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Genetic control of the teratogenic response to acetazolamide in mice Biddle, Frederick Gordon 1973

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GENETIC CONTROL OF THE TERATOGENIC RESPONSE TO ACETAZOLAMIDE IN MICE by FREDERICK GORDON BIDDLE B.Sc, University of Windsor, 1966 M.Sc, University of Windsor, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Field of Genetics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1973 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission for e xtensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Medical Genetics The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date February 10, 1973-ABSTRACT Acetazolamide i s a potent carbonic anhydrase i n h i b i t o r and a known teratogen. In mice, i t Induces a s p e c i f i c postaxial ectrodactyly of the r i g h t forelimb; occasionally the l e f t forelimb i s also a f f e c t e d . No other malformations are produced by this drug except f e t a l death with high doses. The SWV s t r a i n of mice was found to be r e s i s t a n t to any teratogenic or embryotoxic action of acetazolamide. Very high doses resulted i n death of the SWV dam. The teratogenic action and dose-response r e l a t i o n s h i p of acetazolamide were documented f o r the sensitive CBA/J s t r a i n . Mid-day 10 was found to be the embryo sensitive stage f o r the induction of forelimb ectrodactyly. With an appropriate acetazolamide dosage regime of two inj e c t i o n s on day 10 of pregnancy, a genetic analysis was made of the teratogenic s e n s i t i v i t y of the CBA/J s t r a i n and resistance of the SWV s t r a i n . Reciprocal crosses between the two demonstrated that resistance was dominant to s e n s i t i v i t y . No affected SWV.CBA hybrids were found i n SWV dams but a low frequency of ectrodactyly was found among CBA.SWV F 1 hybrids i n CBA/J dams. This d i s p a r i t y may be due to small sample size of the treated SWV.CBA F^ hybrids and was not considered to be a d i f f e r e n t i a l influence of the dams. A backcross program was set up using the CBA/J dam throughout. Both a biometrlcal analysis and an examination of the mean frequencies of ectrodactyly i n each generation suggested that three independently segregating l o c i c ontrolled the t r a i t of embryonic s e n s i t i v i t y to the teratogenic action of acetazolamlde. From th i s model, the predicted BC^ generation mean frequency of ectrodactyly was achieved i n a further breeding program. Also, the CBA/J inbred s t r a i n frequency of ectrodactyly was recovered with two BG2 s i r e s and t h i s suggested that the CBA/J genotype had been recovered. I t was found that induction of ectrodactyly and f e t a l death were two separate actions of acetazolamlde. The embryo transfer technique was used i n a preliminary i n v e s t i g a t i o n of the CBA/J and SWV s t r a i n responses to acetazolamlde. I t was demonstrated that the teratogenic environment does e x i s t i n the SWV dam a f t e r acetazolamide administration and that resistance of thi s s t r a i n i s a property of the embryo. The s e n s i t i v i t y and resistance of the CBA/J and SWV st r a i n s remained with dlchlorphenamide, another ectrodactyly inducing carbonic anhydrase I n h i b i t o r . I t i s suggested that SWV may have a general resistance to this .class of teratogens. The r e s u l t s of t h i s study are discussed i n the l i g h t of other investigations of the mechanism of acetazolamide teratogenesls as well as i n the context of other studies of genetic v a r i a t i o n i n teratogenic responses. i l l TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES v i i LIST OF FIGURES ix LIST OF APPENDICES x ACKNOWLEDGMENTS xi Chapter I. INTRODUCTION 1 I I . GENERAL MATERIALS AND METHODS 10 1. Mice: source and maintainance 10 2 . Time of pregnancy 11 3. Source of teratogenic carbonic anhydrase inhibitors 11 III. TERATOGENIC ACTION OF ACETAZOLAMIDE IN CBA/J AND SWV 12 Materials and Methods 13 1. Acetazolamide administration 13 2 . Examination of fe t a l malformations 13 Results and Discussion , 14 1. Preliminary study of the time of action of acetazolamide in CBA/J and SWV 14 2 . Dose-response of CBA/J and SWV to acetazolamide administered at 10 A.M. and k P.M. on day 10 of pregnancy 17 3. C r i t i c a l time of action of a single intraperitoneal injection of acetazolamlde in CBA/J 20 iv 4 . Range and d i s t r i b u t i o n of d i g i t a l anomalies found i n CBA/J 2 4 Conclusion 25 IV. RESPONSE TO ACETAZOLAMIDE OP RECIPROCAL HYBRID EMBRYOS FROM CROSSES BETWEEN CBA/J AND SWV 26 Materials and Methods 26 Results and Discussion 26 V. ANALYSIS OF THE GENETIC CONTROL OF EMBRYONIC SENSITIVITY TO THE TERATOGENIC ACTION OF ACETAZOLAMIDE 29 Materials and Methods 29 1. Breeding program 29 2 . Administration of acetazolamide and f e t a l examination 31 3 . Genetic analysis of breeding data 31 Results and Discussion 3 3 1. Biometrical estimation of the number of l o c i involved with acetazolamide teratogenic s e n s i t i v i t y 3 3 2 . Estimation of the number of l o c i using the generation mean frequencies of ectrodactyly 40 Conclusion 45 VI. TEST OF THE THREE-LOCUS MODEL 46 Materials and Methods 46 1. Breeding program and teratogenic t e s t i n g 46 Results and Discussion 46 Conclusion 55 VII. EMBRYO TRA.NSFER EXPERIMENTS TO INVESTIGATE THE ACETAZOLAMIDE RESPONSE OF THE CBA/J AND SWV STRAINS 56 v Materials and Methods 5 7 1. Embryo transfer procedure 57 2 . Developmental staging of transferred embryos 5 7 3 . Administration of acetazolamide to dams a f t e r embryo transfer 5 8 Results and Discussion 5 8 VIII. RESPONSE OF THE CBA/J AND SWV STRAINS TO DICHLORPHENAMIDE 6 3 Materials and Methods 64 1. Preparation of dichlorphenamide solution f o r i n j e c t i o n 6 4 2 . Administration of dichlorphenamide and f e t a l examination 64 Results and Discussion 6 4 IX. GENERAL DISCUSSION 6 7 1. S t r a i n v a r i a t i o n s i n teratogenic responses 67 2„ Genetic model f o r acetazolamide-induced ectrodactyly 73 3 . Biochemical basis f o r s t r a i n differences i n response to acetazolamide 79 X. SUMMARY 82 BIBLIOGRAPHY 85 APPENDICES 89 v i LIST OP TABLES Table Page 1. Preliminary Study of Time of Action of Acetazolamide i n CBA/J and SWV 15 2. Dose E f f e c t of Acetazolamide. i n CBA/J and SWV Administered at 10 A.M. and 4 P.M. on Day 10 18 3. Time of Action of a Single Intraperitoneal I n j e c t i o n of Acetazolamide i n CBA/J 21 4. Types and D i s t r i b u t i o n of Acetazolamide-Induced Ectrodactyly Found i n the Time of Action Study with CBA/J Using a Single Intraperitoneal Injection 23 5. E f f e c t of Acetazolamide on Reciprocal F 1 Hybrids between CBA/J and SWV 28 6. CBA/J Progeny Data from CBA/J Males Mated to CBA/J Females 34 7. F]_ Progeny Data from SWV Males Mated to CBA/J Females 35 8. BCi Progeny Data from F^ Males Mated to CBA/J Females 36 9. BC2 Progeny Data from BC^ Males Mated to CBA/J Females 37 10. Means and Variances of CBA/J, SWV, Fj. and BC^ Sires Scores Using the Weighted Arcsine Transformation and the Calculation of the Number of Loci C o n t r o l l i n g the Response to Acetazolamide 41 11. P r o b a b i l i t y that a Fetus i s Genotypically l i k e CBA/J i n the Backcross Study Considering 1, 2, 3 and 4 Independently Segregating Loci 43 12. BC3 Progeny Data from BC 2 Males Mated to CBA/J Females 48 13. Theoretical and Observed Generation Means f o r Ectrodactyly Based on Three-Locus Model i n Backcross Study with CBA/J Dams 51 v i l 1 4 . Development of Embryos In Normal Matlngs and Embryo Transfers Determined by Somite Number 59 1 5 . Summary of Embryo Transfers with CBA/J and SWV Strains and Treatment with Acetazolamide 61 1 6 . E f f e c t of Dichlorphenamide on CBA/J and SWV 66 1 7 . Cortisone-Induced C l e f t Palate i n Progeny of > Backcross Matings with the A and C 5 7 B L Strains 7 1 v l l i LIST OF FIGURES Figure Page 1. Percent ectrodactyly Induced i n CBA/J by acetazolamide administered at d i f f e r e n t gestational times and 500 mg/kg per Injection 16 2. Dose e f f e c t of acetazolamlde In CBA/J and SWV administered at 10 A.M. and 4 P.M. on day 10 19 3. Time of action of a single i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide i n CBA/J 22 4. ' Mating scheme to investigate the genetics of the s e n s i t i v i t y to the teratogenic action of acetazolamide 30 5. Means and d i s t r i b u t i o n s of the percent ectrodactyly scores of the CBA/J, SWV, F« and BC-L s i r e s 39 6. Comparison of the observed mean percent ectrodactyly of the f e t a l generations with the p r o b a b i l i t y that a generation i s genotypically l i k e the CBA/J s t r a i n f o r 1, 2, 3 and 4 l o c i 44 7. Means and percent ectrodactyly scores of the CBA/J, SWV, F i , BCi and BC2 s i r e s plotted against the p r o b a b i l i t y that the f e t a l generation i s genotypically l i k e the CBA/J s t r a i n based on the 3-locus model 49 8. Means and d i s t r i b u t i o n s of the mean weighted arcsine scores of the CBA/J, SWV, F^, BC-j_ and BC 2 s i r e s plotted against the p r o b a b i l i t y that the f e t a l generation i s genotypically l i k e the CBA/J s t r a i n based on the 3-locus model 50 i x LIST OP APPENDICES Appendix p a g e I. Distribution of Acetazolamide-Induced Digital Anomalies in the CBA/J Strain 89 x ACKNOWLEDGMENTS I would l i k e to thank the following members of my thesis committee at the University of B r i t i s h Columbia f o r t h e i r support and guidance during the course of t h i s thesis project: Dr. J. R. M i l l e r , D i v i s i o n of Medical Genetics; Dr. C. W. Roberts, Department of Poultry Science; Dr. H. F. St i c h , Cancer Research Centre. I would l i k e to thank my fellow student, Alan C. Peterson, f o r the many hours that we spent discussing and using the preimplantatlon mouse embryo techniques f o r various studies i n genetics and teratology and f o r the opportunity to use these techniques i n the preliminary i n v e s t i g a t i o n of the s t r a i n v a r i a t i o n s i n response to acetazolamide. I thank the following people, without whose cooperation, t h i s thesis would not be complete: (1) Dr. W. M. Layton, Department of Anatomy-Cytology, Dartmouth Medical School, Hanover, New Hampshire, f o r his many long l e t t e r s of enthusiastic discussion concerning the teratology of acetazolamide and f o r making a v a i l a b l e to me several of his unpublished studies; (2) Dr. K. Takano, Senior S c i e n t i s t , Takeda Chemical Industries Ltd., Takatsuki, Japan, f o r his discussions during the early stages of t h i s thesis while he was a v i s i t i n g s c i e n t i s t with Dr. M i l l e r ; (3) Dr. M. C. Green, The Jackson Laboratory, Bar Harbor, Maine, f o r making a v a i l a b l e to me the r e s u l t s of a preliminary i n v e s t i g a t i o n of the response to acetazolamide of several mouse stra i n s and a confirmation of the response of the CBA/J s t r a i n ; x i (4) Dr. W. D. Dorian, Director, Medical Research Department, Merck Sharp and Dohme (Canada) Ltd., Pointe Claire, Quebec, for his generous g i f t of dichlorphenamidej ( 5 ) Dr. J . E. Baer, Director of Drug Metabolism, Merck Sharp Dohme Research Laboratories, West Point, Pennsylvania, for his instructions for the solution of dichlorphenamide. I acknowledge the support of Medical Research Council Grant No. MT-1062 to Dr. J . R. Miller and the assistance of a Medical Research Council Studentship from 1968 to 1972. x i i CHAPTER I INTRODUCTION Acetazolamide (or Dlamoxv ;) i s a potent carbonic anhydrase i n h i b i t o r which has been used c l i n i c a l l y as a d i u r e t i c i n human patients since 1953. Layton and Hallesy (1965a) found i t to be teratogenic i n rats and mice. The malformation i s a reduction deformity, usually ectrodactyly, l o c a l i z e d i n the postaxlal surface of the forelimb, predominantly the r i g h t forelimb. The p e c u l i a r l o c a l i z a t i o n of the teratogenic l e s i o n i n the forelimb with a predominant r i g h t sidedness i s unique i n both tera t o g e n i c a l l y Induced and spontaneously occurring malformations. There appears to be no developmental basis f o r t h i s one-sided l o c a l i z a t i o n . The present study reports the complete resistance of the SWV s t r a i n of mice to the teratogenic action of acetazolamide, a d e f i n i t i o n of t h i s resistance by comparison with a sensitive s t r a i n CBA/J, and a genetic analysis of t h i s resistance i n a breeding program with the s e n s i t i v e CBA/J s t r a i n . Evidence i s presented to support a three gene model of inheritance, with resistance to t'he teratogenic action of acetazolamide being a dominant t r a i t . Preliminary re s u l t s of these studies were presented to the 12th annual meeting of the Teratology Society (Biddle and M i l l e r , 1972). Maren, Mayer and Wadworth (1954), i n an early report on 1 2 the pharmacology of acetazolamlde, stated, from t h e i r experimence and from personal communications from others, that i t appeared to have no obvious e f f e c t on the embryonic development of rats, guinea pigs, rabbits and dogs. Maren (1965) disputed the findings of Layton and Hallesy (1965a) on the basis that carbonic anhydrase i n h i b i t i o n i s the only known function of acetazolamide (reviewed by Maren, 1967) and maximal p h y s i o l o g i c a l carbonic anhydrase i n h i b i t i o n can be achieved with doses lower than those used by Layton and Hallesy. Possibly, some other unknown function of the drug was responsible f o r i t s t e r a t o g e n i c i t y . Layton and Hallesy (1965b) rebutted Maren's (1965) arguments by s t a t i n g that the high dose of acetazolamide administered i n the d i e t might be required f o r a sustained enzyme i n h i b i t i o n during the embryo sensi t i v e period. If the teratogenic a c t i o n i s d i r e c t l y on the embryo, th i s high dose l e v e l i n the dam might be e s s e n t i a l to maintain a teratogenic l e v e l of the drug i n the embryo. They suggested that s t r u c t u r a l l y d i f f e r e n t carbonic anhydrase i n h i b i t o r s should be screened and, i f they produced similar-anomalies, the causative role of carbonic anhydrase i n h i b i t i o n i n the acetazolamide teratogenesls would be more secure. In 1967, Hallesy and Layton found that dichlorphenamide, a s t r u c t u r a l l y d i f f e r e n t carbonic anhydrase i n h i b i t o r , administered i n the d i e t of r a t s , a l so produced the acetazolamide type of forelimb anomalies. And i n 1968, Wilson, Maren, Takano and E l l i s o n found that ethoxzolamide, 3 s t r u c t u r a l l y d i f f e r e n t from both acetazolamide and dichlorphenamide, produced the acetazolamide type forelimb malformations i n r a t s . Wilson et a l . (1968) investigated more thoroughly the time of action of acetazolamide and found that subcutaneous i n j e c t i o n s of 1,000 mg/kg (divided into 2 i n j e c t i o n s per day) produced the teratogenic l e s i o n on day 10 and 11. This i s the early forelimb stage of rat embryos. When this high dose was administered on day 10, a few extreme cases of adactyly, mlcromelia and amelia of the r i g h t forelimb with severe ectrodactyly of the l e f t forelimb were found. The r i g h t sidedness of the defect s t i l l remained. Wilson and h i s co-workers were not able to demonstrate, by standard enzyme assay, carbonic anhydrase a c t i v i t y i n the rat embryo u n t i l day 13 which i s much beyond the limb sens i t i v e period of day 10 and 11. However, they could detect a c t i v i t y i n the a l l a n t o i c membrane and placenta l a t e on day 11. Therefore, the teratogenic action of carbonic anhydrase i n h i b i t i o n i n the embryo was s t i l l questioned. Takano, Yokota and Nagata (1971) succeeded i n demonstrating the presence of carbonic anhydrase a c t i v i t y i n the fore- and hindllmb buds of the rat embryo by histochemical assays of sections of the limb buds using l i g h t and electron microscopy. With standard enzyme assay, such as that used by Wilson et a l . (1968), of whole embryo preparations, the enzyme a c t i v i t y was probably masked by a large amount of enzyme-free t i s s u e . The a c t i v i t y was 4 present on day 11 in the sections of a l l limb buds and was concentrated in the area between the epidermis and the underlying mesenchyme. It was also localized on the surface of some internal mesenchymal c e l l s . There appeared to be more act i v i t y in the forelimb than in the hindlimb but this was not quantitatively determined. If 500 mg/kg of acetazolamide was administered to the dam 1 hour before i t was k i l l e d , the carbonic anhydrase ac t i v i t y in the embryonic limb buds was greatly diminished or completely inhibited. This suggested that the embryo could be the primary responder to the teratogenic action of acetazolamide. However, the right sidedness of the defect and the immunity of the hindlimbs were s t i l l unexplainable. Layton (1971) demonstrated that hamsters also responded to the teratogenic action of acetazolamide in the same manner as rats and mice0 With single intraperitoneal injections, the time of action was c r i t i c a l l y defined on late day 8 for this species. This is the time at which the forelimb bud f i r s t appears in the hamster. However, in hamsters both forelimbs were affected equally and most of the malformations were b i l a t e r a l . There was s t i l l a postaxlal to preaxial pattern to the severity of the, reduction deformity and administration of acetazolamide later in the sensitive period resulted in more severe ectrodactyly. In addition, a few hindlimbs showed a reduction deformity but most of these were of the preaxial surface and of the l e f t hindlimb. These hindlimb anomalies occurred late in the 5 sensitive period and coincided with the l a t e r appearance of the hlndlimb bud. Vickers (1972) reported an independent examination of acetazolamlde teratogenesis i n r a t s . He presented the f i r s t extensive whole-mount and h i s t o l o g i c a l examinations of the many va r i a t i o n s of the forelimb anomalies i n mature fetuses. Suzuki and Takano (1969) established that day 9 of pregnancy with ICR-JCL mice was the sensitive embryonic stage f o r acetazolamide-induced ectrodactyly i n the mouse. This observation and the v a r i a t i o n s of the time of a c t i o n of acetazolamide that are found with d i f f e r e n t s t r a i n s of mice w i l l be discussed l a t e r i n the present study. In a search f o r the p h y s i o l o g i c a l mechanism of acetazolamlde teratogenesis i n rats, Maren and E l l i s o n were able to define two actions of the drug which were r e q u i s i t e s f o r teratogenesis (Maren and E l l i s o n , 1972a, 1972b, 1972c; E l l i s o n and Maren, 1972a, 1972b). Respiratory a c i d o s i s , potassium depletion and metabolic a c i d o s i s of the dam, which are the p h y s i o l o g i c a l e f f e c t s of the e l e c t r o l y t e l o s s due to carbonic anhydrase I n h i b i t i o n , were themselves ruled out as causative actions. Only carbonic anhydrase i n h i b i t o r s which cause renal loss of potassium produced the teratogenic l e s i o n of acetazolamide. Those i n h i b i t o r s which do not could be made teratogenic by other drugs (theophylline) or dietary r e s t r i c t i o n s which cause potassium deficiency of the dam. These drugs or potassium deficiency by themselves do not produce the acetazolamide-type limb anomalies. 6 Conversely, potassium-retaining drugs (triamterene or amlloride) or potassium supplementation i n conjunction with acetazolamide reduced the frequency and severity of the defects or provided complete protection. However, excessive l e v e l s of potassium or potassium-retaining drugs caused an increase i n the frequency and severity of the acetazolamide defects. Therefore, both carbonic anhydrase i n h i b i t i o n and a potassium Imbalance, at l e a s t i n the dam, are necessary actions to produce the acetazolamide-type l e s i o n . There was a suggestion that t h i s was occurring i n the embryo because term fetuses that had been treated with acetazolamide i n utero and expressed the limb anomalies had a s i g n i f i c a n t l y higher l i v e r potassium concentration than t h e i r normal l i t t e r mates and, i n turn, these were both higher than control fetuses. Experiments with intrauterine administration of acetazolamide suggested that the s i t e of teratogenic action of t h i s and related carbonic anhydrase i n h i b i t o r s i s within the embryonic unit (embryo proper plus embyronic membranes). Scott (1970) administered small quantities of acetazolamide to d i f f e r e n t intrauterine s i t e s of day 11 pregnant r a t s . More of the t y p i c a l acetazolamide malformations were produced when i t was administered close to the embryo, i n or near the decldua capsularis and/or yolk sac and the extraembryonic coelom, than when i t was deposited further away i n the decidua b a s a l i s and/or c h o r i o a l l a n t o i c placenta. No malformations occurred i n embryos at control or non-injected 7 s i t e s . Thus, the maternal organism does not appear to be the s i t e of primary action of t h i s teratogen. If teratogenesis was a systemic e f f e c t of the dam, affected embryos would be expected at non-injected s i t e s . The greater number and more severely affected embryos found when the drug was deposited i n s i t e s c l o s e r to the embryo suggested that the teratogenic ac t i o n of acetazolamide i s d i r e c t l y on the embryo. Intrauterine administration of acetazolamide was undertaken with hamsters by Storch and Layton (Layton, 1972, personal communication). In t h i s case, l t was administered to the uterine lumenal spaces between embryos. More of the acetazolamide-type limb malformations were found i n embryos near the s i t e of a p p l i c a t i o n than i n other parts of the uterus which suggested that the embryonic unit i s the primary responder to the teratogenic a c t i o n of the drug. I f acetazolamide was administered i n t r a p e r i t o n e a l l y at a teratogenic dose l e v e l and amlloride, a drug which blocks potassium excretion, was administered intralumenally to the uterus, the number of malformed embryos i n the amiloride-treated horn was reduced. This agreed with the hypothesis that the a c t i o n of acetazolamlde and the r e s u l t i n g p h y s i o l o g i c a l changes required to create the teratogenic environment i s at the l e v e l of the embryo or embryonic u n i t . To undertake the present study of the resistance of the SWV s t r a i n of mice to the teratogenic a c t i o n of acetazolamide, i t was necessary to define the teratogenic parameters f o r a sensitive s t r a i n . In t h i s case, the CBA/J s t r a i n was chosen. 8 The behaviour of the SWV s t r a i n was then examined i n the l i g h t of thi s information (Chapter I I I ) . The response of the r e c i p r o c a l hybrid embryos from crosses between SWV and CBA/J to acetazolamide, administered at the appropriate time and dosage, was examined. I t was found that embryonic resistance to the teratogenic action i s , dominant to s e n s i t i v i t y (Chapter IV). This led to an appropriate breeding scheme — a repeated backcross program to the sensitive CBA/J s t r a i n dam' — which would discriminate between a single gene and a more complex, polygenic mode of inheritance and, i f the inheritance of s e n s i t i v i t y to acetazolamide was polygenic, i t would permit an estimation of the number of genetic factors involved i n thi s t r a i t . A three-locus model was suggested by two separate methods of analysis of the breeding data (Chapter V) and, within the l i m i t a t i o n s discussed, t h i s was confirmed by further breeding tests of a segregating generation (Chapter VI). Studies on the "acetazolamide syndrome" i n the l i t e r a t u r e , discussed previously, indicated that the teratogenic action was at the l e v e l of the embryo or embryonic membranes. An attempt was made to p a r t i t i o n the embryo and dam by embryo transfers between- the CBA/J and SWV st r a i n s . Teratogenic s e n s i t i v i t y and resistance are properties of the embryonic unit (Chapter VII). The response of the two st r a i n s to dichlorphenamide, a s t r u c t u r a l l y d i f f e r e n t carbonic anhydrase i n h i b i t o r , was examined. The response of these two st r a i n s with dichlorphenamide is the same as with acetazolamide (Chapter VIII). CHAPTER II GENERAL MATERIALS AND METHODS 1. Mice: source and malntalnance. The mice used in this study were Inbred strains of Mus  musculus and were maintained in the Zoology Vivarium at the University of B r i t i s h Columbia. SWV is an inbred strain developed and maintained by Dr. J . R. Miller and i t was at the generation of inbreeding at the time of this study. CBA/J is another inbred strain. At the start of this project, CBA/J mice were from an inbreeding colony at generation Fg_1 Q in this laboratory; however, they originated from the Jackson Laboratory (Bar Harbor, Maine) at inbreeding generation F i j g ^ i ^ ' L a^e r ^n the project, the CBA/J mice were purchased directly from the Jackson Laboratory. There are no spontaneously occurring limb malformations in either the SWV or CBA/J strain. The mice were maintained on an 18 hour light period ( 6 A.M. to 12 midnight) and a 6 hour dark period. They were fed Purina Laboratory Chow and water ad libitum. Females used for timed matlngs were caged in groups of 4 per cage and males were caged singly. Following timed matings, pregnant females were caged in groups of 2 or 3. 10 11 2 . Timing of pregnancy. Females with vaginal signs of proestrus were placed singly with a male at the end of the l i g h t period. If a copulation plug, i n d i c a t i n g mating, was found the following morning, t h i s day was designated as day 0 of pregnancy. 3. Source of teratogenic carbonic anhydrase i n h i b i t o r s . a. Acetazolamlde. The acetazolamide used was parenteral sodium acetazolamide, Diamoxv , manufactured by the Lederle Products Department of Gyanamid of Canada, Ltd., Montreal. I t i s supplied i n 500 mg v i a l s with s u f f i c i e n t sodium hydroxide to s o l u b i l i z e i t i n d i s t i l l e d water with a r e s u l t i n g pH of 9 .2 . The acetazolamide was obtained from the Pharmacy of the Vancouver General Hospital. b. Dichlorphenamide. Dichlorphenamide i s an i n v e s t i g a t i o n a l new drug (not f o r human use) of Merck and Co., Inc. and was supplied through the courtesy of Dr. W. D. Dorian, Di r e c t o r of the Medical Research Department of Merck, Sharp and Dohme Canada Ltd. CHAPTER III TERATOGENIC ACTION OF ACETAZOLAMIDE IN CBA/J AND SWV The teratogenesis of acetazolamide i n mice by the drug-d i e t method throughout pregnancy was presented by Layton and Hallesy (1965&)* They used Manor Farms mice which are a commercially avai l a b l e and g e n e t i c a l l y heterogeneous albino stock. The most frequent deformity was the absence of d i g i t V of the r i g h t forelimb. A time of action study with acetazolamide was made by Suzuki and Takano (1969; Takano, personal communication) with ICR-JCL mice. This i s a random-bred stock of mice maintained by the Japanese Cancer Laboratory and derived from the ICR stocks i n the United States. With two subcutaneous in j e c t i o n s of 500 mg/kg administered at 10 A.M. and 4 P.M., Suzuki and Takano found that day 9 was the sensitive day f o r single day treatment i n t h i s stock of mice. In the present study of the acetazolamide resistance of the SWV s t r a i n , the time of teratogenic action was reinvestigated f o r the sensi t i v e CBA/J s t r a i n since day 9 was found not to be the teratogenically sensitive day. An intra p e r i t o n e a l route f o r administration of acetazolamide was preferred rather than the subcutaneous route of Suzuki and Takano (1969) because a necrotic epidermal l e s i o n was produced by the l a t t e r route that was unaesthetic and made 12 13 the treated animals i r r i t a b l e and d i f f i c u l t to handle. With multiple and single dose administration of acetazolamide, day 10 i s the se n s i t i v e day f o r the CBA/J s t r a i n and, within the dosage l e v e l s employed, the SWV s t r a i n i s r e s i s t a n t to any teratogenic or embryotoxic action of th i s drug. Materials and Methods 1. Acetazolamide administration. fry) The acetazolamide, pharmaceutical Diamox^ ' preweighed to 500 mg per v i a l with sodium hydroxide, was dissolved with 5 ml of s t e r i l e d i s t i l l e d water. The acetazolamide solu t i o n was injected l n t r a p e r i t o n e a l l y into the dam using a 0.25 ml tuberculin syringe and a 26 gauge, \ inch (0.46 x 13 mm) disposable needle. The times of administration during pregnancy and the dosages per kilogram body weight of the dam at the time of i n j e c t i o n are given i n the presentation of the r e s u l t s . The following are the volumes administered per gram mouse and the corresponding mg/kg dosages: 0.0025 ml/g = 250 mg/kg; 0.005 ml/g = 500 mg/kg; 0.0075 ml/g = 750 mg/kg; 0.010 ml/g = 1,000 mg/kg. A l l control l i t t e r s were untreated since the acetazolamlde-lnduced malformations are so s p e c i f i c and do not occur spontaneously i n the st r a i n s of mice used. 2. Examination of f e t a l malformations. A l l pregnancies were terminated on day 18. The dams 1 4 were k i l l e d by cervical dislocation, the uteri were removed and their contents recorded. Resorptions were defined as any trace of resorbing embryonic or placental tissue and a l l dead fetuses, regardless of limb condition, were classified as resorptions. The number of embryo implantations was defined as the sum of the number of resorptions and li v e fetuses. Live fetuses (spontaneously moving) were recorded as to limb condition and other external malformations. A fetus with any limb reduction deformity of the "acetazolamlde type" was defined as having ectrodactyly. Results and Discussion 1 . Preliminary study of the time of action of acetazolamide in CBA/J and SWV. The results of the preliminary study of the time of teratogenic action of acetazolamlde (500 mg/kg per injection) in the CBA/J and SWV strains are presented in Table 1. The 1 0 A.M. and 4 P.M. times for treatment were used for convenience and were the times used by Suzuki and Takano ( 1 9 6 9 ) in their study of ICR-JCL mice. Ectrodactyly was induced by acetazolamlde in CBA/J but not in SWV. The change with time in the frequency of ectrodactyly in CBA/J, using the multiple dosage regime of Table 1 , is graphically presented in Figure 1 . Two injections of 500 mg/kg (at 1 0 A'.M. and 4 P.M.) on each of days 9 t 1 0 and 1 1 of pregnancy resulted in 73% ectrodactyly in live fetuses TABLE 1 Preliminary Study of Time of Action of Acetazolamide in CBA/J and SWV Times of treatment* Affected Resorptions/ Ectrodactyly/ l i t t e r s implants live fetuses No. % No. CBA/J - • 1. day 9 (10 AM, 4 PM) 0/4 9/33 27 0/24 0 2. day 10 (10 AM, 4 PM) V 5 7/35 20 9/28 32 3c day day 10 11 (10 AM, (10 AM) 4 PM) 4/4 5/30 17 8/25 32 4„ day day 10 11 (4 PM) (10 AM) 2/7 9/62 15 3/53 6 5 . day day day 9 10 11 (10 AM, (10 AM, (10 AM, 4 4 4 PM) PM) PM) 2/4 1 V 2 5 56 8/11 73 SWV 1. day day day 9 10 11 (10 AM, (10 AM, (10 AM, 4 4 4 PM) PM) PM) 0/4 9/63 14 0/54 0 * Treatment = 500 mg/kg per injection administered ihtraperitoneally. 8 0 " 7 0 -4 0 -< CO * 30-t I 1 I J i 16 x TJ o ~o o Tj 2 0 -c d) (J °" 10-o-1 1 day 9 day 10 day 11 Time during pregnancy FIGURE 1: Percent ectrodactyly induced in CBA/J by acetazolamide administered at different gestational times and 500 mg/kg per injection. Arrows indicate time of injection and lines connecting arrows designate treated groups. but also a high frequency of resorption (56^). Two injections on day 9 at this dosage did not result in ectrodactyly. This is contrary to the. findings of Suzuki and Takano (1969) who found day 9 to be the most sensitive day with this dosage regime for ICR-JCL mice. Two injections on day 10 resulted in ^ 2% ectrodactyly. Adding another injection at 10 A.M. on day 11 did not change the frequency of ectrodactyly. A single injection at 4 P.M. on day 10 followed by another at 10 A.M. on day 11 resulted in a lower frequency of ectrodactyly ( 6 % ) . Therefore, day 10 was inferred to be the sensitive day for the acetazolamide induction of ectrodactyly in the CBA/J strain of mice. 2. Dose-response of CBA/J and SWV to acetazolamide administered at 10 A.M. and 4 P.M. on day 10 of pregnancy. An examination was made of the dose-response to acetazolamide in CBA/J and SWV using the method of two injections at 10 A.M. and 4 P.M. on day 10. The data are presented in Table 2 and graphically displayed in Figure 2. The data for 500 mg/kg (2 doses on day 10) for CBA/J are repeated from Table 1. In CBA/J, the frequencies of both ectrodactyly and resorption increased with increased dose. In SWV, no ectrodactyly was found and there was no change in the control resorption frequency. However, 3 out of 8 SWV dams died after treatment with the highest dose. No death after treatment occurred in any of the CBA/J dams. 18 TABLE 2 Dose Effect of Acetazolamide In CBA/J and SWV Administered at 10 A.M. and 4 P.M. on Day 10 Dose/ injection (mg/kg) Affected l i t t e r s Resorptions/ implants Ectrodactyly/ liv e fetuses No. % No. % CBA/J 1. 0 0/7 8/62 13 0/54 0 2. 500 4/5 7/35 20 9/28 32 3. 750 9/10 23/85 27 29/62 47 4. 1,000 4/4 18/36 50 11/18 61 SWV 1. 0 0/13 18/173 10 0/156 0 2. 750 0/11 15/162 9 0/147 0 3. l.ooo o/5* 8/75 11 0/67 0 * 3/8 treated SWV dams died after treatment with 1,000 mg/kg per injection. 19 FIGURE 2: Dose e f f e c t of acetazolamide In CBA/J and SWV administered at 10 A.M. and 4 P.M. on day 10. 20 From these observations, i t was concluded that the SWV strain was resistant to any teratogenic or embryotoxlc action of acetazolamide. 3. C r i t i c a l time of action of a single intraperitoneal  injection of acetazolamlde in CBA/J. A c r i t i c a l evaluation of the time of teratogenic action of a single 1,000 mg/kg, intraperitoneal injection of acetazolamide was made with the CBA/J strain. This is presented in Table 3 and Figure 3. From the graphical data in Figure 3. the most sensitive time to a single intraperitoneal injection of acetazolamide appeared to be mid-day 10. This time agrees well with the appearance of the forelimb bud in mice. It also agrees with the c r i t i c a l evaluation of the limb sensitive period for hamster embryos (Layton, 1971) allowing for differences in the rates of embryonic development of mice and hamsters. There is no explanation for the drop in resorption frequency at 4 P.M. on day 10 (Figure 3). It may be due to sampling error; however, i t may prove interesting for later re-evaluation. The distribution of limb anomalies found in the CBA/J fetuses with ectrodactyly in Table 3 Is presented in Table 4. The predominance of affected right forellmbs was obvious. There was also an Indication that, when acetazolamide was administered later in the limb sensitive stage, the right 21 TABLE 3 Time of Action of a Single Intraperitoneal I n j e c t i o n of Acetazolamide i n CBA/J Time of Affected Resorptions/ E c t r o d a c t y l y / treatment* l i t t e r s implants l i v e fetuses No. % No. % 1. day 9 - 10 PM 1/8 18/64 28 1 A 6 2 2 0 day 10 - 4 AM 2/10 27/85 32 3/58 5 3. day 10 - 10 AM 5/10 26/81 32 9/55 16 4. day 10 - 4 PM 5/9 12/77 16 9/65 14 5. day 10 - 10 PM 2/10 24/86 28 3/62 5 * Treatment i s a single i n t r a p e r i t o n e a l i n j e c t i o n of 1,000 mg/kg. 3CH 20H 10-o-day 9 10 PM day 10 4 AM 1 day 10 10 AM 1 day 10 4 PM day 10 10 PM Time during pregnancy of a single i.p. injection (1000 rngy^kg) FIGURE 3: Time of action of a single i n t r a p e r i t o n e a l i n j e c t i o n of acetazolamide i n CBA/J. 23 TABLE 4 Types and Distribution of Acetazolamide-Induced Ectrodactyly Found in the Time of Action Study with CBA/J Using a Single Intraperitoneal Injection Time of Type of Number injection ectrodactyly Female Male . Total 1. day 9 - 10 AM 1. R - V absent 0 1 2. day 10 - 4 AM i . R - V abnormal 1 0 i i . R - V absent 1 1 3. day 10 - 10 AM i . R - V absent 5 4. day 10 - 4 PM i . R - V abnormal 0 1 i i . R - V absent 0 i i i . R L — V absent V absent 2 0 i v . R - I V , V absent 1 l 5. day 10 - 10 AM 1. R - V absent 1 1 11. R L - V absent V absent 1 0 2k forelimb ectrodactyly became more severe and some l e f t forelimb involvement occurred. This increase in severity of the ectrodactyly agrees with Layton1s (1971) findings with hamsters. 4. Range and distribution of d i g i t a l anomalies found in CBA/J. The range of acetazolamide-lnduced forelimb anomalies was extensive. The largest treated sample of CBA/J embryos in this study was made to establish the CBA/J strain response frequency for use in subsequent Interpretation of the genetic analysis (Table 6, Chapter V). Five CBA/J sires were mated to a number of CBA/J females and the dams were treated with 750 mg/kg at 10 A.M. and k P.M. on day 10. There were no obvious differences In the range of anomalies between the 5 sire groups and the fetuses were pooled for examination of the limbs. The range and distribution of d i g i t a l anomalies found in these CBA/J fetuses are presented in Appendix I. It was clear from Appendix I that the most common type of ectrodactyly was an absence of digi t V of the right forelimb. There was a predominance of right sidedness that confirms the general findings of other studies using mice and rats and discussed In Chapter I. There were a few cases in which the l e f t side was the predominant side. The specific localization of the acetazolamide teratogenic lesion remains the most intriguing embryological mystery of this teratogen. 25 Conclusion The CBA/J s t r a i n responds to the teratogenic action of acetazolamlde i n the manner expected from other studies with th i s drug. The ectrodactyly i s of the forelimb and predominantly of the r i g h t side. However, the SWV s t r a i n appears to be completely r e s i s t a n t to the teratogenic and embryotoxic actions of acetazolamide. No ectrodactyly was induced during pregnancy and no change i n the control resorption frequency was found. Administration of acetazolamide l a t e r i n the limb sensitive period of the sensitive CBA/J s t r a i n produces a more severe ectrodactyly of the r i g h t forelimb with some l e f t forelimb involvement. CHAPTER IV RESPONSE TO ACETAZOLAMIDE OF RECIPROCAL Fj_ HYBRID EMBRYOS FROM CROSSES BETWEEN CBA/J AND SWV The difference In the responses of the CBA/J and SWV strai n s to the teratogenic action of acetazolamide i s dramatic. CBA/J i s sensi t i v e while SWV i s completely r e s i s t a n t . The response of the r e c i p r o c a l hybrid embryos was investigated to i n i t i a t e the genetic analysis of t h i s t r a i t . Materials and Methods Reciprocal crosses were made between the CBA/J and SWV s t r a i n s . The dams were treated with acetazolamide on day 10 of pregnancy with two i n t r a p e r i t o n e a l i n j e c t i o n s (at 10 A.M. and 4 P.M.) of 750 mg/kg per i n j e c t i o n . These times were the most sensitive f o r the CBA/J embryo with the multiple i n j e c t i o n regime and t h i s dosage did not r e s u l t i n too great an increase i n the normal resorption frequency (Chapter I I I ) . The fetuses were examined on day i d as described i n Chapter I I I . For the designation of f e t a l o r i g i n , the s t r a i n of the dam i s l i s t e d f i r s t . Results and Discussion The responses of the r e c i p r o c a l F^ hybrid embryos from 26 27 crosses "between CBA/J and SWV are presented in Table 5. In the sample of treated SWV.CBA embryos, there was no induced ectrodactyly and the resorption frequency was essentially that of the control l i t t e r s . In the sample of treated CBA.SWV F^ embryos, 1 out of 117 live fetuses exhibited induced ectrodactyly and the resorption frequency was of the control l e v e l . Comparison of these reciprocal F^ hybrid embryo responses with those of the CBA/J and SWV Inbred strains in Chapter III indicated that embryonic resistance to the teratogenic action of acetazolamlde could be considered to be a dominant t r a i t and sensitivity to be a recessive t r a i t . The fact that 1 affected CBA.SWV F1 fetus was found while no affected SWV.CBA F^ fetuses were observed cannot be taken to be a reciprocal cross difference or evidence for a dam effect. The difference between 1 and 0 affected with these sample sizes was unanalysable. The responses of the two types of F^ embryos were assumed to be equal and essentially zero. Therefore, a backcross program using the sensitive CBA/J strain should quickly recover genes for embryonic sensitivity to the teratogenic action of acetazolamide. TABLE 5 Effect of Acetazolamlde on Reciprocal F. Hybrids between CBA/J and SWV Mating Affected l i t t e r s Resorptions/ implants Ectrodactyly/ liv e fetuses Dam X Sire No. % No. % 1. SWV X CBA/J (treated)* 0/8 10/114 9 0/104 0 2. SWV X CBA/J. (control)** 0/3 6/47 13 0/41 0 3. CBA/J X SWV (treated) 1/14 12/129 9 1/117 1 4. CBA/J X SWV (control) 0/4 5/32 16 0/28 0 * treated = 750 mg/kg, intraperitoneally, at 10 A.M. and 4 P.M. on day 10. ** control = no treatment. CHAPTER V ANALYSIS OF THE GENETIC CONTROL OF EMBRYONIC SENSITIVITY TO THE TERATOGENIC ACTION OF ACETAZOLAMIDE Embryonic resistance to the teratogenic action of acetazolamide is a dominant t r a i t . A backcross breeding program was used to recover genes for sensitivity. The breeding system and some of the approaches to the analyses of the data were modeled on the investigation of the genetic variation in mice to the teratogenicity of 5-fluorouracil by Dagg, Schlager and Doerr (1966). The data collected from crosses with CBA/J and SWV in the preceedlng sections of this thesis were not included in the following analysis. Materials and Methods 1. Breeding program. The backcross program is illustrated in Figure 4. A l l crosses were made to the CBA/J dam to keep the maternal effect ( i f any) constant in each generation. F^ and BC^ males were collected. Then 5 CBA/J males, 5 SWV males, 6 F^ males and 12 BC^ males were each mated to several CBA/J females. The CBA/J dams were subsequently treated with acetazolamide at the CBA/J limb sensitive time 29 30-CBA/J male —CZZ CBA/J female SWV male F^ male 7 BCj_ male FIGURE 4: Mating scheme to Investigate the genetics of the s e n s i t i v i t y to the teratogenic action of acetazolamlde. and the fetuses from terminated pregnancies were scored for the presence or absence of induced ectrodactyly. Since the contribution of the CBA/J dam in each mating was constant, the fetal scores were used to genetically define their respective sires. 2. Administration of acetazolamide and fetal examination. Acetazolamide was administered intraperitoneally to the pregnant CBA/J dams on day 10 at 10 A.M. and 4 P.M. at 750 mg/kg per injection. Pregnancies were terminated on day 18 and uterine contents were examined as described previously. 3. Genetic analysis of breeding data. For each s i r e , a mean frequency of ectrodactyly was calculated from the total number of i t s liv e progeny. The mean frequency of ectrodactyly for the sires of each cross and backcross generation was calculated by averaging the mean frequencies of the sires of each generation. In addition, the ectrodactyly frequency data were transformed for a s t a t i s t i c a l evaluation of the data. With binomial data, in this case the presence or absence of ectrodactyly, the means and variances are related and not independent. Following the approach of Dagg eit a l . (1966), an arcsine transformation of the frequency data was used. In the present study, the Freeman-Tukey arcsine transformation for binomial distributions and small samples 32 (n<50) was used. Tables f o r thi s transformation were presented by Mosteller and Youtz (1961). These tables can be used i n conjunction with the arcsine tables derived by B l i s s (see Snedecor and Cochran, 19^7, pp. 569-571) f o r large samples (n > 50). For each l i t t e r from each s i r e , an arcsine value (x^) was found from the above-mentioned tables of the Freeman-Tukey arcsine transformation. This was based on the number of l i v e fetuses with ectrodactyly and the t o t a l number of l i v e fetuses i n that l i t t e r . This was then weighted by the number of l i v e fetuses ( k ^ i n that l i t t e r . For each s i r e , a mean weighted arcsine value was calculated from the sum of the weighted arcsine divided by the t o t a l number of l i v e fetuses from that s i r e . That i s mean weighted arcsine = ^(k^x^) where k^ i s the number of fetuses i n the _ith l i t t e r and x^ i s the Freeman-Tukey arcsine of the r a t i o of the number of fetuses with ectrodactyly to the t o t a l number of fetuses i n that l i t t e r . Means and variances of each s i r e generation were calculated from the average of the mean weighted arcsines of the s i r e s of that generation. No further weighting of the data was made. The number of genetic f a c t o r s involved i n the t r a i t of s e n s i t i v i t y to the teratogenic a c t i o n of acetazolamide was calculated from the formula derived by Wright (1934). The 3 3 formula employs the means and variances of the two inbred s t r a i n s i r e s , the F^ s i r e s and the BC^ s i r e s . The Freeman-Tukey arcsine transformed data f o r these generations were used. Wright's ( 1 9 3 4 ) formula i s n = Rf , 16 (V BC " V E ) where n = number of l o c i (or segregation index), R = phenotypic range of the two parental st r a i n s (In t h i s case, i t i s the difference between the means of the CBA/J and SWV s i r e s . ) , V B C ^ = variance of the BC 1 s i r e s , and V = an estimate of the non-genetic or environment variance. An independent estimate of the number of genetic factors involved i n the t r a i t of teratogenic s e n s i t i v i t y to acetazolamide was made using the generation means of the untransformed ectrodactyly frequency data. The assumptions made i n t h i s a n a l y s i s are discussed below. The findings of the two approaches to t h i s analysis of the number of genetic factors are compared. Results and Discussion 1 . B lometrlcal estimation of the number of l o c i involved with acetazolamlde teratogenic s e n s i t i v i t y . The relevant progeny data f o r the CBA/J, SWV, and BC^ s i r e s mated to CBA/J females are l i s t e d i n Tables 6, 7, 8 and 9 r e s p e c t i v e l y . These tables include the number of embryo implantations, the number of resorptions, the percent 34 CBA/J Progeny Data from CBA/J Males Mated to CBA/J Females TABLE 6 CBA/J Resorptions/ E c t r o d a c t y l y / male No. implants l i v e fetuses No. % No. % mean weighted arcsine 1. 713 8/45 17.8 17/37 45-9 41.99 2. 714 16/46 34.8 15/30 50.0 45.31 3. 715 12/48 25.0 18/36 50.0 43.46 4. 725 1/44 2.3 23/43 54.5 46.86 5. 726 20/46 43.5 14/26 53.8 47.16 Means 24 .7 50.6 44.96 Summary: 1. Mean percent resorption 24 .7 2. Mean (Xa/) percent ectrodactyly 50.6 3. Mean ( X ) of mean weighted arcsines 44.96 4. Variance (V) of mean weighted arcsines 4.91 35 F 1 Progeny Data from SWV Males Mated to CBA/J Females TABLE 7 male No. Implants l i v e fetuses No. % No. % mean weighted arcsine 1. 1393 14/56 25 .0 0/42 0 10o23 2. 1446 3/45 6.7 0/42 0 9.48 3 . 1463 6/62 9.7 2/56 3 . 6 12.48 4. 1582 9/52 17 .3 1/43 2.3 12.64 5. 1825 10/51 19.6 0/41 0 10.88 Means 15.7 1.2 11.14 Summary: 1. Mean percent resorption 15.7 2. Mean (X^) percent ectrodactyly 1.2 3 . Mean (X) of mean weighted arcsines 11.14 4. Variance (V) of mean weighted arcsines 1.92 3 6 BG Progeny Data from F 1 Males Mated to CBA/J Females T A B L E 8 male No. Implants l i v e fetuses No. % No. % mean weighted arcsine 1 . 1 3 7 / 5 7 1 2 . 3 6 / 5 0 1 2 . 0 1 9 . 0 2 2 . 2 2 2 / 4 7 4 . 3 1 / 4 5 2 . 2 1 1 . 8 6 3 . 23 1 2 / 4 8 2 5 . 0 0 / 3 6 0 1 0 . 8 3 4 . 2 4 8 / 4 0 2 0 . 0 3 / 3 2 9 . 4 1 8 . 5 9 5 . 25 8 / 6 8 1 1 . 8 3 / 6 0 5 . 0 1 4 . 5 7 6 . 26 7 / 6 0 1 1 . 7 5 / 5 3 9 . 4 1 8 . 1 7 Means 1 4 . 2 6 . 3 1 5 . 5 1 Summary: 1 . Mean percent resorption,. 1 4 . 2 2 . Mean ( X ^ ) percent ectrodactyly 6 . 3 3 . Mean ( X ) of mean weighted arcsines 1 5 . 5 1 4 . Variance (V) of mean weighted arcsines 1 3 . 0 0 37 TABLE 9 BC0 Progeny Data from BC, Males Mated to CBA/J Females BCj, male No. Resorptions/ implants No. Ectrodactyly/ live fetuses No. mean weighted arcsine 1. 35 13/45 28.9 11/32 34.4 34.91 2. 36 13/49 26.5 6/36 16.7 24.24 3. 37 23/54 42.6 10/31 32.3 33.97 4. 42 11/50 22.0 5/39 12.8 22.98 5. 48 16/68 23.5 15/52 28.8 32.48 6. 49 16/66 24.2 18/50 36.0 36.98 7. 50 6/52 11.5 6/46 13.0 21.62 8. 51 8/47 17.0 9/39 23.1 29.70 9. 52 13/56 23.1 7/43 16.3 25.47 10. 53 9/70 12.9 13/61 21.3 28.35 11. 68 6/48 12.5 9/42 21.4 27.66 12. 69 5/52 9-6 4/47 8.5 18.11 Means 21.2 22.1 28.04 Summary: 1. Mean , 21.2 2. Mean , 22.1 3. Mean (X*) of mean weighted arcsines. , 28.04 4. Variance (V) of . 33.77 38 resorption, the number of live progeny, the number of progeny with ectrodactyly, the percent ectrodactyly score for live fetuses and mean weighted Freeman-Tukey arcsine transformed ectrodactyly score for each sire in each generation. Also included are the mean frequency or mean percentage of resorption, the mean frequency of ectrodactyly and the mean and variance of the mean weighted Freeman-Tukey arcsine scores for each generation of sires. A discussion of the resorption response to acetazolamide in this backcross program is deferred to Chapter VI. The frequency distribution of ectrodactyly in the progeny of the CBA/J, SWV, F^ and BC^ sires is illustrated in Figure 5* If a single gene determined the embryonic response to acetazolamide, a blmodal distribution would be expected in the ectrodactyly scores of the BC^ sir e s . Some would behave like the F^ sires and the rest would behave like the CBA/J inbred strain sires. This was not the case and a single gene hypothesis was rejected. Wright's (1934) formula for the estimation of the number of segregating genetic factors or l o c i involved in a t r a i t is predicated on the assumption that the genes have equal effect and that they are independently segregating, that i s they are not linked. Further discussions of the formula and i t s derivations can be found in Wright (1934, 1968) and Falconer (I960, pp. 217-219). The calculations for two estimates of the number of l o c i Involved in the t r a i t of teratogenic sensitivity to 60 50H E o < CO U 40H ^ 30-u D "D £ 20" o OJ c CJ \J 1_ CO °- 10-0 ® © 0 © © © © © © © © ©© © © © © ©© © © © CBA/J BC] Fl © © —< ©®© l S W V Sires FIGURE 5: Means and d i s t r i b u t i o n s of the percent ectrodactyly scores of the CBA/J, SWV, F 1 and BC s i r e s . 1 4 0 acetazolamide, using Wright1s (1934) formula, are outlined in Table 10. One estimate was n = 3.44. This was made using the variance of the Fji sires as an estimate of the environmental variance. This approach was suggested by Wright (1934). Excluding sex-linked t r a i t s , the T?^ sires are genetically heterogeneous but they are also genetically homogeneous. Therefore, any variation they exhibit w i l l be due to environment or chance. Another estimate was n = 2.63. This was made using an average of the CBA/J, SWV and F^ sire variances as an estimate of the environmental variance. This method was used by Dagg e_t a l . (1966) and is also valid since these three groups of sires, although they are genetically different, are each genetically homogeneous. These two blometrlcal approaches suggested that there are three l o c i (2.63 -- 3.44) which control embryonic sensitivity to the teratogenic action of acetazolamide. 2. Estimation of the number of l o c i using the generation mean frequencies of ectrodactyly. Since the number of l o c i controlling^the t r a i t of teratogenic sensitivity was estimated to be relatively small, another method of deriving this estimate was tr i e d . It made use of the generation mean frequencies of ectrodactyly as the genetic t r a i t and, naively, i t can be referred to as gene counting. 41 TABLE 10 Means and Variances of CBA/J, SWV, F^, and BC^ Si r e Scores Using the Weighted Arcsine Transformation and the Calc u l a t i o n of the Number of Lo c i C o n t r o l l i n g the Response to Acetazolamlde Sire Mean Variance CBA/J 44.96 4.91 SWV 11.14 1.92 F t 15.51 13.00 BC, 28.04 33.77 Estimation of number of l o c i using Wright's (1934) formula: 1. n = 3.44, using the F^ s i r e variance to estimate Vg. 2. n = 2.63, using the average of the CBA/J, SWV, and F. s i r e variances to estimate V„ l £J 42 Teratogenic sensitivity to acetazolamide is recessive to resistance. On the basis of one locus, the probabilities that the progeny from the SWV, F^ and BC^ sires are genetically like the CBA/J inbred strain are 0, 0.50 and 0.75. respectively. If several independently segregating l o c i are involved and the CBA/J and SWV a l l e l e s at each locus are recessive and dominant, respectively, these probabilities (p^) become (p^1 1, where n i s the number of l o c i . Therefore, the product of the mean frequency of ectrodactyly found in the CBA/J inbred strain (50.6#, Table 6) and the probability that a generation i s like the CBA/J inbred strain becomes the expected generation mean frequency of ectrodactyly. The probabilities that a generation i s like the CBA/J Inbred strain and the respective expected generation mean frequencies of ectrodactyly from these calculations for 1, 2, 3 and 4 l o c i are l i s t e d in Table 11. A third backcross generation using BCg sires i s also included since this information w i l l be important in Chapter VI. Figure 6 i s a graphical comparison of the observed generation mean frequencies of ectrodactyly and the probability that that generation is like the CBA/J strain for 1, 2, 3 and 4 l o c i . The data for three l o c i f i t a straight l i n e . The expected generation means from the three-locus model (Table 11) for the F^ sires (BC^ progeny) and the BC1 sires (BC2 progeny) agreed with those found in Tables 8 and 9» This estimate of three l o c i was in complete TABLE 11 Probability that a Fetus i s Genotypically like CBA/J in the Backcross Study Considering 1, 2, 3 and 4 Independently Segregating Loci Fetus Sire 1-locus 2-locus 3-locus 4-locus model model model model Fl SWV 0 0 0 0 Fl 0.50 (25.3)* 0.25 (12.7) 0.125 (6.3) 0.063 (3.2) BC2 BC1 0.75 (37.8) 0.563 (28.5) 0.422 (21.4) 0.316 (16.0) BC^ BC2 0.875 (44„3) 0.766 (38.8) 0.670 (33.9) 0.586 (29.7) * Numbers in parentheses are the expected percentages of ectrodactyly for each fe t a l generation for the four genetic models and are based on 50.6$ ectrodactyly found in the inbred (homozygous) CBA/J strain. The models assume that resistance i s dominant to sensitivity. 60 1- locus 2 - locus T 1 1 1 1 r 1.0 0.8 0.6 0.4 0.2 0 Probability that fetal generation is like C B A / j FIGURE 6: Comparison of the observed mean percent ectrodactyly of the f e t a l generations with the pr o b a b i l i t y that a generation i s genotypically l i k e the CBA/J s t r a i n f o r 1, 2 , 3 and 4 l o c i . 4 5 agreement with that made with the biometrical approach. This three-locus model also predicts that the mean frequency of ectrodactyly from BC 2 s i r e s backcrossed to CBA/J females (that i s BC^ progeny) w i l l be 33.9% (Table 1 1 ) . This w i l l be tested i n Chapter VI. If three independently segregating l o c i control the t r a i t of embryonic s e n s i t i v i t y to the teratogenic a c t i o n of acetazolamide, i t can be predicted that 1/8 of the BC^ s i r e s should behave l i k e the CBA/J s i r e s . This was not found (see Figure 5 ) . however, only 12 BC^ s i r e s were sampled and th i s absence may be due simply to sampling error. The d i s t r i b u t i o n of BC^ s i r e scores covered that of the F^ si r e s and extended beyond toward that of the CBA/J s i r e s as would be expected with t h i s genetic model. The recovery of a BC^ s i r e with a CBA/J s i r e breeding response w i l l be one way to test t h i s model i n future studies. Conclusion Two d i f f e r e n t approaches to the genetic analysis of the breeding data suggested that three independently segregating l o c i i n the homozygous recessive condition are responsible f o r the t r a i t of teratogenic s e n s i t i v i t y to acetazolamide of the CBA/J embryo when compared with the SWV s t r a i n . One method of analysis predicted that the t h i r d backcross generation, not included i n the experimental design, would have a mean frequency of ectrodactyly of 33.9%. CHAPTER VI TEST OF THE THREE-LOCUS MODEL Two approaches to the genetic analysis of the breeding program with CBA/J and SW suggested a three-locus model for the inheritance of embryonic sensitivity to the teratogenic action of acetazolamide with resistance being a dominant t r a i t . From this hypothesis, the generation mean of the third backcross generation, that i s the mean of the BC^ sire ectrodactyly scores, was predicted to be 33*9%» This prediction was tested in a breeding program. Materials and Methods 1. Breeding program and teratogenic testing. Thirteen BC^ males were selected at random from backcrosses of 5 BC^ sires to CBA/J females. These BCg males were each mated to several different CBA/J females. The CBA/J dams were injected intraperltoneally with acetazolamide at 750 mg/kg per injection at 10 A.M. and 4 P.M. on day 10 of pregnancy. The pregnancies were terminated on day 18 and the uterine contents examined. Results and Discussion The ectrodactyly scores for the 13 BC? sires are lis t e d 46 47 in Table 12. The observed mean frequency of ectrodactyly in BC^ progeny from these BC2 sires was 33,1%. This agreed very favorably with the predicted mean of 33.9$ (Chapter V) derived from the three-locus model. The distribution of the BC2 sires' percent ectrodactyly scores is plotted in Figure 7, and that of the mean weighted Freeman-Tukey arcsine scores is plotted in Figure 8, against the probability that the f e t a l generation (BC^) is like the CBA/J inbred strain from the three-locus model. The relevant distributions for the CBA/J, SWV, and BC^ sires are Included for comparison. From the arcsine scores of the CBA/J sires, the range of the CBA/J phenotype can be defined as the mean arcsine of the CBA/J sires plus and minus two standard deviations. This was 44.96 + 4.43 (or 40.53 — 49.39). Two BC2 sires f e l l within this range (BC2 males # 91 and # 102). Therefore, the CBA/J strain phenotype was recovered. The fact that the arcsine score for BC2 sire # 91 (^9.97) Is slightly greater than the upper lim i t of the a r b i t r a r i l y defined range of the CBA/J sires (49.39) cannot be considered meaningful u n t i l further breeding studies have investigated this observation. The theoretically expected and observed generation means for the SWV, F^, BC^ and BC2 sires are summarized in Table 13* The calculations have been made with the three-locus model and the mean frequency of 50.6$ ectrodactyly from the CBA/J sires. The theoretically expected arcsine scores in this table were taken from the arcsine tables of Bliss 48 TABLE 12 BCo Progeny Data from BC? Males Mated to C B A / J Females B G 3 Resorptions/ Ectrodactyly/ male No. implants l i v e fetuses No. % No. % mean weighted arcsine 1. 89 12/39 30.8 3/27 l l o l 19.49 2. 90 16/52 30.8 14/36 38.9 38.46 3. 91* 8/57 14.0 29/49 59.2 49.97 4. 92 10/54 18.5 11/44 25.0 28.67 5. 93 19/52 36.5 10/33 30.3 34.65 6. 94 8/49 16.3 14/41 34.1 36.69 7. 95 14/55 25.5 15/41 36.6 35.99 8. 96 22/54 40.7 11/32 34.4 36.04 9. 97 9/47 19.1 9/38 23.7 30.20 10. 98 11/53 20.8 15/42 35.7 34.46 11. 99 7/52 13.5 13/45 28.9 33.32 12. 101 7/51 13.7 8/44 19.8 25.97 13. 102* 10/52 19.2 23/42 54.8 48.95 Means 23.0 33.1 34.84 Summary: 1. Mean , . 23.0 2. Mean . . 33.1 3. Mean (X) of mean weighted arcsines. . . 34.84 * Ectrodactyly scores of two BG£ males are i n the range of the C B A / J males. 49 60-50-© 0 © 0 0 © 40-E o < 1 0 30-u 20-D ~D O k . U CD c 10" <D U k . <D Q_ o-Fetus: Sire: 0 © © © © © © © © © © © 0 © 0 © ©O ® 0 © 0 © 1.0 CBA/J CBA/J Probability that T 0.422 B C 2 BC] © © © © © © 0 © 0 © © T 0.670 B C 3 B C 2 fetal generation is like CBA/j 0.125 BCi F1 I 0 h SWV FIGURE 7: Means and percent ectrodactyly scores of the CBA/J, SWV, Fj_, BGj_ and BG 2 s i r e s plotted against the pro b a b i l i t y that, the f e t a l generation i s genotypically l i k e the CBA/J s t r a i n based on the 3-locus model. 50 60-* 50-E D < C Q u 40-£ 30-o "O O o 2 0 -a> OJ c "55 10-© © Fetus: Sire: © 0 0 0 § 00-© © © © © 1.0 CBA/J CBA/J Probability 1 0 .670 BC3 BC2 that fetal © © © © © © © © © © © 0 ®8 © © © 0 0 0 0 0 0.422 BC2 BC] generation 1 0.125 BCi is like CBA/j SWV FIGURE 8: Means and d i s t r i b u t i o n s of the mean weighted arcsine scores of the CBA/J, SWV, Fj_, BC;, and BC£ sir e s plotted against the pr o b a b i l i t y that the f e t a l generation i s genotypically l i k e the CBA/J s t r a i n based on the 3-locus model. TABLE 13 Theoretical and Observed Generation Means for Ectrodactyly Based on Three-Locus Model in Backcross Study with CBA/J Dams Sire Progeny Percent ectrodactyly Arcsine Expected* Observed Expected** Observed*** 1. SWV F1 0 1.2 11.14 2. Fx BC1 6.3 6.3 14.54 15.51 3. BC^ BC2 21.4 22.1 27.56 28.04 4. BC2 BC^ 33.9 33.1 35.61 3^.84 5. CBA/J CBA/J , 50.6 . 44.96 * Theoretical percent ectrodactyly based on observed 50.6$ observed in the CBA/J inbred strain. ** Theoretical arcsine from arcsine tables for large samples (n>50) by Bliss (Snedecor and Cochran, 1967. PP. 569-571) corresponding to the theoretical percent ectrodactyly. *** Observed arcsine is generation mean of the mean weighted Freeman-Tukey arcsine for small samples (n<50) . 52 (see Snedecor and Cochran, 1967. pp. 5&9-571) f o r large sample sizes corresponding to the expected mean percent ectrodactyly for that generation. The. corresponding observed arcsine scores are the generation means of the mean weighted Freeman-Tukey arcsines. There was such close agreement of the observed generation means with the theoretical means that s t a t i s t i c a l comparisons were not considered necessary. Therefore, i t was assumed that the three-locus model is adequate to define the teratogenic sensitivity to acetazolamide of the CBA/J strain when compared with the resistant SWV strain. A discussion of the resorption response after acetazolamide treatment has been l e f t to this point because i t is best discussed in the context of the backcross breeding program. Kalter (1965) stated that "It has often been assumed in teratological studies that f e t a l death is the extreme action of teratogens but not qualitatively a different expression of that action." From his studies of response differences in mouse strains to galactoflavln treatment, he found no correlation between the frequency of fe t a l death and the frequency of malformed fetuses and concluded that galactoflavin-induced fetal death and malformations were unrelated effects. Francis (1971) made an extensive examination of the variations in cleft palate induction and fet a l death due to cortisone with four different inbred strains in a d i a l l e l breeding design. Her data led her to conclude that fetal death was not an extreme form of the 53 teratogenic response but that they are two separate responses to cortisone. In the i n i t i a l examination of the teratogenic responses of CBA/J and SWV to acetazolamlde administered at 750 mg/kg at 10 A.M. and 4 P.M. on day 10 of pregnancy (Table 2, Chapter I I I ) , CBA/J exhibited 27$ resorption and 47$ ectrodactyly while SWV exhibited 9$ resorption with no ectrodactyly. There was d e f i n i t e acetazolamide-induced f e t a l death i n CBA/J but none i n SWV. In the more c r i t i c a l examination of the CBA/J response f o r the backcross program (Table 6, Chapter V), the mean percent resorption f o r the 5 CBA/J s i r e groups was 24.7$ and i t was comparable to the CBA/J response i n the i n i t i a l examination (Table 2 ) . However, when the i n d i v i d u a l s i r e groups were examined, there was considerable v a r i a t i o n i n the resorption responses which ranged from 2.3$ to 43.5$. The ectrodactyly responses, on the other hand, f o r these CBA/J s i r e groups were t i g h t l y clustered around the 50.6$ mean ectrodactyly response. This was one i n d i c a t i o n that f e t a l death and ectrodactyly a f t e r acetazolamlde treatment were not related . There appeared to be a l o g i c a l progression of the mean resorption frequencies when t h i s parameter was examined i n the progenies of the SWV (Table 7, Chapter V), (Table 8, Chapter V), B ^ (Table 9, Chapter V) and BC 2 (Table 12, Chapter VI) s i r e s . I t was 15.7$ f o r the SWV s i r e s and increased slowly to 24.7$ f o r the CBA/J s i r e s discussed 54 above; however, the mean of the sires did not agree since i t was 14.2$. An examination of the resorption frequencies of the progenies from the CBA/J, SWV, F^ and BC^ sires was made using the Freeman-Tukey arcsine transformation. The means and variances from the transformed data were employed in Wright's (1934) formula to see whether some meaningful estimate of the number of genetic factors involved in this resorption frequency difference could be made. The analysis was identical to that used for the investigation of ectrodactyly. An estimate of 0.29 l o c i resulted when the variance of the F^ sires was used to estimate the non-genetic variance. No further manipulation of these data was made since the variance of the resorption frequency found with the CBA/J sires, that is the CBA/J inbred strain, was so great relative to the other sire generations. These data and calculations were not Included in this thesis since they were not germane to the investigation of the ectrodactyly problem; however, they are available from the author. In turn, the resorption frequency and ectrodactyly frequency for each sire group In each backcross generation were examined for a logical association. There was none. In the BC2 sires (Table 12), the BG^ progenies from two BG2 sires (males # 91 and # 102) that behaved like the CBA/J strain with respect to ectrodactyly response, had low resorption frequencies (14.0$ and 19.2$, respectively) relative to the other progeny groups. Therefore, there seemed to be no ass o c i a t i o n of resorption and ectrodactyly responses to acetazolamlde treatment. Since no associations were found, i t was concluded that they were two independent responses to acetazolamlde. Conclusion The p r e d i c t i o n from the three-locus model of the induced-ectrodactyly frequency of t h i r d backcross generation was tested. The breeding r e s u l t was i n close agreement with the p r e d i c t i o n . I t was concluded that the three-locus model, with s e n s i t i v i t y being a recessive t r a i t , would define the s e n s i t i v i t y of the CBA/J s t r a i n to acetazolamide-induced ectrodactyly when compared with the resistance of the SWV s t r a i n . The acetazolamide-induced ectrodactyly and f e t a l death appeared to be independent responses. CHAPTER VII EMBRYO TRANSFER EXPERIMENTS TO INVESTIGATE THE ACETAZOLAMIDE RESPONSE OF THE CBA/J AND SWV STRAINS There i s compelling evidence that suggests acetazolamide acts d i r e c t l y on the embryo. Studies with Intrauterine a p p l i c a t i o n of acetazolamide i n rats (Scott, 1970) and hamsters (Layton, 1972, personal communication) indicated that more ectrodactyly was produced when the teratogen was deposited at s i t e s closer to the embryo. The extensive pharmacological studies of Maren and E l l i s o n (Maren and E l l i s o n , 1972a, 1972b, 1972c; E l l i s o n and Maren, 1972a, 1972b) with rats defined two phys i o l o g i c a l actions that were required, at l e a s t i n the dam, to Induce ectrodactyly i n the embryo: carbonic anhydrase i n h i b i t i o n and a potassium imbalance. They also presented evidence that these actions took place i n the embryo. Suzuki and Takano (19&9) were able to demonstrate that the carbonic anhydrase enzyme was present and active i n the limb bud of the rat embryo at the acetazolamlde-sensltive stage and that i t could be i n h i b i t e d by acetazolamide administered to the dam. In th i s study, no malformed SWV or SWV.CBA F^ embryos were found i n treated SWV dams. I f the sensitive CBA/J embryos, transferred to SWV surrogate dams, responded with ectrodactyly to acetazolamide treatment, t h i s would 56 57 demonstrate that: (1) the teratogenic environment f o r the Induction of ectrodactyly does e x i s t i n SWV dams and (2) the resistance of the SWV s t r a i n to acetazolamide i s a resistance of the SWV embryo proper. Materials and Methods 1. Embryo transfer procedure. The embryo transfer procedure was an asynchronous transfer of day 3 embryos (at the "blastocyst stage) to day 2 pseudopregnant r e c i p i e n t dams. The techniques were established i n t h i s laboratory by Alan C. Peterson f o r use i n the production of a r t i f i c i a l mosaic or chimaerlc mice using the protocols described by Mullen and Whltten (1972). The embryo transfer procedure f o r use i n experimental teratology was more f u l l y described by Takano, Peterson, Biddle and M i l l e r (1972, manuscript i n preparation). 2. Developmental staging of transferred embryos. Normal CBA/J and SWV pregnancies and transfer pregnancies of CBA/J embryos i n SWV dams and SWV embryos i n CBA/J dams were terminated on day 10 between 12 noon and 3 P.M. The developmental age of the embryos was determined by somite number. 58 3. Administration of acetazolamide to dams after embryo  transfer* Acetazolamide was administered intraperitoneally to the recipient dams at 750 vug/kg per injection at 10 A.M. and 4 P.M. on day 10 of their pregnancy. Day 0 in this case was the i n i t i a t i o n of their pseudopregnancy by mating with a vasectomized male in preparation for embryo transfer on day 2. These pregnancies were terminated on day 18 and the embryos were examined as described previously. Results and Discussion The asynchronous transfer of day 3 blastocysts to day 2 pseudopregnant dams in the transfer procedure necessitated the examination of embryonic development later in the transfer pregnancy to determine the time at which to administer the acetazolamide. It was assumed that a morphological rating, based on the somite number, of the transferred embryos that was comparable with those of embryos from normal within-straln matlngs would imply that, at this chronological point in pregnancy, the transferred embryos would have the same teratogenic sensitivity as those in the normal within-strain matings. The means and ranges of the somite number of CBA/J and SWV embryos from normal wlthin-strain matings are presented in Table 14. Their similarity, at least on the basis of this morphological criterion, was an important point to consider 59 TABLE 14 Development of Embryos i n Normal Matlngs and Embryo Transfers Determined by Somite Number Embryo type Somite number, day 10 of pregnancy Mean + s.d. No. embryos Range 1. Normal matings: a. CBA/J b. SWV 26.5 + 1.7 27.4 + 3-3 Transferred embryos: a. CBA/J i n SWV 25.2 b. SWV i n CBA/J 26.8 35 84 5 4 23 21 17 25 31 34 31 29 60 with respect to the response to acetazolamide of these two strains. Their respective sensitivity and resistance cannot "by due to gross differences in morphological development. The means and ranges of the somite number of CBA/J embryos transferred to SWV pseudopregnant dams and SWV embryos transferred to CBA/J pseudopregnant dams are also l i s t e d in Table 14. They were essentially identical to those found in the normal pregnancies at this chronological age. Therefore, the two types of transferred embryos, that were chronologically day 11 on day 10 of the recipient dams' pseudopregnancy, were morphologically similar to day 10 embryos in normal pregnancies. Development had been retarded by approximately one day between the time of asynchronous transfer of the day 3 blastocysts to day 2 pseudopregnant dams unt i l their examination on day 10 of the recipient dams' pseudopregnancy. It was decided to administer the acetazolamide to the recipient dams with transferred embryos at 10 A.M. and 4 P.M. on day 10 of their pseudopregnancy. Because of the limited nature of this study, the only classes of acetazolamide-treated and control transfers with the CBA/J and SWV strains were those l i s t e d in Table 15. The SWV embryos transferred to CBA/J dams were.divided into two groups: one treated with acetazolamide, the other without treatment. A l l CBA/J embryos transferred to SWV dams were treated with acetazolamide. In the two small treatment samples, CBA/J embryos in SWV dams responded with ectrodactyly while SWV embryos in CBA/J dams were resistant 61 TABLE 15 Summary of Embryo Transfers with CBA/J and SWV Strains and Treatment with Acetazolamide Observations SWV embryos/ CBA/J embryos/ CBA/J dams SWV dams treated* c o n t r o l * * treated lo Pregnant per operated r e c i p i e n t dams.......... 8/13 8/14 2 0 Implants per transferred embryos i n pregnant dams..... 23/37 20/41 3. Affected l i t t e r s 0/8 0/8 4. Resorptions per implants .... 1/23 4/20 5. Ectrodactyly per l i v e fetuses. 0/22 0 / l6 12/15 47/56 4/12 15/47 6/32 * Treatment = 750 mg/kg per i n j e c t i o n administered l n t r a p e r l t o n e a l l y a t 10 A.M. and 4 P.M. on day 10 of r e c i p i e n t dams' pregnancy. ** Control = no treatment. 62 to the induction of ectrodactyly. The only v a l i d conclusions possible from these data are: (1) the teratogenic environment a f t e r acetazolamide administration does e x i s t i n the SWV dams because the transferred CBA/J embryos responded with ectrodactyly, and (2) the resistance of the SWV s t r a i n to the acetazolamide Induction of ectrodactyly i s a resistance of the SWV embryo because there were no affected SWV embryos i n treated CBA/J dams. It i s argued that the only comparisons that should be made i n embryo transfer experiments such as t h i s are with those that involve embryo transfers. The l i t t e r s ize i n transfer pregnancies i s made small d e l i b e r a t e l y to insure optimal transfer success a f t e r the extensive studies of McLaren and Mlchle (1956) with t h i s technique. Therefore, the proper control f o r the SWV embryos i n treated CBA/J dams should be CBA/J embryos transferred to CBA/J dams and that f o r the CBA/J embryos transferred to SWV dams should be SWV embryos transferred to SWV dams. CHAPTER VIII RESPONSE OF THE CBA/J AND SWV STRAINS TO DICHLORPHENAMIDE From a number of studies, i t appears that a l l potent i n h i b i t o r s of carbonic anhydrase cause the same teratogenic l e s i o n as acetazolamide. Those that have been tested and proven to be teratogenic are dichlorphenamide (Hallesy and Layton, 1967), ethozzolamide (Wilson et a l . , 1968), methazolamide (Maren, 1971), benzolamide (Maren and E l l i s o n , 1972b) and 2-amino-l , 3,4-thladlazole -5-sulfonamide (CL 53^3) (Maren and E l l i s o n , 1972a). I t was decided to t e s t whether the resistance of the SWV s t r a i n to acetazolamlde-induced ectrodactyly would remain with other teratogenic carbonic anhydrase i n h i b i t o r s . Dichlorphenamide was the only one tested. If the SWV s t r a i n maintained i t s resistance with t h i s ectrodactyly-inducing agent, t h i s would indicate that SWV most l i k e l y possessed a generalized resistance to the teratogenic action of t h i s class of drugs. Also, further biochemical comparison of the SWV s t r a i n with s e n s i t i v e s t r a i n s of mice would uncover the nature of t h i s teratogenic resistance and,' i n turn, point out the cause of the teratogenic s e n s i t i v i t y of other s t r a i n s . 63 Materials and Methods 1. Preparation of dichlorphenamide solution for injection. The following directions for solution of dichlorphenamide were supplied by Dr. John E. Baer (Merck Sharp and Dohme). To 300 mg of dichlorphenamide were added 1.0 ml of 1 N sodium hydroxide and the suspension was warmed unti l i t dissolved. The f i n a l volume was adjusted to 6.0 ml with d i s t i l l e d water resulting in a f i n a l concentration of 5% dichlorphenamide. 2. Administration of dichlorphenamide and fe t a l examination. Dichlorphenamide was administered to pregnant dams at 10 A.M. and k P.M. on day 10 by intraperitoneal injection of 250 mg/kg per injection. With the 5$ solution, 0.005 ml/g mouse was used. The pregnancies were terminated on day 18 and the uterine contents examined as described in Chapter I I I . Results and Discussion Preliminary tests with dichlorphenamide, administered in a single intraperitoneal injection of 500 mg/kg at 1 P.M. on day 10 f a i l e d . This dose resulted in death of both CBA/J and SWV dams. When l t was reduced to 250 mg/kg, administered in the same way, dams of both strains were able to tolerate i t . The CBA/J and SWV dams were also able to tolerate the 250 mg/kg dose of dichlorphenamide administered at 10 A.M. 65 and 4 P.M. on day 10, the same dosage schedule used in most of the acetazolamide studies. The results from this study are li s t e d in Table 16. The CBA/J strain responded with forelimb ectrodactyly while the SWV strain did not. The resorption response was not elevated above the control (no treatment) level found in Table 2 (Chapter I I I ) . Although the acetazolamide dosage regime of 250 mg/kg, administered at 10 A.M. and 4 P.M., was not tried with the CBA/J strain, i t is possible to roughly estimate what the response would be by extrapolation from the graphically displayed dose-response behaviour in Figure 2 (Chapter I I I ) . Approximately 16-17$ ectrodactyly would be expected in CBA/J treated with acetazolamide and this is in the range of the response obtained with dichlorphenamide using this dosage regime. It i s impossible to say unequivocally that dichlorphenamide and acetazolamide have no teratogenic effect on the SWV strain embryo. A l l attempts to use increasingly higher doses to test for teratogenesis were stopped due to death of the SWV dams; however, no obvious f e t a l effects were found in those SWV dams that survived. The only conclusion should be that, within the dosage levels used that were teratogenic to the CBA/J strain, the SWV strain was resistant to any teratogenic or embryotoxlc effects. Since two different potent carbonic anhydrase inhibitors were examined and demonstrated to be teratogenic In the CBA/J strain, the resistance of the SWV strain embryo is most probably a general resistance to this class of teratogens. 66 TABLE 16 Effect of Dichlorphenamide on CBA/J and SWV Strain* Affected Resorptions/ Ectrodactyly/ l i t t e r s implants live fetuses No. % No. CBA/J 3/8 10/72 14 11/72 18 SWV 0/6 9/85 11 0/76 0 * Treatment = 250 mg/kg per injection administered intraperitoneally at 10 A.M. and 4 P.M. on day 10. CHAPTER IX GENERAL DISCUSSION 1. S t r a i n v a r i a t i o n s In teratogenic responses. Other s t r a i n v a r i a t i o n s i n the mouse to d i f f e r e n t teratogenic actions are known and have been reviewed by Smlthberg (1967). The review i s several years old but the state of the a r t has not advanced much i n the intervening years. In examining the studies i n which the nature of these s t r a i n v a r i a t i o n s was Investigated, a very severe c r i t i c i s m can be l a i d . A l l , except Dagg et; a l . (1966) have based t h e i r approaches to the genetic analysis on "one-gene genetics". When they, did not obtain evidence from breeding tests f o r the recovery of the inbred s t r a i n phenotype (high and low frequency responding phenotypes), they stopped t h e i r investigations. Very heavy reliance was placed on f i n d i n g a segregation of discrete response frequencies. Dagg e_t a l . (1966) examined the heritable differences between two st r a i n s of mice which d i f f e r e d In t h e i r responses to induced c l e f t palate and Induced hindlimb malformations by 5 - f l u o r o u r a c i l . Their backcross breeding scheme and t h e i r employment of Wright's (1934) formula f o r the estimation of the number of genetic factors involved i n the t r a i t s were used as guidelines f o r the present 67 68 analysis of the s t r a i n v a r i a t i o n i n acetazolamide-induced ectrodactyly. Dagg and his co-workers estimated that a minimum of three and four l o c i , respectively, were involved i n the induction of c l e f t palate and malformed hlndlimbs by 5 - f l u o r o u r a c i l . However, these were conservative estimates since t h e i r estimates of the non-heritable environmental variances of these t r a i t s were based on averages of variances of the two inbred s t r a i n and F^ s i r e s and not on the F^ s i r e s alone. The F^ s i r e variances f o r the two t r a i t s were very large compared with those of the two Inbred s t r a i n s i r e s and the averaging procedure reduced the estimates of the environmental variances by h a l f . Consequently, i f the estimates of the number of l o c i involved i n the t r a i t s of Induced c l e f t palate and malformed hindfeet were based on the environmental variances derived from the F^ s i r e variances alone, the estimates would be doubled to approximately s i x and eight l o c i , r e s p e c t i v e l y . There i s no j u s t i f i c a t i o n f o r the use of one method of c a l c u l a t i o n over the other since the e f f e c t s of the i n d i v i d u a l genes Is not known and the technique i s based on the assumption that each locus Involved i n a t r a i t has an equal e f f e c t on i t . The conservative estimates of three and four l o c i , however,, tend to bias the i n t e r p r e t a t i o n of these t r a i t s f o r those not i n i t i a t e d i n t h i s blometrical approach to genetic a n a l y s i s . The s t r a i n v a r i a t i o n i n the cortisone induction of c l e f t palate i s a well-known t e r a t o l o g i c a l phenomenon i n mice and best serves to i l l u s t r a t e most of the other 69 investigations that have been made of s t r a i n v a r i a t i o n s i n t e r a t o l o g i c a l responses. Fraser and Falnstat (1951) demonstrated that the A and C 5 7 B L s t r a i n s e x h i b i t a high and low frequency, respectively, of cortisone-induced c l e f t palate. K a l t e r (1954) attempted a genetic analysis of t h i s v a r i a t i o n . He demonstrated that the s u s c e p t i b i l i t y to c l e f t palate induction was not a cytoplasmlcally-transmltted t r a i t and that both the genotype (that i s the inbred strain) of the dam and the genotype of the embryo played a role i n t h i s c l e f t palate induction. However, no meaningful analysis of these t r a i t s was attempted since Kalter f e l t that i n s u f f i c i e n t progeny could be c o l l e c t e d from any one female to c l a s s i f y the dam f o r type of response. He allowed the cortisone-treated pregnancies to go to term and scored the progeny at b i r t h f o r the presence of c l e f t palate. I t would have been r e l a t i v e l y easy to re-mate and treat the dams several times to c o l l e c t s u f f i c i e n t progeny. K a l t e r 1 s breeding design was a backcross to the sensitive A s t r a i n s i r e s . The data from r e c i p r o c a l F^ dams, backcrossed to the A s i r e s , ruled out a maternal cytoplasmlcally-transmltted s e n s i t i v i t y f a c t o r . He then performed one more backcross of BC^ females to the A s i r e s . I f Kalter's data are examined i n the l i g h t of the present study of acetazolamide-induced ectrodactyly, an i n t e r e s t i n g observation r e s u l t s . If the progeny mean frequency response f o r induced c l e f t palate are examined i n a l o g i c a l progression of mating types i n which the cortisone treatment of the dam was begun on day 11, the r e s u l t s i n Table 1 ? are obtained. I t i s postulated that two independent l o c i control the t r a i t of teratogenic s e n s i t i v i t y to cortisone i n the embryo and that resistance i s a dominant (or e s s e n t i a l l y dominant) t r a i t . Based on the 1 0 0 $ c l e f t palate response of the A s t r a i n embryos, the expected BC^ (cross 2, Table 1 7 ) and BCg (cross 3, Table 1 7 ) progeny mean frequencies of c l e f t palate would be 2 5 . 0 $ and 56.3$. r e s p e c t i v e l y . These predicted mean frequencies f o r the two backcross generations are too close to the observed frequencies from Kalter*s (1954) study (Table 1 7 ) • A r e i n v e s t i g a t i o n of t h i s c l e f t palate phenomenon should be made. In the case of cortisone induction of c l e f t palate, the genotype (that i s the inbred strain) of the dam influences the frequency of induced c l e f t palate i n the progeny. For example, i n the case of the se n s i t i v e A and the r e l a t i v e l y r e s i s t a n t C57BL s t r a i n s , the response of the F^ embryo i s greater i n sensitive A s t r a i n dams than i t i s i n the more re s i s t a n t C57BL s t r a i n dams. Both segregation of this t r a i t i n the dam and segregation of the s e n s i t i v i t y factors i n the embryo cannot be examined at the same time. Therefore, f o r the t r a i t of c l e f t palate induction i n the embryo, the genotype of the dam must be kept constant and segregation of the t r a i t must be followed through the s i r e . Unfortunately, K a l t e r 1 s breeding program, by necessity f o r his main study of the dam e f f e c t , was the opposite and segregation was TABLE 17 Cortisone-Induced Cleft Palate In Progeny of Backcross Matings with the A and C57BL Strains* Dam Sire Progeny Cleft palate No. in live fetuses % 1. C57BL A Fl 3/82 3.66 2. P l A 18/71 25.35 3. A BC2 51/91 56.04 4. A A A 36/36 100. * Data are from Kalter (1954) for mating types in which cortisone treatment of the pregnant dam was begun on day 11. followed through the dam. Therefore, the coincidence of the observed and expected generation mean frequencies of Induced c l e f t palate with a two-locus model may be simply f o r t u i t o u s . The important information that i s missing from Kalter's (1954) data i s some measure of the d i s t r i b u t i o n of a f f e c t e d and normal progeny per l i t t e r from which estimates of the genetic and non-genetic variances can be made. From these d i s t r i b u t i o n s , It would be possible to see what spread of v a r i a t i o n was present i n progeny generations where the t r a i t f o r s e n s i t i v i t y would be segregating (crosses 2 and 3 i n Table 1 7 ) . There are further arguments f o r a r e i n v e s t i g a t i o n of the s t r a i n v a r i a t i o n i n cortisone-induced c l e f t palate from the work of Loevy (1963. 1 9 6 8 ) with the sensitive Strong's A s t r a i n and two l e s s - s e n s i t i v e s t r a i n s , G3H and BALB/c. For some reason, unknown to me, Loevy (1963) used the Strong's A and C3H strains i n a breeding program that extended to the examination of an F^ generation treated i n utero with cortisone. From her data, Loevy could only state that "One can only speculate at t h i s time as to the genotypic background needed f o r the expression of induced c l e f t palate. I t c l e a r l y involves more than one gene, and perhaps at l e a s t three." There was absolutely no evidence or analysis made for the l a s t part of her statement. Unfortunately, t h i s did not prevent Smithberg ( I967), i n his review of s t r a i n v a r i a b i l i t y of teratogenic responses, from stat i n g that "Loevy (I963) had estimated that a minimum of 73 three pairs of genes were responsible f o r c l e f t palate." Loevy (1968) examined the cortisone induction of c l e f t palate i n backcross programs between (1) Strong's A s t r a i n and G3H and (2) Strong's A s t r a i n and BALB/c. Her crosses extended only to the BC^ progeny and no analysis was performed other than a demonstration that there are s t r a i n differences i n the induction of c l e f t palate i n the embryo and that the genotype of the dam also influences the embryonic s e n s i t i v i t y . Her conclusions were e s s e n t i a l l y s i m i l a r to those of Kalter (1954). By using other s t r a i n s , she demonstrated that there i s considerable s t r a i n v a r i a t i o n i n teratogenic response to cortisone. However, upon reappraisal of some of her data, there i s l i m i t e d support f o r ei t h e r a one- or two-locus mode of inheritance of the t r a i t of embryonic s e n s i t i v i t y based on the same assumptions used to re-evaluate Kalter's data. 2. Genetic model f o r acetazolamlde-lnduced ectrodactyly. The present study has suggested a three-locus model f o r the inheritance of s e n s i t i v i t y to the induction of ectrodactyly by acetazolamide when the CBA/J s t r a i n i s compared with SWV. I t i s conceivable that other genetic models than that of three l o c i may be possible to define the induction of ectrodactyly by acetazolamide. With the dosage regime used i n the present breeding program with the CBA/J and SWV s t r a i n s , there was a small but d e f i n i t e response of the heterozygous CBA.SWV F embryo but 74 no affected SWV.CBA F^ embryos were found, although, i n t h i s l a t t e r case, the sample size was small. Does t h i s imply a difference i n the teratogenic environment of the CBA/J and SWV dams with the same dosage (that i s a dam effect) or i s i t simply a function of sample size? The embryo transfer study suggested that the teratogenic environment a f t e r acetazolamide administration was present i n the SWV dam but i t was not designed to test subtle differences between CBA/J and SWV dams. The three-locus model Is based on the genes f o r teratogenic resistance being dominant and therefore no affected F^ embryos are expected. If a lower dosage of acetazolamide was used so that no treated CBA.SWV F^ embryos would exhi b i t ectrodactyly, would the three-locus model s t i l l f i t the corresponding induced ectrodactyly data from subsequent backcross generations that would r e s u l t from t h i s lower dosage? These questions must be tested to further investigate the phenomenon of s t r a i n s e n s i t i v i t y and resistance to acetazolamlde. S t r a i n responses to the ectrodactyly induction of acetazolamide, d i f f e r e n t from that of CBA/J, are known from a preliminary study by Cathy C. Azar and Dr. Margaret C. Green of the Jackson Laboratory (Green, 1972, personal communication). Using the acetazolamide dosage regime of 750 mg/kg administered i n t r a p e r i t o n e a l l y at 10 A.M. and 4 P.M., they found that C57BL/6J had approximately the same s e n s i t i v i t y as CBA/J "but C57BL/6J was more sensitive on day 9 than on day 10. They also confirmed the findings of the present study that CBA/J was more sensitive on day 10 than on day 9. Ectrodactyly was found i n the C3H/HeJ, SWR/J (not to be confused with SWV) and DBA/2J s t r a i n s when treated on day 9 and i n the A/J s t r a i n when treated on eith e r day 9 or day 10. The order of decreasing s e n s i t i v i t y was C57BL/6J, C3H/HeJ and SWR/J but i n s u f f i c i e n t data were av a i l a b l e f o r the DBA/2J and A/J st r a i n s to e f f e c t i v e l y rank them. Dr. W. M. Layton, i n personal communication, found that a s t r a i n of mice with the s i t u s inversus mutant i s more sensitive to acetazolamide on day 9 than on day 10. Suzuki and Takano (1969) a l s o found t h i s with the ICR-JCL l i n e of mice. Therefore, considerable s t r a i n v a r i a b i l i t y e x i s t s both i n the frequency of response to acetazolamide and the time of action of acetazolamide. This implies that more genetic v a r i a b i l i t y e x i s t s than simply the CBA/J s e n s i t i v i t y and the SWV resistance and that the i n v e s t i g a t i o n of the genetic differences between these two s t r a i n s , reported i n t h i s study, should only be considered to be the beginning of t h i s a n a l y s i s . Sewal Wright (1934) l a i d a serious charge against many ge n e t i c i s t s , the implications of which have been ignored i n most investigations of the genetics of s t r a i n v a r i a t i o n s i n teratogenic responses. He stated: There are many cases i n slow-breeding animals i n which a gene has been designated 76 on no more basis than dominance i n of a cross between true breeding s t r a i n s , a 3:1 r a t i o i n F? and a 1:1 r a t i o i n the backcross to the recessive s t r a i n . There i s , however, no conclusive evidence f o r or against i t s existence u n t i l breeding tests have been made of the segregating generation. (p. 538) In the present study, both approaches to the genetic analysis of the backcross program suggested three independent l o c i were responsible f o r the t r a i t of teratogenic s e n s i t i v i t y . Predictions from the model were confirmed by a further backcross generation. In addition, two BCg s i r e s produced BC^ progenies from CBA/J dams with ectrodactyly response frequencies comparable to that of the CBA/J s i r e s mated to CBA/J dams and suggested that the CBA/J genotype had been recovered. However, further breeding tests with these two BCg s i r e s would be required to demonstrate that they do breed true and the r e s u l t i n g ectrodactyly response of th e i r progeny was that of the CBA/J s t r a i n and no more. I t would be extremely i n t e r e s t i n g i f , by deliberate s e l e c t i o n , the frequency of acetazolamide-induced ectrodactyly i n a l i n e of mice derived from these two BCg s i r e s could be raised s i g n i f i c a n t l y above that of the CBA/J Inbred s t r a i n . Consideration of the studies of Waddington (1953) with Drosophlla melanogaster, In which he described the phenomenon of "genetic a s s i m i l a t i o n " suggests that t h i s might be possible with the acetazolamide-induced ectrodactyly. Waddington (1953) was able to induce a low frequency of 77 the crossveinless t r a i t "by heat shock of the pupae i n a wild stock that did not exhibit t h i s t r a i t normally. Selection for and against the appearance of the phenocopy rapid l y Increased and decreased, respectively, i t s frequency and, a f t e r lk generations i n the upward selected s t r a i n , f l i e s were found that expressed the t r a i t even when they had not been exposed to the heat shock. Further s e l e c t i o n , without the heat shock, increased and fixed the spontaneous frequency of the crossveinless t r a i t at almost complete penetrance. Therefore, the genes which controlled the expression of a previously induced t r a i t were able to be selected or assimilated and, with continued s e l e c t i o n , they allowed f o r the expression of the t r a i t even i n the absence of the abnormal environmental stimulus. There were no crossveinless f l i e s found i n the o r i g i n a l stock used by Waddington (1953)• However, Bateman (Waddington, 1957, p. 177), who repeated t h i s experiment with a d i f f e r e n t wild stock, checked a l a r g e r number of the s t a r t i n g population and found a very low percentage (less than 1%) with a n a t u r a l l y occurring "low grade crossveinless" phenotype. From these few i n d i v i d u a l s , a crossveinless stock was selected and when tested against, the assimilated crossveinless stock derived from subsequent heat shock of the pupae and s e l e c t i o n of crossveinless adults, the two appeared to be g e n e t i c a l l y i d e n t i c a l . Therefore, the genes for the crossveinless phenotype were already present i n the o r i g i n a l populations but i n low frequency so that only r a r e l y did i n d i v i d u a l s have s u f f i c i e n t of them to express the crossveinless t r a i t . In most other phenocopy t r a i t s studied "by these procedures, there were no abnormal i n d i v i d u a l s i n the s t a r t i n g populations (Waddington, 1957). A s i m i l a r study with the acetazolamide induction of ectrodactyly would provide further information on the nature of t h i s induced t r a i t . If i t i s possible, by s e l e c t i o n , to change the threshold f o r t h i s induced t r a i t beyond that found i n any sensitive Inbred s t r a i n , a more r a t i o n a l discussion can be made of the d i f f e r e n t response frequencies that are found i n d i f f e r e n t s e n s i t i v e inbred s t r a i n s of mice. I t may be that d i f f e r e n t strains have d i f f e r e n t g e n e t i c a l l y determined thresholds f o r t h i s induction of ectrodactyly. I t should be pointed out that genetic a s s i m i l a t i o n , s t a r t i n g with inbred l i n e s of f l i e s , proved unsuccessful (Waddington, 1957). Also, s e l e c t i o n of various quantitative t r a i t s i n Inbred st r a i n s of mice has been unsuccessful (Roderick and Schlager, 1966). Therefore, any attempts to se l e c t a high frequency, acetazolamide-responding s t r a i n of mice would be successful only with an a r t i f i c i a l l y heterogeneous stock derived from outcrossing several d i f f e r e n t inbred s t r a i n s . With respect to the p o s s i b i l i t y of s e l e c t i n g f o r an increase i n the frequency of acetazolamide-induced ectrodactyly, the mutant posta x i a l hemimelia (gene symbol px) should be discussed. Postaxial hemimelia i s a simple, Mendelian recessive t r a i t and i s mapped i n linkage group XI (Searle, 1964). The homozygotes are s t e r i l e but have many c h a r a c t e r i s t i c s i n common with the acetazolamide-induced ectrodactyly syndrome. The most common defect i n px/px homozygotes i s a l o s s of d i g i t V on each forelimb. More severe cases show losses of d i g i t s IV and V and d i g i t s I I I , IV and V. There i s a r i g h t sided predominance to the severity of malformations. In addition, there are ulnar, humeral and scapular defects and a few cases of hindlimb ectrodactyly and syndactyly. The coincidence of the c h a r a c t e r i s t i c s of postaxlal hemimelia and acetazolamide-induced ectrodactyly i s too obvious and warrants a continued and more extensive in v e s t i g a t i o n of the ectrodactyly syndrome that i s induced i n mice by acetazolamide and other s i m i l a r carbonic anhydrase i n h i b i t o r s . 3. Biochemical basis f o r s t r a i n differences i n response to  acetazolamide. The basis f o r the resistance of the SWV s t r a i n to the induction of ectrodactyly by acetazolamide i s unknown. I t i s most probably a general resistance to other potent carbonic anhydrase i n h i b i t o r s since SWV i s r e s i s t a n t to the induction df ectrodactyly by dichlorphenamide. Since i n h i b i t i o n of carbonic anhydrase i s the only known biochemical function of t h i s group of compounds, a good working hypothesis i s that SWV has a r e s i s t a n t form of t h i s enzyme. The hypothesis that the sens i t i v e CBA/J and r e s i s t a n t SWV s t r a i n s may d i f f e r with respect to a sensitive and 80 r e s i s t a n t carbonic anhydrase isoenzyme was tested by looking f o r electrophoretic differences i n t h i s enzyme i n adults of these two s t r a i n s . There are known, g e n e t i c a l l y -determined electrophoretic variants of carbonic anhydrase i n the mouse which were f i r s t i s o l a t e d from the erythrocytes (Biddle and Petras, 1 Q 67; Biddle and Krasny, 1970); however, both CBA/J and SWV have the Pro-lb form of t h i s carbonic anhydrase isoenzyme and t h i s isoenzyme from both strains i s i n h i b i t e d by acetazolamide on the starch gel a f t e r electrophoresis. Another point i s that the C57BL/6J s t r a i n which i s sensitive to the teratogenic action of acetazolamlde (Green, 1972, personal communication) has the Pro-la form of t h i s isoenzyme. Therefore, the Pro -1 carbonic anhydrase locus i s not involved with t h i s teratogenic resistance. This does not eliminate the role of carbonic anhydrase i n t h i s teratogenic action since, i n most mammalian species examined, several forms of the enzyme under separate genetic control have been i d e n t i f i e d (Tashian, S h r e f f l e r and Shows, 1968). One erythrocytic form of the enzyme has high carbon dioxide hydration a c t i v i t y and low esterase a c t i v i t y while the other major form has low carbon dioxide hydration a c t i v i t y and high esterase a c t i v i t y . The ,types of carbonic anhydrase enzymes i n other tissues have not been examined i n the same way; however, examinations of fractionated l i v e r preparations i n several species have revealed several s t r i k i n g f i n d i ngs. Maren, E l l i s o n , F e l l n e r and Graham (1966) found a supernatant f r a c t i o n i n rat l i v e r , amounting to about 81 20% of the t o t a l a c t i v i t y , that was almost r e f r a c t o r y to i n h i b i t i o n by various sulfonamides such as acetazolamide but dog l i v e r did not have t h i s . They reported that mouse (no s t r a i n mentioned) and rabbit l i v e r prepations behaved the same as the r a t l i v e r preparation. I t i s possible that teratogenic resistance of the SWV embryo i s due to resistance of a carbonic anhydrase Isoenzyme. This remains to be tested. D i f f i c u l t y i n t e s t i n g t h i s hypothesis w i l l a r i s e because the enzyme assays used by Wilson, Maren, Takano and E l l i s o n (1968) on r a t embryos f a i l e d to detect any enzyme a c t i v i t y u n t i l several days beyond the sensitive period. However, i t should be possible to r e f i n e the techniques and test t h i s hypothesis. CHAPTER X SUMMARY 1. The SWV Inbred strain of mice was found to be resistant to the teratogenic action of acetazolamlde, a potent carbonic anhydrase inhibitor. 2. The time of teratogenic action was examined in the sensitive CBA/J strain and mid-day 10 of pregnancy was found to be the most sensitive stage for the induction of ectrodactyly which i s the malformation produced by acetazolamide. 3. The dose response to acetazolamide was examined in both the sensitive CBA/J and resistant SWV strains. Within the dosage range examined, the frequency of ectrodactyly in live CBA/J fetuses Increased in an apparent linear fashion with increased dose of acetazolamide while the resorption frequency increased in a curvilinear fashion. No acetazolamide-induced ectrodactyly or fetal death was found with the SWV strain. However, death of the SWV dam occurred at the highest dose examined. 4. Reciprocal crosses with the resistant SWV and sensitive CBA/J strains indicated that resistance to the teratogenic action of acetazolamide i s dominant to sensitivity. 5. Both a metrical analysis of the backcross program 82 83 using the sensi t i v e CBA/J dam and an examination of the generation mean frequency responses i n thi s backcross program suggested that three independently segregating l o c i c o n trolled the t r a i t of embryonic s e n s i t i v i t y to the teratogenic action of acetazolamide. 6. The predicted mean frequency of induced ectrodactyly of the BC^ generation from t h i s three-locus model was demonstrated by further breeding t e s t s . The sensitive CBA/J s t r a i n response frequency was recovered from two BCg s i r e s . 7. No c o r r e l a t i o n was obvious between ectrodactyly induction and f e t a l death a f t e r acetazolamide administration. The two t r a i t s appear to be separate responses to t h i s teratogen. 8. A cursory employment of the embryo transfer procedure with the CBA/J and SWV st r a i n s demonstrated that the teratogenic environment a f t e r acetazolamide administration was present i n the r e s i s t a n t SWV s t r a i n dam and that resistance of t h i s s t r a i n i s a property of the embryo. 9 . The teratogenic s e n s i t i v i t y and resistance of the CBA/J and SWV stra i n s was demonstrated to be the same f o r dichlorphenamide, another potent carbonic anhydrase i n h i b i t o r . 10. The re s u l t s of the above studies were discussed i n the context of other studies of genetic v a r i a t i o n i n teratogenic responses. Further experiments were proposed to test the genetic model presented i n this study f o r 84 acetazolamide-induced ectrodactyly. Variations i n the carbonic anhydrase enzyme were b r i e f l y discussed to gain some perspective of the v a r i a t i o n s i n the teratogenic response to i t s I n h i b i t o r s . BIBLIOGRAPHY Biddle, F. G. and H. Krasny. 1970. The association of carbonic anhydrase a c t i v i t y with the non-hemoglobin erythrocytic protein (Pro-1) of the house mouse. Can. J . Genet. Gytol. 12: 374 (abstract). Biddle, F. G. and J . R. M i l l e r . 1972. Genetic analysis of the resistance to the teratogenic action of acetazolamide i n mice. Teratology j?: 251 (abstract). Biddle, F. G. and M. L. Petras. 1967. The inheritance of a non-hemoglobin erythrocytic protein i n Mus musoulus. Genetics j>7_: 943-949. Dagg, C. P., G. Schlager and A. Doerr. 1966. Polygenic control of the teratogenicity of 5 - f l u o r o u r a c i l i n mice. Genetics jQ: 1101-1117. E l l i s o n , A. G. and T. H. Maren. 1972a. The ef f e c t s of metabolic a l t e r a t i o n s on teratogenesis. Johns Hopkins Med. J . 1J0: 87-94. E l l i s o n , A. C. and T. H. Maren. 1972b. The e f f e c t of potassium metabolism on acetazolamide-induced teratogenesis. Johns Hopkins Med. J . 130: 105-115. Falconer, D. S. 196l. Introduction to Quantitative Genetics. Ronald Press Co., New York. Francis, B. M. 1971• Inheritance of s e n s i t i v i t y to the teratogenic and embryocidal e f f e c t s of cortisone i n four str a i n s of mice. Ph.D. Thesis, University of Michigan, Ann Arbor. Fraser, F. C. and T. D. Fa l n s t a t . 1951. Production of congenital defects i n the off s p r i n g of pregnant mice treated with cortisone. P e d i a t r i c s 8: 527-533. Green, M. C. 1972. The Jackson Laboratory, Bar Harbor, Maine. Personal communication of unpublished data. Hallesy, D. W. and W. M. Layton. 1967. Forelimb deformity of o f f s p r i n g of rats give dichlorphenamide during pregnancy. Proc. Soc. Exp. B i o l . Med. 126: 6-8. Kalter, H. 195^. The inheritance of s u s c e p t i b i l i t y to the teratogenic action of cortisone i n mice. Genetics 39' 185-196. 85 86 Kalter, H. I965. Interplay of I n t r i n s i c and e x t r i n s i c f a c t o r s . Ghapt. 3. In: Teratology; P r i n c i p l e s and Techniques. J . G. Wilson and J . Warkany, ed. Univ. Chicago Press, Chicago. Layton, W. M. 1971. Teratogenic action of acetazolamide i n golden hamsters. Teratology 4: 95-102. Layton, W. M. 1972. Dartmouth Medical School, Hanover, New Hampshire. Personal communication of unpublished data. Layton, W. M. and D. W. Hallesy. 1965a. Deformity of forelimb i n rats: a ssociation with high doses of acetazolamide. Science 149: 306-308. Layton, W. M. and D. W. Hallesy. 1965b. Reply to c r i t i c i s m of T. H. Maren (1965). Science _15_0: 79. Loevy, H. 1963- Genetic influences on Induced c l e f t palate i n d i f f e r e n t s t r a i n s of mice. Anat. Rec. 145: 117-122. Loevy, H. 1968. Cortisone-induced teratogenic e f f e c t s i n mice. Proc. Soc. Exp. B i o l . Med. 128: 841-844. Maren, T. H. 1965. Forelimb deformity i n r a t s : a s s o c i a t i o n with acetazolamide. Science 150 79. Maren, T. H. 1967. Carbonic anhydrase: chemistry, physiology and i n h i b i t i o n . P h y s i o l . Rev. 4£: 595-781. Maren, T. H. 1971. Teratology and carbonic anhydrase i n h i b i t i o n . Arch. Ophthal. 8£: 1-2 ( e d i t o r i a l ) . Maren, T. H. and A. C. E l l i s o n . 1972a. The t e r a t o l o g i c a l e f f e c t of ce r t a i n thiadiazoles related to acetazolamide, with a note on sulfanilamide and thiazide d i u r e t i c s . Johns Hopkins Med. J . 130: 95-104. Maren, T. H. and A. C. E l l i s o n . 1972b. The teratological effect of benzolamide, a new carbonic anhydrase inhibitor. Johns Hopkins Med. J . 130: 116-123. Maren, T. H. and A. C. E l l i s o n . 1972c. The effects of acetazolamide and amilorlde on tissue electrolytes, with a note on the teratogenesis problem. J . Pharmacol. Exp. Ther. 181: 212-218. Maren, T. H., A. C. E l l i s o n , S. K. Fellner and W. B. Graham. 1966. A study of hepatic carbonic anhydrase. Mol. Pharmacol. 2: 144-157. 87 Maren, T. H., E. Mayer and B. C. Wadworth. 1954. Carbonic anhydrase i n h i b i t i o n . I. The pharmacology of Diamox, 2-acetylamino-l,3»4-thiadiazole-5-sulfonamlde. B u l l . Johns Hopkins Hosp. £5: 199-243. McLaren, A. and D. Michie. 1956. Studies on the transfer of f e r t i l i z e d mouse eggs to uterine foster-mothers. I. Factors a f f e c t i n g the implantation and s u r v i v a l of native and transferred eggs. J . Exp. B i o l . 33'- 394-416. Mosteller, F. and C. Youtz. 1961. Tables of the Freeman-Tukey transformations f o r the binomial and Poisson d i s t r i b u t i o n s . Biometrika 48: 433-440. Mullen, R. J . and W. K. Whitten. 1971. Relationship of genotype and degree of chlmerism i n coat color to sex r a t i o s and gametogenesis i n chimeric mice. J . Exp. Zool. V[8: 165-176. Roderick, T. H. and G. Schlager. 1966. Multiple f a c t o r inheritance. Chapt. 9. In: Biology of the Laboratory Mouse. E. L. Green, ed. McGraw-Hill Book Co., New York. Scott, W. J . 1970. E f f e c t s of intrauterine administration of acetazolamide i n r a t s . Teratology 3_\ 261-268. Searle, A. G. 1964. The genetics and morphology of two 'luxoid 1 mutants i n the house mouse. Genet. Res., Camb. £: 171-197. Smithberg, M. 1967. Teratogenesis i n inbred s t r a i n s of mice. Advances i n Teratology 2: 257-288. Snedecor, G. W. and W. G. Cochran. I967. S t a t i s t i c a l Methods. Iowa State Univ. Press, Ames. Suzuki, M. and K. Takano. I969. Teratogenic e f f e c t of acetazolamide i n ICR mice. Cong. Anom. Suppl.: 36 (abstract). Takano, K., A. C. Peterson, F. G. Biddle and J . R. M i l l e r . 1972. The use of embryo transplantation i n experimental teratology: s t r a i n v a r i a t i o n i n triamcinolone-lnduced c l e f t palate. Manuscript i n preparation. Takano, K., S. Yokota and T. Nagata. 1971. Histochemical demonstration of carbonic anhydrase a c t i v i t y i n the r a t limbbud. Anat. Rec. I69: 484 (abstract). Tashian, R. E., D. C. S h r e f f l e r and T. B. Shows. 1968. Genetic and phylogenetlc v a r i a t i o n i n the d i f f e r e n t molecular forms of mammalian erythrocytic carbonic anhydrases. Ann. N. Y. Acad. S c i . 151: 64-77. 88 Vickers, T. H. 1972. Acetazolamlde dysmelia In r a t s . B r i t . J . Exp. Path. $2>. 5-21. Waddington, C. H. 1953. Genetic a s s i m i l a t i o n of an acquired character. Evolution _7_: 118-126. Waddington, G. H. 1957. The Strategy of the Genes. A Discussion of Some Aspects of Theoretical Biology. George A l l e n and Unwin Ltd., London. Wilson, J . G., T. H. Maren, K. Takano and A. E l l i s o n . 1968. Teratogenic action of carbonic anhydrase i n h i b i t o r s i n the r a t . Teratology _1: 51-60. Wright, S. 1934. The r e s u l t of crosses between inbred str a i n s of guinea pigs, d i f f e r i n g i n number of d i g i t s . Genetics 12: 537-551. Wright, S. 1968. Evolution and the Genetics of Populations. Volo I. Genetic and Biometric Foundations. Chapt. 15. The Genetics of Quantitative V a r i a b i l i t y , pp. 373-420. Univ. Chicago Press, Chicago. APPENDIX I D i s t r i b u t i o n of Acetazolamide-induced D i g i t a l Anomalies i n the CBA/J S t r a i n * Class of d i g i t a l anomaly Female Male To t a l 1. Normal 44 41 85 2. R - V abnormal „ 1 3 4 3. L - V abnormal O i l 4. R - V absent ... 31 26 57 5. L - V absent 2 1 3 6. R - V absent L - V absent 0 3 3 7. R - IV,V absent 4 1 5 8. R - III, IV,V absent 2 0 2 9. R - IV,V absent L - V absent 7 0 7 10. R - III abnormal*, IV,V absent L - V absent (* d i g i t III = b i f i d n a i l ) . . . . 1 0 1 11. R - III.IV.V absent L - IV,V absent... o . . . . o 1 0 1 12. R - V absent L - III,IV,V absent 1 0 1 13. R - IV,V absent L - III,IV,V absent 1 0 1 14. R - III,IV,V absent L - III,IV,V absent 1 0 1 Totals 96 76 172 * CBA/J fetuses are from Table 6; R = r i g h t forelimb, L = l e f t forelimb. Acetazolamide was administered i n t r a p e r i t o n e a l l y to the dams at 750 mg/kg at 10 A.M. and 4 P.M. on day 10. 89 

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