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Teratogenic Effect of Lithium Carbonate

in Early Development of Balb/C MouseMOHSEN NOKHBATOLFOGHAHAI1* AND KAZEM PARIVAR2

1Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, Iran2Department of Biology, Science & Research Branch, Faculty of Basic Sciences,

Islamic Azad University, Tehran, Iran

ABSTRACTLithium carbonate is used as a standard treatment for manic depres-

sion. While researchers have investigated the teratogenic effects of lithiumcarbonate on embryos of various animals in later stages of development,very limited work has been done on the probability of effects at early stagesof development. In this study, the teratogenic effect of lithium carbonatewas investigated at earlier preimplantation through implantation stages ofdevelopment of Balb/C mouse embryos. A therapeutic dose (i.e., 300 mg/kgb.w.) was injected into mice intraperitoneally on days 3.5, 4.5, 5.5, and 6.5of pregnancy. Then, on day 15.5 of gestation, embryos were collected fromthe pregnant animals. Among the embryos, 71.7% were healthy, 10.7%resorbed, 3.1% showed lordosis, 8.1% were underdeveloped and 8.4% hadeye malformations. Significant increases (P < 0.05) in the number of he-patic megakaryocytes and nucleated red cells were also observed among ex-perimental embryos. Analysis of maternal serum proteins prepared fromdissected animals showed a significant increase or decrease (P < 0.05) inthe levels of serum proteins albumin,a2 globulin, b globulin, and g globu-lin. This research on early developmental stages suggests that pregnantmothers need to be aware of possible teratogenic effects at early stagesof pregnancy, although it has been thought that the egg envelope can pre-

vent teratogens from entering. In this case, mothers may need to stoplithium carbonate treatment before they make a decision to become preg-nant. Anat Rec, 291:10881096, 2008. 2008 Wiley-Liss, Inc.

Key words: lithium carbonate; teratogenic effect; early devel-opment; Balb/C mouse

Experimental studies have shown for several decadesthat lithium salts cause several teratogenic effects inanimals. In recent years, it has also been shown thatlithium is still teratogenically and toxicologically impor-tant in adults as well as embryos (Sharma and Iqbal,

2005; Zarnescu and Zamfirescu, 2006; Allagui et al.,2006; Tandon et al., 2006; Tsaltas et al., 2007). Exten-sive usage of lithium and its salts in pharmaceuticals,air conditioning, and in several biological and chemicallaboratories increases the probability of this metal beingin close contact to humans. Toxic effects of lithium saltsmay also result from medication. Clinical studies haverecently shown that lithium carbonate causes severalteratogenic effects when patients are treated by long-term administration of the medicine (Lewicki et al.,2006; Garcia et al., 2007; Fujii et al., 2007).

Studies on patients suffering from manic depressionreveal that the majority of these patients are women

and many are of reproductive age (Sharma and Rawat,1986; Kao and Elinson, 1989; Yonkers et al., 1998). Thisraises concerns, because several reports suggest a highrisk of fetal malformations when lithium carbonate isused during pregnancy (Yonkers et al., 1998). Lithiumcarbonate passes through the placenta easily (Smithberget al., 1984), and after a short time, its concentration infetal blood almost equals that in maternal blood serum(Weinstein and Goldfield, 1969).

*Correspondence to: Mohsen Nokhbatolfoghahai, Departmentof Biology, Faculty of Sciences, Shiraz University, Shiraz, Iran71454. Fax: 0098-711-2280916. E-mail: nokhbeh@susc.ac.ir

Received 26 January 2007; Accepted 10 April 2008

DOI 10.1002/ar.20730Published online in Wiley InterScience (www.interscience.wiley.com).

2008 WILEY-LISS, INC.

THE ANATOMICAL RECORD 291:10881096 (2008)

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While findings on embryotoxicity and teratogenicityamong invertebrates show that lithium has detrimentaleffects, the data for vertebrates are dependent on theanimal and strain treated as well as on the dosage andduration of exposure (Smithberg and Dixit, 1982). Thespecific problems also associated with lithium exposure

vary during different stages of gestation (Yonkers et al.,1998). Among vertebrates, numerous reports (Wrightet al., 1971; Allakhverdiev and Smolnikova, 1983; Man-has and Kumar, 1988; Jurand, 1988; Simard et al., 1989;Hansen et al., 1990; Giles and Bannigan, 1997; Schein-feld and Faad, 2004) are available about teratogeniceffects of lithium carbonate, few on humans; most onlaboratory animals. For example, fertilized chick eggswere injected with 0.05 ml of various dilutions of lithiumcarbonate ranging from 0.5 percent to 1 percent(Manhas and Kumar, 1988). The embryos were observedat various intervals on 6th, 8th, 11th, and 13th day afterinjection. The results showed an increased rate of mor-tality and malformations in the chicks injected with lith-ium carbonate. In another investigation, injection of lith-ium carbonate at the rate of 300 mg/kg body weight to

mice on their gestation day 9 caused death in someembryos, growth retardation and central nervous systemmalformation in others (Jurand, 1980). These resultswere later confirmed by other researchers (Jurand,1988; Giles and Bannigan, 1997). There are also reportsof skeletal malformation as a result of lithium carbonatetreatment (Marathe and Thomas, 1986; Sharma andRawat, 1986). In another study, lithium carbonate at therate of 300 mg/kg body weight was injected to Balb/Cmice on days 7.5, 8.5, and 9.5 of pregnancy. These treat-ments caused embryo malformations such as anophthal-mia, microphthalmia, exohepaty, exencephaly, skeletalmalformation, and resorption (Smithberg and Dixit,1982; Parivar and Zeynali, 1995). Smithberg and Dixit(1982) tested the teratogenicity of lithium carbonate in

two inbred strains of mice (Strains 129 and A/J mice).Their results suggested that six times the therapeuticamount of lithium in human serum level is teratogenicin mice. In the human embryo, these teratogenic effectsare targeted at the most vital parts such as heart andblood vessels (Weinstein and Goldfield, 1975). However,in the case of the human embryo, determination of timeintervals (critical periods) during which the applicationof this drug is most detrimental awaits further investi-gation. Due to deleterious teratogenic effects of thismedicine on the human embryo, the aim of the presentstudy was to use the Balb/C mouse as an animal modelfor studying the effects of lithium on mouse embryos notas usual on late development, but on earlier preimplan-tation through implantation stages of development.

Research on early developmental stages is important,because pregnant mothers need to be aware of possibleteratogenic effects resulting from treatment. In thiscase, mothers may need to stop lithium carbonate treat-ment before they make a decision to become pregnant.

MATERIALS AND METHODS

Animals and Treatments

The study was approved by the animal welfare com-mittee of Tehran Training University. Balb/C mice (1012 weeks) were used in our experiments. The mice werekept in polypropylene cages at 21 6 28C under natural

photoperiod (12 hr light and 12 hr dark). Appearance ofa vaginal plug was considered as day zero of pregnancy.The LD50 value was selected based on a previous studyconducted with the same mouse strain and same chemi-cal (Parivar and Zeynali, 1995). To measure the LD50,increasing values of lithium carbonate solution were

injected in a series of six pregnant mice. The experimentwas terminated when approximately 50% of the animalsdie after 48 hr of treatment. The LD50 value wasapproximately 450 mg/kg b.w. and the teratogenic dosewas 300 mg/kg b.w. For studying the effects of lithiumon mouse embryos at early stages of development, 85pregnant mice were taken and divided into threegroupsexperiment, control, and untreated. Mice weretaken on days 3.5, 4.5, 5.5, and 6.5 of pregnancy in eachgroup. The animals of the experimental group, wereinjected with 300 mg/kg body weight of lithium carbon-ate solution in water intraperitoneally. The controlgroup was injected with the same volumes of normal sa-line solution (0.9% w/v of NaCl) only, and the thirdgroup remained untreated. Gestation was terminated onday 15.5, and the uterine contents were removed. After

collection of embryos from the uterus, their amnioticmembranes were removed carefully and the weight ofembryos and their placentas were determined. Measure-ments also were made of the crown-rump (CR) length bycalipers. Embryos were fixed in Bouins solution for atleast 3 hr for morphological and histological studies.Each series of experiments was repeated three times.

Blood Sampling

Mice were anesthetized with ether and maternal bloodsamples from untreated, control, and lithium carbonate-treated animals were taken by cardiac puncture andwas used for serum protein measurements. Serum wasprepared from collected blood and stored at 2208C until

required for later studies.

Sectioning and Tissue Staining

Embryos were prepared for sectioning and stainingaccording to the following procedures: specimens wererinsed in several changes of normal saline and thenfixed in Bouins solution for at least three hours. Theembryos were washed in tap water several times andthen dehydrated in an ascending ethanol series (30%,50%, 70%, 90%, and 100%). The specimens wereprepared for wax embedding using a paraffin bath(57608C), and were sectioned at 7 mm then stained withHematoxylin-Eosin (conventional method). Sections wereexamined over a range of magnifications and photo-

graphed using a Zeiss photomicroscope, M3. Lithium isreported to have a direct effect on vascular developmentin chick embryo (Giles and Bannigan, 1999). To find outif lithium affects blood vessel development in mouseembryos, the vessels in histology sections were chosenrandomly for RBC counting in both experimental andcontrol samples. In each field, the ratio (number) ofnucleated RBCs to nonnucleated RBCs were counted.The same procedure was carried out for the counting ofmegakaryocytes. The slide section of liver at stage 15.5of development was designed for histopathological inves-tigation, specifically to count the number of hepatic meg-akaryocytes as the activity of hematopoiesis is marked

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by presence of these cells in this stage of development.Three measurements of the eye (eye ball, lens diameter,and retina thickness) were recorded in experimental,control, and untreated embryos. The histological serialsections of the head through eye regions were preparedto measure the eye parameters, using light microscopewith graticule and in relevant magnification (Fig. 1).

Analysis of the Main Blood-serum Proteins

Electrophoretic properties of blood serum proteinswere studied according to the method described by themanufacturer (Helena Laboratories, Beaumont, TX). Inthis method, blood serum samples were placed on ace-tate plates, and Desaga electrophoresis equipment wasused to separate the protein bands. The separated bandswere then stained in Coomassie blue. The acetate plateswere scanned, using a densitometer at 525 nm, andgraphs and relative percentages of the five main serumproteins were prepared for statistical analysis at a laterstage.

Statistical Analysis

The data obtained from the numbers and percentagesof the abnormalities in different treatments (Table 1)were analyzed by using Z-test and Bonferroni Correc-tion. Growth parameters such as embryonic weight, pla-centa weight and CR length obtained from differenttreatments (Table 2) were analyzed through multiplecomparison and Tukeys procedure. The data obtainedfrom the relative percentages of the serum proteins (Ta-ble 3) were also analyzed using Tukeys procedure. Alltests were done at 5% level significance.

RESULTS

Macroscopic Observations

Experimental data in comparison to controls anduntreated groups are presented in Table 1. Among 344lithium-treated embryos from 37 litters examined forabnormalities, 247 embryos were healthy; 37 resorbed;11 showed lordosis; 28 were undeveloped; and 21 hadeye malformations. Among 275 control embryos, only 4were resorbed embryos and no other malformationswere seen. No malformation was seen among 293embryos from the untreated mice. The resorbed embryoswere found among all experimental treatments, butwere significantly increased in number in 3.5 and 4.5day injected embryos. Underdeveloped embryos werefound to be significantly increased on days 3.5 and 6.5 ofexperimental treatments. A pronounced body curvature(lordosis) was also observed in some experimentalembryos of days 3.5, 4.5, and 6.5. Figure 2A,B showsmorphological differences between normal and lordosisembryos respectively, in sagittal section. The lithiumcarbonate also significantly decreased the weight of pla-

centa and CR length of experimental embryos comparedwith control embryos except in day 4.5 of injection (Ta-ble 2). Experimental embryos of all days showed a sig-nificant decrease in their weight compared with controland untreated embryos.

Microscopic Studies

Microscopic examination of sections through the eyerevealed a significant decrease in eyeball, and lens diam-eters and retinal thickness (microphtalmia) in the exper-imental mice embryos compared with controls in days3.5 and 5.5 treatments. In the day 6.5 treatment, a sig-nificant decrease was only shown in eyeball diameter.No significant differences in eye malformations wereseen in the day 4.5 treatment. Figure 3 shows the differ-

ences in eye ball diameter generally between normal (A)and malformed (B) embryos. Figure 4 (histogram) showsa comparison between eyeball diameter on differentdays of injection. Other malformations observedincluded: cleft palate in day 3.5, 5.5, and 6.5 treatments;exohepaty (Fig. 5A,B) in the day 5.5 treatment. Bothexohepaty and cleft palate occurred very rarely and allappeared in the embryos with an underdeveloped mal-formation.

A significant increase occurred in the number of he-patic megakaryocytes in all experimental treatmentscompared with control and untreated embryos (Fig. 6).Histological examination of blood showed a significantincrease in the number of undifferentiated nucleated redblood cells in all experimental groups compared with

control groups (not shown).

Electrophoretic Observations

Table 3 shows a comparison between relative percen-tages of five maternal serum proteins (albumin, a1 glob-ulin, a2 globulin, b globulin, and g globulin) in differenttreatments. The main results obtained from electropho-retic studies of serum proteins of pregnant mice on day15.5 of their gestation were as follows: (1) There was asignificant increase in albumin levels in experimentalmice from the 3.5 and 4.5 day treatments. (2) There wasa significant decrease in the value of a1 globulin in the

Fig. 1. Schematic image showing general organization of an eye in

sagittal section (lateral view). ( ____) applied for measuring the length

of eye ball (EB) and lens (L) diameters, also retina (R) thickness.

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TABLE1.

Grossm

alformationsobservedinexperimental,control,anduntreatedembryosafterday3.5,

4,

5.5,and6.5

injection

w

ithlithium

carbonate

Injectiondays

3.5

4.5

5.5

6.5

Treatmenttypes

Observations

Experim

ent

Control

Untreated

Experiment

Control

Untreated

ExperimentC

ontrol

Untreated

Experiment

Con

trol

Untreated

Totalnumberof

pregnantfemales

ineachtreatment

9

6

6

9

6

6

7

6

6

12

6

6

Totalnumber

ofembryos

76

66

72

89

68

74

67

70

76

112

7

1

77

Healthy

embryos(%)

43**,1

1

(56.5)

65

(98.5)

72(100)

68**,

11

(76.3)

66

(97.1)

73

(98.7)

53**,

11

(79.1)

70(100)

76(100)

83**,

11

(74)

7

0

(98

.6)

77(100)

Resorbed

embryos(%)

10**,1

1

(13.2)

1(1.5)

0

16**,

11

(18)

2(2.9)

1(1.3)

2*,

1

(3)

0

0

9**,

11

(8.03)

1(1.4)

0

Embryoswith

lordosis(%)

5**,

11

(6.6)

0

0

3*,

1

(3.4)

0

0

0

0

0

3*,

1

(2.7)

0

0

Underdeveloped

embryos(%)

11**,1

1

(14.5)

0

0

2*,

1

(2.3)

0

0

1*,

1

(1.5)

0

0

14**,

11

(12.5)

0

0

Eye malformation(%)

7**,

11

(9.2)

0

0

0

0

0

11**,

11

(16.4

)

0

0

3*,

1

(2.7

)

0

0

Thehighlightednumbershowssignificantdifferentfrom

otherexperimentvaluesineachrowP