Reproductive and Teratogenic Effects: No More Thalidomides? - ACS

8 Reproductive and Teratogenic Effects: No More Thalidomides? ROCHELLE WOLKOWSKI-TYL

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: August 10, 1981 | doi: 10.1021/bk-1981-0160.ch008

Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709

The term t e r a t o l o g y was f i r s t coined by the f a t h e r and son team Etienne and I s i d o r e Geoffrey S a i n t - H i l a i r e f o r t h e i r i n v e s t i gations of malformations, or monsters ( t e r a s , p l u r a l t e r a t a , from the Greek monster), mostly i n the chick embryo (books published 1822 and 1832). However, human concern with c o n g e n i t a l malformat i o n s i s as ancient as human awareness, mentioned i n B i b l i c a l references and discussed by A r i s t o t l e ( c i t e d i n 136). E a r l i e s t views were that embryos and fetuses were a f f e c t e d s t r u c t u r a l l y by maternal experience during pregnancy; the concept that "maternal impressions" d i r e c t l y a f f e c t e d the unborn gained widespread c r e dence f o r c e n t u r i e s . With the advent of the enlightened s c i e n t i f i c atmosphere i n western Europe i n the nineteenth century, a t t i t u d e s s h i f t e d t o the opinion that the embryo and f e t u s were i n v i o l a t e i n the uterus, untouchable by the environment. The p r e s e n t a t i o n and subsequent a p p r e c i a t i o n of Mendel's Laws of genetics provided the apparent explanation f o r observed abnormal births: a l l flaws arose from genetic mishaps during gametogenesis and the zygote developed based s o l e l y on the incoming genetic information. The twentieth century brought with i t the f i r s t experimental evidence f o r the r o l e of the environment i n production of abnormal o f f s p r i n g . E a r l y experiments on pregnant mammals i n v o l v e d s t u d i e s with i o n i z i n g r a d i a t i o n (43, 66) and sex hormones (33, 34, 69). Studies with d i e t a r y d e f i c i e n c i e s , drugs and chemicals followed rapidly. In 1933, Hale reared pregnant pigs on a vitamin A d e f i c i e n t d i e t and produced anophthalmic p i g l e t s (37, 38). Data were a l s o presented i n 1948 f o r the e f f e c t s of trypan blue and n i t r o g e n mustard on developing r a t embryos (28. 44). The supposed s a f e t y of the human f e t u s In utero was d i r e c t l y challenged by Gregg i n 1941 Ç35) who reported that a German measles epidemic i n A u s t r a l i a r e s u l t e d i n o f f s p r i n g with c a t a r a c t s , deafness and c o n g e n i t a l heart disease. These r e s u l t s were confirmed with the b i r t h of almost 20,000 d e f e c t i v e c h i l d r e n f o l l o w i n g a r u b e l l a epidemic i n

0097-6156/81/0160-0115$10.25/0 © 1981 American Chemical Society Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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the United States i n 1964 (121). The thalidomide d i s a s t e r occurr i n g worldwide i n 1955-1965, u l t i m a t e l y i n v o l v i n g over 8,000 c h i l d r e n i n 28 c o u n t r i e s , was f i r s t reported by Lenz (67, 68) and McBride (75). These events t r i g g e r e d awareness of the v u l n e r a b i l i t y of the i n t r a u t e r i n e occupant to outside i n f l u e n c e s . However, anecdotal evidence f o r r e p r o d u c t i v e e f f e c t s of substances such as lead or mercury on women i n i n d u s t r i a l exposures has been accumulating f o r c e n t u r i e s . The current view i s that embryos and fetuses may be espec i a l l y v u l n e r a b l e to environmental i n s u l t because of q u a l i t a t i v e and/or q u a n t i t a t i v e d i f f e r e n c e s from a d u l t s . These f a c t o r s include: 1. Small c e l l number 2. Rapid rates of p r o l i f e r a t i o n 3. High p r o p o r t i o n of u n d i f f e r e n t i a t e d c e l l s 4. Requirement f o r p r e c i s e temporal and s p a t i a l sequencing of s p e c i f i c c e l l s and c e l l products at the a p p r o p r i a t e p l a c e and time f o r normal d i f f e r e n t i a t i o n , i n c l u d i n g programmed c e l l death 5. Unique metabolism: presence or absence of i n d u c i b l e and/or c o n s t i t u t i v e r e p a i r enzymes, a c t i v a t i n g and d e t o x i f y i n g enzymes, eg. DNA r e p a i r enzymes, the mixed f u n c t i o n oxidases, e t c . 6. Unique t i s s u e s e n s i t i v i t i e s which may be t r a n s i e n t 7. Immaturity of immunosurveillance mechanisms, of s p e c i a l concern f o r the i n d u c t i o n of t r a n s p l a c e n t a l c a r c i n o genesis There i s a l s o the awareness that s e n s i t i v i t y t o environmental i n s u l t , and subsequent expression of that i n s u l t , does not cease with b i r t h . The mammal at term i s not a miniature adult; a part i a l l i s t of systems s t i l l undergoing d i f f e r e n t i a t i o n i n c l u d e : the nervous, endocrine, u r o g e n i t a l , d i g e s t i v e and immune systems. Expression o f an i n s u l t i n c u r r e d i n utero may not develop u n t i l a f t e r b i r t h , i n the human up to ten years of age f o r most detected anomalies, but with a latency of 15-30 years f o r c a r c i n o g e n i c events. A current working d e f i n i t i o n of t e r a t o l o g y , t a k i n g i n t o account the above c o n s i d e r a t i o n s and concerns has been generated by Wilson (136): Teratology i s the study of adverse e f f e c t s of the environment on developing systems; that i s , on germ c e l l s , embryos, fetuses and immature p o s t n a t a l i n d i v i d u a l s . More comprehensively, i t deals with the causes, mechanisms and manifestat i o n s of developmental d e v i a t i o n s of e i t h e r s t r u c t u r a l o r funct i o n a l nature. Agents which a l t e r the r a t e of growth of the fetus or are l e t h a l to the fetus without producing s p e c i f i c anatomic or f u n c t i o n a l anomalies are thought by some to be b e t t e r termed developmental toxins than teratogens (36). Increasing concern i s being r a i s e d as to r e p r o d u c t i v e and t e r a t o g e n i c r i s k s f o r a number of reasons: the increase of women e s p e c i a l l y of c h i l d b e a r i n g age i n the workforce i n n o n - t r a d i t i o n a l

Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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and Teratogenic

Effects

111


jobs, the i n c r e a s i n g l y r a p i d i n t r o d u c t i o n of new chemicals (15002000 new chemicals synthesized or otherwise produced each y e a r ) , and the awareness that r e l a t i v e l y l i t t l e i s known concerning the reproductive and t e r a t o l o g i c a l r i s k involved with exposure to chemicals already i n the workplace; TSCA l i s t s over 60,000 chemic a l s i n current usage. There i s also i n c r e a s i n g evidence from both human and l a b o r a t o r y animal data that the male may mediate teratogenic e f f e c t s on the developing f e t u s . Categories of Teratogenic Agents. Many substances are known to be t e r a t o g e n i c i n one or more species of mammals (Table I ) . The emphasis has been p r i m a r i l y on drugs, with data generated by drug research companies adhering to FDA G u i d e l i n e s (31) f o r reprod u c t i v e t e s t i n g o f drugs, and the awareness that i n our drugpermissive s o c i e t y women consume an average of four drugs, both by p r e s c r i p t i o n and over-the-counter a d m i n i s t r a t i o n , during pregnancy (76, 84, 88). Schardein (98) has l i s t e d over 1200 drugs evaluated as teratogens; Shepard's c a t a l o g (102) l i s t s 600 t e r a t o l o g i c a l agents, only 20 of which are documented as human teratogens. Meyers and Meyers (77) l i s t 527 t e r a t o g e n i c substances but t h e i r l i s t i s based on human err animal data. Human t e r a t o g e n i c agents have been discovered i n i t i a l l y from anecdotal observations, and then more r i g o r o u s l y examined i n epidemiological studies. Suspect human teratogens have been defined one of two ways: c l i n i c i a n s use anecdotal data, animal model researchers have suggested that any agent p o s i t i v e i n two or more mammalian species must be considered a suspect human t e r a togen. Some examples of agents i n both c a t e g o r i e s are presented i n Table I I . According to the N a t i o n a l Foundation (_3) about 7% of a l l l i v e b o r n humans w i l l have b i r t h d e f e c t s . This value may be as high as 10% i f c h i l d r e n are evaluated to age 10 years to i n c l u d e s u b t l e s t r u c t u r a l , f u n c t i o n a l d e f i c i t s such as minimal b r a i n d y s f u n c t i o n . More than 560,000 l i v e s out of approximately three m i l l i o n b i r t h s per year i n the United States are l o s t through i n f a n t death, spontaneous abortion, s t i l l b i r t h s and m i s c a r r i a g e due presumably to d e f e c t i v e f e t a l development. The r e l a t i v e c o n t r i b u t i o n s to human t e r a t o g e n e s i s have been estimated by Wilson (136, 140) as f o l l o w s : known germinal mutations: 20%; chromosomal and gene a b e r r a t i o n s : 3-5%; environmental causes such as radiation: < 1%; i n f e c t i o n s : 2-3%, maternal metabolic imbalance: 1-2%; drugs and environmental chemicals: 4-5%; c o n t r i b u t i o n s from maternal d i e t a r y d e f i c i e n c i e s or excesses and combinations or i n t e r a c t i o n s of drugs and environmental chemicals are unknown. Wilson (136, 140) estimates the c o n t r i b u t i o n from unknown sources as 65-70%. The estimated 20-25% pregnancy l o s s due to chromosomal a b e r r a t i o n s may be even higher due to e a r l y l o s s e s diagnosed as l a t e menstrual b l e e d i n g . Recovered t i s s u e s from spontaneous abort i o n s p r i o r to the t h i r t e e n t h week of g e s t a t i o n e x h i b i t chromosomal anomalies on the order of 560 per 1000 a b o r t i o n s ; the value



a

Teratology

Teratogenic e f f e c t s were u s u a l l y seen only at doses w e l l above t h e r a p e u t i c l e v e l s f o r the drugs, o r above l i k e l y exposure l e v e l s f o r the environmental chemicals.

S a l i c y l a t e s (e.g., a s p i r i n , o i l of wintergreen) C e r t a i n a l k a l o i d s (e.g., c a f f e i n e , n i c o t i n e , c o l c h i c i n e ) T r a n q u i l i z e r s (e.g., meprobamate, chlorpromazine, r e s e r p i n e , diazepam) Antihistamines (e.g., b u c l i z i n e , m e c l i z i n e , c y c l i z i n e ) A n t i b i o t i c s (e.g., chloramphenacol, s t r e p t o n i g r i n , p e n i c i l l i n ) Hypoglycemics (e.g., carbutamide, tolbutamide, hypoglycins) C o r t o i d s (e.g., triamcinolone, c o r t i s o n e ) A l k y l a t i n g agents (e.g., busulfan, chlorambucil, cyclophosphamide, TEM) A n t i m a l a r i a l s (e.g., chloroquine, q u i n a c r i n e , pyrimethamine) A n e s t h e t i c s (e.g., halothane, urethan, n i t r o u s oxide, p e n t o b a r b i t a l ) A n t i m e t a b o l i t e s (e.g., f o l i c a c i d , purine and pyrimidine analogs) Solvents (e.g., benzene, d i m e t h y l s u l f o x i d e , propylene g l y c o l ) P e s t i c i d e s (e.g., a l d r i n , malathion, c a r b a r y l , 2,4,5-T, captan, f o l p e t ) I n d u s t r i a l e f f l u e n t s (e.g., some compounds of Hg, Pb, As, L i , Cd) P l a n t s (e.g., locoweed, l u p i n s , jimsonweed, sweet peas, tobacco s t a l k s ) Miscellaneous (e.g., trypan blue, t r i p a r a n o l , diamox, etc.)

a

TABLE I. SOME TYPES OF DRUGS AND ENVIRONMENTAL CHEMICALS THAT HAVE BEEN SHOWN TO BE TERATOGENIC IN ONE OR MORE SPECIES OF MAMMALS (137)


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8. WOLKOWSKI-TYL

Reproductive

and Teratogenic

TABLE I I .


A.

Effects

119

HUMAN TERATOGENS

Known: alcohol a n t i b i o t i c s ( t e t r a c y c l i n e , sulfonamides, chloramphenicol) a n t i c o n v u l s a n t s (diphenylhydantoin / b a r b i t u r a t e s ) f o l a t e antagonists (aminopterin, methotrexate) lead methylmercury (Minimata disease) smoking s t e r o i d hormones ( o r a l progestins, androgens, estrogens) Thalidomide Vitamin D (excess)

B.

Suspect amphetamines a n t i c o n v u l s a n t s (paramethadione, trimethadione) antihistamines a n t i m a l a r i a l s (quinine, chloroquine) a n t i t h y r o i d drugs, i o d i d e s and i o d i n e l a c k (temporary?) aspirin barbiturates b l i g h t e d potatoes (solanine) folate deficiency hormonal pregnancy t e s t s and c o n t r a c e p t i v e s hypoglycemic agents ( o r a l ) l y s e r g i c a c i d diethylamide (LSD)? operating room environment - probable organic solvents pesticides, fungicides, herbicides P o l y c h l o r i n a t e d biphenyls (PCBs) (Yusho disease) Warfarin (anticoagulant) - probable Vitamin D ( d e f i c i e n c y ) Taken from references 2^5, 7Q, 7j5, 83, 105 and 137


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THE PESTICIDE CHEMIST AND MODERN TOXICOLOGY


at term i s 5 per 1000. Of the c h i l d r e n born l i v e who subsequently d i e i n the f i r s t year of l i f e , approximately 20% of the deaths are a s s o c i a t e d with or caused by b i r t h d e f e c t s , more than any other s i n g l e f a c t o r (136). There i s one f i n a l , almost p l a i n t i v e maxim sometimes termed Karnofsky's law (87) that almost any substance may be t e r a t o g e n i c i f given i n appropriate dose regimens to a g e n e t i c a l l y s u s c e p t i b l e organism at s u s c e p t i b l e stage or stages of embryonic or f e t a l development. Determinants of Teratogenic S u s c e p t i b i l i t y . Factors which i n f l u e n c e the t e r a t o g e n i c response are l i s t e d i n Table I I I . Genetic s u s c e p t i b i l i t y v a r i e s among species, f o r example: a s p i r i n i s t e r a t o g e n i c i n rodents but not i n primates, imipramine i s t e r a t o g e n i c i n r a b b i t s , but not i n humans, thalidomide i s t e r a t o genic i n primates but not i n rodents. D i f f e r e n c e s a l s o e x i s t among s t r a i n s . Inbred mouse s t r a i n s d i f f e r r a d i c a l l y i n t h e i r response to many t e r a t o g e n i c agents, f o r example to c o r t i s o n e i n d u c t i o n of c l e f t p a l a t e (54) and cadmium-induced t e s t i c u l a r and embryotoxicity (144, 145). I n d i v i d u a l s a l s o vary i n t h e i r r e sponse to t e r a t o g e n i c agents i n outbred s t r a i n s and heterogeneous human populations. The current i n t e r p r e t a t i o n i s that teratogens act on a s u s c e p t i b l e genetic locus or l o c i which may c o n t r o l d i s p o s i t i o n of the agent i n c l u d i n g absorption, metabolism, t r a n s port or e x c r e t i o n and/or d i r e c t s u s c e p t i b i l i t y of the target t i s s u e or organ. The teratogen t h e r e f o r e i n c r e a s e s the i n c i d e n c e of p r e v i o u s l y e x i s t i n g malformations; i t s a c t i o n must be viewed against the "background n o i s e " of spontaneous malformation r a t e s , which a l s o vary among species, s t r a i n s and i n d i v i d u a l s . The phocomelic syndrome, induced by thalidomide, occurs at a low r a t e spontaneously i n human populations; approximately 20-80% of the human fetuses exposed, presumably to the a p p r o p r i a t e dose at the appropriate time, developed the malformations (20). This concept of environmentally induced i m i t a t i o n s of genetic anomalies was presented f i r s t by Landauer (63) with experimental evidence of "phenocopies". There i s some s p e c i f i c i t y of agent on the t e r a t o l o g i c a l response (Table IV) w i t h acetazolamide causing perhaps the most s p e c i f i c l e s i o n (74). However, there are almost always e f f e c t s on other systems d e r i v e d i n many cases from d i f f e r e n t primary embryonic germ l a y e r s . The g e s t a t i o n a l stage of the embryo or fetus at the time of environmental i n s u l t appears to be the most c r i t i c a l determining f a c t o r . Figure 1 examines time periods of embryonic and f e t a l development i n humans, mice, and r a t s . The p r e d i f f e r e n t i a t i o n period, from f e r t i l i z a t i o n to establishment of the three primary embryonic germ l a y e r s , i s considered r e f r a c t o r y to t e r a togenic agents (although there are some exceptions such as hypoxia, 125 hypothermia, 104; and actinomycin D, 135). This r e s i s t a n c e has been explained as due to the small, omnipotent c e l l p o p u l a t i o n of the p r e - and immediately p o s t - i m p l a n t a t i o n embryo. C e l l damage


Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981. DETERMINANTS OF TERATOGENIC SUSCEPTIBILITY

Genotype: s p e c i e s , s t r a i n , i n d i v i d u a l S p e c i f i c i t y o f agent G e s t a t i o n a l stage at exposure Dose Route of a d m i n i s t r a t i o n : i n h a l a t i o n , percutaneous absorption, po, i p , se, gavage Duration of a d m i n i s t r a t i o n : short-term, c h r o n i c , i n t e r m i t t e n t D i s p o s i t i o n of agent (maternal, p l a c e n t a l and f e t a l pharmacokineti a. Absorption b. Equilibrium (1) maternal compartments: blood, organs (2) P l a c e n t a l (maternal/placental, p l a c e n t a l / f e t a l ) (3) F e t a l and f e t a l compartments c. Metabolism: a c t i v a t i o n / i n a c t i v a t i o n d. Transport, e s p e c i a l l y t r a n s p l a c e n t a l e. Excretion Animal status a. Age b. Health c. I n t e r a c t i o n s : synergisms, antagonisms, p r o t e c t i o n s

TABLE I I I .



m a s c u l i n i z a t i o n of female

" F e t a l A l c o h o l Syndrome" s m a l l s i z e , shortened p a l p e b r a l f i s s u r e s , stub nose, h i r s u t i s m

b r a i n (exencephaly, hydrocephaly) f a c i a l c l e f t s , eye d e f e c t s , v e r t e b r a e and r i b s , spina b i f i d a

post a x i a l d e f e c t s i n r i g h t forepawf o u r t h and f i f t h d i g i t s and c o r r e s ponding metacarpals

cleft

hydrocephalus, s p i n a b i f i d a , ear, eye, c a r d i o v a s c u l a r d e f e c t s

Androgenic hormones

Alcoho1ism-human (poor n u t r i t i o n ? contaminants?)

Aspirin (salicylic a c i d ) (d 9-11, r a t s )

Acetazolamide (and other c a r b o n i c anhydrase i n h i b i t o r s )

C o r t i s o n e (d 11-14 i n mouse, 23 agents l a s t count)

Trypan blue, rat)

syndactylous (18%) paws

TEM (d 12, r a t )

f o r e (78%) and r e a r

syndactylous f o r e (86%) and r e a r (80%) paws, s h o r t kinky t a i l

Myleran (d 12, r a t )

(d 6-9,

m u s c u l o s k e l e t a l system phocomelia (d 39-45) f a c e

Thalidomide (d 34-50 postmenstruation, human)

palate

c e n t r a l nervous system ( b r a i n , eye, s k u l l )

Excess v i t a m i n A (d 9-16 g e s t a t i o n , r a t )

fetuses

C h a r a c t e r i s t i c Anomaly Effects syndactyly, defects

defects

palate

encephalocele,

cleft

edema

c l e f t palate, s k u l l , t a i l defects, umbilical hernia

heart

c l e f t palate, c a r d i a c anomalies

anorectal stenosis (d. 49-51)

cleft palate, genitourinary

Other

SPECIFICITY OF AGENT ON TERATOGENIC RESPONSE

Agent

TABLE IV.


Reproductive

and Teratogenic

Effects


WOLKOWSKI-TYL


123


124


or death i s e i t h e r c o r r e c t e d f o r by the s u r v i v i n g c e l l s , which r e g u l a t e to produce a normal, a l b e i t small term f e t u s , or the c e l l l o s s i s so devastating that the embryo d i e s . Once implantation and establishment of the primary germ l a y e r s have occurred, the major period of organogenesis begins, a p e r i o d of 8-9 days i n rodents and approximately 40 days i n humans. This i s the p e r i o d of maximal s u s c e p t i b i l i t y to teratogenic agents causing s t r u c t u r a l anomalies. Using data generated from s t u d i e s with actinomycin D and other chemicals, Wilson (133, 134, 135, 136) has described the d i f f e r e n t i a l s u s c e p t i b i l i t i e s of embryonic organ systems to t e r a togenic agents during organogenesis i n the r a t (Table V and VI, F i g u r e 2). In the data from Table VI, f a t a l i t i e s p a r a l l e l m a l f o r mation rate. An increase i n deaths may a l s o obscure the d e t e c t i o n of the abnormality generated which caused the f a t a l i t y , so that the r e l a t i o n s h i p of deaths to anomalous fetuses becomes i n v e r s e . A d m i n i s t r a t i o n of a lower dose of the t e s t agent may be u s e f u l to detect the anomalies r e s p o n s i b l e f o r the f e t a l wastage. From F i g u r e 2, i t i s apparent that a d m i n i s t r a t i o n of an agent on gestat i o n day 10 would a f f e c t eye, b r a i n , heart and a n t e r i o r a x i a l s k e l e t a l development. The same agent, administered on day 15 would a f f e c t palate, u r o g e n i t a l and p o s t e r i o r a x i a l s k e l e t a l development. These times of s p e c i f i c s e n s i t i v i t y need not c o r r e spond to the morphological appearance of the organ or organ system, but to the time of c e l l biochemical commitment: the s h i f t of c e l l s from presumptive to determined s t a t u s . Once h i s t o g e n e s i s has begun: the d i f f e r e n t i a t i o n of t i s s u e s p e c i f i c biochemical and morphological c h a r a c t e r i s t i c s , the conceptus i s termed a fetus and i s viewed as i n c r e a s i n g l y r e f r a c t o r y to teratogenic agents. However, t h i s i s true only of most morphol o g i c a l or s t r u c t u r a l m a n i f e s t a t i o n s . Increasing evidence i n d i cates s u s c e p t i b i l i t y of the fetus to agents causing f u n c t i o n a l d e f i c i t s which presumably have a biochemical or m i c r o - s t r u c t u r a l b a s i s . Those systems not yet complete, e s p e c i a l l y the nervous system, are most v u l n e r a b l e . For example: Vitamin A (118), lead (85), methyl mercury (109, 110) and methyl azoxymethanol (53, 103) a l l cause n e u r o f u n c t i o n a l l e s i o n s when administered during t h i s period. In a d d i t i o n , t r a n s p l a c e n t a l carcinogens, such as d i e t h y l s t i l b e s t e r o l , e t h y l or methylnitrosourea, 7,12-dimethylbenzanthracene and nitrosomethylurethan, act during t h i s period i n humans, rodents and r a b b i t s (91). The l e s i o n i s expressed as a system s p e c i f i c tumor a f t e r a long l a t e n c y i n the p o s t n a t a l mature animal but the only exposure and t h e r e f o r e the i n i t i a t i o n of the l a t e r c a r c i n o g e n i c event occurs jin utero. The route and d u r a t i o n of a d m i n i s t r a t i o n of the agent i s a l s o c r i t i c a l f o r the development of the t e r a t o g e n i c anomaly. Human i n d u s t r i a l exposure i s almost always by i n h a l a t i o n or percutaneous a b s o r p t i o n of fumes, a e r o s o l s or vapors. Consumer or other secondary exposure would be by more v a r i e d routes. Experimental t e r a tology endeavors to d u p l i c a t e the human route of exposure f o r experimental animal models. I n h a l a t i o n presents problems of


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Effects

TABLE V SOME RAT TERATOGENS THAT HAVE L I T T L E EMBRYOTOXIC EFFECT ON THE SIXTH DAY OF GESTATION BUT ARE HIGHLY EFFECTIVE 3 OR A DAYS LATER (136)

Treatment Dose (mg/kg)


Agent

20-day

Day

Total implants

% dead resorbed

fetuses % survivors malformed

5-Fluorodeoxyuridine

20

6 9

114 209

6 10

1 38

Retinoic

20

6 9

95 79

5 44

0 84

6 10

207 88

7 48

5 65

7

1

acid

Actinomycin D

Controls

0.3

(vehicle)

558

Academic Press, Inc.

TABLE V I RELATIONSHIP BETWEEN DEATH AND MALFORMATION ACTINOMYCIN D IN THE RAT

FOR

Dose, mg/kg

Day treated

Fetuses

0.2

7 8 9 10 11

11.5 4.2 32.5 12.3 7.7

1.9 16.0 28.1 4.4 0

0.3

6 7 8 9 10 11

10.3 13.0 84.8 99.2 57.9 12.1

2.8 11.2 26.6 100.0 65.2 0.9

% dead

% malformed

Harper Hospital, Bulletin


126



Figure 2.

Differential sensitivities of embryonic organ systems to teratogens (136)



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127

c o n c e n t r a t i o n monitoring and dose assessment f o r the pregnant female s i n c e pulmonary parameters such as v e n t i l a t i o n , t i d a l volume, e x p i r a t i o n reserve volume, r e s p i r a t o r y t r a c t c a p i l l a r y d i l a t i o n and anatomic c o n s i d e r a t i o n s change r a d i c a l l y during pregnancy (122). The e f f e c t of s t r e s s due to the i n h a l a t i o n exposure i n confined, wire-rack cages i s a l s o a confounding factor. However, c l a s s i c a l t e r a t o l o g i c a l evaluations are being done on chemicals exposed by i n h a l a t i o n , most notably by the group at Dow Chemical Company (99, 100, 101). Exposure by s k i n absorpt i o n i s d i f f i c u l t t o q u a n t i f y and r e q u i r e s a complex a p p l i c a t i o n regimen. In a d d i t i o n , opinion i s mixed as to whether route of entry o f the agent makes a d i f f e r e n c e i n the u l t i m a t e d i s t r i b u t i o n and metabolism of the agent under examination. F i r s t pass organ absorption and metabolism may d i f f e r i f the exposure i s by i n h a l a t i o n to the lung, or o r a l l y to d i g e s t i v e system and l i v e r , although subsequent transport and organ exposure may y i e l d e q u i v a l e n t metabolite patterns. Most t e r a t o l o g y s t u d i e s u s u a l l y employ a d m i n i s t r a t i o n of the t e s t compound i n the feed, by o r a l i n t u b a t i o n o r i n j e c t i o n i n t o the dam. Timing i s important. Experimental exposure before implantat i o n o r during e a r l y organogenesis may r e s u l t i n i n t e r f e r e n c e with implantation o r i n e a r l y embryonic death, r e s u l t i n g i n no term f e t u s e s . Exposure before peak s u s c e p t i b i l i t y or repeated exposure may induce a c t i v a t i n g and/or d e t o x i f y i n g enzymes i n dam, placenta and/or f e t u s . This may r e s u l t i n increased or decreased blood l e v e l s of the a c t i v e metabolite i n the dam, and t h e r e f o r e a l t e r e d exposure to the f e t u s . Conversely, these enzymes may be i n h i b i t e d by accumulation o f metabolite(s) again a l t e r i n g blood l e v e l s of parent compound and m e t a b o l i t e ( s ) . Other e f f e c t s of repeated or e a r l y exposure may be to a l t e r l i v e r o r kidney f u n c t i o n , f o r example, as w e l l as to induce p a t h o l o g i c a l changes i n these organs which w i l l a f f e c t quantity and q u a l i t y of compound reaching the fetus. S a t u r a t i o n of p r o t e i n - b i n d i n g s i t e s may a l s o occur i n the dam to a l t e r transport. A l l of these e f f e c t s may a l t e r the dispos i t i o n parameters l i s t e d i n Table I I I and obscure or change any t e r a t o l o g i c a l e f f e c t s of the agent being examined (136). Dose range and schedule are a l s o c r i t i c a l . Three to four dose l e v e l s are u s u a l l y employed: high dose: t o x i c to the matern a l organism, perhaps l e t h a l to 10-15% of dams, e s s e n t i a l l y t o o b t a i n an e f f e c t , and to e s t a b l i s h target organ(s); mid dose(s): embryotoxic o r embryolethal and a s l i g h t l y lower dose to obtain t e r a t o g e n i c l e v e l with overlap between these two dose l e v e l s ; and low dose: comparable on a body weight b a s i s to p o s s i b l e human exposure l e v e l s or small m u l t i p l e s thereof. T e r a t o l o g i c a l T e s t i n g . Following the reports of the e f f e c t s of thalidomide on fetuses exposed during the f i r s t t r i m e s t e r which appeared i n 1961-1965, (67, 68, 75), the United States Food and Drug A d m i n i s t r a t i o n (FDA) e s t a b l i s h e d Guidelines f o r Reproductive Studies f o r Safety E v a l u a t i o n of Drugs f o r Human Use (31). These



128


are presented diagrammatically i n Figure 3. These g u i d e l i n e s were promulgated "as a routine screen f o r the a p p r a i s a l of s a f e t y of new drugs f o r use during pregnancy and i n women of c h i l d b e a r i n g p o t e n t i a l . " (31). Phase I designated Study of F e r t i l i t y and General Reproductive Performance i n v o l v e s exposure of the males for 10 weeks p r i o r to mating to include exposure during a l l phases of spermatogenesis, estimated as 8 weeks duration i n rodents, and of the females f o r 2 weeks to include oogenesis, a 5 day c y c l e i n rodents. Exposure i s continued i n the females through pregnancy, p a r t u r i t i o n and l a c t a t i o n . One-half the dams are s a c r i f i c e d on g e s t a t i o n day 13 f o r examination of number and d i s t r i b u t i o n of embryos i n u t e r i n e horns, empty implantation s i t e s and r e s o r p t i o n s . The dams allowed to l i t t e r are examined f o r l i t t e r s i z e , s t i l l b o r n and l i v e b i r t h s . Dead pups are examined f o r s k e l e t a l anomalies. L i v e pups are examined f o r gross anomalies and i n d i v i d u a l l y weighed at d e l i v e r y , p o s t n a t a l day 4 and 21. Phase I I , e n t i t l e d T e r a t o l o g i c a l Study, involves treatment during organo genesis g e s t a t i o n a l days 6-15 (mouse) or 7-16 ( r a t ) . Since e v a l u a t i o n i n two species, one other than rodent, i s c a l l e d f o r , parameters f o r the r a b b i t are a l s o i n d i c a t e d . Dams are s a c r i f i c e d 1-2 days before the a n t i c i p a t e d date of p a r t u r i t i o n and fetuses are d e l i v e r e d by cesarean s e c t i o n . Data to be c o l l e c t e d i n c l u d e number of ovarian corpora l u t e a , l i v e and dead f e t u s e s , and e a r l y and l a t e r e s o r p t i o n s . L i v e fetuses are to be weighed and examined f o r e x t e r n a l malformations. In r a t s , one-third of each l i t t e r w i l l be examined f o r s o f t t i s s u e d e f i c i t s by d i s s e c t i o n or the Wilson technique (133, 136), two-thirds preserved and s t a i n e d f o r examination f o r s k e l e t a l anomalies. Rabbit fetuses are to be incubated f o r 24 hours to assess v i a b i l i t y , then a l l fetuses a r e examined f o r e x t e r n a l , v i s c e r a l and s k e l e t a l anomalies. Phase I I I , e n t i t l e d P e r i n a t a l and Postnatal Study, i n v o l v e s exposure of the dam during the f i n a l one-third of g e s t a t i o n and c o n t i n u i n g through p a r t u r i t i o n , and l a c t a t i o n t o weaning. This segment "should d e l i n e a t e e f f e c t s of the drug on l a t e f e t a l development, labor and d e l i v e r y , l a c t a t i o n , neonatal v i a b i l i t y , and growth of the newborn." (31). C r o s s - f o s t e r i n g i s suggested f o r t h i s phase i f s u r v i v a l of test-pups i s impaired. Rearing of pups from t h i s phase and phase I to evaluate reproductive and f e r t i l i t y performance i n these F^ animals i s a l s o suggested as a possibility. These g u i d e l i n e s have survived e s s e n t i a l l y i n t a c t and are now incorporated i n t o proposed U. S. Environmental P r o t e c t i o n Agency (EPA) g u i d e l i n e s (22) as w e l l as recent Interagency Regulatory L i a i s o n Group (IRLG) d r a f t g u i d e l i n e s (79). FDA has f u r t h e r proposed a three generation reproductive study (24) c u r r e n t l y i n use t o evaluate long-term e f f e c t s on reproduction and f e r t i l i t y i n c l u d i n g e f f e c t s on the germinal c e l l l i n e developing i n utero during exposure to the t e s t compound i n the Ρ generation and subsequent generations under continuous exposure to the t e s t substances (Figure 4 ) .


8.

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129

Effects

( F D A , 1966)

Phase I :

Study of Fertility and General Reproduction Performance

weaning

3


3L "

t

1

n

8

^

Examination:

Breeding, fertility, nidation, parturition, neonatal effects

sacrifice one-half lactation,

Teratological Study Ο

9—--C

Rat

Birth

Gross, visceral, skeletal on dead animals

Information o n :

Phase II:

one-hm

•j

_j

Birth

sacrifice

Rabbit

9'

10

C

]

ι |Birth sacrifice

Examination:

Gross, one-third visceral (Wilson sections) two-thirds skeletal (alizarin staining) (rabbit, 24 hr incubation, all fetuses gross, visceral and skeletal)

Information on:

Phase III:

Embryotoxicity,

teratogenicity

Perinatal and Postnatal Study weaning

9'Birth

3£ sacrifice

Examination:

litter size, pup weight,

Information o n :

Figure 3.

Parturition,

e t c . , possible cross-fostering

lactation, neonatal effects

Guidelines for reproductive studies for safety evaluation of drugs for human use (FDA, 1966)



Weigh, obs Wean, s a c r

Figure 4.

Three-generation reproduction study (FDA, 1970)

(FDA A d v i s o r y Committee on P r o t o c o l s f o r S a f e t y Evaluations: Panel on R e p r o d u c t i o n Report on R e p r o d u c t i o n S t u d i e s , 1970)

Ρ

Parent


Autopsy, examine Tissue Histopathologically

8.

WOLKOWSKI-TYL

Reproductive

and Teratogenic

Effects

The FDA Guidelines (24·, 3V) have been the p r o t o c o l used i n almost a l l drug and chemical t e s t s done i n i n d u s t r y . There has been growing concern that these t e s t s are inadequate to i d e n t i f y s u b t l e morphological and f u n c t i o n a l d e f i c i t s expressed pre- or p o s t n a t a l l y . Research to improve the e s t a b l i s h e d t e s t s and to develop new t e s t s and approaches i s gaining momentum. The term s a c r i f i c e , the b a s i s f o r the phase I I Teratology Study t e s t involves counting implantations, r e s o r p t i o n s , dead and l i v e fetuses. L i v e fetuses are then examined f o r s o f t t i s s u e and s k e l e t a l anomalies. Detection of e a r l y implantation s i t e s , not d i s c e r n i b l e by p l a c e n t a l remains, may be v i s u a l i z e d by s t a i n i n g f r e s h u t e r i with ammonium s u l f i d e (61). Examination f o r l i v e f e t u s s o f t t i s s u e anomalies by the Wilson technique (133, 136) i n volves f i x a t i o n of fetuses i n B o u i n s s o l u t i o n f o r d e c a l c i f i c a t i o n , subsequent f r e e hand s e c t i o n s through the head and 1 mm f r e e hand razor cross s e c t i o n s of the trunk. Disadvantages i n c l u d e : d i f f i c u l t i e s i n recognizing c a r d i a c malformations i n s e r i a l c r o s s s e c t i o n s , d i f f i c u l t i e s i n d u p l i c a t i n g the s e c t i o n s from f e t u s to f e t u s and l i t t e r to l i t t e r , and the i n a b i l i t y t o examine skeleton of same f e t u s . A number of m o d i f i c a t i o n s have been suggested (6, 23, 113). The Staples technique e n t a i l s m i c r o d i s s e c t i o n of dec a p i t a t e d fetuses immediately a f t e r cesarean s e c t i o n a t term. Advantages i n c l u d e ease of examination f o r f u n c t i o n a l heart anomalies such as s e p t a l w a l l defects with no d i s t o r t i o n due to f i x a t i o n , with the e v i s c e r a t e d carcass a v a i l a b l e f o r s k e l e t a l examination, and the head preserved i n B o u i n s f o r l a t e r s e c t i o n ing by the Wilson technique. A m o d i f i c a t i o n of Staples' technique has a l s o been suggested (26). Examples of anomalies demonstrable by the Wilson technique i n the f e t a l head region are presented i n F i g u r e 5 (trypan blue: u n i l a t e r a l anophthalmia, hydroxyurea: c l e f t palate). The s k e l e t a l examination involves p r e s e r v a t i o n of the e v i s cerated fetus i n 70-95% ethanol, maceration and c l e a r i n g i n potassium hydroxide and s t a i n i n g with A l i z a r i n Red S, s p e c i f i c f o r calcium and t h e r e f o r e bone (18, 19). Many v e r s i o n s e x i s t (46, 113). T h i s procedure s t a i n s areas of o s s i f i c a t i o n but the r e searcher cannot d i s t i n g u i s h between s i t e s that would have o s s i f i e d i f the f e t u s had continued development, hence delayed o s s i f i c a t i o n , from t o t a l l y absent c a r t i l a g i n o u s anlagen, hence missing bone. Counterstaining with a c a r t i l a g e - s p e c i f i c s t a i n such as a l c i a n blue (49) allows d i s t i n c t i o n between delayed and absent ossification. S k e l e t a l anomalies demonstrable by the c l a s s i c A l i z a r i n technique are presented i n Figure 6 (hydroxyurea: fused r i b s , doubled c e n t r a ) . The b a s i c techniques enumerated above w i l l detect missing, e c t o p i c o r g r o s s l y abnormal organs, m a l p o s i t i o n of or missing major blood v e s s e l s , retarded, abnormal or absent o s s i f i c a t i o n . Embedding, s e c t i o n i n g and s t a i n i n g each fetus would allow detect i o n o f microscopic l e s i o n s but t h i s would r e q u i r e an extended p e r i o d of time and many person-hours and so i s not a p p l i c a b l e to T


131

T




132

Figure 5.

Representative soft tissue anomalies detected in Fischer-344 rat fetuses, gestation day 20, by Wilson sections



8.

WOLKOWSKI-TYL

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Effects

Figure 6. Representative skeletal anomalies detected in Fischer-344 rat fetuses, gestation day 20, by Alizarin Red S staining: control (leît); hydroxyurea, 200 mg/ kg/d administered by gavage on gestation days 7-20 (right,).


133


134

THE


r a p i d screening techniques. In a d d i t i o n , f u n c t i o n a l d e f i c i t s i n terms of biochemical a l t e r a t i o n s and e f f e c t s demonstrable only i n the p o s t n a t a l p e r i o d would not be detected. Detection of these l e s i o n s would be very important f o r human r i s k assessment. In a d d i t i o n , the animal model u t i l i z e d , u s u a l l y r a t , mouse and r a b b i t may not be the most s u i t a b l e t e s t system f o r p a r t i c u l a r agents i f metabolism of these substances d i f f e r s between the t e s t animal and the human. D i f f e r e n c e s i n metabolism may render r e s u l t s i n a t e s t animal system misleading or i r r e l e v a n t to e v a l u a t i o n of drug or chemical r i s k i n humans. At the Chemical Industry I n s t i t u t e of Toxicology, ( C U T ) , a chemical i s f i r s t examined to evaluate parameters of t o x i c o k i n e t i c s , d i s p o s i t i o n and metabolism i n the pregnant r a t and f e t o p l a c e n t a l u n i t i n c l u d i n g e v a l u a t i o n of p l a c e n t a l t r a n s p o r t of the parent compound and/or i d e n t i f i e d metabolites to c h a r a c t e r i z e the system p r i o r to any t e r a t o l o g i c a l t e s t i n g . Once the c h a r a c t e r i s t i c s and l i m i t s of the t e s t system are d e f i n e d , t e r a t o l o g i c a l s t u d i e s or e v a l u a t i o n of reproductive performance are then performed. Whole body autoradiography (WBAR) of the pregnant animal a f t e r exposure to a r a d i o l a b e l l e d t e s t chemical i s v a l u a b l e to assess d i s p o s i t i o n and target organ s p e c i f i c i t y with minimum of person-hours expended. T h i s technique compares f a v o r a b l y with c l a s s i c a l d i s p o s i t i o n s t u d i e s done by r a d i o i s o t o p i c a n a l y s i s on d i s s e c t e d maternal and f e t a l organs (50, 51). Figure 7 presents WBAR r e s u l t s f o r three C U T p r i o r i t y chemicals i l l u s t r a t i n g d i s t r i b u t i o n i n the dam and f e t u s e s . During term s a c r i f i c e , r e l a t i v e f e t a l organ weights may be determined as part of the s o f t t i s s u e examination at s a c r i f i c e (112). Data generated on a C U T p r i o r i t y chemical shown to be t o x i c to the adult spleen are shown i n Table VII. Hematological parameters may also be evaluated such as complete blood count using an automated counting system, and examination of blood smears f o r e v a l u a t i o n of nucleated red blood c e l l s and r e t i c u l o cytes. These l a t t e r c e l l types are very numerous i n the f e t u s and neonate. Touch preparations may a l s o be generated from cut surfaces of f r e s h maternal and f e t a l organs as a r a p i d a l t e r n a t i v e to f i x i n g , embedding and s e c t i o n i n g these t i s s u e s , to evaluate c e l l i n t e g r i t y , d i f f e r e n t i a t i o n and f u n c t i o n . In v i t r o t e s t systems are being considered f o r t e r a t o g e n i c i t y screening C7, 59). These systems i n c l u d e u n i c e l l u l a r organisms, somatic c e l l t i s s u e or organ c u l t u r e , and c u l t u r i n g of i n t a c t i n v e r t e b r a t e , lower v e r t e b r a t e , mammalian and avian embryos. Systems using mammalian embryos i n c l u d e c u l t u r e of pre-implantat i o n or post-implantation embryos and s p e c i f i c organ c u l t u r e s such as p a l a t e or limb bud (73). Preimplantation embryos from mouse (126) or man (114) have been grown s u c c e s s f u l l y i n c u l t u r e up to the b l a s t o c y s t stage. Using techniques developed by D.A.T. New and co-workers (82), postimplantâtion embryos from r a t (9) or mouse (96) have been c u l t u r e d iri v i t r o f o r up to four to f i v e days, with best r e s u l t s obtained from young p o s t i m p l a n t a t i o n embryos at


8.

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maternal

135

Effects

k idm

maternal stomach fetal eternal

bladder

liver

ternal

bladder

maternal maternal


fetal

maternal

maternal maternal

urine

h e p a t i c blood v e s s e l s k.dney

kidney

placenta

liver

lung

maternal

maternal

heart eternal

maternal

h e p a t i c blood v e s s e l s

spleen fetal

laternal

urine

intestine

c a r d i a c blood

maternal

h e p a t i c blood v e s s e l s 50 micron s e c t i o n '

Figure 7. WBARs of pregnant Fischer-344 rats, gestation day 20-21 exposed to various C priority chemicals. (A) WBAR of F-344 dam (gestation day 21) given C-terephthalic acid by gavage; dose =12.5 mg/kg (tracer dose), 30 ^Ci/dose, sacrificed 5.5 h after dose. (B) WBAR of F-344 dam (gestation day 20) given C2,4-dinitrotoluene by gavage; dose = 35 mg/kg, 100 fxCi/dose, sacrificed 6 h after dose. (C) WBAR of F-344 dam (gestation day 21) given C-aniline HCl by gavage for 5 d (gestation day 17-21 ); dose = 100 mg/kg/d, 4 μα/dose, sacrificed 6 h after last dose. 14

14

14

14


136


TABLE VII. 14

A. DAM PARAMETERS

1 day (n = 14 daws)

Hematocrit ± SE


DATA FROM PREGNANT F-344 FEMALES EXPOSED TO C-ANILINE-HCL (100 MG/KG) FOR 1 OR 5 DAYS 1

36.5 ± 0 . 4

Body weight t SE

5 day (n = 11 daws)

1

32.6 l 0.7* (10.68%Ο

214.02 ± 3.47

215.65 l 2.83

Total implantations/daw ± SE

7.9 ± 0.6

8.0 1 0.8

Live fetuses/dam ± SE

7.6 ± 0.6

7.5 t 0.7

% 0rgan/BW ratios ± SE Liver

3.541 ± 0.074

3.348 ± 0.075

Kidneys (2)

0.585 ± 0.010

0.570 ± 0.011

Spleen

0.171 t 0.006

0.247 1 0.010* (44.44%t)

B. FETAL PARAMETERS Hematocrit ± SE

30.2 ± 0 . 6

2

Placenta weight/fetus ± SE

3

Body weight/live fetus ± SE

4

29.2 ± 0.6

0.415 ± 0.009

0.430 ± 0.033

3.485 ± 0.249

4.157 ± 0.111 (19.28%t)

Liver

6.665 ± 0.282

6.301 ± 0.641

Kidneys (2)

0.698 ± 0.033

0.722 ± 0.022

Bladder

0.245 ± 0.014

0.260 ± 0.018

Spleen

0.109 ± 0.014

0.148 ± 0.009* (35.78%t)

^ams sacrificed 1-12 hours after ( l a s t ) gavage and data pooled 2 Hematocrits done on blood pooled from fetuses of one uterine horn. 3

Placenta weight/fetus is determined by dividing total placental weight/litter by number of f e t u s e s / l i t t e r .

4 Body weight/fetus is determined by dividing total weight of entire l i t t e r by number of fetuses in the l i t t e r . ^Organs were pooled from each l i t t e r to obtain enough tissue for weight and radioisotope determinations. Hence the organ/BW ratios represent l i t t e r total weight of each organ divided by the total weight of entire l i t t e r (sum of individual organs plus carcasses) for each dam. *Significantly different from 1 day exposed dams at ρ < 0.05.



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p r i m i t i v e streak or e a r l y head-fold stage. P r e l i m i n a r y r e p o r t s i n d i c a t e that t h i s expiant system i s s e n s i t i v e to known t e r a t o genic agents when they are administered t o r a t s whose serum i s then c o l l e c t e d f o r use as part of the c u l t u r e system f o r t e s t embryos (56). Further work (13) i n d i c a t e s that serum from human s u b j e c t s t r e a t e d with cancer t h e r a p e u t i c or a n t i c o n v u l s i v e agents causes l e t h a l i t y o r t e r a t o g e n i c i t y i n c u l t u r e d r a t embryos. For a review of t h i s promising technique, see Wilson (141). P o s t n a t a l t e s t i n g i s also becoming an important component of teratological testing. I t i s now recognized that i n utero adminis t r a t i o n of many c l a s s i c s t r u c t u r a l teratogens, at lower dosages and/or l a t e r times than u s u a l l y administered f o r production of s t r u c t u r a l e f f e c t s , r e s u l t s i n n e u r o f u n c t i o n a l and endocrine deficits. These l e s i o n s may be of a permanent nature and are d e t e c t a b l e only i n p o s t n a t a l l i f e . Agents so t e s t e d i n c l u d e methyl mercury (32, 108, 111), cadmium (32), Vitamin A (118), lead (60), and 5-azacytidine (94). These f i n d i n g s agree with human data on c h i l d r e n exposed to methyl mercury (2, 39) and lead (5) i n utero. Some agents that have been shown to cause n e u r o l o g i c a l d e f i c i t s had not been considered teratogens a t a l l . A few p r o t o c o l s f o r e v a l u a t i n g p o s t n a t a l development are c u r r e n t l y i n use on a l i m i t e d b a s i s both undergoing and awaiting validation. One such p r o f i l e i n use at C U T i s presented i n F i g u r e 8. Except f o r open f i e l d , which i s run a t C U T on p o s t n a t a l day 29 or 31, one of the most widely used b e h a v i o r a l t e s t s , the other parameters l i s t e d are not v o l i t i o n a l behaviors and a r e perhaps b e t t e r termed developmental landmarks. More s o p h i s t i c a t e d b e h a v i o r a l t e s t s are a l s o being u t i l i z e d (12, 48) with p r e l i m i n a r y attempts to develop and standardize screening methods f o r beh a v i o r a l t e r a t o l o g y (10). Problems e x i s t i n experimental design, s t a t i s t i c a l a n a l y s i s and i n t e r p r e t a t i o n (17). But, t h i s aspect of t e r a t o l o g i c a l t e s t i n g w i l l become an i n c r e a s i n g l y important area of research and required addendum to t e r a t o l o g i c a l t e s t i n g i n the f u t u r e , e s p e c i a l l y i n the l i g h t of increased r e g u l a t o r y agency concern i n t h i s area i n the United States and other c o u n t r i e s world wide. A number of thought-provoking essays have emerged on the e v o l u t i o n (142), current methodology and suggested improvements of t e r a t o l o g i c a l t e s t i n g (7, 16^, 116, 117, 139) and r e p r o d u c t i o n and f e r t i l i t y s t u d i e s (86, 93) which are f r u i t f u l reading beyond the scope of t h i s paper. E x t r a p o l a t i o n to Human Risk Assessment. The d i f f i c u l t y i n e x t r a p o l a t i n g from animal t o x i c i t y data to man i s compounded i n reproduction and t e r a t o l o g y r i s k assessment. Even before the experiment i s begun the choice of animal test system i s important. Is the rodent the best model f o r t h i s chemical? Which genus and s p e c i e s should be used: r a t or mouse? The r a b b i t i s commonly used i n a d d i t i o n to a rodent s i n c e i t i s r e l a t e d to Rodentia but belongs t o the order Lagomorpha and t h e r e f o r e s a t i s f i e s governmental r e g u l a t o r y agency requirements f o r two mammalian s p e c i e s ,


138


0 1

ι 1

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 l? IB 19 20 21 26 1 ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι Ί ι

28

ι

30

ι

ι

32

ι

ι

34

ι ι

3Θ

36

ι ι

ι

ι

ι

40

42

ι ι

ι

44

ι ι

46

ι ι

48

ι

50

ι ι ι

Optning of Vagina

T e s t i s Descent


Mid-Oir Righting Reflex

Opening of Eyelids

Wire Grasping Reflex

Auditory Startle Response

Cliff Avoidance

11

Pilotion Visible

Pinna Detachment

Ο

ilil ι

Surface Righting Reflex

I I I I I I I I I I, 2 3 4 5 6 7 8 9 10 I I 12 13 14 15 16 17 18 19 2 0 21 26

28

30

32

34

36

38

40

42

44

46

48

P O S T - N A T A L AGE IN DAYS

Figure 8.

Acquisition of postnatal developmental landmarks in the Fischer-344 rat


50


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one p r e f e r a b l y non-rodent. Which s t r a i n should be chosen? Should i t be inbred or outbred? Use of an inbred s t r a i n with g e n e t i c a l l y uniform animals allows observed v a r i a t i o n s (anomalies, abnormalities) t o be a s c r i b e d to the environment, i . e . , the agent under t e s t . Use of g e n e t i c a l l y heterogeneous animals, an outbred s t r a i n , allows d e t e c t i o n of t e r a t o g e n i c e f f e c t s i n v o l v i n g chemical-gene i n t e r a c t i o n s and approximates the human genetic s i t u a t i o n with a m u l t i p l i c i t y of genotypes ( h e t e r o z y g o s i t y ) . However, use of an outbred s t r a i n i n c r e a s e s v a r i a b i l i t y , and t h e r e f o r e the number of animals t o be used, and complicates i n t e r p r e t a t i o n of r e s u l t s i n c l u d i n g c a u s a l i t y of observed anomaly. Based on t o x i c o k i n e t i c , d i s p o s i t i o n and metabolism data, the rodent may not be the t e s t animal of choice. T e r a t o l o g i c a l researchers are a l s o using, or contemplating using, the r a b b i t , hamster, guinea p i g , a r m a d i l l o , f e r r e t , dog, miniature swine, c a t , or non-human p r i mates f o r drugs p r e s c r i b e d s p e c i f i c a l l y f o r pregnant women. P r e n a t a l development i n the Rodentia and Lagomorpha d i f f e r s i n s i g n i f i c a n t ways from that i n humans. A l l three have a c h o r i o a l l a n t o i c p l a c e n t a but that of humans i s hemochorial, where the c h o r i o n i c v i l l i of the f e t u s are bathed i n maternal blood and one l a y e r o f s y n c i t i a l trophoblast separates the maternal blood from the f e t a l c a p i l l a r i e s (21). The c h o r i o a l l a n t o i c p l a c e n t a of rodents and lagomorphs i s a complex hemoendothelial type composed of i n t i m a t e l y juxtaposed and modified f e t a l and maternal c e l l s , bathed by a l a b y r i n t h of blood sinuses (55) with three ( r a t , mouse and hamster) o r two ( r a b b i t ) t r o p h o b l a s t i c l a y e r s s e p a r a t i n g maternal blood from f e t a l c a p i l l a r i e s (21). The human and r a t p l a c e n t a a l s o d i f f e r f u n c t i o n a l l y with s e c r e t o r y patterns of p l a c e n t a l lactogen d i f f e r i n g and with the presence i n primates of c h o r i o n i c gonadotropin (80). What e f f e c t i f any these d i f f e r e n c e s have on p l a c e n t a l t r a n s p o r t i s not f u l l y understood. In a d d i t i o n , rodents and lagomorphs a l s o form a y o l k sac placenta immediately a f t e r implantation, which i s the major (only) mechanism f o r nut r i e n t processing and t r a n s p o r t u n t i l g e s t a t i o n day 11-11%, and p e r s i s t s as f u n c t i o n a l , even when the c h o r i o a l l a n t o i c p l a c e n t a forms, almost t o p a r t u r i t i o n . Again, what e f f e c t t h i s has on embryo and f e t a l v u l n e r a b i l i t y i s not yet known, although a t l e a s t one t e r a t o g e n i c agent, trypan blue, appears to a c t s o l e l y on the y o l k sac placenta (8). In m u l t i f e t a l pregnancies there are d i f f e r e n c e s i n blood flow to l e f t and r i g h t u t e r i n e horns and t o implants at o v a r i a n versus c e r v i c a l ends of the u t e r i n e horns. D i f f e r e n t fetuses w i t h i n the same dam have been shown to be at d i f f e r e n t i a l r i s k (119, 145). In a d d i t i o n , f e t a l l o s s i s handled differently: dead implants are not e x p e l l e d i n a spontaneous a b o r t i o n as i n s i n g l e - b i r t h mammals but are resorbed i n s i t u . I t i s not uncommon to recover healthy, v i a b l e f e t u s e s s i d e - b y - s i d e w i t h l a r g e numbers of r e s o r p t i o n s i t e s . Maternal, p l a c e n t a l and f e t a l metabolism of x e n o b i o t i c s may a l s o d i f f e r hence the need f o r p r i o r c h a r a c t e r i z a t i o n , at l e a s t , of the t e s t organism's metabolic c a p a b i l i t i e s o f the substance to be t e s t e d .



140

THE


The placenta i s both a transport and metabolizing organ. Transport i s accomplished by simple d i f f u s i o n , f a c i l i t a t e d d i f f u s i o n , a c t i v e t r a n s p o r t across membranes and by s p e c i a l processes such as p i n o c y t o s i s , phagocytosis and breaks i n the " b a r r i e r " (29). C h a r a c t e r i s t i c s of chemicals showing high t r a n s f e r from maternal blood to placenta i n c l u d e : low molecular weight (< 500 daltons optimal), high l i p i d / w a t e r p a r t i t i o n c o e f f i c i e n t ( l i p o p h i l i c ) , low i o n i z a t i o n at blood pH (pKa) and low binding to plasma p r o t e i n s (4). The placenta contains a f u l l complement of mixed f u n c t i o n oxidases l o c a t e d i n the microsomal and mitochond r i a l s u b c e l l u l a r f r a c t i o n s capable of i n d u c t i o n (eg. benzo(a)pyrene hydroxylase, 24). Metabolism i n the t e s t dam and/or fetus and i t s relevance to the human gravida i s a l s o c r i t i c a l . For example, the parent compound may be t e r a t o g e n i c and i s metabolized to innocuous products as with diphenylhydantoin, an a n t i - s e i z u r e drug used i n the treatment of e p i l e p s y (41). In c o n t r a s t , the parent compound may be harmless and must be metabolized to the proximal t e r a t o g e n i c agent as i n c h l o r c y c l i z i n e , an antihistamine metabolized i n v i v o to the a c t i v e teratogen n o r c h l o r c y c l i z i n e (57, 89). One of the current hypotheses concerning mechanism of thalidomide-induced teratogenesis suggests that thalidomide i s transmitted to the human f e t u s and metabolized to more p o l a r metabolite (s), the p u t a t i v e proximal t e r a t o g e n i c a g e n t ( s ) , which cannot cross the p l a c e n t a back to the maternal organism f o r f u r t h e r metabolism and e x c r e t i o n (58, 129). T h i s sequence may be q u a l i t a t i v e l y or q u a n t i t a t i v e l y d i f f e r e n t i n the i n s e n s i t i v e pregnant rodent. In c o n t r a s t , imipramine, an antidepressant, i s t e r a t o g e n i c i n r a b b i t s where blood l e v e l s of the parent compound stay high. In the human, imipramine i s r a p i d l y metabolized by demethylases and i s not t e r a t o g e n i c (42, 92). Mechanisms of Teratogenesis. Most t o x i c o l o g i s t s have viewed the experimental animal as a "black box" wherein one i n s e r t s t e s t chemicals u s u a l l y at high dose and observes e f f e c t s out. There has r e c e n t l y been a c a l l f o r low-dose exposure, examination of the e f f e c t s and the mechanisms by which they a r i s e and assessment of human r i s k of the e f f e c t s seen i n the t e s t systems (30). Simil a r l y , i n t e r a t o l o g y , the pregnant mammal has been considered a "black box" whereon exposures are done and r e s u l t i n g f e t u s e s examined with l i t t l e or no a t t e n t i o n to mechanisms. Without e l u c i d a t i o n of mechanisms, t e r a t o l o g i s t s are doomed to an endless succession of e m p i r i c a l t e s t i n g screens. Teratology must be concerned with a n t i c i p a t i o n of t e r a t o g e n i c r i s k which r e q u i r e s knowledge of mechanisms. Researchers must be able to e x t r a p o l a t e r e s u l t s from a t e s t compound at ( r e l a t i v e l y ) high dose on a t e s t animal to r i s k assessment to the human at ( u s u a l l y ) lower doses. Given the huge number of p o t e n t i a l teratogens i n use today and new ones e n t e r i n g the environment y e a r l y , t e r a t o l o g i s t s must be able to g e n e r a l i z e from known agents with known mechanisms



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to new agents with s i m i l a r f u n c t i o n a l groups, f o r example, with p r o j e c t e d s i m i l a r e f f e c t s and mechanisms. Landauer, j u s t before h i s death, was beginning to examine the r o l e of f u n c t i o n a l groups on the t e r a t o l o g i c a l response i n the chick embryo (63). The e l u c i d a t i o n o f s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (SARs) has been one of the major t o o l s i n pharmacology and t o x i c o l o g y . Terat o l o g i c a l screens can be improved i f mechanisms are known, by f o c u s s i n g on s p e c i f i c g e s t a t i o n a l times of exposure o r c l o s e examination of c e r t a i n organs expected to be t a r g e t s . One can evaluate the appropriate t e s t model i f the mechanism of a c t i o n i s known. For example, i f the s p e c i f i c biochemical pathways i n v o l v e d were i d e n t i f i e d , the experimental animal with such pathways most s i m i l a r to humans would be the t e s t system of choice. Longer range goals made p o s s i b l e by understanding mechanisms of a c t i o n would include prevention or a m e l i o r a t i o n of the developmental defect p r i o r to f i n a l manifestation by d i v e r t i n g the i n i t i a t i n g mechanism or i n t e r v e n t i o n at some p o i n t ( s ) i n the process of pathogenesis. This could be done by supplementation of d e f i c i e n t enzyme or substrate, r e s t r i c t i o n of d i e t to avoid excess accumulat i o n of a d e l e t e r i o u s metabolite, or augmentation of inadequate transport. One might, i n the future, prevent the "spontaneous" b i r t h defects which r e s u l t from i n t e r a c t i v e or m u l t i p l e causes yet u n i d e n t i f i e d i f the components of the e f f e c t , mechanisms and i n t e r a c t i o n s can be i d e n t i f i e d and the sequence from cause to m a n i f e s t a t i o n i n t e r r u p t e d (136). These i n t e r a c t i o n s , based on information already known, may be with n u t r i t i o n a l status (47), w i t h other teratogens or with a non-teratogen, a s o - c a l l e d "proteratogen" (14, 95, 130). Wilson (140) suggests a number of mechanisms of teratogenesis including: 1. mutations (somatic, that i s n o n - h e r i t a b l e ) ; 2. chromosomal non-disjunctions and breaks; that i s c l a s t o g e n i c events; 3. m i t o t i c i n t e r f e r e n c e ; 4. a l t e r e d n u c l e i c a c i d i n t e g r i t y or f u n c t i o n ; 5. l a c k of precursors and substrates r e q u i r e d f o r b i o s y n t h e s i s ; 6. a l t e r e d energy sources; 7. enzyme i n h i b i t i o n s ; 8. osmolar imbalance; 9. a l t e r e d membrane c h a r a c t e r i s t i c s . A current e s t i m a t i o n i s that 70% o f a l l mutagens are teratogens (40) but not a l l teratogens are mutagens. Thalidomide i s perhaps the best example of a human e p i g e n e t i c teratogenic agent. These a l t e r a t i o n s induced by the teratogen may occHir i n the i n t r a c e l l u l a r compartment i n the nucleus and cytoplasm, at the c e l l surface, i n the e x t r a c e l l u l a r matrix and/or a t the l e v e l of the f e t a l environment: f e t a l organism, p l a c e n t a l or maternal i n t e r a c t i o n s (97). CONCLUSIONS T h i s review has so f a r focussed on the maternal organism as the source to the f e t u s of the teratogenic agent. However, the male has been implicated as the cause of a teratogenic event i n animal studies f o r example, with methadone (106), thalidomide



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(71), lead, n a r c o t i c s , a l c o h o l and c a f f e i n e ( c i t e d i n Science 202:733, 1978). Human male mediation has been s t a t i s t i c a l l y confirmed i n s t u d i e s i n d i c a t i n g increased incidence of spontaneous abortions, s t i l l b i r t h s and c o n g e n i t a l defects from male operating room personnel exposed to waste a n e s t h e t i c gases and vapors (1) and been i m p l i c a t e d i n c o n g e n i t a l heart defects from male production-worker exposure to O r y z a l i n (90). Apparently 1,2-dibromo3-chloropropane (DBCP) i s a human male s t e r i l a n t (127, 128). High c a f f e i n e consumption by the male has a l s o been i m p l i c a t e d i n spontaneous a b o r t i o n , s t i l l b i r t h s , and premature b i r t h s (123). The p u t a t i v e mechanisms may i n c l u d e damage to the sperm, presence of the agent or i t s m e t a b o l i t e ( s ) i n the semen which may a f f e c t the embryo d i r e c t l y or act on the g r a v i d uterus (72), or an i n d i r e c t a c t i o n on the male a f f e c t i n g hormone l e v e l s and perhaps l i b i d o (52, 107). These r e s u l t s have grave i m p l i c a t i o n s f o r production workers of both sexes under r i s k of exposure during c h i l d - s i r i n g or c h i l d - b e a r i n g years. Wilson (136) has suggested c r i t e r i a f o r r e c o g n i z i n g a new t e r a t o g e n i c agent i n humans. These i n c l u d e an abrupt increase i n the incidence of a p a r t i c u l a r defect or a s s o c i a t i o n of defects (syndrome) and coincidence of t h i s increase with a known e n v i r o n mental change such as i n t r o d u c t i o n of a new drug or environmental exposure to other chemicals. The appearance of c h a r a c t e r i s t i c a l l y malformed o f f s p r i n g should be c o r r e l a t e d with known exposure to the environmental change e a r l y i n pregnancy, and there should be absence of other f a c t o r s common to those pregnancies y i e l d i n g i n f a n t s with the c h a r a c t e r i s t i c defect ( s ) . Hunt (45) has made a number of recommendations to increase the data a v a i l a b l e on b i r t h s to i n c l u d e maternal and p a t e r n a l work experience, to encourage a n a l y s i s of data already c o l l e c t e d to i d e n t i f y p o s s i b l e r e l a t i o n s h i p s between occupational h i s t o r y of the mother (and father) and pregnancy outcome, and to encourage and support research on f e t a l development and maternal physiology i n r e l a t i o n to exposure and handling of t o x i c substances. She suggests promotion of information exchange with other c o u n t r i e s e s p e c i a l l y i n Eastern Europe. The importance i s s t r e s s e d of h e a l t h education programs e s p e c i a l l y i n the workplace, c o n s i d e r a t i o n of the pregnant worker i n a l l i n v e s t i g a t i o n s and analyses of o c c u p a t i o n a l s a f e t y and h e a l t h standards, and a concerted e f f o r t to impress c l i n i c i a n s and epidemiologists with the importance of occupational h i s t o r y f o r any study on reproduction from both males and females. The U. S. Department of Health, Education and Welfare has e s t a b l i s h e d a Congenital Malformations S u r v e i l l a n c e published y e a r l y to monitor b i r t h defects i n the United States d i v i d e d i n t o four regions to attempt to detect any a l t e r a t i o n s i n frequency of a dozen major malformation syndromes. T e r a t o l o g i c a l research has made great advances i n the l a s t decade, with r e c o g n i t i o n of the need f o r new approaches, with refinement of a n a l y t i c a l t o o l s , and with the awareness of the importance of s u b t l e s t r u c t u r a l and f u n c t i o n a l a l t e r a t i o n s pre-


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and p o s t n a t a l l y . With input from c l i n i c i a n s and e p i d e m i o l o g i s t s , t o x i c o l o g i s t s , pharmacologists, a n a l y t i c a l chemists and b e h a v i o r a l s c i e n t i s t s , t e r a t o l o g i s t s are working to meet the challenge to guarantee the b i r t h r i g h t of h e a l t h to c h i l d r e n of t h i s chemical age.


ABSTRACT The discipline of Teratology is introduced, including an historical perspective and current definition. Categories of teratogenic agents are discussed including human and animal teratogens. Determinants of teratogenic susceptibility are detailed: specificity of agent, specificity of target and dis position of agent. State-of-the-art teratological testing is presented as well as new trends evolving such as toxicokinetics and metabolism on maternal and fetal tissues, in vitro test systems and postnatal testing procedures. Extrapolation of animal data to human risk assessment is discussed. Possible mechanisms of teratogenesis and site(s) of action are suggested and con clusions tentatively drawn as to the sensitivity of current tera tological testing, new methodology developing and the limitations of available techniques to guarantee the birthright of health to children of this chemical age. Literature Cited 1. Ad Hoc Committee on the Effect of Trace Anesthetics on the Health of Operating Room Personnel: Occupational disease among operating room personnel: A National Study. Anes thesiology, 1974, 41:321-340. 2. Amin-Zaki, L., M. A. Majeed, S. B. Elhassani, T. W. Clarkson, M. R. Greenwood, R. A. Doherty: Prenatal methyl mercury poisoning: Clinical observations over five years. Am. J. Dis. Child, 1979, 133:172-177. 3. Anon., National Foundation/March of Dimes: Facts. National Foundation, New York, 1975. 4. Asling, J. and E. L. Way, Placental transfer of drugs. In Fundamentals of Drug Metabolism and Drug Disposition (Β. N. LaDu, H. G. Mandel and E. L. Way, eds.) Williams and Wilkins; Baltimore, MD, 1971, pp. 88-105. 5. Baloh, R., B. Sturm, B. Green and G. Gleser, Neuropsycho logical effects of chronic asymptomatic increased lead absorption. Arch. Neurol., 1975, 32:326-330.


144


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Reproductive and Teratogenic Effects: No More Thalidomides? - ACS

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