sign in sign up
<<
Home , Inosinic acid, Purine analogue

University of Nebraska Medical Center DigitalCommons@UNMC

MD Theses Special Collections

5-1-1964

Potentiation of 6--Induced anomalies by 4-Hydroxypyrazolo-(3,4-d) in the fetal rat

John R. Bragonier University of Nebraska Medical Center

This manuscript is historical in nature and may not reflect current medical research and practice. Search PubMed for current research.

Follow this and additional works at: https://digitalcommons.unmc.edu/mdtheses

Part of the Medical Education Commons

Recommended Citation Bragonier, John R., "Potentiation of 6-Mercaptopurine-Induced anomalies by 4-Hydroxypyrazolo-(3,4-d) pyrimidine in the fetal rat" (1964). MD Theses. 576. https://digitalcommons.unmc.edu/mdtheses/576

This Thesis is brought to you for free and open access by the Special Collections at DigitalCommons@UNMC. It has been accepted for inclusion in MD Theses by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. POTENTIATION OF 6-MERCAPTOPURINE-INDUCED ANOMALIES BY 4-HYDROXYPYRAZOL0-(3,4-d)-PY~IMIDINE IN THE FSTAL RAT

' J: Robert Bragonier

Submitted in Partial Fulfillment for the Degree of Doctor of Medicine College of Medicine, University of Nebraska February 1, 1Y64 Omaha, Nebraska ACKNOiiJLEDG EidEi'•T~ The author wishes to thank those people who assisted in the preparation of this thesis. He is grateful to the Department of Neurology and Psychiatry for the use of its facilities; to Miss Nadine Roesky for reproduction of the charts; to Mrs. H.eba Benschoter and Vrs. Ada Eichhorn for their help with photography and photographic pro­ cessing; and to Miss Hoesky and ~;r. Richard Serpan for their technical assistance. He is appreciative of his wife Barbara, son Bruce, and daughter Becky for their patience and understanding throughout the preparation of the manuscript. Finally, the author is indebted to Dr. Michael J. Carver for his constant encouragement and guidance. TABLE OF CONTENTS

Introduction 1 Historical Review 2 Experimental Teratology 2 General Considerations 2 Classification of Etiologic Factors 3 Significance of Teratologic

Investigation 6

6-Mercaptopurine 9

General Considerations 9

~i.1etabolism 10 Mechanism of Action 12 4-Hydroxypyrazolo-{ 3, 4-d)-pyrimidine 14

¥aterials and Methods 16

Hesults 19 'I·.1scuss1on . . 22

Summary and Conclusions 24

Figures 25

Bibliography 34 INTHODUCTION That 6-mercaptopurine has an inhibitory effect on certain malignant cells is well documented. In microorganisms and mammalian cells, this ana­ logue is oxidized to 6-thiouric acid by the responsible for of purine bases, oxidase. Certain compounds which inhibit potentiate the antitumor effect of 6-rnercapto­ purine noted above. Six-mercaptopurine has also been shown to pro­ duce malformations in fetal rats when injected during the proper stage of embryonic development. Demonstra­ tion that 4-hydroxypyrazolo-(3,4-d)-pyrimidine, a potent xanthine oxidase inhibitor, is non-teratogenic but capable of potentiating the teratogenic effect of 6-mercaptopurine would draw an interesting parallel between the action of 6-mercaptopurine against rapidly growing, relatively undifferentiated cells, out of control, as in malignancy, and under control, as in the developing fetus. The purpose of this investi­ gation was to demonstrate in this manner that such a parallel does exist, in order to clarify the mode of teratogenic action of 6-mercaptopurine.

1 • HISTOtilCAL HEVIEH Experimental Teratology General Considerations Congenital malformations have in recent years been recognized as one of the leading causes of infant mortality and morbidity. In earlier times the con­ genitally deformed were considered monstrosities, evil, objects of the "wrath of God 11 or "acts of the devil". ¥any were non-viable and chiefly of interest to anato­ mists and pathologists. However, modern medicine has effected such changes in the patterns of human mor­ bidity and mortality tnat ten times as many children now die as a result of congenital malformations as of five contagious diseases once greatly feared ll). This relative increase in the incidence of congenital malformations has placed added emphasis on clinical and experimental investigation in the field. Whenever a medical problem exists, reproduction of the pathologic phenomena in animal experiments is of value. In the past, however, experimental produc­ tion of congenital malformations has generally failed to satisfy those interested in the etiology of anoma­ lies in children, although these investigations have been performed in lower animals for nearly a century. Complaints have commonly been that mammals have a 2. co~pletely different maternal-fetal relationship, or that agents used to produce anomalies are unphysiolog1c and without parallel in the human. The latter criti­ cism was applied to the first teratologic metnod prac­ ticed in mammals during the first decade of this cen­ tury: the administration of x-rays to the pregnant mother! Not until about two decades ago when it became possible to produce malformations experimentally in mammals by maternal dietary deficiencies were these arguments to some degree overcome.

Classification of Etiologic Factor~ The field of experimental mammalian teratology is generally acknowledged to date from Hale's work reported in 1~33 (2); piglets from a sow raised on a vitamin A-deficient diet were born without eyes. Since that time literature in the field has grown quite extensive. Only a brief classification of factors known to cause experimental congenital mal­ formations will be presented here; excellent review articles have been prepared by Kalter and Warkany (lJ,

~arkany and Kalter (3J, Hillemann t4J, and ~ilson (SJ. I. Genetic factors (3,4) A. Gene mutations B. Chromosome abnormalities 1. Chromosome excess

3. 2. Chromosome deficiency 3. Translocation of genes 4. Transduction of genes 5. Deletion of genes 6. Crossing-over of genes c. Embryonic instability, or inheritance of susceptibility to congenital

deformity ( 6)

II. Nutritional factors { 1, 4) A. Vitamin deficiency or antagonism

1. Vitamin A ( 2 )

2. Thiamine

3. Riboflavin ( 7 )

4. Niacin ( 0)

5. Pantothenic acid {~)

6. Cobalamin (!U)

7. Folic acid ( 11-14)

8 • Vitamin c 9. Vitamin J) {15)

1 i.). Vitamin E ( 16) B. Vitamin excess

1. Vitamin A {17) c. Amino acid antagonism 1. Leucine (18)

D. Fasting (6,l~J

4. E. antagonism (20-23) F. Galactose \24) III. Hormonal factors (1,4)

A. Pituitary preparations

1. Anterior pituitary extract a. Growth hormone

b. ACTH 2. Posterior pituitary extract B. Estrogen c. Insulin deficiency and excess D. Thyroid deficiency and excess

E. Corticosteroid hormones (2~,26) IV. Mechanical and physical factors A. Amniocentesis (27) B. Direct physical trauma or mechanical injury (4) c. Hyper- and hypothermia (28) D. Anoxia {2:::;) V. Antibiotics (4) A. 3treptomycin B. Tetracycline c. Penicillin

VI. Inorganic materials (4j A. Carbon monoxide B. Carbon dioxide

5. c. Thallium and lead nitrates D. Boric acid

11 11 E. Heavy water tD 2o) VIL. Organic materials and drugs

A. Trypan Blue dye {3UJ

B. Nicotine t31)

c. 5-Hydroxytryptamine {32, 33) D. (34)

E. (35,22) F. (TEM) l36)

G. Triethylenephosphoramide {TEPAJ {36)

H. Various other drugs, factors and ch emi ca 1 s ( 1 , 4 )

VIII. Radiation (37,3dJ Significance of Teratologic Investigation From the preceding representative but incomplete outline, it can be seen that a large variety of methods are now available for the experimental pro­ duction of congenital malformations. The types of anomalies produced and structures affected are

equally as variable. In addition, malformations are but one manifestation of reproductive failure, and these methods are usually also capable of causing other disturbances such as sterility, abortion, fetal resorption, or neonatal death. It is in fact

6. relatively easy to interrupt pregnancy by environ­ mental interference; it is more difficult to obtain viable offspring with congenital malformations, since this requires an experimental arrangement that permits damaging the embryo without killing it. To achieve such balance, the timing, the type of teratogenic agent, and its dosage must all be coordinated. These varied investigations have demonstrated that each embryologic process has its critical period; damage done by a teratogenic procedure depends largely upon which structures are undergoing critical develop­ ment at the time the procedure is applied. Similarly, damage to those structures cannot be produced after the critical period of development has passed. On the other hand, the investigations have clearly shown that teratogens also have their specificities. Acute agents attack the embryo once and demonstrate that specific malformations are produced at particular embryologic stages, while other agents are chronic, staying with the mother throughout pregnancy and acting only when the embryo is sensitive to them. In addition, agent specificity of different teratogens cannot be denied. While one agent may produce a given anomaly with great effectiveness, another may not, even though the agents are applied at the same

7. gestational stage. The action of chronic teratogens is comparable in some respects to that of genes; these hereditary factors are also present throughout the organogenetic period but appear to act only at certain stages of development. All teratogens (both acute and chronic) have other points of resemblance to genes. For instance, some induced malformations simulate hereditary defects,

11 11 that is, are phenocopies • Just as genes often have 11 pleiotropic 11 effects, so teratogens may produce variable syndromes, or cause malformations in a variety of organs that seem unrelated to each other in post­ natal life. From"these points it may be reasonable to deduce that environmental factors sometimes produce phenocopies by altering developmental processes in the same way gene changes or mutations do. Since little is known about the biochemical reactions of genes that lead to malformations, a knowledge of the reactions that lead to their phenocopies may be of value in elucidating the mode of action of faulty genes. For example, vitamins act as prosthetic groups in engaged in vital metabolic activities; if enzymes par­ ticipate in biochemical reactions that determine channels of embryonic development, then absence of a vitamin may result in the interruption of a biochemical 8. sequence, much as the action of a biochemical mutant does, which eventually leads to abnormal channels of development (1). These methods for producing specific malforma­ tions have thus proved of great value as models whereby general mechanisms leading to congenital malformations in mammals, especially humans, can be studied. 6-Mercaptopurine General Considerations The synthesis of 6-mercaptopurine (6-MPJ was first described by Elion and Hitchings {3Y) in 1Y52. It had originally been studied during the systematic investigation of substituted for antagonists of nucleic acid synthesis (40). Attention was focused on the drug when early reports of its inhibitory effects on microorganisms {41), experimental tumors {42), and mouse {43) appeared, and in 1Y53 temporary remissions in certain cases of acute human following 6-MP therapy were reported (44). Since that time, clinical use of this has been well documented and its biologic and bio­ chemical effects extensively studied (45). The teratogenic action of 6-MP in mammals was first reported by Thiersch in 1~54 {46). Following a single oral ingestion of the drug at varying times

9. in their pregnancy, female rats gave birth to large numbers of stunted or stillborn fetuses. No gross malformations were noted in the survivors, but doses administered were small. Fetuses were most sensitive to the effects of the drug on the seventh and eighth day of gestation. In 1Y56 Didcock and coworkers attempted to inter­ rupt pregnancy in rabbits with intra-amniotic injec­ tions of 6-MF {47). The increased number of abortions noted was accompanied by the unexpected presence of fetuses with congenital malformations; the fetuses were viable at term. A detailed study of the teratogenic effects of antitumor compounds was published by Murphy in 1Y60 (20). She reported observing fetal skeletal malformations following single intraperitoneal injections of 6-VP into pregnant rats on the twelfth day of gestation. Sross anomalies were carefully recorded and consisted of stunting, pointed noses, shortened extremities, fused digits, and rudimentary rear legs and tail. This paper provided the technical basis for the present investigation. Metabolism Six-mercaptopurine closely follows the known pathways for anabolism and catabolism of purine bases.

10. The analogue is a competitive inhibitor of xanthine oxidation (48) and is oxidized to 6-thiouric acid by microorganisms and mammalian cells (4~, 50).

Desulfurization occurs in mice, as indicated by sulfate-s35 excretion and labeling of nucleic acid 14 a d enine• an d • f rom 0-,,1,. Mp -v-1,,;.., " l' 45) ; l a.oe' 1 e d and purine have also been recovered in microorganisms following labeled 6-MP administration (49). In addition to 6-thiouric acid, 6-methylmercaptopurine has been identified as an excretion product of 6-MP metabolism (51), and a considerable amount of the drug is excreted in the urine unchanged or bound as a glucuronide (52,53). Conversion of 6-MP to its ribotide has been reported (54-56), indicating that the analogue undergoes at least the initial steps toward incorporation into nucleic acids. This enzymatic conversion is illus- trated in Chart 1, while Chart 2 summarizes the known and postulated pathways of 6-LlP metabolism (57). SH

N~N~ :-OCQH6 Mg+: ~):) +pp; ~".JlN N I 0-®·® ®-OCHQO H OH OH

OH OH Chart 1. ~nzymatic synthesis of b-mercaptopurine ribo­ by reaction of the base with 5-phospho­ ribosyl-1-. 11. Anabolism MP phosphorylase ~ "''---...... _lkinaseJ ~yrophosphorylase ~ 6-Mercaptopurine M~ --- \ inosinic ~ -----polyphosphates~ acid Catabolism xanthine xanthine oxidase oxidase 6-~ercaptopurine 6-thio- > 6-thiouric xanthine acid L 6-methylmercaptopurine sulfate; hypoxanthine; anabolic and cata­ bolic products of Chart 2. The metabolism of 6-mercaptopurine in vivo

Mechanism of Action Prior to 1Y4d it was assumed that nucleic acid purines were entirely synthesized de nova from formate, glycine, carbon dioxide, and ammonium salts. At that time a report appeared which indicated that preformed purines can be interconverted and incorporated into nucleic acids {5d); it was eventually supported by further investigation t5~,6UJ. The exact mechanism of action of 6-MP is unknown, but the best theory is based on the above premise. Six-mercaptopurine and other analogues of and guanine are believed to interfere with incorporation 12. of preformed purines to form nucleic acids and purine-containing coenzymes (2u). Bacterial data indicate that the primary site of action is at the interconversion, through a hypoxanthine derivative, of guanine and adenine . Formation of the ribonucleotides is competitively inhibited by

6-iJ'P ( 61 j. The apparent inl11bi tion of de ~ purine synthesis by 6-~P may be related to interference with the conversion of to appropriate adenine and guanine derivatives, although there is evidence that, in leukemic blood cells, enzyme inhibi­ tion by 6-MP occurs prior to the formation of inosinic acid (62). Another alternative is that blocking of

~~purine synthesis by 6-f11~F is a manifestation of what is now termed feedback inhibition or end-product inhibition (57). A number of investigators have recognized that unlabeled purines prevent or depress the incorporation into the purine structure of labeled precursors.

Ellis and LeFage (63) have shown that cells susceptible to 6-thioguanine incorporate significant amounts of the purine into their nucleic acids, while resistant cells incorporate only minor amounts. The possibility still remains that in addi­ tion to competitively inhibiting normal nucleic acid 13. synthesis in the manner described above, 6-MP may actually be incorporated in toto in place of adenine or guanine into the nucleic acid molecule, as is thioguanine. No studies have been performed to determine the mechanism of action of 6-MP against developing fetal cells; it has been assumed that the drug acts in a similar manner against normal and malignant cells. The present investigation was designed to test that assumption. 4-Hydroxypyrazolo-(3,4-d)-pyrimidine In an attempt to correlate details of chemical structure with activity, a systematic investigation of purines and their analogues has been under way for a number of years (64,65). In the process, syn­ thesis of the pyrazolo-(3,4-d)-pyrimidine ring system was undertaken to provide new compounds isomeric with various biologically active purines. The pyrazolo­ (3,4-d)- differ from the purines by virtue of a pyrazole rather than imidazole ring attached to the pyrimidine moiety (66). At least one of these compounds, 4-aminopyrazolo-(3,4-d)-pyrimidine, an adenine isomer, has demonstrated significant antitumor activity (67). At the same time in parallel studies, a substantial

14. number of these compounds were found to be inhibitors of xanthine oxidase-catalyzed reactions {6d). Since this enzyme catalyzes the oxidation of 6-MP to 6-thio- (SU), Elion and coworkers decided to deter- mine whether 6-MP oxidation could be inhibited in yivo, and whether the apparent potency and possibly the chemotherapeutic index of 6-MP could be altered by this means (52). Four-hydroxypyrazolo-(3,4-d)-pyri- midine (HPP) was the compound chosen for trial; it had high potency as a xanthine oxidase inhibitor, only moderate toxicity, and no antitumor activity against the test systems (67). Chart 3 provides a comparison of the structures of 6-MP and HPP.

6SH 40H 0-...... s 7 ...... c, 9 3 IN C-N 5 N.-- C-C~ I II ~H 8 I II N 2 2C C-N/ 6 C:-... C-N/ ~N/4 9 "- N....-8 H 3 H 7 I 6-MF HPP Chart 3. Comparison of the structures of 6-MP and HPP. Experiments performed on tumor cells in both the mouse and human demonstrated that inhibition of xanthine oxidase activity is attained in vivo when HPP is administered, and that such inhibition results in a decrease in the metabolic destruction of 6-MP (52).

15. MATEN:IALS AND METHODS Pregnant albino rats (Sprague-Dawley strain) were obtained from the Holtzman Xat Company, Madison, Wis- consin. The day the animals were sperm positive was called gestation day zero. They were housed no more than four to a cage in air conditioned quarters and given Kockland rat chow and tap water ad libitum. On gestation day 12, all animals were injected intraperitoneally with a l.J ml suspension of 6-MP 1 and/or HPP 2 in the approximate dosages listed on the following page in Table I. Amounts of each drug injected were one fifth the values listed, as all rats weighed approximately 200 grams, and prior experiments had demonstrated that more precise dosages were not necessary to assure uniform production of anomalies. The drugs were suspended in carboxymethylcellulose due to their insolubility at a physiologic pH. use of this inert gum as a vehicle assured more uniform dosages; its viscosity minimized error caused by settling out of the drugs. On gestation day 20, 1-2 days before parturition, the rats were sacrificed by cervical dislocation and the fetuses delivered by Caesarean section, a procedure

1. Nutritional Biochemicals Corporation, Cleveland, Ohio. 2. Aldrich Chemical Company, Mil·waukee, '.Visconsin.

16. Table I Dosaae Schedule Total N c>". Dosage No. Paternal Fetuses Drug Injected (mg/kg) Hats Injected Examined Control-No Injection 12 125 Contra 1-CMC-l:- ( Ca rboxymethyl cellulose) 13 114

6-Percaptopurine ( 6-'.\P) 60 15 127 4-Hydroxypyrazolo- ( 3, 4-d )-pyrimidine (HFF) 120 5 48

6-i.~t-' plus 6U I-lFF 120 5 57 15 y 83

HFP 30 lu 101

6-!''l-- plus 15 HFP 3U y SJ6 Totals 7'd 7 51 * All drugs were injected in CMC, lU mg/ml.

necessary as experimental young allowed to deliver nor-

mally were eaten by the mother. The fetuses were examined closely for evidence of malformations or still- birth, and the uteri, for resorption sites. Placentas were separated from the fetuses, and the latter were blotted dry, weighed on a Mettler semi-micro balance, and quick-frozen for subsequent study. NO further processing was done prior to x-ray examination.

Fetuses to be stained were transferred to Y5){, 17. for at least 72 hours to assure adequate fixation. They were then carefully skinned and placed in several changes of l~ potassium hydroxide for clearing. This step required 4-7 days; unless the soft tissues were entirely transl~cent, they would take up stain.

U.Ul~ After clearing 1 the fetuses were placed in

Alizarin ~ed 3 stain in l~ potassium hydroxide until the desired depth of staining was noted {approximately 6-12 hours). Clearing was completed in several changes of 2U parts glycerin: 80 parts l~ potassium hydroxide. The fetuses were then transferred through a graded glycerin series and stored in pure glycerin

{6~).

18. riE..:lJLT::l

Figure 1 illustrates the gross appearance after ethanol fixation of a control fetus and one whose mother r-eceived a teratogenic dose of 6-l~'

Though viable at the ti~e of Caesarean section, the latter fetus demonstrates stunting of growth, genera­ lized throughout the body but propor-tionally more severe at the caudal end. Gross shortening of the tail is noted, together with retardation or absence of digital development, or syndactyly. The digital anomaly is more pronounced on the hind legs; at least two toes commonly appear on the forefeet, whereas complete fusion or lack of differentiation of adja­ cent phalanges and metatarsals is the rule on the hind feet.

The presence of an umbilical hernia can be seen; although appearing inconsistently, this malformation was present in over half the fetuses examined, and the abdominal wall defect was in some instances quite extensive. Other inconsistently appearing defects a re 11 s c r a mb 1 e d 11 s t er nab r a e l 71.J ) and an as a r ca • In many fetuses sternal ossification centers could not be seen, but wnen present they were often deformed and irregular. Anasarca was noted only in those fetuses most severely affected.

19. X-rays were taken at different milliamperage, time, and kilovoltage settings and on several types of film to obtain best possible skeletal detail. The films were printed directly (Figure 2) or photo­ graphed and the resulting negative printed (Figure 3). Neither negative nor positive print adequately demon­ strates fetal structJre due to lack of bone density at this stage of development, but stunting of growth and abnormalities of the ribs and spine can be seen.

Differential staining proved to be the better method for visualization of bony structure. Com­ parison of the stained skeletons of control and experimental fetuses reveals in the latter retardation or absence of ossification of metacarpal, metatarsal, and phalangeal centers (Figures 4,~,7) and absence of one of the two lower leg bones (Figure 7). Both radius and ulna, however, were invariably present (Figure 5). 'P.arked branching and fusing of the ribs can be seen (Figure 5), and a degree of spina bifida is suggested by divergence and irregularity of the lateral pro­ cesses of the lumbar and sacral vertebrae, with dupli­ cation of the central vertebral body (Figure 6). Of note was the consistency of the above findings. Although malformations varied to a small extent in severity (probably due to differences in absolute drug

20. dosage between litters and to variations in fetal susceptibility within the litter), no unaffected fetuses were obtained from maternal animals receiving 6-MP at the teratogenic dosage. At the other extreme, no increase in intrauterine death was apparent, as

judged by the lack of increase in numner of still­ births or uterine resorption sites.

One-fourth the teratogenic dosage of 6-~F (15 mg/kg) caused no apparent change in fetal development;

no malformations could be found (Figure 8), and fetal weight was not significantly different from control values 1 • The same results were obtained after the injection of HPP at 30 and 120 mg/kg. However, when

6-~P (15 mg/kg) and H~P (30 mg/kg) were given together,

~alformations identical to those obtained with the

teratogenic dose of 6-I5P (60 mg/kg) alone were noted

(Figures ~,Y). The combination of the teratogenic dose with HPP (120 mg/kg) resulted not in malformations but in rapid fetal death.

1. Unpublished data. 21. D ISCiJSS ION

Four-hydroxypyrazolo-(3,4-d)-pyri~idine has previously been reported to potentiate the antitumor action of 6-mercaptopurine. The data above indicate that this xanthine oxidase inhibitor has a similar ability to potentiate the teratogenic action of 6-MP. This finding suggests the possibility that 6-MP acts at a similar site in malignant and fetal cells and establishes a precedent for consideration of the mechanism of action in both instances as identical.

That anomalies produced following 6-YP adminis­ tration are due to 6-LlP rather than one of its meta­ bolites (53) has not yet been proven, but this investi­ gation indicates the excretory end product, 6-thiouric acid, and the intermediary metabolite of xanthine oxi­ dation, 6-thioxanthine (57), are not the responsible agents. If they were, addition of HPP would prevent or lessen the anomalies obtained, since 6-thiouric acid and 6-thioxanthine would not be formed enzymati­ cally from 6-MP. On the other hand, if 6-MP or one of its metabolites from pathways other tnan that involving xanthine oxidase were the teratogenic agent, addition of HFF would increase the severity of the anomalies or increase the effectiveness of 6-MP at a lower dosage. Both were the case. When HPP was added 22. to the teratogenic aose of 6-~P, fetal death occurred shortly after administration, as demonstrated by small fetal size and extensive resorption. 6econdly, fetal anomalies obtained with one-fourth the teratogenic dose of 6-MP in combination with HPP resembled those obtained with the teratogenic dose of 6-MP alone. On the basis of these findings, further studies are being carried out to determine the exact nature of 6-MP teratogenesis. It is hoped that this investi­ gation may eventually shed light on the broader pro­ blem of drug-induced teratogenesis in the human. In addition, since a parallel has been established between action of 6-~P against fetal and malignant cells, results obtained from study of 6-MP-induced congenital anomalies may find secondary application to problems in the field of cancer chemotherapy and finally, help to elucidate the larger problem of the nature of malignant growth in general.

23. SUMl~ARY AND CONCLUSIONS Congenital malformations are produced in fetal rats by a single intraperitoneal injection of the purine analogue 6-mercaptopurine into pregnant rats at the critical stage of gestation. Photographs and descriptions of these malformations are presented. Administration of 4-hydroxypyrazolo-lJ,4-dJ­ pyr1mi6ine (a xanthine oxidase inhibitor) with non­ teratogenic doses of 6-mercaptopurine results in similar congenital malformations. Combination of the former drug with teratogenic doses of the latter causes fetal death. It has thus been demonstrated that 4-hydroxypyrazolo-(3,4-d)-pyrimidine potent1ates the teratogenic action of 6-mercaptopurine, as well as its antitumor activity, which has been previously reported.

24. Figure 1. Gross appearance after ethanol fixation of a control and experimental fetus at twenty days of gestation. Stunting of growth, shortening of the tail, retardation or absence of development of hind legs and feet, and umbilical hernia can be clearly seen.

25. CONTROL 6-MP 60 MG /KG

-----~------Figure 2. Direct print taken from an x-ray of a control and experimental fetus. The x-ray film was exposed at 30Umilliamperes, 3U kilovolts for one second. Although indistinct, stunting of growth and abnormalities of the ribs and spine can be seen.

26.

Figure 3. Indirect print of fetuses in Figure 2. The x-ray film was photographed and the resulting negative printed. Original x•ray exposure was 3UU milliamperes, 30 kilovolts, for 2.5 seconds.

27. CONTROL 6-MP 60 MG/KG Figure 4. Differential staining of a control and experimental fetus. This method provides much better visualization of skeletal detail. Back­ lighting was produced by photographing the fetuses on an x-ray view box. -- --:

CONTROL 6-MP 60 MG/KG Figure 5. Close-up view of the experimental fetus pictured in Figure 4. Note the marked branching and fusing of ribs. Both radius and ulna can be identified on the right, but one of the two lower leg bones (probably the fibula) is characteristically absent bilaterally.

Figure 6., Close-up view of the spines of exper1-

mental { 6-f·!}, 6U mg/kg) and control fetus es. The experimental fetus has been bisected transversely to allow viewing of the spine in one plane. Lateral processes of the lumbar and sacral verte- w 0 • brae are irregular and can be seen to diver~e; together with duplication of the vertebral centra, this suggests a degiee of spina bifida. • Figure 7. Hind legs from control and experimental {6-IAP, 6U mg/kg) fetuses. rtetarded or absent development of ossification centers, stunting of growth, and absence of one lower leg bone (probably the fibula) are all visible.

31. ...•

r

r Figure a. Effect of HPP. It is evident that while 6-MP (15 mg/kg) and HPP (3U and 12U mg/kg) had no effect on the fetus, the administration of HPP (30 mg/kg) in combination with 6-MP (15 mg/kg) resulted in anomalies identical in type to those caused by 6-MP (6U mg/kg), but to somewhat less severe a degree.

32. ..

oc CONTROL 6-MP H I i 60 MG/KG 120 MG/KG

,, "'

HPP 6-MP 6-MP 15 MG/KG+ 30 MG/KG 15 MG/ KG HP P 30 MG I KG Figure Y. Fetus whose mother received 6-UF llS

mg/kg) and H~P (30 mg/kg) compared with a control fetus. All skeletal anomalies previously described

with 6-rliP (60 mg/kg) can be identified in this. fetus, such as divergence and irregularity of the lateral processes of the lumbar and sacral verte- w w brae with double vertebral centra, marked branching • and f~sing of the ribs, absence of one of the two lower leg bones, and lack of digital aevelopment, with retardation or absence of ossification of metacarpal, metatarsal, and phalangeal centers. '· .. BIBLIOGnAPEY 1. Kalter, H. and Warkany, J.: Experimental Pro­ duction of Congenital Malformations in Mammals by Metabolic Procedure. Physiol. Reviews 3Y:6Y, 1959.

2. Hale, F.: Pigs Born ~ithout Eyeballs. J. Hered. 24:1U5, 1933. 3. Warkany, J. and Kalter, H.: Congenital Malforma­ tions. New Eng. J. Med. 265:YY3, 1046, 1961. 4. Hillemann, H. H.: The Spectrum of Congenital Defect, Experimental and Clinical. J. Appl. Nutrition .!_1:4, 1~61. s. Wilson, J. G.: Factors Involved in Causing Con­ genital Malformations. Bull. N. Y. Acad. Med. 36:145, 1Y6U. 6. Runner, M. N.: Inheritance of Susceptibility to Congenital Deformity--Embryonic Instability. J. Nat. Cancer Inst. ]2:637, 1~54. 7. Warkany, J. and Nelson, R. C.: Appearance of Skeletal Abnormalities in the Offspring of Rats Reared on a Deficient Diet. Science 92:3d3, 1Y4U. 8. Ingalls, T. H. et al: Acquired Chromosomal Anomalies Induced in Mice by Injection of a Teratogen in Pregnancy. Science 14-!_:810, 1963.

9. Evans, H. M. et al: Multiple Congenital Abnor­ malities from Pantothenic Acid Deficiency in the Rat. Fed. Proc • .!2,:549, lY56. 10. O'Dell, B. L. et al: Vitamin B12' a Factor in Prevention of Hydrocephalus in Infant ~ats. Pro c • Soc • Exp • Biol • Med • 7 6: 3 4::1, l !::i !:> 1 • 11. Evans, H. M. et al: Multiple Congenital Abnor­ malities rtesulting from Acute Folic Acid Deficiency during Gestation. Science lU~: 2YY, 1Y51.

34. 12. Asling, c. w. et al: Congenital Skeletal Abnor­ malities in Fetal ~ats Resulting from Maternal f teroylglutamic Acid Deficiency During Gesta­ tion. Anat. Rec. 12l:T/5, lS155.

13. Thiersch, J. B. and Phillips, F. S.: Effect of 4-Amino-Fteroylglutamic Acid () on Early Pregnancy. Proc. Soc. Exp. Biol. Med. 74:204, 1~50. 14. Thiersch, J. B.: Effect of Certain 2,4-Diamino­ pyrimidine Antagonists of Folic Acid on Pregnancy and aat Fetus. Proc. Soc. Exp. Biol. Med. 87:571, l::i54. 15. Warkany, J.: Effect of Maternal riachitogenic Diet on Skeletal Development of Young Rat. Am. J. Dis. Child. ~~:511, 1~43. 16. Callison, E. c. and Orent-Keiles, E.: Abnor­ malities of the Eye Occurring in Young Vita­ min E-deficient ~ats. Proc. Soc. Exp. Biol. Med. 76:2!:>'5, lS15l. 17. Cohlan, s. Q.: Excessive Intake of Vitamin A as a Cause of Congenital Anomalies in the kat. Science 117:535, 1~53. 18. Thiersch, J. B.: Effect of 6-Diazo-5-oxo-L-nor­ leucine (DON) on ~at Litter in Utero. Proc. Soc. Exr. Biol. Med. Y4:33, 1957. l~. t~unner, r-~. N. and J: 1'iller, J. R.: Congenital Deformity in the Mouse as a Consequence of Fasting. Anat. Hee. 124:437, 1Y56.

2u. ~r,urphy, M. L.: Teratogenic :affects of Turnor­ inhibiting Chemicals in the Foetal Hat. (In: ~olstenholme, G. E. W. and O'Connor, c. M., eds., Ciba Foundation Symposium on Congenital Malformations, Little, Brown and Company, Boston, 1Y60, p. 7t>).

21. Thiersch, J. B.: Effect of 2-6 Diaminopurine (2-6 DF): 6-Chloropurine (ClP) and Thio­ guanine (ThG) on Rat Litter in Jtero. Proc. Soc. Exp. Biol. Med. !:J4:4U, l!::i57.

35. 22. Murphy, ~r,. L. and Karnofsky, D. ;\.: Effect of Azaserine and Other Growth-Inhibiting Agents on Fetal Development of the Rat. Cancer ~: 955, 1~56.

23. Thiersch, J. B.: .Effect of o-0iazo-acetyl-L­ serine on rtat Litter. Proc. Soc. Exp. Biol. Afed • :::14: 27, lY.:>7 • 24. Mandrey, J.: Development of Cataract in the Embryonic Lens of the Albino ~at. Anat. Hee. 76:suppl. 2, Y2, 1Y40. 25. Robson, J. M. and Sharaf, A. A.: Effect of Adreno­ corticotroph1c Hormone (ACTH) and Cortisone on Pregnancy. J. Physiol. 116:236, 1952. 26. Gunberg, D. L.: Some tffects of Exogenous Hydro­ cort1sone on ~regnancy in the xat. Anat. i-{eC. 12'.:::1:133, 1::::1.:il.

27. Gulienetti, K. et al: "~mniot1c fluid Volume and Experimentally-Induced Congenital Malforma­ tions. Biol. Neonat. 4:3UU, 1Y62. 2o. Esu, C.-Y.: Influence of Temperature on Develop­ ment of itat Eimbryos. Anat. rl.ec. 100:7si, 1!;14d.

2::,1. Ingalls, T. H. et al: Experimental Production of Congenital Anomalies. Ti~ing ana Oegree of Anoxia as Factors Causing Fetal Deaths and Congenital ,:womalies. New Eng. J. P"ed. 247: 75u, lsi52. --

JO. Lyngdoh, o.: Production of Congenital Abnorma­ lities in Offspring of Trypan Blue Injected rm ts • An at • t( e c • 10 6: 2 d 1 , l ~ 5U • 31. Nishimura, H. and Nakai, K.: Developmental Anoma­ lies in Offspring of Pregnant Mice Treated with tHcotine. Science 127:877, 1~5d.

32. Robson, J. ~. and Sullivan, F. k.: ~echanism of Lethal Action of ~-Hydroxytryptamine on the Fetus. J. t::noocr1nol, 25::i5J, 1~63.

33. ~oulson, E. et al: Teratogenic ~ffect of j-Hydroxy­ tryptamine in Hice • .::icience 141:717, 1~63.

36. 34. Chang, !''. C.: Artificial froduction of Monstro­ sities in the Rabbit. i~ature 154:150, 1Y44.

35. Danforth, c. :-i. and Center, E.: Nitrogen f'~ustard as a Teratogenic Agent in the ~ouse. Froc. Soc. Exp. Biol. Nled. 36:705, 1954.

11 11 36. Thiersch, J. B.: i:ffect of 2,4,6-Triamino- s - triazine (TR), 2,4,6- 11 Tris 11 -(ethyleneimino)­ ns11-triazine rrEl-1) anci i".,N 1 ,N 11 -Triethylene­ phosphoramide (TBPA) on ,,at Litter in ~Jtero. Proc. Soc. Exp. Biol. Med. ~4:36, 1957.

37. Hicks, s. P.: So~e 2ffects of Ionizing ~adiation and i'1etabo 1 i c Inhibition on the Dev el oping r-~ammalian Nervous ~:iystem. J. Pediat. 40: 4d9, 1952. -

3d. ~ilson, J. G.: Jifferentiation and rteaction of Hat Embryos to ~adiation. J. Cell. Comp. Fhysiol. 43:suppl. 1, 11, 1Y54.

3Y. Elion, G. B. and :utchings, G. H.: ~tudies on Condensed fyrimidine Synthesis. IX. The Synthesis of Some 6-Substituted ~urines. J. Am. Chem. Soc. 74:411, 1~52.

40. Hitchings,·~. H. et al: Studies on ,J.nalogs of Furines and ~yrimidines. Ann. N. Y. Acad. Sci. 52:1318,lYSU.

41. Elion, G. B. et al: Antagonists of Nucleic Acid Derivatives. VI. Furines. J. Biol. Chem. 19 2 : 50 5' 19 51 • 42. Clarke, '-'· A. et al: 6-1,1ercaptopurine: .t:ffects in Vause Sarcoma 160 and in ~ormal Animals. Cancer i{es. Q: 595, 1953.

43. Law, L. ~.: rtesistance in Leukemic Cells to an Adenine Antagonist, 6-~ercaptopurine. froc. :3oc. Exp. Biol t::ed. d4: 40!::!, l!:1SJ.

44. Burchenal, J. H. et al: Clinical Evaluation of a New Antimetabolite, 6-hlercaptopurine, in the Treatment of Leukemia and Allied Liseases. Blood. B:Y65, 1~53.

37. 45. Miner, 1-i. w. ana Rhoads, c. P. (eds.): 6-!·.~er­ c apt op u r i n e • Ann • N • Y • Ac ad • S c i • 60 : 183, lY!:>4. 46. Thiersch, J. B.: The Effect of 6-Percaptopurine on the kat Fetus and on ~eproduction of the ;{at. Ann. u. Y. Acad. Sci. 60:220, 1954. 47. Didcock, K. et al: The Action of Some Nucleo­ toxic Substances on Fregnancy. Brit. J. Pharm • .U:.:437, 1Y56.

4d. Silberman, H. k. and ',lyngaarden, J. B.: 6-Mer­ captopurine as Substrate and Inhibitor ot Xanthine Ox1dase. Bioch1m. Biophys. Acta 47:178, 1Y61.

4~. Carey, H. H. and rdandel, H. G.: The l'vietabolism of 6-~ercaptopurine by Bacillus cereus. Biochem. Pharmacol. ~:64, 1~6u.

SU. cilion, G. B. et al: Studies on Condensed Pyrimi­ dine Systems. XXI. The Isolation and Synthesis of 6-Mercapto-2,8-purinediol l6-Thiouric Acid). J. Am. Chem. Soc. 31:3042, 195!::1.

51. Sarcione, E. J. and Stutzman, L.: 6-"1ethylmer­ captopurine: Identification as a J.'letaboli te of 6-Vercaptopurine in vivo and its Activity in vitro. Proc. Soc-.-EX'P:-Biol. Ped. lJl: 766, 195!:>. . --

52. Elion, G. B. et al: Potentiation by Inhibition of Drug Degradation: 6-Substituted Furines and Xanthine Oxidase. Biochem. Pharm. 12: 8~, 1~63.

53. Elion, G. B. et al: rrnlationship between Meta­ bolic Fates and Antitumor Activities of . Cancer Res. 23: 1207, 1~63.

54. Lukens, L. N. and Herrington, K. A.: Enzymatic Formation of 6-Mercaptopurine dibotide. Biochim. Biophys •.'{eta 24: 432, 1957.

55. ~~ay, J. L. and Parks, i~. E.: Enzymatic Synthesis of 5 1 -Phosphate Nucleotides of furine Ana­ logues. J. Biol. Chem. 231:467, 1~5d.

38. 56. Paterson, A. R. F.: The Formation of 6-T·'ercapto­ purine Hiboside fhosphate in Ascites Tumor Cells. Can. J. Diochem. Fhysiol. lZ_:lOll, 1Y59.

57. Brockman, R. ~.: Biochemical Aspects of Mercapto­ purine Inhibition and Resistance. Cancer Res. 23:11Yl, 1963.

5d. Brown, G. B. et al: The Utilization of Adenine for Nucleic Acid Synthesis and as a Pre­ cursor for Guanine. J. Biol. Chem. 172: 469, 1948.

5~. Bendich, A. et al: On the riole of 2,6-Diamino­ purine in the of Nucleic Acid Guanine. J. Biol. Chem. 185:423, 1Y50.

60. Brown, G. B.: The Biosynthesis of Nucleic Acids as a Basis for an Approach to Chemotherapy. A.nn. i~. Y. Acad. Sci. 60:185, 1954.

61. Handschumacher, H. E. and ~Jelch, A. D.: Agents which Influence Nucleic Acid Metab6lism. (In: Chargaff, E. and Davidson, J. N., eds., The Nucleic Acids, vol. 3, Academic Press, Inc., New York, 1960, p. 453).

62. Wilmanns, W.: Zurn Wirkungsmechanismus von 6-Mer­ captopurin. Klin ••lochschr. 4J:ll70, l.si62.

63. Ellis, D. B. and LeFage, G. A.: Biochemical Studies of Kesistance to 6-Thioguanine. Cancer Res. 23:436, 1963.

64. Clarke, D. A. et al: Structure-Activity Relation­ ships among Purines ~elated to 6-~ercapto­ purine. Cancer Res. ld:445, 195~.

6~. Montgomery, J. A.: The ~elation of Anticancer Activity to Chemical Structure. Cancer rtes. lY: 447, 1Y5Y.

66. Robins, R. K.: Potential Purine Antagonists. I. Synthesis of 3ome 4,6-Substituted Pyrazolo­ (3,4-d)-pyrimidines. J. Am. Chem. Soc. 78: 413, 1YS6.

39. 67. Skipper, H. E. et al: Structure-Activity Kela­ tionships Observed on Screening a Series of Pyrazolopyrimidines against Experimental Neoplasms. Cancer xes. !Z.:579, 1~57. 6d. Feigelson, P. et al: Pyrazolopyrimidines as Inhibitors and Substrates of Xanthine Oxidase. J. Biol. Chem. 226:Y~3, 1~57.

6~. Dawson, A. B.: A Note on the Staining of the Skeleton of Cleared Specimens with Alizarin Red s. Stain Technol. 1:123, 1~26. 70. rncColl, J. D. et al: Drug Induced Skeletal Mal­ formations in the kat. Experientia 1~:183, 1963.

40.