The Fetal Alcohol Syndrome In Mice: An Animal Model GERALD F. CHERNOFF Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5
ABSTRACT CBA and C3H female mice were maintained on liquid dietsMetrecal plus ethanol -containing 15-3596ethanol-derived calories. These diets, which resulted in alcohol blood levels of 73-398 mg/100 ml blood in nonpregnant females, were the sole sustenance for the females for a t least 30 days before and throughout gestation. Females were killed on day 18 of gestation and offspring examined for skeletal and soft tissue anomalies. Prenatal death and maldevelopment increased with the level of alcohol intake. Deficient occiput ossification, neural anomalies, and low fetal weight occurred with low ethanol diets, and cardiac and eye-lid dysmorphology with higher ethanol diets. This pattern of malformations, which exhibited both a dose-response effect and strain differences in susceptibility, indicated that chronic maternal alcoholism is embryolethal and teratogenic in mice. Recently a number of clinical reports have suggested a relation between chronic maternal alcoholism and a specific pattern of anomalies in offspring of such women (Jones and Smith, '73; Jones et al., '73; Ferrier et al., '73; Jones et al., '74; Hall and Orenstein, '74; Palmer et al., '74; Jones and Smith, '75; Tenbrinck and Buchin, '75). This pattern, called the fetal alcohol syndrome, is characterized by developmental and psychomotor delay, preand postnatal growth deficiency, impaired intellectual performance, and craniofacial, cardiac, and joint defects. Questions have been raised about the origin of the anomalies (Rosett, '74; Bianchine and Taylor, '741, and since a detailed investigation into their pathogenesis is not feasible in human beings, it was desirable to develop an animal counterpart of the human syndrome. Many investigations of the effect of ethanol on prenatal development in animals were made in the past, but their results were contradictory and unconvincing (for recent reviews see Sandor and Elias, '68; Green, '74; Warner and Rosett, '75). In addition more recent studies (Sandor, '68; Sandor and Elias, '68; Sandor and Amels, '71; Schwetz et al., '75; Kronick, '76;) had variable results, possibly because of the differences in experimental technique. In these and most earlier studies various doses of ethanol were fed or injected a t different gestational periods, and thus TERATOLOGY, 15: 223-230
failed to provide the continual administration that occurs in human chronic alcoholism. In the experiment reported here an attempt was made to simulate the condition of human chronic alcoholism in female mice and then observe the offspring of these females for congenital defects. MATERIALS AND METHODS
CBA/J and C3H/lgM1mice were maintained on a 12-hour light cycle in the Zoology Vivarium, University of British Columbia. The females were housed two per cage in standard plastic cages and were fed ad libitum the diets listed in table 1, which are defined by the percent of ethanol-derived calories (EDC) each contains (Freund, '69; Freund and Walker, '71). The solid-food controls, received Purina Lab Chow, containing 4.25 cal/g, and tap water. The liquid diet preparation consisted of chocolate Metrecal (Mead Johnson, Evansville, Indiana), containing 0.95 cal/ml, and Vitamin Diet Fortification Mixture (Nutritional Biochemicals, Cleveland), 3 g/l. Diet 0 contained the liquid diet plus isocaloric amounts of sucrose (87% v/v, 3.5 cal/ml) for ethanol. Diet 15 consisted of the basic liquid diet plus 95% v/v ethyl alcohol (5.25 cal/ml), Received Apr. 22, '76. Accepted Jan. 12, '77. 'This work was presented in part at the 15th Annual Meeting of the Teratology Society, May, 1975. *Supported hy Medical Research Council of Canada, Grant No. MA 1062 to Dr. James R. Miller.
223
224
GERALD F. CHERNOFF
added such that the final preparation contained 15%EDC. Diet 20 contained 20%EDC, diet 25, 25% EDC, etc. The liquid diets were prepared fresh daily and were the only source of calories for the animals fed them. The preparation, composition, and documentation of nutritional adequacy of the liquid diets used were noted in detail previously (Walker and Freund, '71); each treatment diet contained several times the minimum daily requirements of all nutrients, based on previous recommendations for mice (Walker and Zornetzer, '74). Animals received the liquids from inverted 50-ml plastic syringes with the needle end sealed, through standard glass drinking tubes with 1.5-mmopenings that extended to approximately 3 cm above the cage floor. Daily fluid consumption and caloric intake were determined by measuring the amount of fluid left each day a t between 10 A.M. and 12 noon. Caloric intake for animals on Lab Chow was determined using metabolic cages. Blood alcohol levels were measured in 2 - 4 samples collected from a tail vein and analyzed by gas chromatography (Freund, '67). To avoid weight loss and sickness that might have resulted from an abrupt introduction to high ethanol doses animals were introduced to the diets in stages. Virgin females, 60 to 100 days old, were given diet 0 for ten days and then diet 15 or 20 for 10 days, followed by the next higher ethanol diet for ten days, until ten females were in each diet group. The females were then maintained on their respective group diets for a t least 30 days, after which they were mated to sober males. At the time of the initial mating attempt all females had been on liquid diets 80 days, and on their respective group diet from 30 to 80 days, depending on the diet group, i.e., diet 0, 80 days; diet 15, 70 days; diet 35, 30 days. To keep the males sober and the females alcoholic, matings were restricted to 1.5 hours, during which the mating pair was deprived of food and water. The presence of a copulation plug was taken as indicating day 1 of gestation. Females were kept on their respective diets throughout gestation, weighed and killed on day 18, and the contents of the uterus examined. Resorptions, dead fetuses, external malformations, and individual live fetal weights were recorded. One-third of the live animals were randomly assigned for alizarin red S skeletal staining (Crary, '62) and the remainder fixed in Bouin's solution for subsequent examination by freehand razor
sectioning. Maternal livers were removed, weighed, and fixed for sectioning and staining with HE. RESULTS
Effectsof alcohol on nonpregnant females The effects of different amounts of EDC on caloric intake, liver weight, and blood alcohol levels in nonpregnant females (table 2) were analyzed by analysis of variance. C3H mice were not put on diet 15 and CBA's could not be maintained on diet 35 beyond ten days. Daily caloric intake, averaged over a 30-day period, was not significantly different among diets within strains. The mean ratio of liver weight to total body weight after 60 days on treatment was not significantly different within strains. This absence of abnormal liver size was supported by the absence of pathology in liver sections examined histologically. Blood was taken from nonpregnant females after ten days on their respective diets, and blood alcohol levels were determined. There was a significant dose-related linear increase ( P S 0.05) within strains, and a significant difference (PS 0.05) between strains on the same diet. These findings suggested a strain difference in the amount of ethanol required to achieve a particular blood alcohol level. Effects of maternal alcoholism on the fetus Table 3 shows the effects of variable amounts of EDC on implantation, resorption, and fetal weight. An analysis of variance indicated that the mean number of implants did not differ significantly within strains; however the rate of resorption increased dramatically (0-100%)with increasing amounts of EDC. With diet 30 the CBA females carried no offspring through to day 18, even though they gained weight earlier in pregnancy. The same was true for the C3Hs on diet 35. From the dose-response curves (fig. 1)it appeared that CBA are more sensitive to increasing EDC when measured by resorption rate, an increased sensitivity also reflected in mean fetal weight (fig. 2). The most common skeletal abnormality observed was an incomplete or apparently missing supraoccipital bone (table 4). This occurred in both strains even on the lowest alcohol-containing diet. At higher EDC diets, apparently missing sternebra and rib anomalies, including fusion and misalignment, were produced. Examination of soft tissue revealed a high
225
FETAL ALCOHOL SYNDROME TABLE 1
Composition of diets Ethanol (95%vlv)
Metrecal
Diet (% EDC) Calories
mlllOO
Calories
%
Lab chow 0 15 20 25 30 35
m11100
%
-
-
-
-
100
15 20 25 30 35
3.1 4.3 5.7 7.2 8.9
85 80 75 70 65
100.0 96.9 95.7 94.3 92.8 91.1
' All diets administered ad libitum. TABLE 2
Effect of diets on daily caloric intake, liver weight, and blood alcohol levels in nonpregnant female mice Strain
CBA fn= 3 females for each diet) CH3 (n= 3 females for each diet)
Diet (% EDC) '
Cal intake
Lab chow 0 15 20 25 30 Lab chow 0 20 25 30 35
141-1.5 2022.4 2 0 1 1.8 18t2.5 1521.3 16t1.6
Liverlwtltotal wt
Blood EtOH
-
x k SEM 0.071t0.002 0.057 t0.001 0.06120.003 0.072t0.001 0.071t0.003 0.065 t0.003 0.062 20.002 0.054rt0.002 0.062C0.003 0.07110.001 0.068t0.001 0.067 20.002
16k1.6
20k1.8 19k1.7 1 7 2 1.8 1711.6 1621.5
0 0 7 3 t 5.8 1212 4.6 1742 6.5 3 1 5 t 8.9 0 0 1032 5.5 1602 5.0 2892 6.8 3 9 8 t 11.9
'See text for details.
TABLE 3
Effect of diets on implants, resorptions, and fetal weight Strain
Diet (% EDC)
CBA
Lab chow 0 15 20 25 30 Lab chow 0 20 25 30 35
CH3
No. litters
No. implants
10
48 56 40 44 52
n 1
% 2
0 23 32 38
-
0 57 72 73 100
-
110
8
73 68 52 61
0
7 0 0 30 72 100
1.14 1.27 0.77 0.50 0.58
10
10 8 10 10 10 10 10 8 10 10
-
Fetal wt (g)
Resorptions
0 16 44
-
-
x tSEM 0.97 0.005 0.95 0.025 0.64 0.040 0.33 0.022 0.51 0.081
-
0.032 0.018
0.041 0.37 0.56
'See text for details.
percentage of brain anomalies for both strains (36 and 78%at the lowest ethanol containing diets (table 5)). These anomalies included dilated or immature cerebral ventricles and absence of the corpus callosum. Cardiac anom-
alies, mainly ventricular septa1 defects and hemopericardium, were observed at t h e lowest EDC diet and the percentage rose with increasing EDC levels. Open eyelids occurred in 100%of the CBA in the two higher treat-
226
GERALD F. CHERNOFF
100
50
I
I
I
I
I
15 20 25 30 o/o Ethanol-dQrivQd CaloriQs
35
Fig. 1 Dose-response curve of resorption rate.
o-----o
CBA C3H
I I
I
15
0 o/o
I
I
8
20
25
30
Ethanol- dQrivQd calories
Fig. 2 Dose-response curve of mean fetal weight (bars represent 95%confidence limits).
227
FETAL ALCOHOL SYNDROME TABLE 4
Types and frequency of skeletal anomalies Strain
Diet ( X EDC) '
No fetuses cleared
Oce1put
Sternum
Ribs
Lab chow 0 15 20 25 Lab chow 0 20 25 30
15 18 6 4 5 35 24 22 12 6
1 0 6 4 5 2 1 18 12 6
0 0 3 4 5 0
0 0 0 3 3 0
0
0
6 5 4
5 5 6
Total abnormal
%
CBA
CH3
1
7 0
100 100 100 6
4 82 100 100
See text for details TABLE 5
Types and frequency of soft-tissue anomalies Strain
Diet (36 EDC) '
CBA
Lab chow
C3H
15 20 25 Lab chow
0
0
20 25 30 I
No. fetuses sectioned
32 38 11 8 9 67 49 46 24 11
Dilated brain ventricles
Eyelids open
0 0
0
4 8 9
0 8 9
0
1 36 24 11
0
-
Exencephaly
Heart
Gastrosehisis
0 0 0
0
0
0
0
2 7 8 0 0 18 17 10
0 2 1 0 0 3 9 9
0 3 0 0 4 7 9
Total abnormal
9: 0 0 36 100 100 0 2 78 100 100
See text for details.
ment diets, but could not be evaluated in the C3H since this strain is genetically open lidded a t birth. Exencephaly and gastroschisis were observed in both strains a t higher EDC diets. DISCUSSION
Recent studies on the teratogenicity of ethanol resulted in a variety of abnormalities. In chicken egg studies, where ethanol was injected into the air space a t early stages of incubation, the major effect was growth retardation with generalized malformations of the central nervous system leading to increased chick mortality (Sandor and Elias, '68; Sandor, '68).Injection iv of rats with ethanol on gestation days 6 to 8 caused dysmorphology in CNS anlagen, increased resorption, and retardation of skeletal development (Sandor and Amels, "71).Injecting mice ip in midgestation produced offspring with coloboma of the iris
and ectrodactyly, as well as an increased resorption rate (Kronick, '76). Finally, the administration of ethanol in the drinking water of mice over a 10-day period in mid to late gestation resulted in small fetuses with an increased rate of minor skeletal variants (Schwetz et al., '75). Although these studies all reported abnormalities associated with the fetal alcohol syndrome, the full expression of the syndrome was not observed. This is not surprising since the array of malformations produced in offspring is partly dependent on the period of gestation during which the agent is administered. Hence the duration of administration of a teratogenic agent would also be relevant. This has been demonstrated, e.g., with vitamin A deficiency where relatively brief treatment tended to cause only a portion of the total syndrome caused by extended treatment (Wilson et al., '53). Therefore, to simu-
228
GERALD F. CHERNOFF TABLE 6
Criteria for an animal model of the fetal alcohol syndrome 1. Oral route of administration 2. Even circadian distribution of ethanol consumption 3. Blood alcohol levels greater than 100 mg/100 ml blood 4. Diet of at least 25%ethanol derived calories 5. Behavioral manifestation of intoxication 6. Physical addiction 7. Good nutrition and caloric intake 8. Intoxication prior to and during gestation
late human alcoholism and possibly reproduce a fetal alcohol syndrome in animals, there should be continuous exposure to ethanol throughout gestation. It is now recognized among researchers in alcohol addiction that an animal model, it it is to be effective in delineating the etiology, prevention, and cure of a human disorder, should meet criteria observed in the human condition Falk et al., '72; Mello, '73; Lester and Freed, '73). Similarly, when attempting to establish the teratogenicity of chronic alcoholism, one should try to mimic the mode and duration of human alcohol consumption and apply these findings to the animal population being studied. Paralleling criteria for a diagnosis of human alcoholism (Criteria Committee, '72; Ulleland, '72), and the pattern of consumption in mothers of children with the fetal alcohol syndrome, an ideal set of criteria was established and is shown in table 6. The first 6 requirements are those commonly used to establish a diagnosis of human alcoholism. The final two requirements are specifically applicable to a teratologic model. Since the effects of maternal chronic alcoholism as opposed to the effects of ethanol per se are under question, it is necessary that the ethanol treatment take place prior to, as well as during gestation. By doing this, allowance is made for the many adaptive changes in hepatic cellular metabolism following chronic ethanol consumption (Lieber, '75), which might have secondary teratogenic effects contributing to the full fetal alcohol syndrome. Finally, the nutritional and caloric intake must be carefully controlled so that the teratogenic effect of the maternal chronic alcoholism can be kept separate from maternal malnutrition (Wilson, '73: pp. 40-42) or fasting (Runner and Miller, '56). With the protocol used it was possible to meet the criteria listed. Furthermore, with
the strains of mice used, it was possible to obtain blood alcohol levels in excess of 100 mg/100 ml blood with diets containing less than 25%EDC. This finding suggests that the quantitative blood alcohol level is a more reliable indication of the maternal physiological state than the amount of ethanol in the diet, since the former is influenced by maternal rates of metabolism which are ultimately under genetic control. It was found that day-18 mouse fetuses had a pattern of malformations similar to those observed in children with the fetal alcohol syndrome. These similarities included prenatal growth defiiciency evidenced by low fetal weight and incomplete ossification, neural and cardiac anomalies, skeletal dysmorphogenesis, and prenatal wastage. Together with the clinical reports on this syndrome the evidence from this study strongly supports the dangers of maternal chronic alcoholism as a teratogenic agent, or perhaps more correctly, a teratogenic activity. In this study a dose-response curve ranging from embryolethality a t high doses (blood alcohol equivalent of 300 mg/100 ml blood) to brain malformations a t the lowest dose (blood alcohol equivalent of 73 mg/100 ml blood) was demonstrated. This supports a hypothesis that ethanol or its metabolic by-products act on various developing systems throughout gestation. Considering its molecular weight (46.07) and high lipid solubility it is conceivable that the earliest effects of ethanol may be on the blastocyst, with later effects on the embryo and fetus. Although validation of this interpretation awaits further study, it clearly demonstrates the need to consider the long term effects of maternal alcoholism a s opposed to exposure only during organogenesis. Another point of interest is that the lowest dose producing abnormalities is below the minimum blood level required for a diagnosis of human chronic alcoholism. The full expression of anomalies in the fetal alcohol syndrome has only been reported in the offspring of chronic alcoholic women, but the possibility thus exists that a milder form of the syndrome may be found in the offspring of women who have been moderate to heavy drinkers during pregnancy. CBA/J mice were more sensitive to the induction of the syndrome than CSHAgM1. Considering the difference in blood alcohol levels between strains on the same dose i t is possible that the difference in susceptability
FETAL ALCOHOL SYNDROME
is due to the rate of ethanol metabolism. Two liver enzyme systems, alcohol dehydrogenase (ADH) in the cytosol fraction and the inducible microsomal ethanol oxidizing system (MEOS) in the microsomal fraction, are responsible for ethanol metabolism in both man and mouse (Lieber and DeCarli, '70). The ADH system is known to be under genetic control in the mouse (Sheppard et al., '68); MEOS has not been investigated from this point of view. ACKNOWLEDGMENTS
The author wishes to express appreciation to Doctor James R. Miller for his enthusiasm and guidance throughout this study, Ms. Jean McLeod for the daily well-being of the mice, and Ms. Shiela Manning for the illustrations. LITERATURE CITED Bianchine, J. W., and B. D. Taylor 1974 Noonan syndrome and the fetal alcohol syndrome. Lancet, 1: 933. Crary, D. D. 1962 Modified benzyl alcohol clearing of alizarin-stained specimens without loss of flexibility. Stain Tech., 37: 124-125. Critera Committee, National Council on Alcoholism 1972 Criteria for the diagnosis of alcoholism. Ann. Int. Med., 77: 249-258. Falk, J. L., H. H. Samson and G. Winger 1972 Behavioral maintenance of high concentrations of blood ethanol and physical dependence in the rat. Science, 177: 811-813. Ferrier, P. E., I. Nicod and S. Ferrier 1973 Fetal alcohol syndrome. Lancet, 2: 218. Freund, G. 1967 Exchangeable injection port cartridge for gas chromatographic determination of volatile substances in aqueous fluids. Anal. Chem., 39: 545-546. 1969 Alcohol withdrawal syndrome in mice. Arch. Neur., 21: 315-320. Freund, G., and D. W. Walker 1971 Impairment of avoidance learning by prolonged ethanol consumption in mice. J. Pharmacol. Exp. Ther., 179: 284-292. Green, H. G. 1974 Infants of alcoholic mothers. Am. J. Obst. Gyn., 118: 713-716. Hall, B. D., and W. A. Orenstein 1974 Noonan's phenotype in a n offspring of a n alcoholic mother. Lancet, I : 680. Jones, K. L., and D. W. Smith 1973 Recognition of the fetal alcohol syndrome in early infancy. Lancet, 2: 999-1001. 1975 The fetal alcohol syndrome. Teratology, 12: 11-26. Jones, K. L., and D. W. Smith 1973 Recognition of the fetal alcohol syndrome in early infancy. Lancet, 2: 999-1001. 1975 The fetal alcohol syndrome. Teratology, 12: 11-26. Jones, K. L., D. W. Smith, A. P. Streissguth and N. C. Myrianthopoulous 1974 Outcome in offspring of chronic alcoholic women. Lancet, 1: 1076-1078.
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229
Jones, K. L., D. W. Smith, C. W. Ulleland and A. P. Streissguth 1973 Pattern of malformation in offspring of chronic alcoholic women. Lancet, 1: 1267-1271. Kronick, J. B. 1976 Teratogenic effects of ethyl alcohol administered to mice. Am. J. Obst. Gyn., 124: 676-680. Lester, D., and E. X. Freed 1973 Criteria for a n animal model of alcoholism. Pharmacol. Biochem. Beh., I : 103107. Lieber, C. S. 1975 Interference of ethanol in hepatic cellular metabolism. Ann. N.Y. Acad. Sci., 252; 24-50. Lieber, C. S., and L. M. DeCarli 1970 Hepatic microsomal ethanol-oxidizing system: in vitro characteristics and adaptive properties in vivo. J. Biol. Chem., 245: 25052512. Mello, N. K. 1973 A review of methods to induce alcohol addiction in animals. Pharmacol. Biochem. Beh., I: 89101. Palmer, R. H., E. M. Quellette, L. Warner and S. R. Leichtman 1974 Congenital malformations in offspring of chronic alcoholic mother. Pediatrics, 53: 490-494. Rosett, H. L. 1974 Maternal alcoholism and intellectual development of offspring. Lancet, 2: 218. Runner, M. N., and J. R. Miller 1956 Congenital deformity in the mouse as a consequence of fasting. Anat. Rec., 124: 437-438 (Abstract). Sandor, S. 1968 The infiuence of aethyl-alcohol on the developing chick embryo. Rev. Roum. Embryol. Cytol., Ser. Embryol., 5: 167-171. Sandor, S., and D. Amels 1971 The action of aethanol on the prenatal development of albino rats. Rev. Roum. Embryol. Cytol., Ser. Embryol., 8: 105-118. Sandor, S., and S. Elias 1968 The influence of aethylalcohol on the development of the chick embryo. Rev. Roum. Embryol. Cytol., Ser. Embryol., 5: 51-76. Schwetz, B. A., B. K. Leong and R. E. Staples 1975 Teratology studies on inhaled carbon monoxide and imbibed ethanol in laboratory animals. Teratology, 11: 33A (Abstract). Sheppard, J., P. Albersheim and G. McClearn 1968 Enzyme activities and ethanol preference in mice. Biochem. Genet., 2: 205-212. Tenbrick, M. S., and S. Y. Buchin 1975 Fetal alcohol syndrome. J. Am. Med. Ass., 232: 1144-1147. Ulleland, C. N. 1972 The offspring of alcoholic mothers. Ann. N.Y. Acad. Sci., 197: 167-169. Walker, D. W., and G. Freund 1971 Impairment of shuttle box avoidance learning following prolonged alcohol consumption in rats. Physiol. Beh., 7: 773-778. Walker, D. W., and S. F. Zornetzer 1974 Alcohol withdrawal in mice: EEG and behavioral correlates. EEG Clin. Neurophysiol, 36: 233-243. Warner, R. H., and H. L. Rosett 1975 The effects of drinking on offspring: an historical survey of the American and British literature. J. Stud. Alc., 36: 1395-1420. Wilson, J. G. 1973 Environmental and Birth Defects. Academic Press, New York. Wilson, J. G., C. B. Roth and J. Warkany 1953 Analysis of the syndrome of malformations induced by vitamin A deficiency. Effects of restoration of vitamin A a t various times during gestation. Am. J. Anat. 92: 189-217.
ABSTRACT CBA and C3H female mice were maintained on liquid dietsMetrecal plus ethanol -containing 15-3596ethanol-derived calories. These diets, which resulted in alcohol blood levels of 73-398 mg/100 ml blood in nonpregnant females, were the sole sustenance for the females for a t least 30 days before and throughout gestation. Females were killed on day 18 of gestation and offspring examined for skeletal and soft tissue anomalies. Prenatal death and maldevelopment increased with the level of alcohol intake. Deficient occiput ossification, neural anomalies, and low fetal weight occurred with low ethanol diets, and cardiac and eye-lid dysmorphology with higher ethanol diets. This pattern of malformations, which exhibited both a dose-response effect and strain differences in susceptibility, indicated that chronic maternal alcoholism is embryolethal and teratogenic in mice. Recently a number of clinical reports have suggested a relation between chronic maternal alcoholism and a specific pattern of anomalies in offspring of such women (Jones and Smith, '73; Jones et al., '73; Ferrier et al., '73; Jones et al., '74; Hall and Orenstein, '74; Palmer et al., '74; Jones and Smith, '75; Tenbrinck and Buchin, '75). This pattern, called the fetal alcohol syndrome, is characterized by developmental and psychomotor delay, preand postnatal growth deficiency, impaired intellectual performance, and craniofacial, cardiac, and joint defects. Questions have been raised about the origin of the anomalies (Rosett, '74; Bianchine and Taylor, '741, and since a detailed investigation into their pathogenesis is not feasible in human beings, it was desirable to develop an animal counterpart of the human syndrome. Many investigations of the effect of ethanol on prenatal development in animals were made in the past, but their results were contradictory and unconvincing (for recent reviews see Sandor and Elias, '68; Green, '74; Warner and Rosett, '75). In addition more recent studies (Sandor, '68; Sandor and Elias, '68; Sandor and Amels, '71; Schwetz et al., '75; Kronick, '76;) had variable results, possibly because of the differences in experimental technique. In these and most earlier studies various doses of ethanol were fed or injected a t different gestational periods, and thus TERATOLOGY, 15: 223-230
failed to provide the continual administration that occurs in human chronic alcoholism. In the experiment reported here an attempt was made to simulate the condition of human chronic alcoholism in female mice and then observe the offspring of these females for congenital defects. MATERIALS AND METHODS
CBA/J and C3H/lgM1mice were maintained on a 12-hour light cycle in the Zoology Vivarium, University of British Columbia. The females were housed two per cage in standard plastic cages and were fed ad libitum the diets listed in table 1, which are defined by the percent of ethanol-derived calories (EDC) each contains (Freund, '69; Freund and Walker, '71). The solid-food controls, received Purina Lab Chow, containing 4.25 cal/g, and tap water. The liquid diet preparation consisted of chocolate Metrecal (Mead Johnson, Evansville, Indiana), containing 0.95 cal/ml, and Vitamin Diet Fortification Mixture (Nutritional Biochemicals, Cleveland), 3 g/l. Diet 0 contained the liquid diet plus isocaloric amounts of sucrose (87% v/v, 3.5 cal/ml) for ethanol. Diet 15 consisted of the basic liquid diet plus 95% v/v ethyl alcohol (5.25 cal/ml), Received Apr. 22, '76. Accepted Jan. 12, '77. 'This work was presented in part at the 15th Annual Meeting of the Teratology Society, May, 1975. *Supported hy Medical Research Council of Canada, Grant No. MA 1062 to Dr. James R. Miller.
223
224
GERALD F. CHERNOFF
added such that the final preparation contained 15%EDC. Diet 20 contained 20%EDC, diet 25, 25% EDC, etc. The liquid diets were prepared fresh daily and were the only source of calories for the animals fed them. The preparation, composition, and documentation of nutritional adequacy of the liquid diets used were noted in detail previously (Walker and Freund, '71); each treatment diet contained several times the minimum daily requirements of all nutrients, based on previous recommendations for mice (Walker and Zornetzer, '74). Animals received the liquids from inverted 50-ml plastic syringes with the needle end sealed, through standard glass drinking tubes with 1.5-mmopenings that extended to approximately 3 cm above the cage floor. Daily fluid consumption and caloric intake were determined by measuring the amount of fluid left each day a t between 10 A.M. and 12 noon. Caloric intake for animals on Lab Chow was determined using metabolic cages. Blood alcohol levels were measured in 2 - 4 samples collected from a tail vein and analyzed by gas chromatography (Freund, '67). To avoid weight loss and sickness that might have resulted from an abrupt introduction to high ethanol doses animals were introduced to the diets in stages. Virgin females, 60 to 100 days old, were given diet 0 for ten days and then diet 15 or 20 for 10 days, followed by the next higher ethanol diet for ten days, until ten females were in each diet group. The females were then maintained on their respective group diets for a t least 30 days, after which they were mated to sober males. At the time of the initial mating attempt all females had been on liquid diets 80 days, and on their respective group diet from 30 to 80 days, depending on the diet group, i.e., diet 0, 80 days; diet 15, 70 days; diet 35, 30 days. To keep the males sober and the females alcoholic, matings were restricted to 1.5 hours, during which the mating pair was deprived of food and water. The presence of a copulation plug was taken as indicating day 1 of gestation. Females were kept on their respective diets throughout gestation, weighed and killed on day 18, and the contents of the uterus examined. Resorptions, dead fetuses, external malformations, and individual live fetal weights were recorded. One-third of the live animals were randomly assigned for alizarin red S skeletal staining (Crary, '62) and the remainder fixed in Bouin's solution for subsequent examination by freehand razor
sectioning. Maternal livers were removed, weighed, and fixed for sectioning and staining with HE. RESULTS
Effectsof alcohol on nonpregnant females The effects of different amounts of EDC on caloric intake, liver weight, and blood alcohol levels in nonpregnant females (table 2) were analyzed by analysis of variance. C3H mice were not put on diet 15 and CBA's could not be maintained on diet 35 beyond ten days. Daily caloric intake, averaged over a 30-day period, was not significantly different among diets within strains. The mean ratio of liver weight to total body weight after 60 days on treatment was not significantly different within strains. This absence of abnormal liver size was supported by the absence of pathology in liver sections examined histologically. Blood was taken from nonpregnant females after ten days on their respective diets, and blood alcohol levels were determined. There was a significant dose-related linear increase ( P S 0.05) within strains, and a significant difference (PS 0.05) between strains on the same diet. These findings suggested a strain difference in the amount of ethanol required to achieve a particular blood alcohol level. Effects of maternal alcoholism on the fetus Table 3 shows the effects of variable amounts of EDC on implantation, resorption, and fetal weight. An analysis of variance indicated that the mean number of implants did not differ significantly within strains; however the rate of resorption increased dramatically (0-100%)with increasing amounts of EDC. With diet 30 the CBA females carried no offspring through to day 18, even though they gained weight earlier in pregnancy. The same was true for the C3Hs on diet 35. From the dose-response curves (fig. 1)it appeared that CBA are more sensitive to increasing EDC when measured by resorption rate, an increased sensitivity also reflected in mean fetal weight (fig. 2). The most common skeletal abnormality observed was an incomplete or apparently missing supraoccipital bone (table 4). This occurred in both strains even on the lowest alcohol-containing diet. At higher EDC diets, apparently missing sternebra and rib anomalies, including fusion and misalignment, were produced. Examination of soft tissue revealed a high
225
FETAL ALCOHOL SYNDROME TABLE 1
Composition of diets Ethanol (95%vlv)
Metrecal
Diet (% EDC) Calories
mlllOO
Calories
%
Lab chow 0 15 20 25 30 35
m11100
%
-
-
-
-
100
15 20 25 30 35
3.1 4.3 5.7 7.2 8.9
85 80 75 70 65
100.0 96.9 95.7 94.3 92.8 91.1
' All diets administered ad libitum. TABLE 2
Effect of diets on daily caloric intake, liver weight, and blood alcohol levels in nonpregnant female mice Strain
CBA fn= 3 females for each diet) CH3 (n= 3 females for each diet)
Diet (% EDC) '
Cal intake
Lab chow 0 15 20 25 30 Lab chow 0 20 25 30 35
141-1.5 2022.4 2 0 1 1.8 18t2.5 1521.3 16t1.6
Liverlwtltotal wt
Blood EtOH
-
x k SEM 0.071t0.002 0.057 t0.001 0.06120.003 0.072t0.001 0.071t0.003 0.065 t0.003 0.062 20.002 0.054rt0.002 0.062C0.003 0.07110.001 0.068t0.001 0.067 20.002
16k1.6
20k1.8 19k1.7 1 7 2 1.8 1711.6 1621.5
0 0 7 3 t 5.8 1212 4.6 1742 6.5 3 1 5 t 8.9 0 0 1032 5.5 1602 5.0 2892 6.8 3 9 8 t 11.9
'See text for details.
TABLE 3
Effect of diets on implants, resorptions, and fetal weight Strain
Diet (% EDC)
CBA
Lab chow 0 15 20 25 30 Lab chow 0 20 25 30 35
CH3
No. litters
No. implants
10
48 56 40 44 52
n 1
% 2
0 23 32 38
-
0 57 72 73 100
-
110
8
73 68 52 61
0
7 0 0 30 72 100
1.14 1.27 0.77 0.50 0.58
10
10 8 10 10 10 10 10 8 10 10
-
Fetal wt (g)
Resorptions
0 16 44
-
-
x tSEM 0.97 0.005 0.95 0.025 0.64 0.040 0.33 0.022 0.51 0.081
-
0.032 0.018
0.041 0.37 0.56
'See text for details.
percentage of brain anomalies for both strains (36 and 78%at the lowest ethanol containing diets (table 5)). These anomalies included dilated or immature cerebral ventricles and absence of the corpus callosum. Cardiac anom-
alies, mainly ventricular septa1 defects and hemopericardium, were observed at t h e lowest EDC diet and the percentage rose with increasing EDC levels. Open eyelids occurred in 100%of the CBA in the two higher treat-
226
GERALD F. CHERNOFF
100
50
I
I
I
I
I
15 20 25 30 o/o Ethanol-dQrivQd CaloriQs
35
Fig. 1 Dose-response curve of resorption rate.
o-----o
CBA C3H
I I
I
15
0 o/o
I
I
8
20
25
30
Ethanol- dQrivQd calories
Fig. 2 Dose-response curve of mean fetal weight (bars represent 95%confidence limits).
227
FETAL ALCOHOL SYNDROME TABLE 4
Types and frequency of skeletal anomalies Strain
Diet ( X EDC) '
No fetuses cleared
Oce1put
Sternum
Ribs
Lab chow 0 15 20 25 Lab chow 0 20 25 30
15 18 6 4 5 35 24 22 12 6
1 0 6 4 5 2 1 18 12 6
0 0 3 4 5 0
0 0 0 3 3 0
0
0
6 5 4
5 5 6
Total abnormal
%
CBA
CH3
1
7 0
100 100 100 6
4 82 100 100
See text for details TABLE 5
Types and frequency of soft-tissue anomalies Strain
Diet (36 EDC) '
CBA
Lab chow
C3H
15 20 25 Lab chow
0
0
20 25 30 I
No. fetuses sectioned
32 38 11 8 9 67 49 46 24 11
Dilated brain ventricles
Eyelids open
0 0
0
4 8 9
0 8 9
0
1 36 24 11
0
-
Exencephaly
Heart
Gastrosehisis
0 0 0
0
0
0
0
2 7 8 0 0 18 17 10
0 2 1 0 0 3 9 9
0 3 0 0 4 7 9
Total abnormal
9: 0 0 36 100 100 0 2 78 100 100
See text for details.
ment diets, but could not be evaluated in the C3H since this strain is genetically open lidded a t birth. Exencephaly and gastroschisis were observed in both strains a t higher EDC diets. DISCUSSION
Recent studies on the teratogenicity of ethanol resulted in a variety of abnormalities. In chicken egg studies, where ethanol was injected into the air space a t early stages of incubation, the major effect was growth retardation with generalized malformations of the central nervous system leading to increased chick mortality (Sandor and Elias, '68; Sandor, '68).Injection iv of rats with ethanol on gestation days 6 to 8 caused dysmorphology in CNS anlagen, increased resorption, and retardation of skeletal development (Sandor and Amels, "71).Injecting mice ip in midgestation produced offspring with coloboma of the iris
and ectrodactyly, as well as an increased resorption rate (Kronick, '76). Finally, the administration of ethanol in the drinking water of mice over a 10-day period in mid to late gestation resulted in small fetuses with an increased rate of minor skeletal variants (Schwetz et al., '75). Although these studies all reported abnormalities associated with the fetal alcohol syndrome, the full expression of the syndrome was not observed. This is not surprising since the array of malformations produced in offspring is partly dependent on the period of gestation during which the agent is administered. Hence the duration of administration of a teratogenic agent would also be relevant. This has been demonstrated, e.g., with vitamin A deficiency where relatively brief treatment tended to cause only a portion of the total syndrome caused by extended treatment (Wilson et al., '53). Therefore, to simu-
228
GERALD F. CHERNOFF TABLE 6
Criteria for an animal model of the fetal alcohol syndrome 1. Oral route of administration 2. Even circadian distribution of ethanol consumption 3. Blood alcohol levels greater than 100 mg/100 ml blood 4. Diet of at least 25%ethanol derived calories 5. Behavioral manifestation of intoxication 6. Physical addiction 7. Good nutrition and caloric intake 8. Intoxication prior to and during gestation
late human alcoholism and possibly reproduce a fetal alcohol syndrome in animals, there should be continuous exposure to ethanol throughout gestation. It is now recognized among researchers in alcohol addiction that an animal model, it it is to be effective in delineating the etiology, prevention, and cure of a human disorder, should meet criteria observed in the human condition Falk et al., '72; Mello, '73; Lester and Freed, '73). Similarly, when attempting to establish the teratogenicity of chronic alcoholism, one should try to mimic the mode and duration of human alcohol consumption and apply these findings to the animal population being studied. Paralleling criteria for a diagnosis of human alcoholism (Criteria Committee, '72; Ulleland, '72), and the pattern of consumption in mothers of children with the fetal alcohol syndrome, an ideal set of criteria was established and is shown in table 6. The first 6 requirements are those commonly used to establish a diagnosis of human alcoholism. The final two requirements are specifically applicable to a teratologic model. Since the effects of maternal chronic alcoholism as opposed to the effects of ethanol per se are under question, it is necessary that the ethanol treatment take place prior to, as well as during gestation. By doing this, allowance is made for the many adaptive changes in hepatic cellular metabolism following chronic ethanol consumption (Lieber, '75), which might have secondary teratogenic effects contributing to the full fetal alcohol syndrome. Finally, the nutritional and caloric intake must be carefully controlled so that the teratogenic effect of the maternal chronic alcoholism can be kept separate from maternal malnutrition (Wilson, '73: pp. 40-42) or fasting (Runner and Miller, '56). With the protocol used it was possible to meet the criteria listed. Furthermore, with
the strains of mice used, it was possible to obtain blood alcohol levels in excess of 100 mg/100 ml blood with diets containing less than 25%EDC. This finding suggests that the quantitative blood alcohol level is a more reliable indication of the maternal physiological state than the amount of ethanol in the diet, since the former is influenced by maternal rates of metabolism which are ultimately under genetic control. It was found that day-18 mouse fetuses had a pattern of malformations similar to those observed in children with the fetal alcohol syndrome. These similarities included prenatal growth defiiciency evidenced by low fetal weight and incomplete ossification, neural and cardiac anomalies, skeletal dysmorphogenesis, and prenatal wastage. Together with the clinical reports on this syndrome the evidence from this study strongly supports the dangers of maternal chronic alcoholism as a teratogenic agent, or perhaps more correctly, a teratogenic activity. In this study a dose-response curve ranging from embryolethality a t high doses (blood alcohol equivalent of 300 mg/100 ml blood) to brain malformations a t the lowest dose (blood alcohol equivalent of 73 mg/100 ml blood) was demonstrated. This supports a hypothesis that ethanol or its metabolic by-products act on various developing systems throughout gestation. Considering its molecular weight (46.07) and high lipid solubility it is conceivable that the earliest effects of ethanol may be on the blastocyst, with later effects on the embryo and fetus. Although validation of this interpretation awaits further study, it clearly demonstrates the need to consider the long term effects of maternal alcoholism a s opposed to exposure only during organogenesis. Another point of interest is that the lowest dose producing abnormalities is below the minimum blood level required for a diagnosis of human chronic alcoholism. The full expression of anomalies in the fetal alcohol syndrome has only been reported in the offspring of chronic alcoholic women, but the possibility thus exists that a milder form of the syndrome may be found in the offspring of women who have been moderate to heavy drinkers during pregnancy. CBA/J mice were more sensitive to the induction of the syndrome than CSHAgM1. Considering the difference in blood alcohol levels between strains on the same dose i t is possible that the difference in susceptability
FETAL ALCOHOL SYNDROME
is due to the rate of ethanol metabolism. Two liver enzyme systems, alcohol dehydrogenase (ADH) in the cytosol fraction and the inducible microsomal ethanol oxidizing system (MEOS) in the microsomal fraction, are responsible for ethanol metabolism in both man and mouse (Lieber and DeCarli, '70). The ADH system is known to be under genetic control in the mouse (Sheppard et al., '68); MEOS has not been investigated from this point of view. ACKNOWLEDGMENTS
The author wishes to express appreciation to Doctor James R. Miller for his enthusiasm and guidance throughout this study, Ms. Jean McLeod for the daily well-being of the mice, and Ms. Shiela Manning for the illustrations. LITERATURE CITED Bianchine, J. W., and B. D. Taylor 1974 Noonan syndrome and the fetal alcohol syndrome. Lancet, 1: 933. Crary, D. D. 1962 Modified benzyl alcohol clearing of alizarin-stained specimens without loss of flexibility. Stain Tech., 37: 124-125. Critera Committee, National Council on Alcoholism 1972 Criteria for the diagnosis of alcoholism. Ann. Int. Med., 77: 249-258. Falk, J. L., H. H. Samson and G. Winger 1972 Behavioral maintenance of high concentrations of blood ethanol and physical dependence in the rat. Science, 177: 811-813. Ferrier, P. E., I. Nicod and S. Ferrier 1973 Fetal alcohol syndrome. Lancet, 2: 218. Freund, G. 1967 Exchangeable injection port cartridge for gas chromatographic determination of volatile substances in aqueous fluids. Anal. Chem., 39: 545-546. 1969 Alcohol withdrawal syndrome in mice. Arch. Neur., 21: 315-320. Freund, G., and D. W. Walker 1971 Impairment of avoidance learning by prolonged ethanol consumption in mice. J. Pharmacol. Exp. Ther., 179: 284-292. Green, H. G. 1974 Infants of alcoholic mothers. Am. J. Obst. Gyn., 118: 713-716. Hall, B. D., and W. A. Orenstein 1974 Noonan's phenotype in a n offspring of a n alcoholic mother. Lancet, I : 680. Jones, K. L., and D. W. Smith 1973 Recognition of the fetal alcohol syndrome in early infancy. Lancet, 2: 999-1001. 1975 The fetal alcohol syndrome. Teratology, 12: 11-26. Jones, K. L., and D. W. Smith 1973 Recognition of the fetal alcohol syndrome in early infancy. Lancet, 2: 999-1001. 1975 The fetal alcohol syndrome. Teratology, 12: 11-26. Jones, K. L., D. W. Smith, A. P. Streissguth and N. C. Myrianthopoulous 1974 Outcome in offspring of chronic alcoholic women. Lancet, 1: 1076-1078.
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Jones, K. L., D. W. Smith, C. W. Ulleland and A. P. Streissguth 1973 Pattern of malformation in offspring of chronic alcoholic women. Lancet, 1: 1267-1271. Kronick, J. B. 1976 Teratogenic effects of ethyl alcohol administered to mice. Am. J. Obst. Gyn., 124: 676-680. Lester, D., and E. X. Freed 1973 Criteria for a n animal model of alcoholism. Pharmacol. Biochem. Beh., I : 103107. Lieber, C. S. 1975 Interference of ethanol in hepatic cellular metabolism. Ann. N.Y. Acad. Sci., 252; 24-50. Lieber, C. S., and L. M. DeCarli 1970 Hepatic microsomal ethanol-oxidizing system: in vitro characteristics and adaptive properties in vivo. J. Biol. Chem., 245: 25052512. Mello, N. K. 1973 A review of methods to induce alcohol addiction in animals. Pharmacol. Biochem. Beh., I: 89101. Palmer, R. H., E. M. Quellette, L. Warner and S. R. Leichtman 1974 Congenital malformations in offspring of chronic alcoholic mother. Pediatrics, 53: 490-494. Rosett, H. L. 1974 Maternal alcoholism and intellectual development of offspring. Lancet, 2: 218. Runner, M. N., and J. R. Miller 1956 Congenital deformity in the mouse as a consequence of fasting. Anat. Rec., 124: 437-438 (Abstract). Sandor, S. 1968 The infiuence of aethyl-alcohol on the developing chick embryo. Rev. Roum. Embryol. Cytol., Ser. Embryol., 5: 167-171. Sandor, S., and D. Amels 1971 The action of aethanol on the prenatal development of albino rats. Rev. Roum. Embryol. Cytol., Ser. Embryol., 8: 105-118. Sandor, S., and S. Elias 1968 The influence of aethylalcohol on the development of the chick embryo. Rev. Roum. Embryol. Cytol., Ser. Embryol., 5: 51-76. Schwetz, B. A., B. K. Leong and R. E. Staples 1975 Teratology studies on inhaled carbon monoxide and imbibed ethanol in laboratory animals. Teratology, 11: 33A (Abstract). Sheppard, J., P. Albersheim and G. McClearn 1968 Enzyme activities and ethanol preference in mice. Biochem. Genet., 2: 205-212. Tenbrick, M. S., and S. Y. Buchin 1975 Fetal alcohol syndrome. J. Am. Med. Ass., 232: 1144-1147. Ulleland, C. N. 1972 The offspring of alcoholic mothers. Ann. N.Y. Acad. Sci., 197: 167-169. Walker, D. W., and G. Freund 1971 Impairment of shuttle box avoidance learning following prolonged alcohol consumption in rats. Physiol. Beh., 7: 773-778. Walker, D. W., and S. F. Zornetzer 1974 Alcohol withdrawal in mice: EEG and behavioral correlates. EEG Clin. Neurophysiol, 36: 233-243. Warner, R. H., and H. L. Rosett 1975 The effects of drinking on offspring: an historical survey of the American and British literature. J. Stud. Alc., 36: 1395-1420. Wilson, J. G. 1973 Environmental and Birth Defects. Academic Press, New York. Wilson, J. G., C. B. Roth and J. Warkany 1953 Analysis of the syndrome of malformations induced by vitamin A deficiency. Effects of restoration of vitamin A a t various times during gestation. Am. J. Anat. 92: 189-217.
