TERATOGENESIS IN INBRED STRAINS OP MICE

Major Professor

V* y *r^

^inor/Professox^. ^

• \

Director of the Department of ^Jology

Deanl of the Graduate School

TERATOGENESIS IN INBRED STRAINS OP MICE

THESIS

Presented to the Graduate Council of the

North Texas State University In Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF ARTS

By

Robert A. Morgan, E. A.

Denton, Texas

Augustt 1966

TABLE OP CONTENTS

Pag®

LIST OP TABLES lv

Chapter

I. INTRODUCTION 1 History of Teratogenesis Statement of Problem

II, MATERIALS AND METHODS 12

Selection of Mouse Strains Preparation of Thalidomide Experimental Procedure

III. RESULTS 21

IV. HIS CUSS IOM 31

APPENDIX A k2

APPENDIX B 50

BIBLIOGRAPHY 56

ill

Lic-p OF *A*-LES

Table Pace

I. Results of Administering Throe Dosage Levels of Thalidomide to Pour Wouse Strains 22

II. Results of Administering Distilled Water to Four Strains of Mice 23

III. Comparison of Per Cent Resorptions Due to Administration of Thalidomide to Pour Strains of Mice. . • . . . . . . 2k

IV. Scale for Rating Mice According to Extent of Deformity 26

V. Effect of Thalidomide Administration on Mean Deformity Score 27

VI. Mean Differences in Abnormality Scores in Pour Strains of wj.Ce 28

VII. Analysis of Vsrience in Mean Number of Mice Per Litter Due to Level of Thalidomide . . . . 2*>

VIII. Analysis of Variance in Mean Deformity Score Per Group Due to Level of Thalidomide . . . . 29

IX. Analysis of Variance Due to Route of Thalidomide Injection 30

iv

CHAPTER I

INTRODUCTION

History of Teratogenesis

The first experimental production of congenital ab-

normalities in manna la was accomplished by Altmann (1),

who utilised x-irradiatlon of the pregnant mother. Since

this work, which was performed in the first decade of this

century, various methods have been employed to bring about

deformations in the offspring of treated mothers. The fol-

lowing ia a review of some of the more Important work, and

la by no means exhaustive.

Diets deficient in the various vitamins have been proved

teratogenic in laboratory anlmala. The earliest study with

a vitamin deficient diet was conducted by Hale (26), who fed

a aow a regimen lacking In vitamin A, with the resultant

birth of a litter of eyeless offspring. Vitamin A deficiency

has since been found to be teratogenic in the rat (li|) and the

rabbit (39). Vfarkany and Nelson (8l, 82, 83, 8I4.) obtained

malformations in the progeny of female rats fed a fciboflavin-

deficlent diet. Other workers (21, 3k$ 53) have subsequently

corroborated these findings and expanded upon them somewhat.

The rat has been a popular animal for use in teratogenic

studies of vitamin deficiencies, as diets lacking in folic

acid (6I4.), pantothenic acid (55)» vitamin E (65), thiamin (5U),

X

•nd frltttaln D (80), have all caused congenital malform-

ations in the rat. Niacin antagonists have been shown to

causa developmental anomalies in the chick embryo (141, U2)

and In the mouse '62).

While moat of the above studies Involve diets deficient

in aorae particular vitamin, excesses of vitamin A can alao

cause gross deformities In the rat (85) and mouse (51).

Actadnistratlon of various other amenta to the present fe-

msle laboratory animal has* resulted In deformed offspring.

6-mercaptopurlne, a nucleic acid antagonist'(73)» and an ato

dye, trypan blue (77) ars teratogenic in the rat (7l|» 75# 76)

and In the mouae (33* 67* 66), while cortisone has been

found to eauae cleft palate in the mouse (11, 22, 26, 29,

30, 31# 32# 63) snd the rabbit (20).

In recent yeara, certain drugs used by humans have been

shown to be teratogenic in laboratory animals, a sulfon-

amide, aulphamoprine, causes skull abnormalities in mice

and rats (13)* Acetazolamide (k5)» Ancoloxan (6), and

phsnobarbltal (50) each cause developmental anomalies in

rats. Tetracyclines have been statiatioally implicated

as teratogenic in humans (9, 78) «nd definitely ahown to

cause limb and beak deformities in chick embryos (2, 10).

Even acetylsalicyllc acid, taken in large quantltiea, is

teratogenic in the mouse and the rat (57)*

In 1962 Somera (70) administered thalidomide, a tran-

quilizer, to pregnant white rabbits. There were deformed

fetuaei in three of the four litters delivered. This

tranquilizer had already been strongly suspected tc be the

eauaal agant of a rsssh of limb deformities in humans (lj.9, 60) .

Following Soners1 work, other experimenters produced skeletal

deformities In mice (17# 25, 56), monkeys (16, I4.fi), rats

(11, 37. 58), rabbits (1^, 72, 87), and the chick (3, 5,

6, 15, 19, 36, 52, 66) . Thalidomide has subsequently been

found to produce abnormal growth in a marine red alga (72),

but not in higher plants (59) .

Skeletal malformations due to ingestion of this tran-

quilizer by the pregnant human have been reported in West

Germany (U7, 61) , England (38, W>, 69, 71) , Scotland (22) ,

Taiwan (88) , Japan (28) , Africa (86) , and the Hetherlands

(18 ) . The drug was never placed on the American market;

thus the United States had little direct contact with the

trag«0y. nevertheless, the deformities due to thalidomide

have had the effect of emphasizing the fact that congenital

malformations do occur, and can be caused by seemingly

harmleaa agents.

With the above facts in mind, the Food and Drug Admin-

istration has established 8 program so that all new drugs

are now teated for effect on reproduction in the rat (35).

Both male and female rats are fed a diet containing sub-

toxic levels of the drug in question. These rats are mated,

and the offspring assayed for abnormalities. This procedure

haa three major shortcomings. The first is that the treated

fiaele rats are permitted to give birth to a litter of younp:,

tte Membera of whieh are then examined for abnormalitiee.

thla method of teetinr completely neglects the fact that

ptdmts oftm eat their dead or deformed offapring, thereby

leavinn no evidence of malformed young. The second fault ia

that, regardlass of the drug toeing tested, the pregnant rata

i n a uniform diet* Evidence of diet-teratogen inter-

action plays • major rola In level of malformation (1*0, l|3,

79) makee thia procedure fall aooiatfhat abort of the ideal,

the third fault la that the testing program, aa now estab-

lished, neglecta the likelihood that genetica may be an

important, if not the primary, factor in determining aua-

ce»tibillty to varloua teratogenic agents {?).

Statement of Problem

In view of the fact that the program now being utilised

to teat drugs for poeaible teratogenic effecta does not con-

sider genetic variability among teator atocka, it ia impor-

tant that a atudy te conducted to investigate the influence

that genotype may have on auaceptibility to thalidomide

teratogeneaia. The purpoae of this inveatigation ia to

determine if differencea exist between four inbred atraina

of *lee in auaceptibility to the drug thalidomide.

CHAPTER BIELIO-RAFHY

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It* Boone, M. A., Mary Hammond, and B. D. Earnett, "The Effect of Thalidomide on Chickens and Embryos," Poultry Science. XXXXIII (1%U), 1+73-

5* Boylen, Joseph, Helen Korne, and Vllllard Johnson, "Teratogenic Effects of Thalidomide and Related Substances," Lancet. II (1963), 552^557•

6. Cameron, J. M., "Thalidomide and Congenital Abnor-malities," Lancet, II (1962), 937.

7. Cargener, Norman, "Teratogeneala," Lancet. II (1963)*

6* Carter, M. P. and F. Wilson, "Ancoloxin and Foetal Abnormalities," British Medical Journal, VCCCIXXX (1962), 1609.

9. • , "Antibiotics and Con-genital Abnormalities,** Lancet. II (1963)» 1267.

10. , "Tetracycline and Con-

fenitai Abnormalities," British Medical Journal, CCCI (1962), 729.

11. Christie, 0. A., "Thalidomide end Congenital Abnor-malities," Lancet, II (1962), 21»9.

12* Clark, Karin H., "Histological Investigation of Cor-tisone Induced Cleft Palate in Mice." Genetice,

(1956), 79U-798.

13. Clegg, Hugh, "Teratc.enic Effects of Sulfonamides," British ypdlcal Journal, VCCCCXXVIII (1^65), IJ42.

lit* Cohlan, Sidney Q., "ixcesslva Intake of Vitamin A as a Cause of Congenital Anomalies in the Rat," Science, CXVIII (1°S3),

15. De Bock, C. A. »nd .. Peters, "Effect of Thalidomide on Development ol t e Chick Embryo," Nature, CIC (1963)» 12014.-1206.

16. Delahunt, C. S. sni L. J. Lassen, "Thalidomide syn-drome In Monkeys," Science, CXXXXVI (1964), 1300-130$.

17. Di Paolo, Joseph, "Congenital Malformations in Strain A Mice," Journal of the American Medical Association, CLXXXIII U % M , TT9^TI?I~

18. Dljkhuls, H. J., B. Bekker, and W. van Duyne, "Thalido-mide and Congenital Abnormalities," Lancet, I (1963), 1375.

19. Ehmann, B., "Teratogenic Effects o; Thalidomide," Lancet, I (1963),' 772.

20. Falnstat, Theodore, "Cortisone-Induced cleft Palate in Rabbits," Endocrinology, LV (1954)* 503.

21. Fenton, P. F. and G. R. Covgill, "The Nutrition of the Mouse." Journal of nutrition. XXXIV (191*7), 273-283.

22* Fox, Theodore, "Congenital Abnormalities Due to Thal-idomide," Lancet,' II (1962), 986.

23. Fraser, F. C., H. Kslter, B. E. Walker, and T. D. Falnstat, "The Experimental Production of Cleft Palate with Cor-tisone «md other Hormones," Journal of Cellular and Comparative Physiology, ajuuIIX ( J , •~~mm

2k' Giroud, A., H. Tuchmann-Dupleias, and L. Merceir-Parot, •Thalidomide and Congenital Abnormalities," Lancet, II (1962), 296-299.

25* Hagen, Odette, "Dru^s and Congenital Abnormalities," Lancet, I (1963), 501.

26* Rale, Fred, "Pigs Born Without Eyeball*,* Journal of Heredity, XXIV (1933), 105-106.

27* Jacoba, Richard M., "Histochemical Study of Morpho-genesis and Teratofrenesis of the Palate in Mouse Embryos," Anatomical Record, CIL (1961*), 691-690.

28. Kajli, Tadaahi, "Thalidomide and Congenital Abnormali-ti«s," Lancet, II (1°62), 151.

29* Kalter, Harold, "Factors Influencing the Frequency of Cortisone-Induced Cleft Palate in Mice," Journal of Experimental Zoology, CXXXIV (1957), W - U F T I

30. , "Modification of Teratogenic Action of Cortisone In Mice by Maternal Age. Maternal Weight, and Litter Sise," American Journal of Physiology, CLXXXV (1956). 65-BF:

31. , "The Inheritance of Susceptibility in the Teratogenic Action of Cortisone in Mice," Oenetica. XXXIX (195U)# 185.

32* , and P. C. Frsser, nTti9 Modification of tne Teratogenic Action of Cortisone by Parity," Science, CVIXI (1953), 625-626.

33. , "Production of Congen-ltai uereota in tne orrsprmg of Pregnant Mice Treated with Compound P.* Mature. CLXIX (1952). U30-1*32.

3i|.. Xalter, Harold and Joseph Warkany, "fongenltal Malfor-mations in Inbred Strains of Mice Induced by Ribo-flavin-Deficient, Galactoflavin-Containing Diets,"

lal of Experimental Zoology, CXXXVI (1Q57)»

35* Kelsey, Francis 0., "Problems Rsised for the PDA by the Occurrence of Thalidomide ttabryopathy in Germany, 1960-1961," American Journal of Pablic Health, LV (1965), 1*23-555":

36. Semper, P., "Thalidomide and Congenital Abnormalities," Lancet, II (1962), 836.

37* King, C. T. 0. and P. J. Kendriok, "Teratogenic Effects of Thalidomide in the Sprsgue Dawley Rat," Lancet, II (1962), 1116.

38. Kohler, G. G., H. M. Fiacher, and P. M. Dunn, "Thalido-mide and Congenital Abnormalltiea," Lancet, I (1Q62), 326-327.

39* Laming, G. B., G. V. Salisbury, R. L. Hays, and K. A. Kendall, "The Effect of Incipient Vitamin A Defici-ency on Reproduction in the Rabbit," Journal of Nutrition, LII (1°5U), 217-236.

i|.0. Landauer, Walter, "The Effect of Nicotinamide and alpha-Ketoglutaric Acid on the Teratogenic Action of Insulin," Journal of Experimental Zoology, CIX (1948), 283-290.

, "niacin Antagoniats and Chick Devel-opment," journal of Experimental Zoology. CXXXVI (1957), 171*183. * "•

k2* , and E. M. Clark, "The Interaction in Teratogenic Activity of the Two Niacin Analoga 3-Acetylpyridine and 6-Aminonicotinamide,tt Journal of Experimental Zoology, CXIX (1952), 221-25T;

1)3* , and M. B. Rhode a, "Further Obser-vations on the Teratogenic Nature of Insulin and its Modification by Supplementary Treatment," Journal of Experimental Zoology, CLI (1962), 253-258.

Larson* Valdemar, M % e Teratogenic Effects of Thalido-mide, Imapr amine HC1 and laiApr amine N-Oxide HC1 an tihlte Danish Rabbits," Acta Physiologies Scandinavia, LIX (1963), 87. *

kS* Lay ton, W. M. and D. V. ffallesy, "Deformity of Pore-limb in Rata,* Science, CXXXXIX (1965), 306-307.

46* Leek, Ian and E. L. M. Millar, "Short-Term Changes in the Incidence of Malformations," British Journal of Preven-tive Medicine, XVII (1963), $TT"^

k7» Lens, W., "Thalidomide and CoagiMital Abnormalities," Lancet, I (1962), 1*5.

J|£. Lttoey, J. P. and R. Behnan, "Thalidomides Effect Upon Pregnancy in the Rheaua Monkey," Science, CXXXIX (1963).

J|.9. Mo Bride, P., "Thalidomide and Congenital Abnormalities," Lancet. II (1961), 1358.

50. Mo Ooll, J. D., M. Globus, and S. Robinson, "Drug-Indu4«d Skeletal Malformationa in the Rat," Experlentla. XIX (1963)* 323.

51. Murakami, U. and Y. Kameyama, "Malformations of the Mouse Caused by Hypervitaminosis A of the Mother During Pregnancy," Archives of Environmental Health. XI (1965), 732-7ET:

52. Haber, E. C. and E. J. Largent, "Malformations Induded by Thalidomide in the Developing Chick Embryo," Poultry Science, XXXXII (196^), 1293-12914..

53* Nelson, M. M.# C. D. Baird, H. V. Wright, and H. M. Evans, "Multiple Congenital Abnormalitiea in the Rat Resulting from Riboflavin Deficiency Induced by the Antimetabolite Galactoflavin," Journal of Nutrition. LVIII (1965), 125-145. '

54* » *nd H. M. Evans, "Relationship of Thiamine to production in the Rat/ Journal of Nutrition, LV (1955), 151-163.

55* « C. D. Baird, end H. M. Evans, "Terato-

fenic ffffeots of Pantothenic Acid Deficiency in the at» Journal of Nutrition. LXII (1957), 3^5-405.

56. Niahiraura. Hideo. Takaahl Tanamura, snd Maaaaki Suzuki, Effects of Thalidomide Administered to Pregnant Mice Upon the Development of Their Offspring," Acta School

P p l T * r ' l t T — 1 H o t o - * 3 £ * m i

ST. Obblnk, H., J4 Kl.ln, and L* K. Dalderup, "Effact of Acetylsalleylie Acid on Postal Mice and Rata." Lancet, I (1961*), 565.

Thalidomide on the Rat Foetus/ Experientla!CXIX°f (1963), 645*

59« Psrups, E. V, and X. Hoffman, "Ansence of Growth Effects of Thalidomide on Higher Plants," Nature. CCV (1965) . 1241.

60. Peterson, C. E. and H. R. Wiedemann, "Thalidomide and Petal Abnormalitiea," Lancet, I (1963), 174.

61. Pfeiffer, R. A . and W. Koaenow, "Thalidomide and Con-genital Abnormalities," Lancet. I (1962), 45-46.

62. Pin sky, L., "The Production of Sfcsleta* Malformalitie a in the Offapring of Pregnant Mice Treated with 6-Aminonicotinamide," Physiological Review, XXXIX (1959), 69-113.

m and Angelo Di George, "Cleft Palate in the Mouae: a Teratogenic Index of Glucocorticoid Potency." Solenoe. CXXXXVII (1965), 402-404.

64. Richardson, C. R. and A. 0. Hogan, "Diet of Mother and Hydrocephalus in Female Rata," Journal of Nutrition, XXXII (191*6), 459-465.

10

65- Shut*, I., "The Relation of Deficiency of Vitamin B to the Anti-Proteolytic Factor Pound in the Serure of the Aborting Woman," Journal of Obstetric® and Gynecology of the BrltlaE~5SpIre~xmHI (193RT

66. Smith, T. E., V. 0. Berndt, P. 0' Leary , and D. Cavanaugh, "The in vitro Inhibitory Effect of Thalidomide on the Chick~Ewfcryo7" -American Zoologist, II (1962), 559.

67* Sadthberg, M., "Teratogenic Effects of some Hypoglycemic Substances in Mice," Anatomical Record, CXXXVI (1960), 280.

68* , Ind H. M. Runner, "Teratogenic Effects of Hypoglycemic Treatment in Inbred Strains of Mice," American Journal of Anatomy, CXIII (1963), 172,

69* Smithella, R. V. tad M. B. Lend, "Thalidomide and Mal-formations in Liverpool," Lancet, I (1962), 1270.

70, 8oners, O. P.. "Thalidomide and Congenital Abnormalities," Lancet, I (1962), 912-913.

71* Speirs, A. L., "Thalidomide and Congenital Abnormalities," Lanoet. I (1962), 303.

72. Spencer, K. B. V., "Thalidomide and Congenital Abnormal-ities," Lanoet, II (1962), 100.

73* Thiersch, J. B., "Effect of 6-Mercaptopurine on Rat Fetus and on Reproduction of Rats," Annals New York Academy of Solence, LX,(1954), 220-227 •

74* Tuehmann-Dupleiss, H. and L. Merceir-Parot, '"Influence of Three Hypoglycemic Sulfamides, Carbutamlde BZ55, Tolbutamide Do60, and Chlorpropamide on the Pregnancy and Prenatal Development of the Rat," Anatomical Record, CXXXVI (I960), 294.

75. , "Oral Antl-ai acetic Druga ana Teratogenicity," Lancet, II (1963), 408.

76. , "Production of Congenital Eye Malformations, Especially in Rat Fetuses," Anatomical Record, CXXXVI (I960), 294.

77. Turbow, M. M., "Teratogenic Effect of Trypan Blue on Rat Embryos Cultivated in vitro," Nature, CCVI (1965), 179.

11

78. Walford, P., "Antibiotics and Congenital Malformations," Lancet, II (1963), 298-2Q9.

79. Warburton, D., D. Tr?>sler, A. Naylor, J. R. Miller, and F. C. Proaer, "Htfalls In Tests for Teratogenicity," Lancet. II 'lG6?)f 1117.

fiO. Warkany, Joseph, "iffe:t of Maternal Rschitogenic Diet on Skeletal Development of Young Rats Reared on a Deficient Diet," American Journal of Diseases of Children. LXVI (1043).

8l« . and R. C. Nelson, "Appearance of Skele-tal Abnormalities in the Offspring of Rats Reared on a Deficient Diet," Science, LXXXXII (1<>40), 3p3-384.

82. , "Congenital Malfor-mations Induced in Wats by Maternal Nutritional Deficiency," Journal of Nutrition, XXIII (1QU2). 321-333.

83* i "Skeletal Abnorroal-ities in the Offspring oi" «ata Reard on Deficient Diets," Anatomical Record. LXXIX (1041), 83-101.

81 . , and b. Schraffenberger, ^congenital nairormations induced in Rats by Maternal Nutritional Deficiency," American Journal of Diseases of Children. L/.IV (1942), 237-244.

85. Wilson, J. G., "Teratogenic Interaction of Minimal Doses of Hypervitaminosis A and Trypan blue in the Rat," Anatomical Record, CXXXXII (1962), 292.

86. Wright, 0. B., "Thalidomide and Foetal Abnormalities," British Medical Journal. VCCLXXXXIV (1962), 1758.

87. Wueat, H. M., IS. B. Sigg, and I. Fratta, "Pharmacological Properties and Teratogenic Action of 2-Hexahydrophthal-imide, Glutsri-nide ana 2-Fhthelimido-N-Methyl Glutar-imide," Life Science. Ill (1961*), 721-724.

88. Yang, T*u S*Uj "Drugs and Congenital Abnormalities," Lancet, I (1963(, 501.

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CHAPTER II

"ATS:*.I ALS and methods

Selection of Mouae Strains

Inbred strains of nice are becoming increasingly popu-

lar as laboratory animals for use in genetic studies. In

order for mice to qualify as an inbred strain, they must be

the descendants of a minimum of twenty generations of inde-

pendent brother to sister matings. Parent to offspring matinps

may be substituted if the parent utilized is always the younger

of the two parents (13). Inbreeding has the effect of pro-

ducing genetic homogeneity; that is, Inbred mice are theoret-

ically homozygous at alnost every locus. Due to their high

degree of genetic homogeneity, mice of a particular strain

will usually exhibit much lass variation in response to a

given treatment than will randomly bred mice. Furthermore,

mloe of • particular strain may respond to a given treatment

in a markedly different manner than mice of seme other

strain* There are many well established inbred strains of

mice, whose responses to various parameters, such as irradia-

tion and contact with different viruses and carcinogens, are

well known. Practically nothing la known concerning the sus-

ceptibility of different mouse strains to teratogenic agenta.

12

13

With respect to the teratogenic effects of vita-nin

deficiencies on inbred mice, Kalter and Warkany (7) found

C57BL/6J mice to fee much ore resistant to a riboflavin-

deficient diet th*n ce of strains 12°/j and A/j. Experi-

ments reported by Praser et (2) indicate that A/J mice

•re very susceptible to niacin antagonists, while Ingalls

et si. (6) reported a difference between strains A/j and

C57BI/6J with respect to susceptibility to hypoxla-induced

teratogenesis.

Several workers have reported teratogenic effects of the

tranquilizer, thalidomide, In inbred strains of mice. Ha^en

(5) found that thalidomide Increased the resorption rate in

strain A/J but not In strain C57BL/6J. Di Paolo (1) reported

resorptions and skeletal deformities In strain A/J mice,

using thalidomide as the teratogenic agent. Glroud £t al.

(It) administered thalidomide to Swiss Albinos and the Black

and A/BC strains of mice and reported deformities In the pro-

geny, but did not report any differences between strains.

Silveatrini and Garau (11) reported nalformltlea in strains

CP} and Swiss upon administration of thalidomide. Again, no

differences between strains ware noted. Various other workers,

including Fratta et al. (3), Seller (10), Somers (lk), and

Woollaa ( 1 5 ) , failed to obtain abnormalities in offspring of

inbred nice administered thalidomide.

With the exception o f Hagen ( 5 ) , none of the above-

mentioned experimenters have reported any differences between

Ik

the Inbred strains utilized with reapect to susceptibility

to the teratogenic effects of thalidomide. Furthermore,

Hagen (5) reported a difference in resorption rate, not in

actual deformities.

Since different inbred strains of mice are known to

exhibit very different reactions to varioua treatments, it

seema feasible that some strains would show different sus-

ceptibilities to teratogenic apents. In order to test this

possibility, four inbred strains were chosen to be tested

for susceptibility to the teratogenic effects of thalidomide*

C57BL/6J and A'J were chosen because the work of Kalter and

Warkany (7) showed these two strains to have different sus-

ceptibilities to the teratogenic effects of a riboflavin-

deficient diet and also because of the results reported by

Hagen (5). 129/J ^ice were chosen for use in this study

beoauae of their high susceptibility to the riboflavin

deficient diet used by Kalter and Warkany (7)> C3HeB/PeJ,

not mentioned above, was chosen for this investigation

because they are phenotypically different from the other

three strains and hate been widely uaed in research.

Freparetion of Thalidomide

Thalidomide, 2, 3 dioxo-piperidyl phthalimide, for

use in this inveatigation was prepared according to the

wethod utilized by Chemie Grunthal In preparing the drug for

pharmaceutical use.

1*

Phthalylglutamic anhydride was prepared,using the

method of King and Kldd (0). Forty grams of phthalic an-

hydride find forty «$rams of L-?lutamlc acid were refluxed

In one hundred thirty mlliUlnars of dry pyridine for two

hours. Dry pyridine we. [repar;d by adding barium oxide

to stock pyridine «nd heating it gently. The clepr solution

was then evaporated under reduced pressure and ninety milli-

liters of acetic anhydride added. The solution was boiled

for three minutes and then concentrated under reduced pres-

sure. Anhydrous ether was added and the phthalylglutamic

anhydride, which precipitated at this point, dried under

reduced pressure.

Two grams of phthalylglutamic anhydride and two grams

of urea were placed in a live hundred milliliter round bot-

tom flask containing one hundred fifty milliliters of xylene.

The mixture was then refluxed for twenty-four hours, an

electric hotplate being used as the heat source. The heat

source was removed froT beneath the flask and the mixture

allowed to stop boiling. The still-hot liquid portion was

then decanted into a two hundred fifty milliliter Erlenmeyer

flask which was placed in an ice bath. Needles of thalido-

mide appeared upon cooling. The melting point of the

crystalline thalidomide was two hundred seventy degrees

Centigrade. This corresponds to the expected melting

point of two hundred seventy degrees Centigrade.

16

Experimental Procedure

Mice of four strains, CS7B1/6J, 12Q/J, A/j, snd C3HeE/PeJ

were caged separated, wit-' npprox4mateiy five females and one

male in each pan. e.-nfilea w»ir« inspected each morning for

the presence of " . r.rinai pi^g. Discovery of the vaginal

plug, a shiny mass o: mucous blocking the female's vagina,

was taken to be evidence of pregnancy. The date of discov-

ery of auch a vaginal flu? was considered to be the first

day of pregnancy, as mice normally breed between ten P. M.

and two A. M. (12)

On the sixth day, the pregnant females were weighed,

their ears punched for identification, and the mice of each

strain divided into eirht groups of approximately equal num-

ber. Three of the groups of mice from each strain were riven

intraperitoneal injections of a 0.5 per cent suspension of

thalidomide in sterile distilled water, at a dosae level of

twenty-five, one hundred, or one hundred fifty milligrams

of thalidomide per kilogrom tody weight. One group from

each strain was injected with sterile distilled water alone,

thus serving as a control group. Three other groups were

given a $ per cent suspension of thalidomide by oral intubation,

Again, the dosage levels were twenty-five, one hundred, or

one hundred fifty milligrams per kilogram body weight. The

final group of mice from each strain was administered dis-

tilled water by oral intubation, thus serving as an additional

17

control group. Intraperitoneal injection was accomplished

by use of e tuberculin syringe end sterile twenty-Fauge

needles. Oral intubation w a pccomplished uainp' a tuberculin

syringe and a blunt el^hteen-gauge needle. Treatments were

administered on the sixth through the *-enth days of pregnancy.

On the eighteenth day, the females were killed by

etheri?at'on and their uteri removed and examined for signs

of pregnancy. Fetuses present were eviscerated and stained

with Alizarin Red bone stain. The stainin? procedure is

given below. After staining, the fetuses were examined for

developmental abnormalities.

Alizarin Red Staining Technique

1. Fix fetuses in 95 per cent ethyl alcohol for

one week.

2. Place in 1 per cent potassium hydroxide for one

day.

3. Wash in tap water three times.

4. Place in 95 par cent ethyl alcohol. Change alcohol

twice. Leave fetuses in alcohol a total of six hours.

5. Place in diethyl ether for one to lour hours, or

until *11 fat is dissolved.

6. Place in <>5 P®r cent ethyl alcohol for one ni»ht.

7. Change the concentration of alcohol gradually

until the fetuses *re in tap water, "ftien add 1 per cent

18

potBsaium hydroxide. The fetuses should be in the potassium

hydroxide, for about on© week.

8. Wash in tap water three times find gradually add

ethyl alcohol until the concentration reaches 70 per cent.

9. Add enough Alizarin Red staining solution to make

the total stain concentration about 1 per cent.

10. When the bones are stained 8 dark red, place the

fetuses in 1 per cent potassium hydroxide.

11. Preserve the fetuses in 70 per cent ethyl alcohol

with a few drops of formalin added.

1. D1 Paolo, Joseph, "^onrenitsl Malformation in Strain A Mice." Journal o 1 the American Radical Associa-tion, C „ . X * V T T T , 13°-1U1.

2. Fraaer, F. C., M . Goldstein, and Merrille Feiner, "Teratogenic Effects of a '"wo Hour Inactivation of Nicotinamide Activity in the Mouse," American Journal of the Diseases of Children, CXIV (1061),

3. Pratta, I. <»>., E. f. Sicg, and K. Maiorena, "Terato-genic Effects of Thalidomide in Rabbits, Rats, Hams-ters, °nd vice,n Toxicology and Arplied fharmacology, VII (1^65), 26fi-2*5T

ij.. Giroud, A., H. Tuchmann-Dupleiss, and L. Merceir-Farot, "Thalidomide end Congenital Abnormalities," Lancet, I (1<>63), 298-299.

5. Hagen, Odette, "Drugs and Congenital Abnormalities," Lancet, I (1963), 501.

6. Ingalls, T. H., F. R. Avis, F. J. Curley, and H. M. Temin, "Genetic Determinants of Hypoxia-Induced Congenital Anomalies," Journal of Heredity, XXXXIV (1953). 185.

7. Ralter, Harold and Joseph Warkany. "Congenital Malfor-mations in Inbred Strains of Mioe Induced by Ribo-flavin Deficient, Galactoflavin-Contalnin^ Diets," Journal of Experimental Zoology, CXXXVI (1957)» 531-553."""

8. King, R. and J. Kidd, "Biochemical Preparations," Journal of the Chemical Society, WMCCCXV (19U9), T J I F :

9. Seller, Mary J., "^alidomide and Congenital Abnor-malities," Lancet, II (1962), 2^9.

10. Silveatrini, b. and A. Garau, "Malformazioni Feteli Frodotte Dalle Talidomide Nel Topo," Bollettino Chimico Farmaceutlco, CIII (196U), B04-81U.

19

20

11. Snell, 0. D., £. Fekette, K. P. Hummel, and L. W. Law, "The Relation of Mating, Ovulation, and the Eatroua Smear in the Houae Mou8e to Time of Day," Anatomical Record, LXXVI (19i*0), 39-5$.

12. , Joan Statts, M. P. Lyon, L. C. Dunn, H. Gruneberg, P.Herwlg, and W. £. Heston, "Standard-ized Nomenclature for Inbred Strains of Mice, Second Listing," Cancer Research, XX (1060), ll|5-l69.

13. Somers, G. F., "Thalidomide and Congenital Abnormalities," Lancet, I (1962), 912-913.

Ill* Woollam, D. H. M., "Thalidomide disaster Considered as an' Experiment in Mammallian Teratology," British Medical Journal, II (1962), 236-237*

CHAPTER I I I

RESULTS

During th« course of this investigation, a total of

three hundred fifty-six pregnant female mice of four strains

were administered either thalidomide or distilled water.

Routes of administration were divided equally between oral

intubation and intraperitoneal injection. Results of admin-

istering thalidomide to two hundred sixty-eight pregnant mice

is presented in Table I. This table shows the number of mice

given each treatment and the number of young born and resorb-

ed in each case. Table II lists the eighty-eight pregnant

females receiving water and indicates the mean number of

young recovered in each of the four strains investigated.

Examination of Table reveals that 86.2 per cent of the

thalidanldo-treated females failed to carry their fetusoa

full terra iind presumably resorted them. In comparison, Table

II shows that only 62.5 per cent of the control group females

resorbed their young. A statistical comparison of each treat*

ment group with its corresponding control is shown in Table

III.

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'"ABLE III

COMPARISON OF PER CENT RESORT TONS DUB TO ADMINISTRATION OF THALIDOMIDE TO POUR STRAINS OF MICE

Strain Thalidomide 3roup

Control Group

Chi-Squared

P

A/J 80.7 50.0 23.29 .001

C3HeB/FeJ 90.2 53.3 33.36 .001

C57BL/6J 85.2 81.1 1.29 .30

129/J 82.8 1*2.8

r 1+1.83 .001

Uae of the Chi-square teat revealed that thalidomide

increaaed the reaorption rate significantly in strains

C3HeE/FeJ, A / j , and 129/J, but not in strain C57&1/6J.

Examination of the fetuses after staining with Alizarin

led revealed various abnormalities in the treated group,

including stunting of growth, retardation of oasification,

shortening of long bones, absence of digits, anophthalmia,

microphthalmia, lateral curvature of the spine, exencephaly,

encephalocoel, hydrocephaly, cleft palate, and hare lip. Ey

far the most widely observed anomaly was general retardation

of development.

2?

The progeny of ••eh of the trtitcd mother mice are

11ated in Appendix A and th«lr abnormalities indicated.

A 11at of th« eontrol group progeny and their deformitiea

la given In Appendix B.

In order to facilitate eoniparlaon of the extent of ab-

normalltlea between the four atraina of mice, eaoh fetua

waa assigned a number between one and ten, with a larger

number corresponding to a greater degree of deformation.

9 » rating syatem uaed in arriving at theae numbera la given

ia Table I?.

The mean deformity aoore for eaoh atrain and within

atraina la ahown In Table Y. Thia table ahowa that atrain

129/J had higher deformity aoorea than the other atraina.

In order to teat the algnlfloanoe of thia difference, the

dlffereneea between atraina were compared statistically,

ualng the "atudent* a t" teat. The difference between the

M a n deformity aoorea for each of the atraina of mice ia

given in Table VI. The atatlatloal comparison indicatea

that atrain 129/J waa significantly more euaceptlble to

the teratogenic effeota of thalidomide than were any of the

other three atraina. the small dlffereneea between strains

C57BI/6J, C3SeB/PeJ"# and A/J were not atatlatieally signifi-

cant.

Table VII ahowa the reaults of an analyaia of variance

In the mean number of mice per litter due to the levels of

thalidomide adminletered. The variance due to dosage level

26

TABLE IV

SCALE FOR RATIHI *ICF ACCORDING TO EXTENT OF DEFORMITY

Condition of Mouse Score ^Iven

Normal . 1

Cleft Palate or Anophthalmia; Otherwise

Normal 2

Small Size but Normal In Other Reapecta 3

Retarded Ossification; Partially Reaorbed; Otherwise Normal U

Reterded Slcull Development; Otherwise Normal 5

Hydrocephalua; Curvature of the Spine;

Normal in Other Reapecta 6

Extreme Swill Size; Normal Ossification 7

Multiple Deformities; Vsrloua Combinetiona

of the Above 8

Extreme Shortening of Bonea; Missing Bones . . . . . 9

Grossly Malformed; Bonea Barely Vialble 10

27

TAFLE V

EFFECT OF THALIDOMIDE AD'THSTRATOK (T MEAN DS'r'OR1*!TY STORES

Strain Treatment Mean Deformity No. Mice tig /kg Score Recovered

C57BL/6J 25 2.3U 26 C57BL/6J 100 3.62 24

1.80 22. Total 2.^8 65

129/J 25 9.10 20 100 5.93 i 29 Igo 1.00 9 Total 6.26 35

C3H®B/P«J 25 1,00 9 100 1.00 17 l£0 2^6 il Total 1.73 k9

VJ 2$ 2.02 50 100 1.00 8 150 kill 27 Total 2.78 85

Orand Total 3.23 277

2fi

TAELE VI

MEAN DIFFERENCES IN AFNORMALITY SCORES IN FOUR STRAINS OF

Strain A/J C3Heh/Pe.T 12 9/J

C57BL/6J .30 .75 3.78*«*

129/J 3.48-tHH* lj.,53 ***

C3HeB/PeJ 1.05

««*lndlc<ites .001 level of significance by t test

is insignificant, indicating that increasing the level of

thalidomide administered from twenty-five milligrams per

kilogram boijr weight to one hundred fifty milligrams per

kilogram body weight had no significant affect on the number

of mioe per litter.

Table VIII shows an analysia of variance in mesh

deformity score due to the level of thalidomide administered.

Differences in thalidomide dosage level did not significantly

effect the variance, indicating that the three levels of

thalidomide were not different with reapect to the extent

to which they caused deformities.

29

TAPLE VII

ANALYSIS OF VARIANCE T'J MKAf! T'^ER OF V10L ISR LIFTER DT,E TC L:-;VSL 0Y HALIDOYIDE

Source 1 « 8utn of Squares Mean Square

Dose Levels 2 8.70 I+.35

Error 9 26.30 2.92

Total 11 35.00

F • • • I.I4.8 p • • . .20

TABLE VIII

ANALYSIS OP VARIANCE IN MEAN DEFORMITY SCORE PER GROUP DUE TO LEVEL OF THALIDOMIDE

Source df Sum of Squares Mean Square

Dose Levels 1

2 2.72 1.36

Error 9 66.38 .. . „ |

7.1*0

Total 11* 69.10

F . . . 0.018 P . . > • • 20

3C

An analysis of variance in mean deformity score due to

route of administration of thalidomide is shovm in Table IX.

Progeny born to nothers with thalidomide ora H y *«d

higher deformity scores than did the progeny of mothers p-iven

thalidomide by intrspsritonesl injection. The level of

significance of this difference is only .20, so that it is

doubtful that there is actually a real difference due to

route of administration. The RB an number of mice per litter

was not significantly effected by route of administration,

Indicating that raaorption rata is independent of adminis-

tration route.

TABLE IX

ANALYSIS OP VARIANCE DUE TO ROUTE OP THALIDOMIDE INJECTION

Souroa df Sun of Squares Mean Square

Routes 1 12.U9 12.1*9

Error 6 26.07 U.35

Total 7 3P.56

P . . . 2.Q8 P , a a * •20

CHAJThR IV

DISCUSSION

The primary mode of action by which thalidomide exerts

Its teratogenic effects la unknown. Several workera have

postulated possible mechanisms for the damage done the

developing fetus by thalidomide, but no explanation has

thus far been brought forward with enough supporting evidence

to warrant widespread acceptance.

7fte thalidomide syndrome in man and laboratory animals

resembles very closely the teratogenic effects of hypovita-

mlnosls B« For this reason, some workers feel that thalido-

mide may act as a vitamin B antagonist (9). T!here is some

evldeno* to support this opinion. Robertson (11*) reported

that adainlatratlon of thalidomide to the human interferes

with vitamin B metabolism la some cases. Kemper (8) , work-

ing with the chick embryo, reported striking aimllaritles

between the deformities obtained with vitamin B deficiencies

and with thalidomide.

More apecifioally, Roath (13) haa reported reaulta

indicating that thalidomide may act aa either a glutamlne

or nicotinic acid antagonist. Williams (15) haa shown that

soara metabolites of thalidomide will inhibit the enzyaw

glutamlne synthetase. Working with the ohiek, Peliaatl (3)

and Boylen (1) reported that glutamlne counteracted the

31

32

teratogenic effect® of thalidomide. Prank et al. (U) reported

reversal of the growth Inhibitory effects of thalidomide on

protozoans upon addition of either nicotinic acid or nicoti-

namide to the growth media.

All of the nbove results: point to the likelihood that

thalidomide exerts ita teratogenic effact by functioning as

either a vitamin 6 antagonist or a glutamine antagonist.

Again, although the evidence Indicates that this may be the

mode of action of thalidomide, this is by no means generally

accepted. The evidence is not sufficiently complete for a

definite conclusion to be reached.

Whatever the mechanism affected by thalidomide, it has

been well established that the drug does cause congenital

malformations. Accumulated evidence does not prove that

thalidomide alone brines on these malformations, otherwise

all Individuals or laboratory animals treated with thalido-

mide ahould react in the same manner, their diets pnd general

nutritional levela feeing equal. Since this is obviously not

the caae, then each treated individual or animal must possess

some mechanism which affects Its susceptibility to the tera-

togenic effects of thalidomide. Refering to the works cited

above* this mechanism might be vitamin B metabolism. What-

ever the mechanism Is, it is very likely under genetic

control (2). Demonstratlem of differences in susceptibility

to the teratogenic effects of thalidomide between genetically

h onto gene ou 11 inbred strains of mice would certainly support

33

tha likelihood that genetics playa • role in determination

Oi* reaponae to teratogenic agents.

The comparison shown in Table III indicates that there

la a difference existlnr between strains C57BL/6J and atraina

129/J# C3HeB/PeJ, and A/J with respect to increase in resorp-

tion rata due to the administration of thalidomide. The

control group mice of strain C57BL/6J had a higher resorption

rata than the other three 8traina. Administration of thali-

domide did not significantly increase the raaorptlon rate

In strain C57BL/6J, while the resorption rates of atraina

129/J, C|HeB/FeJ, and A/J were increased significantly.

lhaae results indicate that C57BL/6J mice are resistant to

the eabryocidal effecta of thalidomide. This la oonsiatent

with the reaulta reported by Hsgen ($), who found C57BL/6J

mloa to ba leas susceptible than A/j mice to increase of

resorption rate due to thalidomide.

Examination of Table VI raveala that strain 129/J mice

ara significantly more ausceptible to the actual terato-

genic affects of thslldomide than are atraina C57BL/6J,

C3HeB/FeJ, and A/j. In thla table the relative suacepti-

bl11tiea are shown aa mean differenoea in abnormality aeorea.

There era email dlfferencea in susceptibility between strains

C3HeB/PeJ# A/J, and C57BL/6J. These differences are too small

to ba statlatlcally significant and could be due to chance.

In this studyt varying the doaaga level by a faotor of

aix had no significant effect on either the embryoeidal or

3U

teratogenic effect® of thalidomide. This agrees with ^he

results obtained by MoCafferty et al. (12), who found no

differences in percentage of deformities in mice treated

with different dosage levels of thalidomide. MacKenrie and

McGrsth (10) rerorted that, in the rat, the amount of thal-

idomide absorbed is independent of the dosage level adminis-

tered. This would naturally rule out any dosage effects, as

long as the level is above the minimum necessary. Whether

limited absorption ability in the mouse Is the reason that

dotage level does not effect the embryopathic activity of

thalidomide has not been established.

In this investigation, route of administration of tha-

lidomide had no significant effect on the mean number of

mica par litter. The mice given the drug by oral Intubation

did give birth to progeny with higher mean deformity scores

than did the group to which the thalidomide was administered

lntraperltoneally. This difference in mean deformity scores

waa tested statistically, and waa of doubtful significance,

the probability lying between the .1 end .2. Additional

study, with larger number of mica, would be necessary to

determine if there ia a real effect in mean deformity score

due to the route of administration of thalidomide*

The results obtained in this study lead to the conclu-

aion that differences in susceptibility to thalidomide exist

between certain of the four strains of mice utilized, and

35

that susceptibility to embryocldal effects, reflected by

resorption rate, and to teratogenic effects, reflected by

abnormality scores, do not necessarily coincide. For example,

strain C3HeB/PeJ, which was susceptible to the increase in

resorption rate due to thalidomide, was least susceptible to

the teratogenic effects. On the other hand, mice of strain

129/J were highly susceptible to both effects of the drug.

The fact that susceptibility to teratogenic effects and

embryocldal effects do not felwaya coincide indicates that thee

genetic mechanism involved in resorption might differ from

the one Involved in teratogenesis.

Obviously, there is much to be learned concerning gene-

tic determinants in susceptibility to teratogenesis. At the

beginning of this thesis, it was hypothesized that the pro-

gram now being utilized by the Pood and Drug Administration

to screen new drugs for teratogenic effects is inadequate

and that one of the program,1* chief weak points 1s its fail-

ure to consider the effect of test animals• genetic back-

ground cm susceptibility to teratogenesis. There is a

definite need for more knowledge In this area. Inbred mice,

due to their genetic homogeneity, are ideal animals for

screening of possible teratogenic drugs. New drugs could

be tested for teratogenic effect using more than one strain

of miee. Naturally, if strains of mice were ohosen for

testing that ere known to be susceptible to teratogenic

agents, then the tests would be more senaitibe than if

realatant strains were chosen. This is assuming, of course,

36

that there are strains of mice which are hypersusceptible

to teratogenic agents In general. It is equally feasible

to postulate that different strains of mice will exhibit

different susceptlbi1'ties to different drugs, so that a

battery of mice strains, each suscept'ble to a particular

type of teratogenic agent, would have to be used In order

for such a testing program to be meaningful.

Kalter and Warkany f?) found that mice of strain

C57EL/6J were more resistant to the teratogenic effects

of a rlboflavln-deficlent diet than mice of strain A/j.

they then found that mice of strain C57BL/6J utilized

riboflavin more efficiently than those of strain A/j, SO

that the hypersusceptiblllty to a riboflavin deficient

diet's teratogenic effects waa probably due, In strain A/J,

to simple inability to utilize riboflavin efficiently.

Assuming that thalidomide exerta Its teratogenic

effeota by some mechanism akin to vitamin E antagonism, it

would be of value if strains of mice susoeptlble to thali-

domide teratogeneals, such aa 129/J, were tested for errors

in vitamin B metabolism. If It oould be establlahed that

the thalidomide-auaceptlble mice had faulty vitamin B

metabolism whereas resistant mice did not, then this would • *

serve as evidence that thalidomide functions aa a vitamin

B antagonist. The reverae experiment could alao be run,

In which miee known to have faulty metabolism of a parti-

cular vitamin, such as one of the B group, would be tested

37

This would not have to Involve a random choice of drug and

vitamins, as structural similarities between suspected

teratogens and vitamins coQld be noted. For example,

Pelisati (3) noted a similarity between riboflavin and

thalidomide. Riboflavin deficiency was subsequently found

to aceentuate the teratogenic effects of thalidomide, in-

dicating that thalidomide might function as a riboflavin,

vitamin Bg, antagonist. Metabolic studies failed to yield

evidence of any riboflavin antagonism by thalidomide.

Although these results were negative, this is an example of

the type of work whieh can be done in the study of terato-

gen! city.

Another worthwhile project would be to test several of

tha more videly used inbred strains of mice for susceptibility

to various teratogenic agents. Again, teratogenic effects

of diets deficient in the different vitamins could be con-

sidered* When enough is learned concerning the relative

sensitivities of different mouse strains to teratogens, then

much can be eluoldatod about the actual causes of teratogenic

•ffeota and the Inheritance of ausceptibllity to these ter-

atogenic effeots. To use the results obtslned in this study

aa an example, strains 129/J and C39eB/FeJ could be crossed.

Since 129/J mice are susceptible to the teratogenic aotion

of thalidomide, while C3HeB/FeJ mice are relatively resiatant,

and alnoe inbred mice are homozygoua at almost all loot,

than the progeny would presumably be heterozygous for whstever

38

genes control susceptibility to thalidomide teratoe-eneais.

The progeny could then t.•? *estsd for susceptibility *o the

teratogenic effects of

Assuminr th«t sus-ep t i t ill ty or resistance is determined

by a single t ene pair, if thf proeeny exhibited susceptibility

to thalidomide, then the f-ene causln* susceptibility would be

dominant to the allele causing resistance to the teratorenic

effects. Conversely, if the proeeny proved to be resistant

to the teratogenic effects, then the allele causing suscep-

tibility would be assumed to be recessive, if some of the

progeny were resistant and some susceptible, then the inheri-

tence of susceptibility to thalidomide teratogenesis would

probably be due to a dominant gene with incomplete penetrance.

Of course, the susceptibility to thalidomide terato-

genesis could be controlled by several gene pairs. If these

were additive genes, then the progeny would probaily be inter-

mediate between the parents. By backcrossing the hybrids, it

would be possible to estimate, statistically, the number of

genes Involved. It soon becomes obvious that studies con-

cerned with the inheritance of susceptibility or resistance

to teratogens will require large numbers of mice if they are

to be meaningful.

Another study suggested ty the results of this investi-

gation Involves the embryocidal rather than the teratogenic

action of thalidomide. It has been reported that the

39

chief cause of apparent resorptions in the armadillo is

damare to the preimplantation blastocyst (11). It could

be hypothesized that resistance to the erabryocidal effects of

thalidomide is due to early Implantation of the developing

blastocyst on the uterine wall. If it could be shown that

implantation in strains resistant to the embryocidal effects

of thalidomide, C57BL/6J for example, occurs earlier than in

more susceptible strains of mice, then this would indicate

that resistance to the embryocidal effects of thalidomide

could be due to early Implantation. This would be an example

of an apparent plelotropic effect; the genetic mechanism

controlling time of implantation of the uterine wall thus

controlling susceptibility or resistance to the increase of

resorption rate induced by thalidomide. It should be empha-

sised that this is merely a proposal of a study which could

be conducted. No evidence exists, at present, that time of

lmplanatlon has any effect on relative susceptibility of

different Inbred mice strains to thalidomide.

At this time, too little is known concerning the res-

ponses of sny of the mouse strains to any teratogenic agent.

Homburger et al. (6) have reported differences in suscepti-

bility to thalidomide teratogenesis between inbred strains

of hamsters; however, hamsters sre not used widely in

research as are inbred mice. For this reason, it seems prac-

tical that further study should be done concerning the effects

of various teratogens on different inbred mouse strains.

CHAPTER BIBLIOGRAPHY

1. BoyIon, J., H. K. Horn®, and W. J. Johnson, "Teratogenic Effects of 'ihalidomide and its Metabolites on the Developing Chick iimbryo," Caned 1 an Journa I of Biochemistry, XXXXII (196Jj.J, 55'2-553."

2. Carpener, Norman, "Teratogenssia," Lancet» II (1^63), 885«

3. Felisati, Dino. Wrperatogenio Action of Thalidomide," Lancet, II (I'M), 72^-725.

I±, frank, Oscar, H. baker, H. Ziffer, and C. M. Leevy, "Metabolic Lesions Induced by Thalidomide," Journal of Laboratory and Clinical Medicine, LX (1962), 89.

5. Hagen, Odette. "Drugs end Congenital Abnormalities," Lancet, I *1963), 501.

6. Horoburger, P., S. Chaube, M. Eppenberger, P. D. Bog-donoff, and C. W. Nixon, "Susceptibility of Certain Inbred Strains of Hamsters to Teratogenic Effects of Thalidomide," Toxicology and Applied Pharmacology* VII (1965), 686-693.

7. ICalter, Harold and Joseph Warkany, "Congenital Malfor-mations in Inbred Strains of Mice Induced by Ribo-flavin Deficient. GalactoflavIn-Containing Diets," Journal of Experimental Zoology, CXXXVI (1957)*

8. Kemper, Von Fritz, "Thalidamid und Entwicklung von Hunerembryoen, ArzneimitteIforachung, XII (1962), 836. ~

9. Leek, Ian °nd E. M. Millar, "Incidenoe of Malformations Since the Introduction of Thalidomide," British Medical Journal, II (1962), 16-20.

10, MacKensie, C. and W. R, McGrath, "Absorption of Thalidomide in the Rat," Proceedings of the Society of Experimental Biology and Medicine,"7lTTl%?), 511-515.

11. Marin-Padllla, Miguel and Kurt Benirschke, "Thalldomide-Induced Alterations in the Blastocyst and Flaoemta of the Armadillo, 'Dasypua novemcinctus mexiconos* including a Choriocarcinoma," American Journal of Pathology. XXXXIII (1963), 999-1009.

kl

12. MeCafferty, R. E., M. L. Wood, *nd W. H. Knisely, "Morphological and Physiological Effects of Thali-domide and Trypan Elue on Uteri and Concepti of Gravid Mice," American Journal of Obstetrics and Gynecology. LXXXU ( 1 % M , ^U-2gQ~

13. Roath, S., M. Elves, end M. C. G. Israels, "Effects of Thalidomide and its Derivatives on Human Leucocytes Cultured in vitro," Lancet, I (1°63), 2^9-2$0.

14. Robertson, W. P., "Thalidomide and Vitamin B Deficiency,' British Medical Journal, I (1962), 792-7^3.

15. Williams, R. T., "Teratogenic Effects of Thalidomide and Related Substances," Lancet, II (1963), 723-721+.

APPENDIX A

ABNORMALITIES OBSKHVrr TV wIOE TRSATED WITH THALIDOMIDE

Abnormality Symbol Uaed

HOD* A

Cleft Palate B

Anophthalmia C

Reduced Site D

Retarded Oaaification E

Partial Reaorption F

Underdeveloped Cranium G

Hydrocephaly H

Curvature of the Spine I

Shortened Bonea J

Kxtreae Reduced Size K

Mlsalng Bones L

1+3

Strain Littar No. Mousa !9o. Abnormalities Obsorvad

C3H«B/P«J C3HeB/P«J C3ReB/P®J C3HaB/F*J C3HeB/P«J C3HeB/P»J C3HeB/PeJ 2 C3HaB/PaJ 2 C3H©B/F»J 2 C3HaB/P»J 2 C3H«B/P«J 2 C3HaB/PaJ 2 C3H«B/P«J 2 C3H»B/P»J 3 C3H«B/P»J 3 C3H«B/P«J 3 C3HaB/PaJ 3 C3HeB/F«J 3 C3HaB/PaJ 3 C3HtB/P»J 3 C3HtB/FaJ 3 C3HaB/PaJ 3 C3H«B/P«J k C3H«B/P«J If C3HaB/PaJ 1+ C3H«B/F»J if C3HaB/PaJ ^ C3H6B/P«J k C3H«B/P«J if C3HaB/PaJ 4 C3H«B/P»J '+ C3H»B/P» J it C3B»B/P»J ^ C3H«B/PeJ 5 C3H»B/PeJ 5 C3H»B/P»J 5 C3H»B/P«J 5 C3H«B/F«J 5 C3HaB/PaJ 5 C3EaB/PaJ 6 C3B«B/P*J 6 C3H«B/P»J 6 C3H«B/P«J 6 C3H«B/P«J 6 C3H«B/F»J 6 C3HaB/PaJ 6 C3H#B/P«J 6 C3HaB/PaJ 6 C3HaB/PaJ 6

1 2 3

't 6 1 2 3 if 5 6 7 1 2 3

if 6

1 9 1 2 3

I 6

1 9 10 11 1 2

1 6 1 2 3

I 6

9 10

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A h K K X K K A* A A A A A A A A A

1*4

Strain Litter No, Mouse No. Abnormalities Observed

129/ J 129/J 129/ J 129/J

ISA m 189/J J?9/J 129/J 129/J 129/J 129/J

HI; 129/J ^9/J R9/J [9/J h / j J9/J i9 /J

189/J

9/J

$

M S ;

19/J N / j ? / /

m

1 J,L

C H, J,L ** L

k D,L

5 D,L 2 l D,H,L 2 2 D,L 2 3 D,L 2 U D,L 2 5 D,L 2 6 D,H,L 2 7 S,H,L 3 1 D,H,L 3 2 D,L

k 1 D,H,L

k 2 D,H,L

k 3 D,H,L

k D,H,L

5 1 D,H,L

5 2 D,L

5 3 D,L 5 4 D,H,L

5 5 D,H,L 6 l D,H,K 6 2 D,H,K 6 3 D #H,K

7 1 A 7 2 A

7 3 A 7 A

7 A

7 6 A

7 7

7 e

A A

7 o / A

6 1 D,L 8 2 D,L 8 3 D,L 8 4 D,L 8 5 D,I,L 8 6 D,L 8 7 D,L 8 e D,L

kS

S t r a i n L i t t e r No. Mouse No. A b n o r m a l i t i e s Observed

129/J 9 1 D,L 1 2 9 / j 9 2 D,L 129/J 9 J D, J ,H,L 129/J 9 k Da JjHgXJ 129/J 9 5 D,L 12<VJ 9 6 D,L 129/J 10 1 A 129/J 10 2 A 129/J 10 3 A 129/J 10 k A 129/J 10 5 A 129/J 10 6 A 129/J 10 7 A 129/J 11 1 A 129/J 11 2 A 129/J 11 3 A

C57BL/6J C57BL/6J C57BL/6J C57BL/6J CS7BL/6J CS7BL/6J C57BI./6J

j 7 8 L / 6 J S7BL/6J

U / 6 J J L / f J

C ^ 7 B t / 6 j C57BL/6J C57BL/6J C57BI /6 J C5TBL/6J CgTBi./6J CS7BL/6J

f i t 6 J L / 6 J L / 6 J

1 1 A 1 2 A 1 3 A 1 k A 1 5 A 1 6 A 2 1 A 2 2 A 2 3 A 2 A 2 5 A 2 6 A 3 1 A 3 2 A 3 3 A 3 4 A 3 5 A 3 6 A 3 7 A 3 8 A 3 9 A 3 10 A

1*6

S t r a i n Litter No. WT ouae No. Abnomal lties Observed

C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57Bt/6j C57BL/6J CS7BL/6J C57BL/6J C57BL/6J C57?l/6J GS791/6J C|75t/6J C5TBI/6J C§7BI/6J <*5fBL/6J C57BL/6J C57BL/6J C5TPL/6J CSTet/6j

J# CS7B1./6J C§7fcl/6J (557BL/6J C579L/6J

if C57Bt/6j C57BL/6J C57BL/6J C57BL/6J C57BL/6J

§?P& C57PI/6J $ £ & C57BL/6J C57BL/6J C57BI/6J

W

U 1+ k k k I 5 5 5 5 5 5 6 6 6 6 6 6 6 6

f

I 8 8 8 8 8 8 8 8 9 9 9 9 9

1 2 3

i 6 7 1 2

A A C A A A A A A A A A C A A A A A A A A A A A A A A

G 0 a

47

Strain Litter No. Mouse No. Abnormalities Observed

C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J CS7BL/6J C57BL/6J G$7BL/6J CS7BL/6J C57BL/6J" C57BL/6J C57BL/6J CS7BL/6J C57Bi/6J

9 6 G, J 9 7

i G 9 P G 9 9 G 10 1 D,.T 10 2 D 10 3 D,G 1C k

**

> D

10 k **

> A 11 1 ? 11 2 G, J, K 11 G,J,K 12 1 K 12 2 K 12 3 G,K 12 k K 12 $ G,K 12 6 G,K 12 7 G,K 12 a G,K

A/J A/J A/J A/J

A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J $

A/J

1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3

it if

it

1 2 3

1 6 ?

1 2 3

6 7 8 1 2 1 2 3

A A A A A E A B B A A A A A A A

4?

Strain Litter Ncu Mt^sa No* Abnormalities Observed

A/J I4. o A A/J k 7 A A/J J ft A A/J i* 9 A A / J £ 10 A A/J 5 1 S A/J 5 2 A A/J 5 3 A A/J 5 4 A A/J 5 5 E A/J 5 6 E A/J 5 7 A A/J 5 8 A A/J 5 9 S A/J 6 1 a A/J 6 2 E A/J 6 3 A A/J 6 4 A A/J 6 5 S A/J 6 6 K,3 A/J 7 1 S,K A/J 7 2 E,H,K A/J 7 3 E,K A/J Y k. E,K A/J 7 5 E,K A/J 7 6 E,K A/J 7 7 E,K A/J a 1 A A/J 8 2 A A/J 3 3 A A/J 3 k A A/J 3 $ A A/J <3 . 6 A A/J 9 1 E,I,K A/J 9 2 E,K A/J 9 3 E,K A/J 9 I E,H,I A/J 9 5 P A/J 10 1 B kft 10 2 A A/J 10 3 A A/J 10 h A A/J 10 5 A A/J 10 6 D,E

+9

Strain Litter No. Mouse Nc. Abnormalities Observed

A/j 1 A A/J 11 2 A A/J 11 3 A A/J 11 4 A A/J 11 5 A A/J 11 6 A A/j 11 7 A A/J 11 s A A/J 11 9 A A/J 11 10 D A/J 11 11 D A/J 12 1 D A/J 12 2 D A/J 12 3 D A/J 12 fc D A/J 12 5 D A/J 12 6 D A/J 12 7 D

AFP3NDIX h

ABNORMALITIES •VhSSRVEO T?3 COKTHOL MICE

Strain Litter No, Mouse Ho. Abnormalities Observed

129/J 129/j 129/J 129/J 129/j 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129/J 129^J

1 1 A 1 2 A 1 3 A 2 1 A 2 2 A 2 3 A 2 k A 2 5 A 2 6 A 3 1 A 3 2 A 3 3 A 3 1+ A 3 5 A k 1 A k 2 A 5 1 5 2 5 3 5 4 5 5 6 5 7 5 8 5 9 6 1 6 2 7 1 7 2 7 3 8 1 8 2 8 3 8 8 8 6 8 7

$0

51

Strain Litter No. Mouse Ho. Abnormalities Observed

A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A/J A / J A/J A/J A/J

A/J A/J A// A/J A/J A/J A/J A/J A / J A/J A/J A / J A/J A/J A/J A/J A/J A / J A/J A/J

1 1 B 1 2 A 1 3 A 2 1 C,D 2 2 C,D 2 3 C,D 2 5 C,D 2 5 C,D 3 1 P 3 2 * 3 3 A 3 if A 3 A 3 6 A 3 1 A 3 B A 3 9 A it 1 A i* 2 A 4 3 A U if A

5 o

A 5 o A 7 A

If 8 A 9 A

5 1 A 5 2 A 5 3 A 5 k A 5 $ A 5 6 A 5 7 A 6 1 A 6 2 A 6 3 A 6 A 6 5 A 6 6 A 6 7 A 6 a A 6 9 A

?.

Strain Litter No. w>!se No. Abnormalities Observed

A / J 7 1 A A/J 7 £ A A / J 7 3 A A / J 7 & A A / J 7 5 A A / J 6 A A / J

8 1 A

A / J 8 2 A A / J 8 3

* J H

A / J 8 U A A / J 8 5 A A / J 8 6 A A / J 8 7 A A / J 8 8 A a/J 9 1 A A / J 9 2 A A / J 9 3 A A / J 9 $ A A / J 9 5 A A / J 9 6 A A / J 1 0 1 E A / J 1 0 2 A A / J 1 0 3 A A / J 1 0 k A A / J 1 0 5 A A / J 1 0 6 A A / J 1 0 7 A A / J 1 0 8 A A / J 1 1 1 A A / J 1 1 2 A A / J 1 1 3 A A / J 1 1 K A A / J 1 1 5 A A / J 1 1 6 A A / J 1 1 7 A

C3H«B/P«J 1 1 E C#H*B/FW 1 2 A C3H«B/P#J 1 3 A C3H«B/PeJ 1 4 A C3H«B/PeJ 1 5 A C3H«B/P«J 2 1 A C3H»B/P»J 2 2 A C3H«B/P«J 2 3 A C3B«B/P«J Z k A

Stra in L i t t e r No. Mouse No. Abnormalities Observed

A <3 A

A

C3H«B/PeJ 2 5 C3HeB/PeJ 2 6 C3HeB/FeJ 2 7 C3HeB/P«J 2 6 A C3HeB/PeJ 3 l A C3HeB/PeJ 3 2 a G3HeB/PeJ 3 3 a C3HeB/PeJ 3 k A C3H«B/PeJ 3 5 a C3H«B/PeJ 3 6 A C3HeB/P»J 3 7 a C3HeB/Pej 3 B A C3HeB/FeJ 4 1 A C3H«B/Pe J k 2 A C3HeB/PeJ 4 3 A C3HeB/P«J U k A C3H»B/PeJ k 5 a C3HeB/PeJ k 6 a C3HeB/PeJ 1+ 7 V C3HeB/PeJ 4 * J C3HeB/P*J I* 9 a C3HeB/p«j 5 1 a C3H«B/P«J 5 2 a C3H»B/FeJ 5 3 a C3HeB/PeJ 5 k a C3H«B/PeJ 5 5 a C3H«B/PeJ 5 6 a C3HeB/PfJ 5 7 A C3HeB/PeJ 5 6 A C3HeB/PeJ 5 9 J C3H«B/PeJ 5 10 ? C3H«B/F*J 5 11 A C3H«B/P*J 6 1 A C3HeB/P»J 6 2 a C3HeB/PeJ 6 3 A C33«B/PeJ 6 L 7 C3HeB/PeJ 6 5 A C3HeB/PeJ 6 6 . A C3HeB/Pej 6 7 V C3HeB/p*j 6 8 * C3HeB/Pej 6 9 A C3HeB/pej 7 x * C3HeB/PeJ 7 2 a C3HeB/P»J 7 3 A C3H»B/Pej 7 J

S t r a i n L i t t e r No. Mouse Mo. Abnormal i t ies Observed

C3KeB/PaJ 7 <5 A OH®b/PeJ 7 6 A C3H«B/PeJ 7 7 A C3HeB/P®J 7 p A

C5>7BL/6 J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C$7BL/6J C57BL/6J C57BL/6J C57BL/6 J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57Bl# t J C57BL/6J C57BL/6J C57BL/6J C57BL/6J CJ>7BL/6J

1 1 1 1 1 ?

2 2 2 2 2

! 5 5 5 5 5 5 5 6 6 6 6 6

1 2 3 *

1 2 3

1 6 7 1 2

i 6

1 2

1 6 1 2

I 6

c A A A A A A A A A A A C C A A A A A A A A

1 2 3 *

It y>

Strain Litter No. mOU3« NC . Abnormalitiea Observed

C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C573L/6J

1 ? 3 I* 5 6 I

A A A A A A A A

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