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Teratogenic effects of lithium and ethanol in the developing fetus
Alcohol. Vol. 3, pp. 101-106, 1986. ©AnkhoInternationalInc. Printed in the U.S.A.
0741-8329/86$3.00 + .00
Teratogenic Effects of Lithium and Ethanol in the Developing Fetus A R C H A N A S H A R M A .1 A N D A R U N K. R A W A T ? 2
Central Drug Research Institute, Lucknow* and Midwest Institute fi~r Treatment and Study o f Alcoholism University o f Toledo,+ Toledo OH 43613
SHARMA, A. AND A. K. RAWAT. Teratogenic effects of lithium and ethanol in the developingfetus. ALCOHOL 3(2) 101-106, 1986.--Prolonged administration of either lithium (7 mg/kg body wt.) or ethanol (30% of daily caloric intake) for 10 days to pregnant rats results in several anatomical abnormalities in the fetus, lntragastric administration of lithium carbonate to pregnant rats immediately after confirmation of pregnancy resulted in high incidence of cleft palate, growth retardation, brain liquification and pulpy brain, hepatomegaly and digital abnormalities, when compared to the salinetreated controls. Furthermore, lithium administration during gestation also resulted in other less frequently observed abnormalities in the fetus, e.g., cardiomegaly, hydronephrosis, ankle-joint defects, syndactyly, defected ribs and sternum ossification defects. Chronic ethanol consumption by pregnant rats during early gestation also resulted in several anatomical abnormalities of prenatal growth retardation, resorption and still births, cleft palate, hydrocephaly and hydronephrosis. The severity and frequency of several of the fetal abnormalities were compounded when lithium and ethanol were administered simultaneously. The possible mechanisms of lithium and ethanol teratogenicity and their synergistic effects have been explained on a biochemical basis. Lithium
Ethanol
Developing fetus
Teratogenic effects
drugs in adult mammalian system, much information is needed in the perinatal systems. In this study therefore, the effects of prolonged lithium, ethanol, or lithium and ethanol administration have been investigated on the developing fetus.
ADVERSE effects of several maternally administered drugs on the fetus and the neonate have been observed [5,14]. In fact, highly lipid soluble drugs cross the placenta in amounts that are directly proportional to the maternal placental blood flow [5]. Furthermore, nearly all drugs administered to lactating mothers are detectable in maternal milk. The teratogenic potential of several psychotropic drugs is well known [5,13]. The teratogenic potential of lithium salts was suggested in early 1950's by the work of Needham [6] and others by employing the isolated invertebrate systems and in chick embryo. However, more recent studies have been equivocal, since both negative and positive findings have been reported relating to the teratogenicity of lithium (cf. [14, 16--18]). As lithium carbonate is widely being used in the treatment of manic-depressive disorders and other disorders of psychomotor excitement; the question of its teratogenic potential has become increasingly important. This is especially true as the population receiving lithium has expanded to include more women in the fertile range. Furthermore, in recent years lithium has been increasingly deployed in the treatment of alcoholics (cf. [14]) with, or without various dysthymic disorders. Additionally, it is already well known that chronic alcohol consumption by pregnant women during gestation exerts a number of toxic effects on the developing fetus [I, 9-12]. The condition has been termed "Fetal Alcohol Syndrome." Although several studies have appeared regarding the interactions between ethanol and other
METHOD
Chemicals Alazarin-red, hematoxylin and DPX mount were the products of Aldrich Chemical Laboratories. Dithionite aniline hydrochloride, aminopyrine, potassium cyanide and formaldehyde were of reagent grade and were obtained locally. Ethanol and lithium carbonate were obtained from the local pharmacy. Liquid Sustacal was obtained from Mead Johnson Co., Evansville, IN.
Animals Albino rats were maintained on laboratory chow and water ad lib. Animals were kept for mating, upon confirmation of pregnancy, the dams were divided into control and experimental groups. Lithium carbonate (7 mg/kg) was administered intragastrically for 10 days immediately upon confirmation of pregnancy. The control group was given I N saline. In experiments where ethanol was given, it was given in liquid diet (30% of total calories) for 10 days. The Sustacal
~Present address: University of Pennsylvania, Philadelphia. '-'Requests for reprints should be addressed to Prof. A. K. Rawat, D. Sc., Midwest Institute for Treatment and Study of Alcoholism, P.O. Box 5888, Toledo, OH 43613.
I01
102
SHARMA AND RAWAT TABLE 1 HIGH INCIDENCE TERATOGENIC EFFECTS OF LITHIUM
Treatment Lithium (7 mg/kg) for 10 days
Types of Abnormalities Reduction in size Cleft palate Hepatomegaly Brain liquification Other brain defects Lower digit defects Upper digit defects
Abnormalities % of Control 29.4 (77) 10.2 (46) 15.1 (46) 13.0 (46) 7.1 (46) 61.0 (37) 49.0 (30)
Effects of prolonged lithium carbonate administration during early gestation have been studied on the developing fetus compared to the corresponding control fetuses from the saline-treated group. High teratogenic defects were observed by using macroscopic, slicing and skeletal studies as described in the Method section. The results are expressed as percent of control with the number of observations in parentheses. N.D. denotes not detectable.
liquid diet and nutritional details have been published previously from this laboratory (9-11). In experiments where ethanol and lithium were given simultaneously, ethanol was given in liquid diet and lithium was given intragastrically. The corresponding control group received isocaloric Sustacal-sucrose liquid diet and saline intragastrically. FIG. 1. Effect of lithium in producing clubbed foot in rat fetus.
Preparation of Tissue fi~r Teratologicul ILramination The methods for teratological studies were essentially similar to those recommended by the World Health Organization (W.H.O.) Scientific Group [19]. The abdomen of pregnant near term animals was opened carefully. The number of resorptions were counted, the uterus was carefully opened and the alive and dead fetuses were counted. The fetuses and placentae were removed and washed in 1 N cold saline. Each fetus and placenta was weighed individually, and the samples were stored in 7(1% alcohol. The samples were immediately divided for macroscopic, skeletal, slicing and other examinations. Macroscopic or gross anatomical studies. The fetuses which were fixed in 70% alcohol were checked for external morphological abnormalities. The fetuses with abnormalities and their corresponding controls were further processed for skeletal tests with Alazarin-red. The details of the procedure utilizing Mall's solution and Alazarin-red stain have been described in detail elsewhere [21. Sectioning or slicing studies. The fetuses used for slicing studies were placed in supine position in a Petri dish and were sectioned. The details of sectioning technique have been described in detail [20]. The techniques of tissue fixation, washing, dehydration, embedding and staining were essentially the same as used by Wilson [20]. RESULTS
Teratogenie EfJ~,cts of Lithium Carbonate Intragastric lithium carbonate administration to pregnant rats for 10 days immediately upon confirmation of pregnancy produced several teratogenic abnormalities in the developing fetus. These teratogenic effects could be generally divided into two catagories; (t) those teratogenic effects which are
observed more frequently and are therefore named as high incidence teratogenic effects, and (2) those effects which are observed less frequently and are therefore named as low incidence teratogenic effects. The overall frequency of teratogenic effects is recorded on the basis of three examination techniques, namely, gross anatomical examination, slicing studies and microscopic studies. Growth retardation. Chronic lithium carbonate administration during early gestation resulted in a significant reduction in total body weights of the fetus and the fetal body size, compared to the corresponding controls (Table 1). The observed deleterious effect of lithium on the fetus during this early gestational age are not surprising since it is the period of most of the organogenesis. Placentae from the lithiumtreated group also showed a significant decrease in the weights of the placentae compared to the corresponding controls. Furthermore prolonged lithium-intake by pregnant dams during early gestation also resulted in about 15% higher rates of fetal resorption compared to the corresponding controis. Cleft palate. A high incidence of cleft palate abnormalities were observed in the fetuses from the lithium-treated group compared to the corresponding saline-treated control fetuses (Table 1). Bruin liquification. As shown in Table 1, chronic lithium carbonate administration during early gestation was observed to produce fetal brain liquification defect, in which the solid brain matter was replaced by milky white liquid. However, the brains from the saline-treated control group showed normal healthy brain tissue. Hepatomegaly. Slicing studies showed that lithium administration during early gestation resulted in high incidence
L I T H I U M AND E T H A N O L IN THE F E T U S
103 TABLE 2 LOW INCIDENCETERATOGENICEFFECTSOF LITHIUM Types of Study
Types of Abnormalities
Abnormalities % of Control
Macroscopic
Wrist drop Clubbed foot Broad snout Hydrocephaly Inverted claw
1.5 (88) 2.5 (88) 4.2 (100) 4.6 (66) 3.4 (77)
Slicing
Cardiomegaly Hydronephrosis
3.1 (46) 3.0 (77)
Skeletal
Ankle-joint Syndactyly Defected ribs Sternum
5.4 5.5 7.5 3.1
(30) (37) (40) (30)
Effects of prolonged (10 days) lithium carbonate (7 mg/kg) administration during early gestation have been studied on the developing fetus compared to the corresponding control fetuses from the saline-treated group. Low incidence teratogenic defects were observed by using macroscopic, slicing and skeletal studies as described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
FIG. 2. Normal claw in the fetus from saline-treated control.
of hepatomegaly. Such enlargement of the liver was not observed in the saline-treated control group (Table 1). Digital abnormalities. Skeletal studies conducted upon the fetuses from the lithium-treated group showed that the fetuses from this group had high incidence of nonossification of upper and lower digits. The non-ossification of the bones was confirmed by the lack of Alazarin-red reaction, showing lack of calcium in the bones. The incidence of non-ossification of digits was not observed in the fetuses from the saline-treated control group. The frequency of digital abnormalities in the lithium-treated fetuses compared to the control fetuses is shown in Table 1.
Low Incidence Teratogenic Effects of Lithium
FIG. 3. Effect of lithium in producing inverted claw in rat fetus.
Prolonged lithium carbonate administration during early pregnancy resulted in other specific low incidences of teratogenic abnormalities. These abnormalities are observed during teratological examinations conducted by employing macroscopic, slicing, skeletal or microscopic techniques. The macroscopic or gross anatomical studies were conducted using 77 fetuses from the lithium-treated group and 100 fetuses from the control group. The macroscopic studies showed that administration of lithium carbonate during early gestation resulted in fetal abnormalities of wrist drop, clubbed foot, broad snout, and inverted claw (Figs. 1, 2, 3). In addition in some cases a reduction in the size of external pinna and superficial hemorrhage on the head and neck of some fetus from the lithium-treated group was observed. Such abnormalities were not encountered in the control saline-treated group. Cardiomegaly. The fetuses from dams receiving lithium intragastrically for 10 days immediately after conception, showed greater incidence of enlargement of heart or cardiomegaly, compared to the corresponding controls (Table 2). The incidence of cardiomegaly observed in the fetuses from the lithium-treated group is not surprising, previous
104
SHARMA AND RAWAT TABLE 3 TERATOGEN1CEFFECTSOF ETHANOLCONSUMPTION Treatment Ethanol-fed (Blood alcohol) 190 mg %
Types of Abnormalities Growth retardation Cleft palate Resorptions Hydrocephaly Hydronephrosis Ankle-joint Syndactyly
Abnormalities % of Control 69.8 (20) 11. I (20) 14.6 (20) 3.5 (20) 3.1 (20) 6.8 (20) 6. l (20)
Effects of prolonged (10 days) ethanol (30% caloric intake/day) consumption by pregnant dams from the first day of conception have been investigated on the developing fetus compared to the fetuses from the sucrose-fed control dams. The details of the techniques have been described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
TABLE 4 TERATOGEN1CITYOF LITHIUMAND ETHANOLCOMBINATION Treatment FIG. 4. Effect of lithium in producing syndactyly in rat fetus.
studies [14] have also shown a similar effect of lithium on the fetal heart. Hydronephrosis. As shown in Table 2, fetuses from lithium-treated dams showed hydronephrosis or swollen and enlarged kidneys. Such kidney defects were not observed in the fetuses from dams receiving saline, as shown in Table 2.
Skeletal Abnormalities Skeletal examinations were conducted on the fetuses from dams receiving lithium carbonate (7 mg/kg body weight) for 10 days immediately after confirmation of conception and on the corresponding saline-treated controls. The following skeleton defects were observed in the lithiumtreated group: Ankle-joint defects. As shown in Fig. 3, chronic lithium administration during early gestation resulted in several ankle-joint defects, when compared to the corresponding controls. The presence of inverted claw in the lithium-treated group suggests that in all probability the ankle-joint defect might have been contributed to by the presence of inverted claw abnormality. Syndactyly. As shown in Fig. 4, the fetuses from the lithium-treated group showed syndactyly. This defect was absent in the fetuses from the control saline-treated group. Defected ribs. The fetuses from the lithium-treated group showed that their ribs were shorter compared to the corresponding fetuses from the control group. The most pronounced reduction in the size of the rib observed was that in the last or the so called floating rib. Sternum ossification defect. Fetuses from the lithiumtreated group showed either the absence, or the lack of os-
Lithium (7 mg/kg, 10 days) and Ethanol (30% caloric intake/day, 10 days)
Types of Abnormalities Growth retardation Cleft palate Hepatomegaly Resorption Hydrocephaly Hydronephrosis Ankle-joint Syndactyly
Abnormalities % of Control 81.3 (20) 20.4 (20) 18.3 (20) 23.1 (20) 8.2 (20) 6.0 (20) 11.3 (20) 12.0 (20)
Effects of simultaneous lithium and ethanol administration have been investigated on the developing fetus compared to the fetuses from the corresponding control dams treated with saline and sucrose. The details of experimental techniques have been described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
sification of the sternum when compared to the corresponding saline-treated control.
Teratogenic L~[fects of Ethanol Prolonged ethanol consumption by pregnant rats for 10 days immediately after confirmation of conception results in several anatomical, behavioral and metabolic abnormalities. Several of the anatomical abnormalities observed in the fetuses from ethanol-fed dams are similar in nature to those observed upon chronic lithium intake by the pregnant dams. The fetal abnormalities observed in the fetuses and newborns from ethanol consuming dams have been collectively described as "Fetal Alcohol Syndrome." The details of specific teratogenic effects in the fetuses from ethanol consuming dams are as follows: Prenatal growth retardation. As shown in Table 3, prolonged ethanol consumption by pregnant dams resulted in a significant decrease in the body size and weights of the
L I T H I U M AND E T H A N O L IN THE FETUS fetuses when compared to the corresponding controls from the sucrose-fed dams. In experiments in which pregnant dams received both lithium and ethanol a further decrease in the size and weights of the fetuses was observed when compared to either the lithium alone or ethanol alone treated groups (Table 4). The body size and weight of fetuses from the ethanol-lithium treated groups were significantly lower than those of the corresponding sucrose-saline treated controis (Table 4). Resorption and still births. The incidence of resorption and still births was significantly higher in the fetuses from the ethanol consuming dams compared to the corresponding sucrose-fed controls. However, in the dams taking ethanol and lithium simultaneously the incidence was significantly higher when compared to either ethanol alone or lithium alone (Tables 3, 4). Cleft palate. Prolonged ethanol intake during early gestation resulted in an increased incidence of cleft palate when compared to the fetuses from the sucrose-fed dams (Table 3). Administration of lithium and ethanol simultaneously to pregnant dams resulted in a significantly higher incidence of cleft palate compared to either lithium alone or ethanol alone (Table 4). Hydrocephaly and lowered brain weights. The fetuses from the ethanol consuming dams showed hydrocephaly and lowered brain weights compared to the fetuses from sucrose-fed dams. The incidence of hydrocephaly and decreased brain weights was much higher when ethanol and lithium were administered to the pregnant rats, compared to either the ethanol-treated or lithium-treated groups (Table 4). Hydronephrosis. The incidence of hydronephrosis, or enlarged kidney was significantly higher in the fetuses from the ethanol-fed dams compared to the corresponding control fetuses from the sucrose-fed dams. Administration of lithium and ethanol simultaneously to the pregnant dams had a pronounced effect on the incidence of hydronephrosis as shown in Table 4. Limb and joint defects. Several defects of limb and joint are also observed in the fetuses from the ethanol-fed dams such as microcephaly, shortened fingers, and syndactyly. All of the above mentioned abnormalities are increased significantly in severity and frequency when the pregnant dams are given both lithium and ethanol simultaneously as shown in Table 4. DISCUSSION A large number of drugs when administered maternally have been observed to cross the feto-placental barrier and have been detected in the fetal tissue and circulation [5]. In view of the fact that lithium carbonate is a drug of choice in the treatment of manic-depressive disorder and other disorders of psychomotor excitement, the study of its teratogenicity is of obvious importance. Secondly, in recent years since lithium has been also deployed in the treatment of alcoholics the study of teratogenicity of lithium and ethanol has become of added importance. Previous studies from this laboratory [9-12] have shown that prolonged ethanol administration either during gestation or lactation exerts a number of injurious and toxic effects on the developing fetus and newborn. In humans hundreds of cases of the so called "Fetal Alcohol Syndrome" have been identified and reported from different geographical areas of the world [1, 3, 4, 7]. Present study therefore examines the teratogenic effects of lithium, ethanol and the synergistic effects of
105 ethanol and lithium combination. As far as the present authors are aware no reports are available in the literature in which the teratogenic effects of the lithium-ethanol combination might have been examined. The generalized growth retardation effect of lithium on the fetus when administered to the rats, observed in the present study (Table I) is in agreement with the findings of other investigators [17] who observed similar effects of lithium. As shown in Table 4, it is not surprising that the lithium-ethanol combination had a synergistic effect on the fetal body size and weight. It has been previously observed that administration of ethanol alone to the pregnant dams results in prenatal and postnatal growth retardation [10]. The teratogenic effect of cleft palate observed upon lithium administration in the present study is one of the frequently encountered anatomical abnormalities. Studies from other laboratories [14, 16-18] have also shown that lithium administration during early gestation results in cleft palate abnormalities. The observations recorded in Table 4, showing an increased incidence of cleft palate abnormalities in the lithium-ethanol treated group compared to lithium alone are not surprising. Prolonged ethanol consumption during early gestation has been shown to result in cleft palate in fetuses from both human and animal species [8]. It is clear from this observation (Table 4) that as far as cleft palate abnormality is concerned lithium and ethanol have a synergistic teratological effect. As demonstrated by slicing studies lithium administration alone resulted in higher incidence of hepatomegaly or enlargement of the liver compared to the corresponding controls (Table 1). However, simultaneous administration of lithium and ethanol did not significantly increase the incidence of hepatomegaly. A lack of a synergistic effect of lithium and ethanol in regards to hepatomegaly is suprising as the administration of ethanol alone to pregnant rats leads to lipid, triglyceride and cholesterol accumulation in the fetal liver, as we have previously reported. However, it is possible that decreased contents of RNA and DNA observed [10] in the fetal liver from the ethanol-fed dams along with an inhibited protein synthesis [10] may counteract the increase in liver size which might result from lipid accumulation. The observations that prolonged lithium administration to pregnant rats resulted in brain defects such the pulpy brain and liquification of brain of the fetus (Table 1) have been also made previously by other investigators [16--18]. In this context it is therefore noteworthy that when lithium and ethanol were administered simultaneously to pregnant rats an increase in the incidence of hydrocephaly was observed. Ethanol administration alone has been previously observed to lower the brain weights of fetuses [11] and increase the incidence of hydrocephaly [8]. Hydrocephalus has also been observed in "Fetal Alcohol Syndrome" human infants. It is quite likely that the decrease in brain weights and hydrocephaly may have been a consequence of such drastic alterations in biochemical pathways of the brain as the synthesis of macromolecules. A pronounced inhibition of fetal brain RNA, DNA [11] and protein synthesis [9,11] has been previously reported from this laboratory, in the fetuses from the ethanol-fed dams compared to fetuses from the pair-fed control dams. Furthermore, it has been shown that this effect of nucleic acid synthesis inhibition is a direct consequence of ethanol and not a consequence of the hypothermic effect of ethanol [12]. It is evident from the observations in Table 4 that the incidence of hydronephrosis was significantly increased
106
SHARMA AND RAWAT
w h e n lithium and ethanol were given simultaneously compared to lithium alone. Administration of lithium alone to pregnant rats has been previously o b s e r v e d to produce hydronephrosis in the fetus [18]. It is not very surprising that in the adult human subjects lithium accumulation in the kidney cortex has been o b s e r v e d to result in various kidney diseases. It is one of the reasons that in patients with kidney disease prescribing of lithium is not r e c o m m e n d e d , and furt h e r m o r e in clinical situations blood levels of lithium are routinely monitored to p r e v e n t lithium from reaching toxic levels. Studies by Randal [8] have shown that ethanol administration to pregnant mice produces hydronephrosis in the fetus. In this study lithium and ethanol had a synergistic effect in producing hydronephrosis in the developing fetus (Table 4). In conclusion, administration of either lithium or ethanol
to pregnant rats during early gestation, immediately after confirmation of pregnancy results in a n u m b e r of anatomical abnormalities. The severity and f r e q u e n c e of o c c u r r e n c e of these teratogenic effects is significantly increased when lithium and ethanol were administered simultaneously. The possible biochemical basis of the teratogenic effects of lithium, ethanol, and the synergistic effects of lithium and ethanol have been discussed. ACKNOWLEDGEMENTS Part of the studies presented in this paper have been submitted in the Ph.D. Thesis by one of us (A.S.). We wish to thank Ms. Penny Plessner for technical assistance. The financial assistance for this study to A.S. was provided by the Indian Council for Medical Research and by the Ohio Department of Mental Health and Midwest Institute Research Fund to A.K.R.
REFERENCES
1. Bary, R. G. G. and S. O'Nuallain. Case report. Fetal alcoholism. Ir Med Sc'i 144: 286-288, 1975. 2. Chaube, S. Procedures for staining rat skeleton with Alazrinred. In: Drugs as Teratogens, edited by J. Shardein. New York: C.R.C. Press, 1978, pp. 68-78. 3. Collins, E. and G. Turner. Six children affected by Fetal Alcohol Syndrome. Med J Aust 2: 606-608, 1978. 4. da Silava, V. A., R. R. Laranjeira, F. M. Dolnikoff, H. Grinfeld and J. Msur. Alcohol consumption during pregnancy and newborn outcome: A study in Brazil. Neurobeha v Toxicol Teratol 3: 16%172, 1981. 5. Mirkin, B. L. and S. Singh. In: Perinatal Pharmtwology and Therapeutics, edited by B. L. Milkin. New York: Academic Press, 1976, pp. 1-69. 6. Nedham, J. Biochemistry and Morphogenesis. Ph.D. Thesis, London: Cambridge University, 1952. 7. Olegard, R., K. G. Sabel, M. Aronsson, B. Sandin, P. R. Johansson, C. Carlson, M. Kyllerman, K. lverson and A. Herbek. Effects on the child of alcohol abuse during pregnancy. Acta Paediatr S t a n d [Suppl] 275:112-121, 1979. 8. Randall, C. L. and W. J. Taylor. Prenatal ethanol exposure in mice: Teratogenic effects. Teratology 19: 305-312, 1979. 9. Rawat, A. K. Ribosomal protein synthesis in the fetal and neonatal rat brain as influenced by maternal ethanol consumption. Res Commun Chem Pathol Pharmacol 12: 723-732, 1975.
10. Rawat, A. K. Effects of maternal ethanol consumption on fetal and neonatal rat hepatic protein synthesis. Biofhem .I 160: 653661, 1976. I 1. Rawat, A. K. Inhibition of ribonucleic acid synthesis in developing brain by ethanol. Res ('ommun Subs't Abuse 2: 331-342, 1981. 12. Rawat, A. K. Nucleic acid and protein synthesis inhibition in the developing brain by ethanol in the absence of hypothermia. Neurobehav Toxic'ol Teratol 7: 161-166, 1985. 13. Schardien, J. L. Antipsychotic drugs. In: Drugs as Teratogens. edited by J. L. Schardien. New York: C.R.C. Press, 1978. 14. Schou, F. J., M. D. Goldfield, M. R. Weinstein and B. Villeunve. Lithium and pregnancy. Br Med J 2: 135-143, 1973. 15. Schou, M. The range of clinical uses of lithium. In: Lithium itt Medi~'al Practice, edited by F. N. Johnson and S. Johnson. Baltimore: University Park Press, 1979, pp. 21-39. 16. Smith, R. L., S. Fabro, H. Shumacher and R. T. Williams. Embryopathic A c'tivities ~f Drugs. Boston: Little Brown Press. 1965. 17. Smithberg, M. and P. K. Dixit. Teratogenic effects of lithium in mice. Teratology 26: 239--245, 1982. 18. Szab, K. T. Teratogenic effects of lithium carbonate in fetal mice. Teratology 225: 73-85, 1970. 19. W.H.O. Scientific Group. Principles for Testing o f Drugs ji~r Teratogenicity. W.H.O. Tech. Report Ser. No. 364, 1967. 20. Wilson, J. G. Teratogenic interactions of chemical agents in rats..I Pharma('ol Exp ?'her 144: 429-438, 1964.
0741-8329/86$3.00 + .00
Teratogenic Effects of Lithium and Ethanol in the Developing Fetus A R C H A N A S H A R M A .1 A N D A R U N K. R A W A T ? 2
Central Drug Research Institute, Lucknow* and Midwest Institute fi~r Treatment and Study o f Alcoholism University o f Toledo,+ Toledo OH 43613
SHARMA, A. AND A. K. RAWAT. Teratogenic effects of lithium and ethanol in the developingfetus. ALCOHOL 3(2) 101-106, 1986.--Prolonged administration of either lithium (7 mg/kg body wt.) or ethanol (30% of daily caloric intake) for 10 days to pregnant rats results in several anatomical abnormalities in the fetus, lntragastric administration of lithium carbonate to pregnant rats immediately after confirmation of pregnancy resulted in high incidence of cleft palate, growth retardation, brain liquification and pulpy brain, hepatomegaly and digital abnormalities, when compared to the salinetreated controls. Furthermore, lithium administration during gestation also resulted in other less frequently observed abnormalities in the fetus, e.g., cardiomegaly, hydronephrosis, ankle-joint defects, syndactyly, defected ribs and sternum ossification defects. Chronic ethanol consumption by pregnant rats during early gestation also resulted in several anatomical abnormalities of prenatal growth retardation, resorption and still births, cleft palate, hydrocephaly and hydronephrosis. The severity and frequency of several of the fetal abnormalities were compounded when lithium and ethanol were administered simultaneously. The possible mechanisms of lithium and ethanol teratogenicity and their synergistic effects have been explained on a biochemical basis. Lithium
Ethanol
Developing fetus
Teratogenic effects
drugs in adult mammalian system, much information is needed in the perinatal systems. In this study therefore, the effects of prolonged lithium, ethanol, or lithium and ethanol administration have been investigated on the developing fetus.
ADVERSE effects of several maternally administered drugs on the fetus and the neonate have been observed [5,14]. In fact, highly lipid soluble drugs cross the placenta in amounts that are directly proportional to the maternal placental blood flow [5]. Furthermore, nearly all drugs administered to lactating mothers are detectable in maternal milk. The teratogenic potential of several psychotropic drugs is well known [5,13]. The teratogenic potential of lithium salts was suggested in early 1950's by the work of Needham [6] and others by employing the isolated invertebrate systems and in chick embryo. However, more recent studies have been equivocal, since both negative and positive findings have been reported relating to the teratogenicity of lithium (cf. [14, 16--18]). As lithium carbonate is widely being used in the treatment of manic-depressive disorders and other disorders of psychomotor excitement; the question of its teratogenic potential has become increasingly important. This is especially true as the population receiving lithium has expanded to include more women in the fertile range. Furthermore, in recent years lithium has been increasingly deployed in the treatment of alcoholics (cf. [14]) with, or without various dysthymic disorders. Additionally, it is already well known that chronic alcohol consumption by pregnant women during gestation exerts a number of toxic effects on the developing fetus [I, 9-12]. The condition has been termed "Fetal Alcohol Syndrome." Although several studies have appeared regarding the interactions between ethanol and other
METHOD
Chemicals Alazarin-red, hematoxylin and DPX mount were the products of Aldrich Chemical Laboratories. Dithionite aniline hydrochloride, aminopyrine, potassium cyanide and formaldehyde were of reagent grade and were obtained locally. Ethanol and lithium carbonate were obtained from the local pharmacy. Liquid Sustacal was obtained from Mead Johnson Co., Evansville, IN.
Animals Albino rats were maintained on laboratory chow and water ad lib. Animals were kept for mating, upon confirmation of pregnancy, the dams were divided into control and experimental groups. Lithium carbonate (7 mg/kg) was administered intragastrically for 10 days immediately upon confirmation of pregnancy. The control group was given I N saline. In experiments where ethanol was given, it was given in liquid diet (30% of total calories) for 10 days. The Sustacal
~Present address: University of Pennsylvania, Philadelphia. '-'Requests for reprints should be addressed to Prof. A. K. Rawat, D. Sc., Midwest Institute for Treatment and Study of Alcoholism, P.O. Box 5888, Toledo, OH 43613.
I01
102
SHARMA AND RAWAT TABLE 1 HIGH INCIDENCE TERATOGENIC EFFECTS OF LITHIUM
Treatment Lithium (7 mg/kg) for 10 days
Types of Abnormalities Reduction in size Cleft palate Hepatomegaly Brain liquification Other brain defects Lower digit defects Upper digit defects
Abnormalities % of Control 29.4 (77) 10.2 (46) 15.1 (46) 13.0 (46) 7.1 (46) 61.0 (37) 49.0 (30)
Effects of prolonged lithium carbonate administration during early gestation have been studied on the developing fetus compared to the corresponding control fetuses from the saline-treated group. High teratogenic defects were observed by using macroscopic, slicing and skeletal studies as described in the Method section. The results are expressed as percent of control with the number of observations in parentheses. N.D. denotes not detectable.
liquid diet and nutritional details have been published previously from this laboratory (9-11). In experiments where ethanol and lithium were given simultaneously, ethanol was given in liquid diet and lithium was given intragastrically. The corresponding control group received isocaloric Sustacal-sucrose liquid diet and saline intragastrically. FIG. 1. Effect of lithium in producing clubbed foot in rat fetus.
Preparation of Tissue fi~r Teratologicul ILramination The methods for teratological studies were essentially similar to those recommended by the World Health Organization (W.H.O.) Scientific Group [19]. The abdomen of pregnant near term animals was opened carefully. The number of resorptions were counted, the uterus was carefully opened and the alive and dead fetuses were counted. The fetuses and placentae were removed and washed in 1 N cold saline. Each fetus and placenta was weighed individually, and the samples were stored in 7(1% alcohol. The samples were immediately divided for macroscopic, skeletal, slicing and other examinations. Macroscopic or gross anatomical studies. The fetuses which were fixed in 70% alcohol were checked for external morphological abnormalities. The fetuses with abnormalities and their corresponding controls were further processed for skeletal tests with Alazarin-red. The details of the procedure utilizing Mall's solution and Alazarin-red stain have been described in detail elsewhere [21. Sectioning or slicing studies. The fetuses used for slicing studies were placed in supine position in a Petri dish and were sectioned. The details of sectioning technique have been described in detail [20]. The techniques of tissue fixation, washing, dehydration, embedding and staining were essentially the same as used by Wilson [20]. RESULTS
Teratogenie EfJ~,cts of Lithium Carbonate Intragastric lithium carbonate administration to pregnant rats for 10 days immediately upon confirmation of pregnancy produced several teratogenic abnormalities in the developing fetus. These teratogenic effects could be generally divided into two catagories; (t) those teratogenic effects which are
observed more frequently and are therefore named as high incidence teratogenic effects, and (2) those effects which are observed less frequently and are therefore named as low incidence teratogenic effects. The overall frequency of teratogenic effects is recorded on the basis of three examination techniques, namely, gross anatomical examination, slicing studies and microscopic studies. Growth retardation. Chronic lithium carbonate administration during early gestation resulted in a significant reduction in total body weights of the fetus and the fetal body size, compared to the corresponding controls (Table 1). The observed deleterious effect of lithium on the fetus during this early gestational age are not surprising since it is the period of most of the organogenesis. Placentae from the lithiumtreated group also showed a significant decrease in the weights of the placentae compared to the corresponding controls. Furthermore prolonged lithium-intake by pregnant dams during early gestation also resulted in about 15% higher rates of fetal resorption compared to the corresponding controis. Cleft palate. A high incidence of cleft palate abnormalities were observed in the fetuses from the lithium-treated group compared to the corresponding saline-treated control fetuses (Table 1). Bruin liquification. As shown in Table 1, chronic lithium carbonate administration during early gestation was observed to produce fetal brain liquification defect, in which the solid brain matter was replaced by milky white liquid. However, the brains from the saline-treated control group showed normal healthy brain tissue. Hepatomegaly. Slicing studies showed that lithium administration during early gestation resulted in high incidence
L I T H I U M AND E T H A N O L IN THE F E T U S
103 TABLE 2 LOW INCIDENCETERATOGENICEFFECTSOF LITHIUM Types of Study
Types of Abnormalities
Abnormalities % of Control
Macroscopic
Wrist drop Clubbed foot Broad snout Hydrocephaly Inverted claw
1.5 (88) 2.5 (88) 4.2 (100) 4.6 (66) 3.4 (77)
Slicing
Cardiomegaly Hydronephrosis
3.1 (46) 3.0 (77)
Skeletal
Ankle-joint Syndactyly Defected ribs Sternum
5.4 5.5 7.5 3.1
(30) (37) (40) (30)
Effects of prolonged (10 days) lithium carbonate (7 mg/kg) administration during early gestation have been studied on the developing fetus compared to the corresponding control fetuses from the saline-treated group. Low incidence teratogenic defects were observed by using macroscopic, slicing and skeletal studies as described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
FIG. 2. Normal claw in the fetus from saline-treated control.
of hepatomegaly. Such enlargement of the liver was not observed in the saline-treated control group (Table 1). Digital abnormalities. Skeletal studies conducted upon the fetuses from the lithium-treated group showed that the fetuses from this group had high incidence of nonossification of upper and lower digits. The non-ossification of the bones was confirmed by the lack of Alazarin-red reaction, showing lack of calcium in the bones. The incidence of non-ossification of digits was not observed in the fetuses from the saline-treated control group. The frequency of digital abnormalities in the lithium-treated fetuses compared to the control fetuses is shown in Table 1.
Low Incidence Teratogenic Effects of Lithium
FIG. 3. Effect of lithium in producing inverted claw in rat fetus.
Prolonged lithium carbonate administration during early pregnancy resulted in other specific low incidences of teratogenic abnormalities. These abnormalities are observed during teratological examinations conducted by employing macroscopic, slicing, skeletal or microscopic techniques. The macroscopic or gross anatomical studies were conducted using 77 fetuses from the lithium-treated group and 100 fetuses from the control group. The macroscopic studies showed that administration of lithium carbonate during early gestation resulted in fetal abnormalities of wrist drop, clubbed foot, broad snout, and inverted claw (Figs. 1, 2, 3). In addition in some cases a reduction in the size of external pinna and superficial hemorrhage on the head and neck of some fetus from the lithium-treated group was observed. Such abnormalities were not encountered in the control saline-treated group. Cardiomegaly. The fetuses from dams receiving lithium intragastrically for 10 days immediately after conception, showed greater incidence of enlargement of heart or cardiomegaly, compared to the corresponding controls (Table 2). The incidence of cardiomegaly observed in the fetuses from the lithium-treated group is not surprising, previous
104
SHARMA AND RAWAT TABLE 3 TERATOGEN1CEFFECTSOF ETHANOLCONSUMPTION Treatment Ethanol-fed (Blood alcohol) 190 mg %
Types of Abnormalities Growth retardation Cleft palate Resorptions Hydrocephaly Hydronephrosis Ankle-joint Syndactyly
Abnormalities % of Control 69.8 (20) 11. I (20) 14.6 (20) 3.5 (20) 3.1 (20) 6.8 (20) 6. l (20)
Effects of prolonged (10 days) ethanol (30% caloric intake/day) consumption by pregnant dams from the first day of conception have been investigated on the developing fetus compared to the fetuses from the sucrose-fed control dams. The details of the techniques have been described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
TABLE 4 TERATOGEN1CITYOF LITHIUMAND ETHANOLCOMBINATION Treatment FIG. 4. Effect of lithium in producing syndactyly in rat fetus.
studies [14] have also shown a similar effect of lithium on the fetal heart. Hydronephrosis. As shown in Table 2, fetuses from lithium-treated dams showed hydronephrosis or swollen and enlarged kidneys. Such kidney defects were not observed in the fetuses from dams receiving saline, as shown in Table 2.
Skeletal Abnormalities Skeletal examinations were conducted on the fetuses from dams receiving lithium carbonate (7 mg/kg body weight) for 10 days immediately after confirmation of conception and on the corresponding saline-treated controls. The following skeleton defects were observed in the lithiumtreated group: Ankle-joint defects. As shown in Fig. 3, chronic lithium administration during early gestation resulted in several ankle-joint defects, when compared to the corresponding controls. The presence of inverted claw in the lithium-treated group suggests that in all probability the ankle-joint defect might have been contributed to by the presence of inverted claw abnormality. Syndactyly. As shown in Fig. 4, the fetuses from the lithium-treated group showed syndactyly. This defect was absent in the fetuses from the control saline-treated group. Defected ribs. The fetuses from the lithium-treated group showed that their ribs were shorter compared to the corresponding fetuses from the control group. The most pronounced reduction in the size of the rib observed was that in the last or the so called floating rib. Sternum ossification defect. Fetuses from the lithiumtreated group showed either the absence, or the lack of os-
Lithium (7 mg/kg, 10 days) and Ethanol (30% caloric intake/day, 10 days)
Types of Abnormalities Growth retardation Cleft palate Hepatomegaly Resorption Hydrocephaly Hydronephrosis Ankle-joint Syndactyly
Abnormalities % of Control 81.3 (20) 20.4 (20) 18.3 (20) 23.1 (20) 8.2 (20) 6.0 (20) 11.3 (20) 12.0 (20)
Effects of simultaneous lithium and ethanol administration have been investigated on the developing fetus compared to the fetuses from the corresponding control dams treated with saline and sucrose. The details of experimental techniques have been described in the Method section. The results are expressed as percent of control with the number of observations in parentheses.
sification of the sternum when compared to the corresponding saline-treated control.
Teratogenic L~[fects of Ethanol Prolonged ethanol consumption by pregnant rats for 10 days immediately after confirmation of conception results in several anatomical, behavioral and metabolic abnormalities. Several of the anatomical abnormalities observed in the fetuses from ethanol-fed dams are similar in nature to those observed upon chronic lithium intake by the pregnant dams. The fetal abnormalities observed in the fetuses and newborns from ethanol consuming dams have been collectively described as "Fetal Alcohol Syndrome." The details of specific teratogenic effects in the fetuses from ethanol consuming dams are as follows: Prenatal growth retardation. As shown in Table 3, prolonged ethanol consumption by pregnant dams resulted in a significant decrease in the body size and weights of the
L I T H I U M AND E T H A N O L IN THE FETUS fetuses when compared to the corresponding controls from the sucrose-fed dams. In experiments in which pregnant dams received both lithium and ethanol a further decrease in the size and weights of the fetuses was observed when compared to either the lithium alone or ethanol alone treated groups (Table 4). The body size and weight of fetuses from the ethanol-lithium treated groups were significantly lower than those of the corresponding sucrose-saline treated controis (Table 4). Resorption and still births. The incidence of resorption and still births was significantly higher in the fetuses from the ethanol consuming dams compared to the corresponding sucrose-fed controls. However, in the dams taking ethanol and lithium simultaneously the incidence was significantly higher when compared to either ethanol alone or lithium alone (Tables 3, 4). Cleft palate. Prolonged ethanol intake during early gestation resulted in an increased incidence of cleft palate when compared to the fetuses from the sucrose-fed dams (Table 3). Administration of lithium and ethanol simultaneously to pregnant dams resulted in a significantly higher incidence of cleft palate compared to either lithium alone or ethanol alone (Table 4). Hydrocephaly and lowered brain weights. The fetuses from the ethanol consuming dams showed hydrocephaly and lowered brain weights compared to the fetuses from sucrose-fed dams. The incidence of hydrocephaly and decreased brain weights was much higher when ethanol and lithium were administered to the pregnant rats, compared to either the ethanol-treated or lithium-treated groups (Table 4). Hydronephrosis. The incidence of hydronephrosis, or enlarged kidney was significantly higher in the fetuses from the ethanol-fed dams compared to the corresponding control fetuses from the sucrose-fed dams. Administration of lithium and ethanol simultaneously to the pregnant dams had a pronounced effect on the incidence of hydronephrosis as shown in Table 4. Limb and joint defects. Several defects of limb and joint are also observed in the fetuses from the ethanol-fed dams such as microcephaly, shortened fingers, and syndactyly. All of the above mentioned abnormalities are increased significantly in severity and frequency when the pregnant dams are given both lithium and ethanol simultaneously as shown in Table 4. DISCUSSION A large number of drugs when administered maternally have been observed to cross the feto-placental barrier and have been detected in the fetal tissue and circulation [5]. In view of the fact that lithium carbonate is a drug of choice in the treatment of manic-depressive disorder and other disorders of psychomotor excitement, the study of its teratogenicity is of obvious importance. Secondly, in recent years since lithium has been also deployed in the treatment of alcoholics the study of teratogenicity of lithium and ethanol has become of added importance. Previous studies from this laboratory [9-12] have shown that prolonged ethanol administration either during gestation or lactation exerts a number of injurious and toxic effects on the developing fetus and newborn. In humans hundreds of cases of the so called "Fetal Alcohol Syndrome" have been identified and reported from different geographical areas of the world [1, 3, 4, 7]. Present study therefore examines the teratogenic effects of lithium, ethanol and the synergistic effects of
105 ethanol and lithium combination. As far as the present authors are aware no reports are available in the literature in which the teratogenic effects of the lithium-ethanol combination might have been examined. The generalized growth retardation effect of lithium on the fetus when administered to the rats, observed in the present study (Table I) is in agreement with the findings of other investigators [17] who observed similar effects of lithium. As shown in Table 4, it is not surprising that the lithium-ethanol combination had a synergistic effect on the fetal body size and weight. It has been previously observed that administration of ethanol alone to the pregnant dams results in prenatal and postnatal growth retardation [10]. The teratogenic effect of cleft palate observed upon lithium administration in the present study is one of the frequently encountered anatomical abnormalities. Studies from other laboratories [14, 16-18] have also shown that lithium administration during early gestation results in cleft palate abnormalities. The observations recorded in Table 4, showing an increased incidence of cleft palate abnormalities in the lithium-ethanol treated group compared to lithium alone are not surprising. Prolonged ethanol consumption during early gestation has been shown to result in cleft palate in fetuses from both human and animal species [8]. It is clear from this observation (Table 4) that as far as cleft palate abnormality is concerned lithium and ethanol have a synergistic teratological effect. As demonstrated by slicing studies lithium administration alone resulted in higher incidence of hepatomegaly or enlargement of the liver compared to the corresponding controls (Table 1). However, simultaneous administration of lithium and ethanol did not significantly increase the incidence of hepatomegaly. A lack of a synergistic effect of lithium and ethanol in regards to hepatomegaly is suprising as the administration of ethanol alone to pregnant rats leads to lipid, triglyceride and cholesterol accumulation in the fetal liver, as we have previously reported. However, it is possible that decreased contents of RNA and DNA observed [10] in the fetal liver from the ethanol-fed dams along with an inhibited protein synthesis [10] may counteract the increase in liver size which might result from lipid accumulation. The observations that prolonged lithium administration to pregnant rats resulted in brain defects such the pulpy brain and liquification of brain of the fetus (Table 1) have been also made previously by other investigators [16--18]. In this context it is therefore noteworthy that when lithium and ethanol were administered simultaneously to pregnant rats an increase in the incidence of hydrocephaly was observed. Ethanol administration alone has been previously observed to lower the brain weights of fetuses [11] and increase the incidence of hydrocephaly [8]. Hydrocephalus has also been observed in "Fetal Alcohol Syndrome" human infants. It is quite likely that the decrease in brain weights and hydrocephaly may have been a consequence of such drastic alterations in biochemical pathways of the brain as the synthesis of macromolecules. A pronounced inhibition of fetal brain RNA, DNA [11] and protein synthesis [9,11] has been previously reported from this laboratory, in the fetuses from the ethanol-fed dams compared to fetuses from the pair-fed control dams. Furthermore, it has been shown that this effect of nucleic acid synthesis inhibition is a direct consequence of ethanol and not a consequence of the hypothermic effect of ethanol [12]. It is evident from the observations in Table 4 that the incidence of hydronephrosis was significantly increased
106
SHARMA AND RAWAT
w h e n lithium and ethanol were given simultaneously compared to lithium alone. Administration of lithium alone to pregnant rats has been previously o b s e r v e d to produce hydronephrosis in the fetus [18]. It is not very surprising that in the adult human subjects lithium accumulation in the kidney cortex has been o b s e r v e d to result in various kidney diseases. It is one of the reasons that in patients with kidney disease prescribing of lithium is not r e c o m m e n d e d , and furt h e r m o r e in clinical situations blood levels of lithium are routinely monitored to p r e v e n t lithium from reaching toxic levels. Studies by Randal [8] have shown that ethanol administration to pregnant mice produces hydronephrosis in the fetus. In this study lithium and ethanol had a synergistic effect in producing hydronephrosis in the developing fetus (Table 4). In conclusion, administration of either lithium or ethanol
to pregnant rats during early gestation, immediately after confirmation of pregnancy results in a n u m b e r of anatomical abnormalities. The severity and f r e q u e n c e of o c c u r r e n c e of these teratogenic effects is significantly increased when lithium and ethanol were administered simultaneously. The possible biochemical basis of the teratogenic effects of lithium, ethanol, and the synergistic effects of lithium and ethanol have been discussed. ACKNOWLEDGEMENTS Part of the studies presented in this paper have been submitted in the Ph.D. Thesis by one of us (A.S.). We wish to thank Ms. Penny Plessner for technical assistance. The financial assistance for this study to A.S. was provided by the Indian Council for Medical Research and by the Ohio Department of Mental Health and Midwest Institute Research Fund to A.K.R.
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