warned not to confuse the fetal alcohol syndrome with other similar syndromes (84). The fetal alcohol syndrome has been adequately identified, characterized, and publicized to both the professional and layman, and is finally receiving some credence. The important issue now is to determine its etiology.
Ethanol, per se, is most often implicated as the toxic agent in producing the anomalies (64,84). As indicated earlier, it rapidly crosses the placenta and reaches the fetus in amounts approximating those in maternal blood. It is, therefore, a likely prospect, although a variety of alternative possibilities exist.
The role of malnutrition-induced anomalies is casually refuted, although alcoholic women are often either malnourished or undernourished, lacking adequate vitamins, minerals and protein (56). The argument is that the pattern of sustained growth deficiency is not similar to that reported in children of malnourished mothers. Recent animal experiments performed under controlled nutritional conditions have more convincingly ruled out nutritional factors as the primary teratogen by demonstrating embryopathology and toxicity in the presence-of adequate diet, but the possibility still remains (32,34,73).
Social and environmental factors may also be responsible for the anomalies observed in children of alcoholic mothers. For example, strenuous work during gestation or accidental falls may result in defective children as well as premature births. Premature birth may, in fact, be responsible for some of the deficiencies observed. Proper prenatal care is usually minimal in the majority of women alcoholics and since the fetus is abnormally small, the exact duration of gestation may not be accuratelyebtermined.
Maternal age and parity may also be significant variables. Many of the mothers reported on were over thirty-five years old (64). Multiple drug use must also be considered as another viable alternative explanation for the anomalies observed.
The importance given acetaldehyde, ethanol's major metabolite, is minimal, even though acetaldehyde in high doses is toxic and may be responsible for effects previously attributed to ethanol (85). Kesaniemi and Sippel (8) recently reported a placental barrier to acetaldehyde in the rat. These authors also reported significantly higher circulating levels of acetaldehyde in pregnant rats as opposed to nonpregnant rats (19). They propose that the placenta metabolizes acetaldehyde before it reaches the fetus and protects it from possible toxicity. These experiments were performed in the near-term fetus, however.
In the rodent the placenta continually changes with increasing gestation. What may hold true for the last trimester when the chorioallantoic placenta is the functioning placenta may not necessarily be true for earlier stages of gestation characterized primarily by the yolk-sac placenta. In light of the recent results from animal models, teratogenic effects of ethanol are evident with second trimester exposure alone (34, 71,73). Determination of acetaldehyde levels in fetuses at this time period is critical, before acetaldehyde can validly be rejected as the teratogenic agent. Direct or indirect toxic effects produced by acetaldehyde remains a viable alternative for the anomalies observed in the fetal alcohol syndrome.
The teratogenic effects of a drug need not be a result of direct embryotoxicity; indirect effects can be equally deleterious. Alterations in the normal maternal-fetal interaction may be critical to normal development.
Ethanol may render the pregnant mother protein and/or vitamin deficient. Folic acid levels may be low. Acid-base balance may be impaired. Blood pressure, and therefore oxygen transfer, may be expected to change with high doses. Any of these variables can result in anomalous development (86-89).
Probably the most important indirect drug effect is the effect on the transport mechanisms of the placenta. Placental dysfunction or morphological changes most probably result from chronic alcohol abuse and high acetaldehyde levels, although this issue has yet to be examined.
The mechanism of action, critical period, and toxic dose level responsible for abnormal development in children of alcoholic women are all within the realm of scientific experimentation. Unlike other teratogens (e.g. tryphan blue) the basic actions of alcohol and to a lesser degree, acetaldehyde, are documented (90). Prospective studies in the clinical setting, with adequate control groups, can offer answers to basic questions from verbal self-report data regarding history, duration, and pattern of drug use as well as environmental factors that animal models will not adequately simulate. Animal models, on the other hand, should soon offer insight into the possible biochemical and physiological etiology of ethanol-induced changes in organogenesis. The ultimate goal of identification is subsequent intervention and prevention of ethanol's deleterious effect on the developing fetus.
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