comparison of teratogenic chemicals in the...

COMPARISON OF TERATOGENIC CHEMICALS INTHE RAT AND CHICK EMBRYOS

An Exhibit with Additions for Publication

By M. Lois Murphy, M.D., C. P. Dagg, Ph.D., and David A. Karnofsky, M.D.Embryology Section, Divixion of Experimental Chemotherapy, Sloan-Kettering Institute

f or Cancer Research, New York

Presented at the Annual Meeting, October 8-11, 1956.This research was supported, in part, by grants from the American Cancer Society, Inc., the Albert

and Mary Lasker Foundation, and grant C-675 from the National Cancer Institute of the National

Institutes of Health, U. S. Public Health Service.

ADDRESS: (M.L.M.) 444 East 68th Street, New York, New York.

701

T HE CHICK EMBRYO 15 used widely in

studying drugs for teratogenic activity.

Chemicals, which have been injected at the

same period of incubation of the chick

eml)ryo, may produce separate and char-

acteristic patterns of developmental ab-

normalities. The effects of many drugs,

such as insulin, azasenine, sulfanilamide,

4-aminopteroyl glutamic acid, 8-azaguanine,

physostigmine, thallium, lead, boric acid

and cortisone, have been neviewed.1�

The chick embryo in the egg is an

isolated and independent system, whereas

mammalian embryos are usually intimately

involved with the maternal host which may

detoxify, excrete or otherwise protect the

fetus against noxious chemicals. It was,

therefore, of interest to determine the effects

of drugs, teratogenic in the chick embryo,

on the mammalian fetus. The rat was

selected because of its availability and be-

cause there is considerable information on

rat embryology and teratology.’’ Because

some drugs, apparently inactive in the chick

embryo, have produced consistent de-

velopmental abnormalities in the rat fetus,

the pregnant rat also has been used for

the initial study of selected compounds.

The objectives of these studies were:

1) to determine and compare the terato-

genie action of drugs on the chick and rat

embryos; 2) to determine the consistency

and specificity of action of each drug; 3) to

determine the relation between the time

during gestation when a drug is intro-

duced and the occurrence of specific ab-

normalities; 4) to determine the relation-

ship of the dose of a drug toxic to tile

mother, the dose toxic to the embryo, and

the dose producing consistent develop-

mental defects, and 5) to detect compounds

which protect the embryo from the terato-

genie action of a chemical.

Observations on five distinctive com-

pounds are presented in this exhibit.

MATERIALS AND METHODS

White Leghorn eggs were obtained from a

commercial source and incubated at 38#{176}C.The

test chemicals, at various dose levels, were

routinely injected into the yolk sac of the 4-

day-old embryos. The embryos surviving the

treatment were killed at 18 days of incuba-

tion, or 14 days after the injection of the drug.

Previously unmated female rats, Wistar

strain, were placed in cages with males and

watched for 12 hours. Those observed in copu-

lation were isolated and regarded as zero days

pregnant. At various times, ranging from 7 to

16 days of gestation, groups of rats received asingle intraperitoneal injection at various dose

levels of the test drugs. The pregnant ratswere sacrificed on the twenty-first day of ges-

tation, the uteri examined, the implantation

sites counted, and the surviving fetuses re-

moved for study.

The chick embryos and rat fetuses were cx-

amined grossly, weighed and then cleared

and the skeleton stained with alizarin so that

it could be examined macroscopically in the

whole animal.

The test chemicals were dissolved in saline

or suspended in carboxymethylcellulose (CMC)immediately before injection.

by guest on June 2, 2018www.aappublications.org/newsDownloaded from

702 �vlURPHY - COMPARISON OF TERATOGENIC CHEMICALS

FIG. 1.


AMERICAN ACADEMY OF PEDIATRICS - PROCEEDINGS 703

DEVELOPMENT OF THE CHICK AND

RAT EMBRYOS (FIG. I)

CmcK E�fBYnos:’ ‘#{176} The incubation

period of the cilick embryo is 21 days.

Growth and differentiation proceeds nap-

idly, and many detailed descriptions are

available. Tile classical monograph on chick

embryology is by Lillie and Hamilton.”

Hamburger and ‘ 2 have prepared

an atlas of the stages of embryonic de-

velopment, and related them to incubation

time. By 18 to 24 hours the primitive streak

has appeared, heart beat begins about the

end of second day (9 somite stage), the tail-

bud appears at about 33� days of incubation,

and the initiation of endocrine function and

the end of metamorphosis” begins about

the eigllth to ninth days.1’ Cartilage ap-

pears at tile fifth day, and active calcifica-

tion of bone begins about the eleventh day.

When the chick hatches it weighs about

30 gin, has feathers and is able to feed

itself.

RAT E�IBRYos:’4” Tile rat, from the time

of fertilization, has a gestation period of

about 22 days. Implantation begins about

the seventh day, and embryonic develop-

ment is, in general, about 6 to 8 days be-

ilind that of tile chick embryo. Thus, tile

primitive streak appears at 83� to 9 days,

the heart begms to beat about the ninth

daY, tile tailbud appears at 113k days, and

the “end of metamorphosis” appears at 16

to 17 days.13 Ossification begins on the

sixteenth day. Thus, the 4-day-old chick

embryo is at about the same stage as the

11 to 12-day-old rat fetus, and the 8-day

chick embryo is equivalent in development

to tile 15 to 16-day rat fetus. These com-

parative periods are important, because it

is noted that teratogenic effects to be de-

scribed are most commonly produced in

the chick embryo treated on the fourth day,

and the rat fetus on the tenth to twelfth

days. At birth the rat fetus weighs 4 to

5 gm which is equal to the 12-day chick

embryo, and its development is far less

advanced than the chick.

NITROGEN MUSTARD (FIG. 2)

Methyl-bis (�-chloroethyl)amine hydro-

chloride (HN2), a nitrogen mustard, is a

crystalline substance, soluble in water.

Closely related structurally and pharmaco-

logically to the war-gas, mustard gas, it was

intensively studied during World War II

as a potential chemical warfare �

In the past 10 years it has been widely used

in the treatment of neoplastic disease in

man; principally in tumors of the lymph

nodes and blood forming organs.’#{176} HN2

has wide and clearly defined patterns of

biologic effects, which have been reviewed

on several occasions.”” It is carcinogenic,

mutagenic, and in mammals toxic doses

produce severe depression of the bone mar-

row, with decrease in the formed elements

of the blood, involution of lymphatic tissue,

and injury to the intestinal mucosa; these

effects are similar to those produced by total

body irradiation. HN2 inhibits the growth

of transplantable tumors, and produces

characteristic histologic changes in mouse

tumors, and in mouse and human tumors

growing on the chonioallantoic membrane

(CAM) of the chick embryo.’2 Teratogenic

effects in tile rat fetus have been described

by Haskins.”

Teratogenic Effects

CHIcic EMBRYO: The LD,0 of HN2 is

variable and doses in the range of 0.01

mg/egg may be lethal for tile 4-day em-

bryo. Death usually occurs within 1 to 5

days after injection, and those surviving

in the dose range of the LD,0 are usually

normal. An occasional embryo, however,

shows weight inhibition, crossed beak, and

thin legs.

RAT EMBRYO: The LD,0 of single doses

of HN2 given intrapenitoneally to the preg-

nant rat is in the range of 2.0 mg/kg; this

is in tile same range as the LD,0 in the

nonpnegnant nat (1.9 mg/kg by the sub-

cutaneous route and 1.1 mg/kg intrave-

nously). Pregnant rats were given single in-

jections of HN2 intraperitoneally ranging


HN2 (nitrogenmustard)

CH2 CH2CI

CH2 CH2CI

(1

S

I

S 114

MA TERNAL L05fl 2.0 MG. /KG.1N) II 04}’

Fic. 2.

/8 VAY EMBRYO (IN1! 4 04)’)EFFECTIVE DOSE 0.0/ M6�/EGGABNORMALITIES INFREQUENT

Cross beakThin Iegc

ABNORMALITIES INFREQUENT

Head flattenedMandible shortRibs angulated

Rear extremities rotatedLens calcified

TERA TOGENIC DOSE 0. 5 MG/KG./N�i /2 04)’

SACRIFICED 21 VA)’ABNORMALITIES FREQUENT

Cranial circular defectParietal, interparietal and supraoccipital

Ribs angulated

Vertebral bodiesdouble,Tl1 and L3and4Scapulae curvedForetoes syndactylous, metacarpols

missing



from 0.3 to 0.5 mg/kg on the seventh

througil the fourteenth days of gestation.

The rats treated on tile seventh day showed

no evidence of pregnancy when sacrificed

on the twenty-first day. Pregnant rats

treated once on given days from the ninth

to the fourteenth, while showing only slight

increase in the incidence of resonbing im-

plantation sites, had many stunted fetuses

with a variety of gross abnormalities. The

fetal abnormalities were inconstant in rats

treated on the eleventh day of gestation.

The fetuses were smaller than normal, the

skull was sometimes flattened with a small

jaw. The cleared skeleton showed elongated

and flattened head, short mandible, angu-

lated nibs, and occasionally calcified lenses

and rotated pelvis and rear extremities. The

most striking effects were seen in the

fetuses of the pregnant rats treated on the

twelfth day. Grossly there were encephalo-

celes at the crown of the head and cleft

palates were a rare occurrence. Syndactyly

on ectrodactyly of the front as well as the

rear toes was sometimes seen. The cleared

skeleton, even in specimens without gross

evidence of encephalocele, showed a circu-

Ian cranial defect involving the occipital,

frontal, parietal and interparietal bones.

The nibs were angulated, with nibs 12 and

13 sometimes missing, the vertebral canal

showed widening in the cervical area. The

vertebral bodies in tile lower thoracic and

upper lumbar regions were not fused and

gave tile appearance of double ossification

centers. The scapulae were curved toward

the body. Ossification centers in the digits

appeared to be delayed although occasional

fusion of metacarpals or metatarsals was

found.

Discussion

HN2 has an inconstant effect on the

chick embryo injected on the fourth day.

It apparently is acutely toxic to the embryo

and the surviving embryos show rare and

inconstant abnormalities. In this respect

HN2 differs from radiation, which produces

consistent developmental defects in chick

embryos treated on the fourth day.’4 This

difference may be related to the fact that

tissue recovery after nitrogen mustard seems

to be considerably more rapid than after

irradiation. The rat fetus is apparently sus-

ceptible to the action of HN2 at about one-

fourth or one-sixth the dose toxic to the

mother. The effects are most striking when

the embryo is treated on the twelfth day,

hut abnormalities can also be produced at

higher doses when the embryos are treated

on the fourteenth to sixteenth day. The

skeletal effects produced have not yet been

compared with those produced by irradiat-

ing the pregnant rat.

3 3-DIMETHYL- I -PHENYLTRIAZENE

(TRIAZENE) (FIG. 3)

This triazene is an oily substance, which

is prepared for injection by suspending it

in 0.5% CMC or by dissolving it in peanut

oil. Clarke et al.25 demonstrated that it in-

hibited the growth of transplantable San-

coma-180 in mice; in subsequent studies

it prolonged survival in mice with trans-

plantable leukemia,’6 and produced his-

tologic changes and loss of viability in

human and mouse tumors growing on the

CAM of the chick embryo.” In the mouse2’

and rabbit this tniazene produced depres-

sion of the bone marrow and a decrease in

the formed elements of the blood. The

histologic changes in the blood-forming

organs and intestinal tract of the rabbit,

and in tumors growing on the CAM of the

chick embryo were similar to those pro-

duced by nitrogen mustard.” Other dis-

tinctive actions of the triazene have been

described: micnomelia in the chick embryo,

prevented by nicotinamide;27 and a “waltz-

ing” syndrome in Swiss mice, prevented by

nicotinic acid.”

Teratogenic Effects

CHIcic EMBRYO:’7 The yolk sac of 4-day

embryos was injected with the triazene sus-

pended in CMC; the LD,0 was in the range

of 0.5 to 1 mg/egg, and embryos surviving

these doses showed consistent develop-

mental abnormalities. The general effects

were weight inhibition, edema and occa-

sionally short, clubbed feathers. Specific

effects occurred in the appendicular skele-


.

f

4

MA TERNAL L050 /80 MG. /K6� TERA TOGENIC DOSE 30 MG/KG.IA/i II 04 Y /N1 /2 04 Y

FIG. 3.

7W/A ZENE

CH3

f\-N=NN\=_/

CH3

, - . -% -

/8 #{248}� EM8RYO (lAid. 4 DAY)EFFECTIVE DOSE 0. 5 MG /EGGABNORMALITIES FREQUENT

Parrot beakM icromeliaTibiotarsus bentTarsometatarsus bent

ABNORMALITIES FREQUENT

Scapula small, narrow, curvedHumerus and radius smallUlno, tibia and femur missing

SACRIFICED 2/ 04)’ABNORMALITIES FREQUENT

Maxilla and mandible shortZygoma missingToes syndactylousRibs small or missing


MIERICAN ACADEMY OF PEDIATRICS - PROCEEDINGS 707

toIl, Witil niicronielia, bending of the tibio-

tarsus and occasionally the tarsometatansus.

Beak abnormalities ranged from I)arrot-

beak to shortening of the lower beak or

both upper dildi lower beaks. Nicotinamicle,

5 mg/egg, given at tile same time as the

triazene protected against the skeletal de-

fects, but did not prevent stunting or

mortality

RAT E�IBilYos: Tile LD30 of single doses

of tile tniazene (in Ieantit oil) given intra-

peritoneally ill the pregnant rat was in the

range of 180 to 200 mg/kg; death often

occurred several days after an LD,0 dose.

Pregnant rats were given single doses of

30 mg/kg of the tniazene on days 6, 9, 11,

12, 14, or 16 of gestation. Rats treated on

the sixth clay showed no abnormal fetuses,

and only a slight increase in fetal re-

sorptions; there were few fetal abnormal-

ities in rats treated on the ninth, and the

fourteenth and sixteenth day. The fetuses

of rats treated Ofl tile eleventh or twelfth

days sho�ved consistent and severe gross

and skeletal abnormalities; few resorptions

occurred, however, and the abnormal

fetuses survived to 21 clays.

The embryos treated at 11 days were

sttiiitecl and occasionally edematous, and

the extremities were shortened. The front

paws did Ilot rotate normally and ne-

sembled seal flippers. The cleared skeleton

typically had normal digits. The front cx-

treniities showed a small curved or absent

llumerus an(l tile hind legs showed a small

or absent femur, absent tibia and small

pelvic bones. The scapula was small, curved

or the spinous process rotated and don-

gated laterally and downward to resemble

a double scapula. Tile vertebral bodies ap-

peared double from Ti to L3; the sterne-

brae were double or fused cephalocaudal.

The 12-day embryos were strikingly

different. The paws appeared short and

pointed, the nose was pointed, the lower

jaw often appeared to be absent, the eyes

were open and cleft palates were a con-

sistent finding. In the cleaned skeletons, the

skull had a normal premaxilla, but the

zvgomatie processes of the maxilla and

squamosal bones were absent giving 110

infnaorbital bony framework. The mandible

was small with little, if any, angle or tem-

1)oral portion. Tile vertebrae were usuallynormal but the ribs varied from being fused

to form a continuous bony plate like a vest

of armor, to complete absence of nibs. The

bones of tile arms and legs and of the

scapula and pelvis were normal but the

metatarsals, metacarpals and phalanges

showed absence or fusion.

Discussion

The micromelia produced by the tniazene

in tue chick embryo is similar to that caused

by many other drugs; insulin, sulfanilamide,

physostigmine, and 6-aminonicotinamide,

tile micromelic action of these drugs is also

prevented by nicotinamide.

The rat fetus is susceptible to the action

of tile tniazene at about one-sixth of the

dose toxic to the mother; the effects vary-

ing with the duration of pregnancy at the

time of injection. While the triazene has

some of the biologic activities of an alky-

lating agent, HN2 did not produce the

specific effects on the extremities or the

mandible of the rat fetus seen after this

tniazene. The specificity of the teratogenic

action of various alkylating agents in the rat

requires more detailed exploration.

2-ETHYLAM INO- I ,3I4-THIADIAZOLE(THIADIAZOLE) (FIG. 4)

2-Ethylamino-1,3,4-thiadiazole is a water

soluble chemical. Olesen et al.2M demon-

strated that the thiadiazole inhibited the

growth of transplantable Sarcoma-180. This

observation has been confirmed in other

transplantable mouse tumors, and including

leukemia.” The mechanism of action of this

drug is not known. It is of considerable

interest, Ilowever, that it produces a marked

rise in uric acid excretion in man, pre-

sumably due to an increase in de novo

punine syntilesis since there is no evidence

of tissue destruction.’#{176}

Teratogenic Effects

CHICK EMBRYO: The yolk sac of the 4-

day embryo was injected with tile thia-


H�’�-NHC2 H5

4 4 p

-

MA TERNAL L050 200 MG. /K�IN�J 9DAY


Vertebral arches and bodies:Cephalocaudat fusion and fragment-ation involving C4 thru L4

Ribs fused and branched I thru 3

Fic. 4.

TH/�4D/A ZOL E

ie OAY EMBRYO (/N� 4 04)’)EFFECTIVE DOSE /3. 5 MG/EGGABNORMALITIES INFREQUENT

Upper beak shortened

TERA TOGENIC DOSE 100 MG /KG.IMI II DAY

SACRIFICED 2/ DAYABNORMALITIES FREQUENT

Vertebral arches and bodies:Cepholocaudal fusion 18 to end ofvertebral column at level of sacrumSacral and caudal vertabrae missing

Ribs fused and branched 8 thru 13

Sternobrae fused or bifurcated



diazole; the LD,0 is in the range of 13.5

mg/egg. Generally the embryos surviving

this dose showed no developmental ab-

normalities, although occasional embryos

surviving to the eighteenth day showed a

silortening of the upper beak.

RAT EMBRYOS: The LD50 of single doses

of tile thiadiazole in the pregnant rat is

in tile range of 200 mg/kg. Pregnant rats

received single doses of 50 to 100 mg/kg

of tile tlliadiazole on days 9, 11 or 12 of

gestation. Motllers treated on the ninth day

showed no increase in the resorbing im-

plailtatioll sites and gross fetal abnormal-ities were infrequent. Those affected, how-

ever, were reduced in weight, had pointed

lower jaws and lorclosis of the spine. In

the cleared skeleton, tile cervical, thoracic

and first tilree lumbar vertebrae were dis-

torteci by double ossification centers of the

bodies or cephalocaudal fusion of the

arclles. Tile remaining lumbar, sacnal, and

caudal vertebrae were normal. The ribs

were branched and fused and the total

number appeared to be reduced.

Rats treated on the eleventh day of

gestation showed about 50% resorbing fe-

tuses, and the surviving ones were small

and had gross skeletal abnormalities. The

fetuses ilad absent or short tails and syn-

dactyly on ectrodactyly of the front and

hind paws. The cleared skeletons showed

branching and fusion of the eighth through

the tilirteenth ribs; the lower thoracic and

lumbar vertebral arches and bodies showed

hemivertebrae, cephalocaudal fusion and

fragmentation and usually complete ab-

sence of the sacral and caudal vertebrae.

Ill rats treated on the twelfth day of

gestation with doses of 50, 100 or 200

mg/kg of the thiadiazole, fetal resorption

was noted only in the group receiving 200

mg/kg. Abnormalities were infrequent in

the 50 mg/kg group but were usual at

doses of 100 and 200 mg/kg. Tile affected

fetuses showed cleft palate, harelip, en-

cephalocele, syndactvly and ectrodactyly,

small pelvis and shortening of the tails.

This latter occurred particularly in the

group receiving 200 mg/kg. The cleaned

skeletons showed circular defects of the

frontal, panietal, intenpanietal and occipital

bones. The clavicles were often minute and

the spinous process of the scapula was ro-

tated from its normal position. The extremi-

ties showed syndactyly and shortened or

absent fibulae and ulnae. Those receiving

the higher dosage also showed retarded

ossification of the pelvic bones and less than

four caudal vertebral bodies.

Discussion

The thiadiazole does not appear to Ilave

a consistent effect on development of the

chick embryo. The rat fetus, however, was

susceptible to its action in doses in the

range of one-fourth to one-half of that lethal

to the mother. Some of the gross skeletal

abnormalities were similar to those seen

with nitrogen mustard and the triazene. The

most distinctive feature, however, was the

absence of the tail in the fetuses of the

rats treated on the eleventh day of gesta-

tion.

6-AM INONICOTINAM IDE

6-Aminonicotinamide is a crystalline ma-

tenial which is insoluble in water. It is pre-

pared for injection by suspending it in 0.5%

CMC in saline. It appears to be a highly

effective antagonist of nicotinamide and

approximately 10 times more active than

3-acetylpynidine in mice.” Simultaneous ad-

ministration of nicotinamide will protect

mice against eight times the LD,0 dose of

6-aminonicotinamide. In Sancoma-i80 in

mice 30 mg/kg was lethal while 8 mg/kg

gave some toxicity without any evidence of

tumor regression.

Teratogenic Effects

CHIcic EMBRYO: The yolk sac of the 4-day

embryo was injected with 6-aminonicotin-

amide and the LD,0 was in the range of

0.01 mg/egg. Gross skeletal abnormalities

occurred regularly with a characteristic

syndrome including parrot beak, micro-

melia, bending of tibiotarsus and tarsometa-

tarus. These effects could be prevented by

the simultaneous administration of nicotin-

amide.


6-AM/NON/CO TIN-

H2N

U0

Fic. 5.

4 M/12E

MA TERNAL LDi#{231}/t61 II 04’


Size retardedSkull-wide suture lines

Ossification retarded. Vertebral canal widened

Vertebral bodies doubleKyphosis and scoliosisFibula missing

/8 04 Y EMBRYO (INI 4 DAY)EFFECTIVE DOSE 001 MG /EGGABNORMALITIES FREQUENT

Parrot beakM icromeliaTibiotarsus bentTarsometatarsus bent

/5 MG. /K&� TERA TOGENIC DOSE 8 MG. /KG.IN�J 12 DAY

SACRIFICED 2/ DAYABNORMALITIES FREQUENT

Size retardedCleft palate : palatine missing


AMERICAN ACADEMY OF PEDIATRICS - PROCEEDiNGS 711

RAT E�siis�Yos: There appears to be some

differences ill tile LD31 of 6-aminonicotin-

amide at different stages of gestation for

ti1(� rat. Tile LD� close is in tile range of

8 to 15 nig/kg, intraperitoneally. Rats

treate(I on the tentil clay of gestation with

15 nlg/kg showed resorbing implantation

sites Witil 110 viable fetuses at 21 clays. With

8 ing/kg Oil the eleventh or twelfth claY of

gestation, nlaternal survival occurred regu-

larly with ai)Out 50% fetal resorption. The

remaining fetuses were stunted and showed

gross abnormalities. Fetuses of mothers

treate(1 with 8 mg/kg on the tenth clay of

gestation Silo\Vedl syndactvlv and ectro-

(lactyly, club feet, harelip and cleft palate.

The cleared skeleton showed marked re-

tardation of ossification, tile ribs were fused

and l)railched, the vertebral columns

curved, the veretebral canals widened and

ossification centers of the digits were not

visible.

The fetuses treated on tile eleventh day

of gestation showed an inhibition in size

and weigllt, bilateral harelip and cleft

palate or absent palatal structures, syn-

dactvly and often lumbar lordosis. Skeletal

preparations showed retarded ossificationof all bony structures, wide cranial sutures,

and rotated vertebral column with occa-

sional fused or branched ribs and syn-

dactylv.

The fetuses of motilers treated on the

twelfth day showed weight and size inilibi-

tion, syndactyly, and ilarelips were more

complete on tile right with the cleft palates

often involving only tile posterior portion

of the palate. Cleared specimens showed

(lelaved ossification.

The toxic and teratogenic effects of 6-

aminonicotinamide on the mother and fetus

cai� l)C prevented by the administration of

100 mg/kg of nicotinamide just prior to the

injection of 6-aminonicotinamide.

Discussion

6-Aminonicotinamide produces a micro-

melic syndrome in the chick embryo which

is prevented by nicotinamide. This syn-

drome is similar to that produced by a

variety of other agents and is similarly

prevented in most instances by nicotin-

amide. In the rat fetus there is little differ-

ence between the dose of 6-aminonicotin-

amicle toxic to tile mother and to the fetus;

dosage which caused consistent injury to

tile fetuses were also debilitating or lethal

to tile mother. Fetal development was

severely affected with stunting, defects in

ossification and skeletal deformities.

AZASERINE (FIG. 6)

O-diazoacetyl-l-senine (azasenine) is a

wllite crystalline powder soluble in water.

It proved to be the active ingredient of a

filtrate of Streptomyces which was found

to inhibit tile growth of tile mouse Sarcoma-

180. Azasenine is inhibitory to a wide spec-

trum of transplantable nat and mouse tu-

mors; it produces mutations in E. coli, and

it causes developmental abnormalities of

Drosophila and tile chick embryo.32 It has

been studied in the chemotherapy of hu-

man cancer.” The teratogenic effects of

azasenine in tile chick embryo can be pre-

vented by the simultaneous administration

of adenine on 4-amino-5-imidazole-carbox-

amide.” Azasenine is of particular interest

in teratogenesis because of its specific in-

hibitory action on purine synthesis.

Teratogenic Effects

CHICK EMBRYO : � Azaserine was injected

into tile yolk sac of the 4-day embryo; the

LD� was in the range of 0.15 to 0.2 mg/egg.

The majority of the injected embryos died

around the twelfth day of development, hut

some survived to the sixteenth (lay. Tile

affected embryos showed a characteristic

picture of inhibition of growth, paleness,

edema and impaired feather formation.

There were also gross abnormalities of tile

head and the extremities. The cleared skele-

ton siloWed a well-developed axial skeleton

in contrast to defective limbs and beak. The

facial defects ranged from a shortened

lower beak, to parrot beak to absence of the

lateral elements of the maxilla, usually

associated with a greatly reduced mandible

and cleft palate. The appendicular defects


Fic. 6.

AZA SEP/NE

0. II

N2CHC-0-CH2 CHCOOH

NH2

.‘ ,

18 DAY EMBRYO (INJ 4 DAY)EFFECTIVE DOSE 0.25 MG. /EGGABNORMALITIES FREQUENT

Facial coloboma with cleft palateMandible shortenedWing: distal defect

Single, small metacarpalTarsometotorsols short and bentEctrodactylyHypophalangy

I

,

%. S

MA TERNAL L050 75 MG. /KG.IN�J /0 DAY

ABNORMALITIES FREQUENTCleft palateVertebral bodies doubleVertebral arches and bodies:

Cephalocaudal fusion 14 thru L3HemivertebroeRibs fused

TERA TOGENIC DOSE � MG/KG.lAid 12 DAY

ABNORMALITIES FREQUENTVertebral bodies double T6 thru L4Ischium smallFemoro missingFibulae missing



consisted principally of shortening of the

tansometatarsals with ectrodactyly ranging

from tile loss of one to all four toes. An

analogue of azasenine, 6-diazo-5-oxo-1-

norleucine (DON) gave approximately the

same effects but is about 50 times more

active by weight.

RAT EMBRYOS:36 Tile LD,0 of azasenine

in tile pregnant rat ranges between 75 and

100 mg/kg by the intrapenitoneal route.

Pregnant rats received single doses of 2.5

mg/kg of azaserine from the seventh to

fourteenth day of gestation. Those treated

on tile seventh and fourteenth days de-

livered normally.

Pregnant rats treated on the ninth day

Silowed 100% resorption of the implanta-

tiOll sites. Rats treated on days 10, 11

and 12 showed approximately 50% ne-

sorption with 80% of the surviving embryos

showing abnormalities, although weight in-

hibition was not marked. Embryos treated

on the tenth day occasionally showed cleft

palate and harelips. The cleaned skeleton

revealed fused or branched ribs, abnonmali-

ties of the lower tiloracic vertebral bodies

and arches manifested as cephalocaudal

fusions of the bodies on hemivertebrae.

Tile 11-day-treated embryos showed cleft

palate, bifid or fused sternebrae, small or

absent radii, and vertebral abnormalities

similar to the tenth day, but involving the

lumbar instead of the thoracic vertebrae.

In those treated at 12 days, the skeletal ab-

Ilornlalities were similar to those at ii days;

l)ut included small on absent pelvic bones,

femur or fibula. The extremities showed

syndactyly on absent toes.

DON was far more active by weight than

azaserine; pregnant rats treated on the tenth

day with 0.2 mg/kg intraperitoneally

showed complete fetal resorption, and 50%

resorption occurred at 0.1 mg/kg. Surviving

animals did not show the incidence of de-

velopmental abnormalities produced by

azasenine.

Discussion

Azasenine, a llighly specific antimetabo-

lite, produced severe developmental ab-

normalities in the chick embryo and rat

fetus. The abnormalities of the appendicu-

lan skeleton in the rat fetus and chick em-

bryo appeared to be related.

ACKNOWLEDGMENTS

We are pleased to acknowledge the fol-

lowing sources of compounds used in this

study:

Merck & Co., Inc., methyl, bis(�-chloro-

etyl)amine; Chemical-Biological Coordina-

tion Center, National Research Council,

and Dr. C. S. Rondestvedt, Jr., Univen-

sity of Michigan, tniazene, 3,3-dimethyl-l-

phenyl-; Dr. J. M. Ruegseggen, Ledenle Lab-

oratories, American Cyanamid Company,

i,3,4-thiadiazole,2-ethylamino-; Squibb In-

stitute for Medical Research, nicotinamide,

6-amino; Parke, Davis and Company, azas-

enine and DON; Dr. Earl Balis, Sloan-

Kettering Institute for Cancer Research,

imidazole, 4-amino-5-carboxamido-.

REFERENCES

1 . Ancel, P. : La Chimioteratogenese chez lesVertebres. Paris, Doin, 1950.

2. Karnofsky, D. A. : The use of the develop-

ing chick embryo in pharmacologic re-search. Stanford M. Bull., 13:247, 1955.

3. Landauer, W. : On the chemical produc-tion of developmental abnormalities andof phenocopies in chicken embryos. J.Cell. & Comp. Physiol., 43:261, 1954.

4. Zwilling, E. : Teratogenesis, in Analysis ofDevelopment, edited by Willier, B. H.,

Weiss, P. A., and Hamburger, Viktor.

Philadelphia, Saunders, 1955.5. Hicks, S. P. : Developmental malformations

produced by radiation: A timetable of

their development. Am. J. Roentgenol.,69:272, 1953.

6. Nelson, M. M. : Mammalian fetal develop-ment and antimetabolites, in Antimetab-

olites and Cancer, Rhoads, C. P., ed.Washington, Am . Assoc. Advancement

Sc., 1955.7. Warkany, J. : Congenital malformations in-

duced by maternal dietary deficiency;experiments and their interpretation.

Harvey Lect., Series 1952-53, 48:89,

1954.8. Wilson, J. G. : Influence on the offspring

of altered physiologic states duringpregnancy in the rat. Ann. New YorkAcad. Sc., 57:517, 1954.

9. Miner, R. W., ed. : The chick embryo in


714 MURPHY - COMPARISON OF TERATOGENIC CHEMICALS

biological research. Ann. New YorkAcad. Sc., 55: 275, 1952.

10. Romanoff, A. L., and Romanoff, A. J. : TheAvian Egg. New York, Wiley, 1949.

ii. Lillie, F. R. : Development of the Chick,

revised by H. L. Hamilton. New York,

Holt, 1952.

12. Hamburger, V., and Hamilton, H. L. : A

series of Ilormal stages in the develop-

meiit of the chick embryo. J. Morphol.,

88:49, 1951.13. Witschi, E. : Development of Vertebrates.

Philadelphia, Saunders, 1956.

14. Long, J. A., and Burlingame, P. L. : Thedevelopment of the external form of therat, with some observations on the originof tile extraembryOllic coelom and foetal

meml)ranes. Zoolog�’, 43: 143, 1938.15. Nicholas, J. S. : The Normal Stages in the

Development of the Rat. Osborn Zo-

ological Laboratory, Yale University

(Thesis), 1935, private printing.

16. Strong, R. NI. : The order, time and rateof ossification of the albino rat (Mus

Norvegicus Albinus) skeleton. Am. J.Anat., 36:313, 1926.

17. Henneberg, Bruno: Normentafeln zur

Entwicklungsgeschichte der Wander-ratte, Vol. 15 only (Keibel, F.:

Normentafeln zur Entwicklungsgeschi-chter der %Virbeltiere). Jena, Fischer,1937.

18. Karnofsky, D. A. : Nitrogen mustards in

the treatment of neoplastic disease.Advances lilt. Med., 4:1, 1950.

19. Philips, F. S. : Recent contributions to the

pharmacolog�’ of bis(2-haloethyl)amines

and sulfides. J. Pharmacol. & Exper.Therap. (Part II), 99:281, 1950.

20. Alexander, P. : The reactions of carcinogens

with macromolecules. Advances CancerRes., 2:2, 1954.

21. Ross, W. C. J. : The chemistry of cytotoxicalkylating agents. Advances Cancer Res.,1:397, 1953.

22. Dagg, C. P., Karnofskv, D. A., Toolan,

Helen W., and Roddy, Jacqueline:Serial passage of human tumors in chick

embryo; growth inhibition by nitrogen

mustard. Proc. Soc. Exper. Biol. & Med.,87:223, 1954.

23. Haskin, D. : Some effects of nitrogen

mustard on the development of external

body form in the fetal rat. Anat. Rec.,493:511, 1948.

24. Karnofskv, D. A., Patterson, P. A., andRidgwav, L. P. : Toxicity of roentgen

rays to chick embryo. Am. J. Roent-genol., 64:280, 1950.

25. Clarke, D. A., Barclay, R. K., Stock, C. C.,

and Rondestvedt, C. S., Jr. : Triazenes asinhibitors of mouse sarcoma 180. Proc.

Soc. Exper. Biol. & Med., 90:484, 1955.26. Burchenal, J. H., Dagg, M. K., Beyer, M.,

and Stock, C. C. : Chemotherapy ofleukemia. VII. Effect of substitutedtriazenes on transplanted mouse leu-kemia. Proc. Soc. Exper. Biol. & Med.,

91:398, 1956.27. Dagg, C. P., Karnofskv, D. A., Stock,

C. C., Lacon, C. R., and Roddy, Jacque-line: Effects of certain tniazenes on chick

embryos and on tumors explanted to

the chorioallantois. Proc. Soc. Exper.

Biol. & Med., 90:489, 1955.28. Oleson, J. J., Sloboda, A., Troy, W. P.,

Halliday, S. L., Landes, M. J., Angier,R. B., Semb, J., Cyr, K., and Williams,

J. H. : The carcinostatic activity of some2-amino-1,3,4-thiadiazoles. J. Am. Chem.Soc., 77:6713, 1955.

29. Burchenal, J. H., and Dagg, M. K. : Effectsof 6-diazo-5-oxy-L-norleucine and 2-

ethylaminothiadiazole on strains of trans-planted mouse leukemia. (Abstract.)Proc. Am. A. Cancer Res., 2:97, 1956.

30. Krakoff, I. H., and Magill, G. B. : Effects of

2-ethylamino-1,3,4 thiadiazole HC1 onuric acid production in mall. Proc. Soc.Exper. Biol. & Med., 91 :470, 1956.

31. Johnson, W. J., and McColI, J. D. : 6-aminomcotinamide-a potent nicotina-

mide antagonist. Science, 122:834, 1955.

32. Gelihorn, A., and Hirschberg, E., eds.:Investigation of Diverse Sy’stems forCancer Chemotherapy Screening. Chi-cago, Univ. Chicago Press, 1955, suppl.3.

33. Ellison, R. R., Karnofsky, D. A., Stern-

berg, S. S., Murphy, M. L., andBurchenal, J. H. : Clinical trials of o-diazoacetvl-L-serine (Azaserine) in neo-plastic disease. Cancer, 7:801, 1954.

34. Dagg, C. P., and Karnofskv, D. A. : Pro-

tection against teratogenic action ofazaserine and DON. Federation Proc.,15:238, 1956.

35. Dagg, C. P., and Karnofsky, D. A. : Terato-genie effects of azaserine on the chickembryo. J. Exper. Zool., 130:555, 1955.

36. Murphy, M. L., and Karnofskv, D. A.:Effect of azaserine and other growth-inhibiting agents on fetal developmentof the rat. Cancer, 9:955, 1956.


1957;19;701Pediatrics M. Lois Murphy, C. P. Dagg and David A. Karnofsky

EMBRYOS: An Exhibit with Additions for PublicationCOMPARISON OF TERATOGENIC CHEMICALS IN THE RAT AND CHICK

ServicesUpdated Information &

http://pediatrics.aappublications.org/content/19/4/701including high resolution figures, can be found at:

Permissions & Licensing

http://www.aappublications.org/site/misc/Permissions.xhtmlentirety can be found online at: Information about reproducing this article in parts (figures, tables) or in its

Reprintshttp://www.aappublications.org/site/misc/reprints.xhtmlInformation about ordering reprints can be found online:


http://http://pediatrics.aappublications.org/content/19/4/701

http://www.aappublications.org/site/misc/Permissions.xhtml

http://www.aappublications.org/site/misc/reprints.xhtml

1957;19;701Pediatrics M. Lois Murphy, C. P. Dagg and David A. Karnofsky

EMBRYOS: An Exhibit with Additions for PublicationCOMPARISON OF TERATOGENIC CHEMICALS IN THE RAT AND CHICK

http://pediatrics.aappublications.org/content/19/4/701the World Wide Web at:

The online version of this article, along with updated information and services, is located on

Copyright © 1957 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 1073-0397. American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007.been published continuously since 1948. Pediatrics is owned, published, and trademarked by the Pediatrics is the official journal of the American Academy of Pediatrics. A monthly publication, it has


http://pediatrics.aappublications.org/content/19/4/701

comparison of teratogenic chemicals in the...

Documents