Structure-activity and dose-response relationships in the neural and behavioral teratogenesis of retinoids

Neurotoxicology and Teratology, Vol. 15, pp. 193-202, 1993 0892-0362/93 $6.00 + .00 Printed in the U.S.A. All rights reserved. Copyright © 1993 Pergamon Press Ltd.

MINIREVIEW

Structure-Activity and Dose-Response Relationships in the Neural and Behavioral

Teratogenesis of Retinoids

J A N E A D A M S

Department o f Psychology, University o f Massachusetts, 100 Morrissey Boulevard, Boston, M A 02125-3393

Received 4 September 1992; Accepted 4 J a n u a r y 1993

ADAMS, J. Structure-activity and dose-response relationships in the neural and behavioral teratogenesis of retinoids. NEUROTOXICOL TERATOL 15(3) 193-202 1993.-The therapeutic use in the human teratogenic range of retinoid compounds (Accutane; Tigason) for the treatment of dermatologic disorders has refocused attention on the teratogenicity of vitamin A-related compounds. This article reviews rodent data on three retinoids: vitamin A (retinol), all-trans retinoic acid (tretinoin), and 13-cis retinoic acid (isotretinoin) with respect to effects on central nervous system (CNS) malformations and postnatal death, growth reduction, and behavioral dysfunction. All three retinoids have been shown to effect these endpoints in a dose-dependent manner whereby smaller doses are necessary to produce each consecutive endpoint at the same stage of gestation. Whereas gestation days (GD) 8-10 are the most sensitive for the induction of gross CNS malformation, GD 11-13 appear most sensitive in effecting postnatal endpoints in rats with normal gross morphology. With respect to structure-activity relationships, retinol appears to have less than 1/4 the potency of all-trans retinoic acid, and isotretinoin may have much less than 1/8 the potency of all-trans retinoic acid in disrupting development with respect to all endpoints. The systematic relationships across endpoints argue for common controlling variables. For these retinoids, all-trans retinoic acid and its metabolites are proposed as the active agents of developmental toxicity.

Retinoids Isotretinoin Adverse effects Vitamin A Teratogenicity Retinoic acid Neural development

Dermatologic disorders

THE extensive therapeutic use of two synthetic retinoids (isotretinoin and etretinate) for the treatment of dermatologic disorders has reawakened interest in the spectrum of teratogenic effects associated with members of the vitamin A family of compounds. The retinoids (especially vitamin A) have a rich history of use in classical studies of structural as well as functional teratogenesis and, historically, have been used as tools to examine basic principles and tenets of these disciplines and their interrelationships (see comprehensive reviews of vitamin A teratogenesis in refs. 16 and 24 and of functional teratogenesis in ref. 55). More recently, studies of natural and synthetic analogues have provided further information to be assimilated into this framework. Extensive work by Kochhar and others (13,14,25-30) suggests all-trans retinoic acid as the putative agent for the induction of teratogenesis by members of the retinoid family. This working hypothesis provides both the foundation and the opportunity to examine the generality of structure-activity relationships derived from the teratogenesis of the palate and limb with respect to their applicability to

abnormalities of the central nervous system (CNS), both anatomic and functional. Newer data also provide the opportunity to reexamine, across compounds, basic principles of dose-response relationships in neural and behavioral teratogenesis. Several questions can be addressed: (a) Do the rules which govern structure-activity relationships for one malformation apply to that of an "embryologically distinct malfor- marion"?, Co) Do these rules generalize to the expression of functional teratogenesis in animals who appear free of detectable malformations?, and (c) Are dose-response and period- response relationships between anatomic and functional endpoints similar across structural analogues? While affLrmative answers to these questions are endorsed by the theoretical framework in which we all operate as neurobehavioral teratol- ogists, an empirical demonstration is nevertheless in order. The purpose of this review is to examine available studies to this end. We first review chemical, biologic, and teratogenic characteristics of pertinent retinoids. The discussion is then organized around the three questions stated above.

193

194 ADAMS

RETINOIDS: VITAMIN A AND ITS ANALOGUES

A thorough discussion of the biochemical and physiological functions of retinoid compounds is presented by Marcus and Coulton (38). Briefly, vitamin A is a lipid soluble compound available from naturally-occurring dietary sources which are approximately half retinol or retinol esters and half carotenoids. The term vitamin A, however, is used generically to represent compounds possessing the biological properties of retinol. The term, retinoid, represents retinol plus its natural derivatives, most of which are lipid soluble, and synthetic analogues, some of which are water soluble. At physiological levels, retinoids are well-known to stimulate the growth of epithelial tissues and to be essential to normal mammalian reproduction and embryonic development (35,48). Whereas more than 2000 compounds have now been identified in this family (5), pertinent research application defines three retinoids of importance to this review: vitamin A (retinol), tretinoin (all-trans retinoic acid), and isotretinoin (13-cis retinoic acid). Conversion methods have been developed to provide commonality across various sources of vitamin A through the expression of dose levels in retinol equivalents. It has been common to represent vitamin A content in IUs where 1 IU of vitamin A equals 0.33 mg of all-trans retinol (10,000 IU = 3.3 mg all-trans retinol). In the body, retinol is converted to retinoic acid through oxidation processes and retinoic acid can be further isomerized to create 13-cis retinoic acid (and vice-versa). All-trans retinoic acid (tretinoin) is considered to be the primary active form of vitamin A in all tissues except the retina. Different functions of vitamin A are mediated by different forms of the molecule. Depending on the function, all-trans retinoic acid has been shown to be 10-100 times more potent than retinol in various systems evaluated in vitro.

ALL-TRANS RETINOIC ACID, THE PUTATIVE AGENT OF RETINOID TERATOGENESIS

All of the retinoid compounds (this precludes beta caro- tene) are teratogenic at some level and this is currently believed to be due to their conversion to aU-trans retinoic acid and metabolites (13,25,29). Through studies examining the quanti- tative relationships in mice between teratologic and pharmacokinetic properties of various retinoids (28-30) and deductions from information across species (50,57), Kochhar et al. (29) have provided compelling evidence that all-trans retinoic acid and its metabolites act as the agents of teratogenesis. Conse- quently, the teratogenic effects of retinol and isotretinoin are believed to be mediated through metabolic conversion to this retinoic acid. In rodent models, both compounds have a lower teratogenic potency than all-trans retinoic acid, with isotretinoin being the least potent of many retinoid compounds. How- ever, isotretinoin (trade name Accutane, Hoffman-LaRoche, Inc.) is the most problematic teratogen due to its therapeutic use at teratogenic levels for the treatment of severe recalcitrant cystic ache and other dermatologic problems.

Marked interspecies differences in the metabolism and teratogenicity of isotretinoin have been described with humans being more sensitive to the drug's teratogenic properties. For example, isotretinoin is teratogenic in humans at therapeutic doses of 0.5 to 1.5 mg/kg/day but only marginally teratogenic in the mouse at single doses as high as 200 mg/kg (13,14,28). In rodents, the low isomerization rate of isotretinoin to all- trans retinoic acid and the low level of placental transfer of isotretinoin is believed to account for reduced potency relative to humans (14,25,30).

On the basis of prospective data, isotretinoin was first reported to be a human teratogen in 1985 by Lammer and co- workers (31). Risks associated with human exposure during cmbryogenesis include a 40% risk for spontaneous abortion, 4%-5% perinatal mortality, 16% premature birth, and 25% major malformation (31,32). The characteristic pattern of malformations involves craniofacial, cardiac, thymic, and CNS structures. Major malformations of the CNS include cerebdlar hypoplasia, agenesis of the vcrmis, malformation- induced hydrocephalus, and cortical abnormalities. Longitu- dinal follow-up of the children, both with and without malformations, has shown a 47% overall incidence of reduced general mental ability (borderline to mentally retarded range) with mental deficiency noted in many children who are free of major malformations (2,3). Nevertheless, the drug is currently prescribed to approximately 60,000170,000 U.S. women of child-bearing age per year in conjunction with an unprece- dented utilization of education and informed consent for fe- male patients.

Just as the putative agent of retinoid teratogenesis has been proposed, inroads have also been made toward an understand- ing of its actual mechanism. Retinoic acid is known to play an important role in the morphogenesis of the embryo, especially for limb and nervous system development (15,35,58). Retinol and retinoic acid have been shown to serve as endogenous ligands for cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). The exact functions of these cytosolic binding proteins are unknown, but they have been proposed to serve as substrates to bind, store, and/or perhaps transport retinoids to the nucleus (58,59). Once in the nucleus, retinoic acid interacts with nuclear receptors for retinoic acid (retinoic acid receptors; RARs) and following this interaction, the receptor can modify rates of gene transcription by binding to specific sequences on the cell's DNA (for review, see ref. 58). Such receptor interactions are known to play a regulatory role on DNA function in the developing nervous system of both the mouse and the rat embryo (35). High levels of CRABP have been found in neural crest cells targeted for both CNS structures and the branchial arches (59). These structures are of primary relevance to the teratogenic profile of retinoids and, indeed, have been proposed as primary targets for human retinoid teratogenesis (31). Because retinoids are known to inhibit the migration of neural crest cells, Maden et al. (35) have proposed that neural crest cells are targets for retinoid teratogenicity due to the binding of retinoic acid to CRABP and the subsequent disturbance of normal migration. Thus, the normal role of retinoids during embryogenesis is believed to be disturbed by the high levels which follow exogenous administration and teratogenicity of relevant structures results.

PROPOSED STRUCTURED-ACTIVITY RELATIONSHIPS FOR THE INDUCTION OF PALATAL AND SKELETAL

TERATOGENESIS IN THE MOUSE

Kochhar et al. (29) have proposed that retinol is 1/4 as potent of a teratogen as is all-trans retinoic acid for the production of cleft palate and limb defects, prominent markers of teratogenesis in mice exposed on GD l0 or 11. In this research, a 200 mg/kg dose of retinol was noted to be equivalent to 50-100 mg/kg all-tram retinoic acid in terms of teratogenic expression (less than 10%0 resorption rate with 80%- 100% abnormal fetuses). For isotretinoin, doses of > 100 rag/ kg are necessary to produce malformations in mice during the same treatment periods, and single doses of 400 mg/kg pro-

RETINOID TERATOGENICITY OF THE CNS 195

duce only 33-38% malformed fetuses along with a less than 10% resorption rate (derived from ref. 28). Individual doses of 200 ms /ks isotretinoin given three times at 8-h intervals between days 10.5-11 appear to achieve comparable terato- genie expression to that of single doses of 50-100 ms/kg all- trans retinoic acid and 200 ms /ks retinol (derived from ref. 28). It is safest to equate these doses by viewing the comparable teratogenic dose of isotretinoin as simply >400 ms/ks. Therefore, on the basis of data from the ICR mouse exposed on GD 10 or I1, it is proposed that retinol may have 1/2- 1/4th the potency of all-trans retinoic acid (200 ms retinol: 50 or 100 ms all-trans), and 13-cis retinoic acid may have approximately 1/4-1/8 the potency of all-trans retinoic acid (400 ms isotretinoin: 50 or 100 nag all-trans).

GENERALITY OF STRUCTURE-ACTIVITY RELATIONSHIPS ACROSS ANATOMIC ENDPOINTS OF TERATOGENESIS

This review examines the generality of the structure-activity relationships for the teratogenic potency of retinoids with respect to validity for neural and functional endpoints. Given that functional studies have almost exclusively been conducted in rats, only rat data have been evaluated. Thus, structure- activity relationships are examined with the confoundment of a species change. Relationships for different exposure periods are examined for four endpoints of abnormal development: CNS malformation, postnatal death, postnatal growth, and behavior. Summarial or deductive dose-response relationships which are presented in the following discussion have been tabulated in Table 3 (see later).

Table 1 presents studies of retinoid teratogenesis in which CNS malformations were addressed. In all cases, detection was based on gross observation and the gross malformation, exencephaly, was used as the primary endpoint. Exencephaly is maximally produced following exposures on GD 8-10 in the rat (ref. 24; GD 7-8 in the mouse; 41). Earlier studies often expressed dose levels on a per dam basis so estimates of dosage levels per kg body weight have been made. In these estimates, an average rat weight of 0.25 kg was used. In all cases in all tables, the route of administration was oral. All treatment periods have been placed on an equal basis such that the day of sperm detection has been designated as GD 0.

Restricting our focus to the induction of exencephaly by vitamin A, it is shown in Table 1 that doses at or above 120,000 IU vitamin A/kg administered on GD 8-10 appear capable of producing this gross CNS malformation in rats and that these doses also produce high levels of resorption. This dose is approximately 40 mg in retinol equivalents. After GD 10, much higher doses (240,000 IU) are necessary to produce CNS teratogenesis.

Following a review of the literature for multiple malformation endpoints, The Teratology Society (7) expressed consen- sus opinion that a dose of 50 ms retinol/kg is the lowest teratogenic dose in the rat. Given the variations in compounds, strains of rats, and observational techniques, this may be a reasonable figure specific to gross CNS malformation as well. However, it is also possible that the CNS may be more sensitive to vitamin A than are other developing systems.

Inspection of Table 1 with respect to the effects of all-trans retinoic acid reveals that doses of I0 ms/kg are capable of producing CNS abnormalities during the period of greatest vulnerability, with embryotoxicity produced by 20-40 mg/kg on GD 9-11 or 10-12. Thus, retinol appears to be about 1/4 as potent as all-trans retinoic acid (40 ms /ks retinol: 10 nag/ kg all-trans retinoic acid) with respect to the induction of gross

CNS malformation during the most susceptible stage of gestation.

While data are limited for doses which produce comparable levels of teratogenesis at later embryonic stages, it seems reasonable to compare the effects produced by a 240,000 IU/kg dose of vitamin A on GD 9-11 or 10-12 to what may occur at doses between 10 and 20 ms /ks all-trans retinoic acid on GD 9-11 and 20-40 ms /ks on GD 10-12. Crudely then, retinol may demonstrate 1/4 to 1/8 the potency of all-trans retinoic acid for inducing teratogenesis on GD 9-11, and 1/2-1/4 the potency for producing teratosenesis on GD 10-12. Therefore, these values are comparable to estimates of the structure- activity relationships in mice for frank palatal or skeletal teratogenesis.

As evident in Table 1, the rat has not been the species of choice for studies of isotretinoin teratogenesis. On the basis of unpublished findings (4), it has been suggested that the lowest teratogenic dose of isotretinoin in the rat is 75 mg/kg/ day when given on GD 8-10 (8). In light of the data shown in Table 1, this estimate seems valid for comparable levels of teratogenesis expressed by a 10 ms /ks dose of all-trans retinoic acid. Thus, isotretinoin may have approximately 1/8 the teratogenic potency of all-trans retinoic acid in rats following administration on GD 8-10 (75 ms /ks isotretinoin: I0 ms /ks all-trans retinoic acid). This relative potency is equivalent to that estimated for mice for the induction of cleft palate or limb defects.

In conclusion, the structure-activity relationships for the potency of retinol, all-trans retinoic acid, and 13-cis retinoic acid in the mouse for palatal and skeletal teratogenesis appear to apply to CNS teratogenesis during the most vulnerable period for the induction of exencephaly in the rat: retinol is approximately 1/4 as potent as all-trans retinoic acid and isotretinoin is approximately 1/8. This structure-activity relationship appears to hold for retinol for the production of teratogenesis following dosing at repeated points in later embryogenesis but cannot be directly ascertained for isotretinoin. Nevertheless, as shown later in Table 3 and as clarified by later discussion, isotretinoin teratogenicity on GD 11-13 may be expected to be > 150 ms/ks. This extrapolation provides a relative potency estimate of 1/8 when compared to the teratogenicity produced by all-trans retinoic acid treatment on GD 10-12, a less than perfect comparator.

GENERALITY OF STRUCTURE-ACTIVITY RELATIONSHIPS FOR POSTNATAL ENDPOINTS OF ABNORMAL DEVELOPMENT

The typical postnatal endpoints of abnormal development which have been measured are growth as defined by weight and behavioral performance. Table 2 presents studies that have addressed these endpoints following prenatal retinoid exposure. Let us first consider structure-activity relationships defined by growth reduction and then discuss the relationships for behavioral dysfunction.

Postnatal Survival and Growth

Generally, postnatal studies have selected sub-lethal doses and have shown that neither survival nor weight is affected by exposures to doses up to and including 100,000 IU/kg of vitamin A (< 33 retinol equivalents) when given prior to GD 11. The Fischer rat appears to be more sensitive than other strains, however, and a dose of 100,000 IU/ks on GD 8-10 is lethal in this strain.

196 ADAMS

TABLE 1 CNS MALFORMATIONS INDUCED BY EMBRYONIC EXPOSURE TO RETINOID COMPOUNDS

Compound Species Dose* Time? Lethality CNS Malform. Other Malform. Study

Vitamin A Oily Wistar 240000 d5-7 not reported 9% exencephaly eye defects 18

IU/kg cleft

Oily Fischer 100000 dS-10 for pre- and postnatal not evaluated not evaluated 51 344 rat IU/kg postnatal death

study

Aqueous Wistar 120(R~ d8-10 44% resorption 26% exencephaly eye & fimb defects 33 IU/kg cleft palate

Aqueous Wistar 160000 d8-10 63% resorption 19O70 exencephaly eye & fimb defects 33 IU/kg cleft palate

Aqueous Wistar 200000 d8-10 70°70 resorption 13% exencephaly eye & limb defects 33 IU/kg cleft palate

Aqueous Wistar 240000 d8-10 79% resorption 19% exencephaly eye & fimb defects 33 IU/kg cleft palate

Oily Wistar 240000 d8-10 not reported 53% exencephaly eye defects 18 IU/kg 3o70 Spina Bifida cleft palate

Oily Long-Evans 240000 d9-11 54% resorption 15% exencephaly eye defects 27 IU/kg cleft palate

Oily Long-Evans 240000 d10-12 22°70 resorption not produced eye defects 27 IU/kg cleft palate

Oily Wistar 240000 d10-12 not reported 5070 exencephaly eye defects 18 IU/kg 3% Spina Bifida cleft palate

Aqueous Wistar 140,000 > dl0 not reported not produced cleft palate 33 IU/kg

Aqueous Wistar 140000 d2, 3, 4, 90°70 pregnancy loss 54°70 exencephaly eye defects 10 IU/kg to 16 claft palate

All-trans Charles 5 mg/kg d8-10 not affected not produced not produced 46 retinoic acid River CD Exp. 1

Charles l0 mg/kg d9 increased resorption low incidence eye defects 45 River CD exencephaly cleft palate

skeletal defects

Long-Evans 20 mg/kg d9-11 95 % resorption Anencephaly cleft palate 26

Long-Evans 40 mg/kg d9-11 89o70 resorption Sacral Spina Bifida cleft palate 26 Exencephaly skeletal defects

Long-Evans 20 mg/kg dl0-12 49% resorption not produced eye defects 26

Long-Evans 40 mg/kg dl0-12 8% resorption Spina Bifida eye defects 26 cleft palate skeletal defects

13-cis retinoic rat 75 mg/kg d8-10 yes yes yes 4 acid

rat 150 mg/kg d7-15 yes yes yes 6

*In some cases where doses were expressed on a per dam basis, these values are estimated for an average dam weight of 250 g; tAll dosing intervals have been put on a common gestational stage basis where GD 0 is the day of detection of insemination.

As shown in Table 2, GD 11-13 appear to represent a particularly sensitive period with respect to the induction of postnatal death or weight reduction. A dose of 80,000 IU/ kg (26.4 retinol equivalents) given during this interval causes postnatal weight reduction but does not affect weight when given earlier or later in gestation. It is extrapolated that higher doses might be lethal to postnatal survival. After GD 13, doses of 240,000 IU/kg (80 retinol equivalents) or greater have been

shown to affect weight or survival in Wistar rats, but data are very limited. Specific to exposures on GD 14-16 (a time period examined for all-trans retinoic acid), doses greater than 80,000 IU/kg (26 retinol equivalents) appear necessary to effect viability or growth: how much greater cannot be determined.

As shown in Table 2, all-trans retinoic acid does not affect survival or weight when administered at 5 mg/kg on GD 8- 10, but this dose is perhaps on the threshold for reducing

R E T I N O I D T E R A T O G E N I C I T Y OF T H E CNS 197

T A B L E 2

POSTNATAL FUNCTIONAL ALTERATIONS FOLLOWING PRENATAL EXPOSURE TO RETINOIDS

Weight Alterations Compound Species Dose* Timer Reduced Behaviors Evaluated Present Study

Vitamin A Oily Sprague Dawley 80,000 IU/kg d5-7 no preweaning T maze no 54

postweaning Act. no Biel maze no

Aqueous Sprague Dawley 85,000 IU/kg d7-9 yes 15 measures of physical & yes, on 12 indices 39 motor development

Oily Fischer 344 10,000 IU/kg d8-10 no open field no 51 shock avoid, yes



Aqueous Sprague Dawley 40,000 IU/kg dS-10 no Neg. geotaxis no 1 ultrasonic distress voc. no

Oily Sprague Dawley 80,000 IU/kg d8-10 no preweaning T maze no 54 postweaning act. yes Biel maze yes

Aqueous Sprague Dawley 80,000 IU/kg d8-10 no Neg. geotaxis no 1 ultrasonic distress roe. yes

Oily Sprague Dawley 100,000 IU/kg dS-10 no Biel maze yes 9

Oily Wistar 100,000 IU/kg d8-10 no puzzle box maze yes:~ 12 Biel maze yes~

Oily Fischer 344 100,000 IU/kg dS-10 yes, lethal not evaluated - 51 dose

Oily Sprague Dawley 80,000 IU/kg dl 1-13 yes preweaning T maze yes 54 postweaning act. no Biel maze yes

Oily Wistar 240,000 IU/kg d13-14 yes operant disc. (auditory) yes 21

Oily Sprague Dawley 80,000 IU/kg di4-16 no preweaning T maze no 54 postweaning act. no Biel maze no

Oily Wistar 360,000 IU/kg d16-17 no, but operant disc. (auditory) yes 20 reduced survival

Oily Sprague Dawley 80,000 IU/kg d17-19

Oily Sprague Dawley 40,000 IU/kg

Oily Sprague Dawley 80,000; 160,000 IU/kg

no

d7~20 yes

d6-20 no

prewcaning T maze no 54 postweaning act. no Biel maze yes

motor dev. yes 53 postweaning act. yes Biel maze no active avoid, yes passive avoid, no rotorod yes

phys. landmarks no 49 righting reflex yes, both dose

groups

negative geotax, yes, high dose only swimming devel, yes, both dose

groups open field-pre & yes, both during 49 postweaning pre-weaning

yes, high dose only during postweaning

(continued)

1 98 A D A M S

TABLE 2 Continued

Weight Alterations Compound Species Dose* Timet Reduced Behaviors Evaluated Present Study

All-trans retinoic acid

13-cis retinoic acid

Sprague Dawley 5 mg/kg dg-10 no reflex dev. yes 46 Experiment 1 preweaning act. yes

M-maze no postweaning act. no

Sprague Dawley 2.5 mg/kg dll-13 yes reflex dev. yes 46 Experiment 2 preweaning act. no

M-maze yes postweaning act. no

Sprague Dawley 5 mg/kg dl 1-13 yes, lethal not examined - 46 Experiment 1 dose

Sprague Dawley 5 mg/kg dl 1-13 no, but reflex dev. yes 46 Experiment 2 reduced preweaning act. no

survival M-maze no postweaning act. no

Sprague Dawley 2 mg/kg d14-16 no reflex dev. no 46 Experiment 3 prewcaning act. no

M-maze no post-weaning act. no Amphetamine-challenge act. no

Sprague Dawley 2.5 mg/kg d14-16 yes reflex dev. yes 46 Experiment 2 preweaning act. no


Sprague Dawley 4 mg/kg d14-16 no reflex dev. yes 46 Experiment 3 preweaning act. no

M-maze no postweaning act. yes amphetamine-challenge act. yes

Sprague Dawley 5 mg/kg d14-16 yes reflex dev. yes 46 Experiment 1 preweaning act. yes


Sprague Dawley 5 mg/kg d14-16 no reflex dev. yes 46 Experiment 2 preweaning act. yes


Sprague Dawley 6 mg/kg d14-16 yes, males reflex dev. yes 46 Experiment 3 preweaning act. yes

M-maze yes active avoid, yes amphetamine-challenge act. yes running wheel yes

Wistar 50 mg/kg d I I - 13

Wistar 50 mg/kg dl 1-13

no dev. milestones no 22 ultrasonic vocalization yes postweaning act. no running wheel no water T-maze no active avoid, no

no ultrasonic vocalization yes 23

(continued)

RETINOID TERATOGENICITY OF THE CNS

TABLE 2 Continued

199

Weight Alterations Compound Species Dose* Timer Reduced Behaviors Evaluated Present Study

13-cis retinoic acid

Wistar 100 mg/kg dl 1-13 no

Wistar 100 mg/kg dl 1-13 no

Wistar 125 mg/kg dl 1-13 yes, lethal dose

Wistar 150 mg/kg dl 1-13 yes, lethal dose

dev. milestones yes 22 ultrasonic vocalization no postwcaning act. yes running wheel yes water T maze no active avoid, yes

ultrasonic vocalization yes 23

not evaluated - 22

not evaluated - 22

*In some cases where doses were expressed on a per dam basis, these values are estimated for an average dam weight of 250 g; tAll dosing intervals have been put on a common gestational stage basis where GD 0 is the day of detection of insemination; ~Behavioral differences were present only in treated animals raised in an enriched environment postnatally.

postnatal survival and reliably reduces postnatal weight when given on GD 11-13. Therefore, evidence is again seen which implicates GD 11-13 as particularly sensitive to retinoid effects on survival or growth. Exposures of up to 6 mg/kg on GD 14-16 do not affect survival of the pups and appear to show marginal effects on weight.

Studies using isotretinoin to examine postnatal ¢ndpoints have been restricted to exposures on GD 11-13. Data presented in Table 2 show that doses of 50 or 100 mg/kg do not affect survival or weight when administered at this time, but doses of 125 or 150 mg/kg are lethal to postnatal survival of apparently normal animals. These doses appear to compare to the effects on postnatal viability of a 5 mg/kg dose of all-trans retinoic acid given at this time. This suggests a potency for isotretinoin of at least 1/25th that of all-trans retinoic acid in effecting postnatal survival following treatment at this later stage of organogenesis (125-150 mg/kg isotretinoin: 5 mg/kg ail-trans retinoic acid). It would be expected that doses higher than 150 mg/kg would be necessary to produce CNS malformations at this time.

In summary, GD 11-13 appear to be particularly vulnerable to the induction of abnormal development defined by postnatal death or weight reduction. During this interval, a vitamin A dose of 80,000 IU (26.4 retinol equivalents) produces weight reduction in Sprague-Dawley rats and it is presumed that a higher dose might be lethal. A dose between 2.5 and 5 mg/kg of all-trans retinoic acid may have comparable activity with respect to weight reduction. A dose of isotretinoin that produces comparable effects may lie between 100 and 125 mg/ kg in Wistar rats, a less sensitive strain than the Sprague- Dawley with respect to vitamin A teratogenesis (44). As summarized in Table 3, these data therefore suggest general con- formity for retinol to the structure-activity relationships demonstrated in rats for frank teratogenesis but possible devi- ation from this model for isotretinoin. With respect to postnatal lethality and growth reduction induced by treatment on GD 11-13, retinol is about 1/5 as potent as all-trans retinoic acid and isotretinoin may have less than 1/25th the potency of all-trans retinoic acid. This marked decrease in relative potency for isotretinoin at doses that mediate postnatal sur- vivai and growth but not at the higher dose necessary to induce

frank malformation, suggests that different pharmacokinetics are operative as a function of dosage level. Clearly more data are needed to substantiate this extrapolated relationship.

Behavioral Performance

Inspection of Table 2 reveals that a range of behaviors have been examined in studies of the behavioral teratogenesis of retinoid compounds. Note that all three retinoids have been shown to alter motor development, activity, and learning- related processes as shown in Table 2. The use of different behavioral endpoints, however, increases the complexity of identifying comparable levels of functional disruption across compounds as well as doses. With apologies for offending the sensibilities of the readers who are well aware that different behaviors index the integrity of different underlying structures and pathways and may therefore have different stage as well as exposure vulnerabilities, I am nevertheless going to enter the arena of pretending that numerous behavioral endpoints represent the integrity of some type of unitary substrate. This is more acceptable when multiple behaviors have been shown to be altered than when deficits have been detected on a single behavioral endpoint.

Inspection of Table 2 reveals that 80,000-100,000 IU/kg (26-33 retinol equivalents) of vitamin A given to Sprague- Dawley rats on GD 8-10 or 11-13 disrupts postnatal behavior, although this dose range does not have general adverse conse- quences when given before or after this period. Note that this regimen is lethal to Fischer rats (see Table 1) who show behavioral disruptions following doses as low as 10,000 IU/ kg following treatment on GD 8-10.

After GD 13, much higher doses have been used to demonstrate that growth and behavior can be disrupted following treatments in late embryogenesis or early in the fetal period, and therefore outside of the sensitive period for gross CNS teratogenesis. The lowest effective dose for behavioral alter- ation following treatment on GD 14-16 is above 80,000 IU/ kg (26 retinol units), but its value is unknown.

With respect to all-trans retinoic acid, the data presented in Table 2 demonstrate that 5 mg/kg doses of this retinoid produce behavioral disruptions following treatment on GD 8-

200 ADAMS

TABLE 3 STRUCTURE-ACTIVITY AND DOSE-RESPONSE RELATIONSHIPS FOR PRENATAL RETINOID-INDUCED DEVELOPMENTAL TOXICITY

Retinol* Time of Potency Lowest 13-cis Potency Administration Lowest Effective Relative to Effective Dose Lowest Effective Relative to (Gestational Day) Endpoint Dose of Rctinol All-Trans RA of All-Trans RA Dose of 13-cis RA All-Trans RA

8-10 CNS malf./resorption 40 mg/kg 1/4 10 mg/kg Postnatal death > 33 mg/kg 1/6 > 5 mg/kg Postnatal weight reduction > 33 mg/kg 1/6 > 5 mg/kg Postnatal behavioral _> 26 mg/kg 1/5 5 mg/kg

dysfunction

9-11 or 10--12 CNS malf./resorption 80 mg/kg 1/4 20 mg/kg

11 - 13 CNS malf./resorption - - - Postnatal death (> 26 mg/kg) 1/5 > 5 mg/kg Postnatal weight reduction > 26 1/5 < 5 mg/kg Postnatal behavioral -< 26 1 / 10 < 2.5 mg/kg

dysfunction

14-16 CNS malf./resorption - - - Postnatal death > 26 mg/kg ~ > 6 mg/kg Postnatal weight reduction > 26 mg/kg ~ ~ 6 mg/kg Postnatal behavioral > 26 mg/kg ~/ ~ 2.5 mg/kg

dysfunction

> 75 _< 1/8

(> 150 rng/kg)t (1/8) > 125 mg/kg 1/25 > 100 mg/kg 1/20 < 100 mg/kg 1/40

*A dose which is 1/4 the relative potency would need to be administered at a level 4 times higher than that for all-trans retinoic acid to produce comparable effects; 1"Values are extrapolated from marked effects on other endpoints; ~tNot estimated due to a very limited database and consequent poor definition of the lowest dose of retinol that would be effective.

I0. These doses are lethal when given on GD 11-13 when a 2.5 mg/kg dose results in postnatal behavioral disruption. On GD 14-16, doses of 2.5 to 6 mg/kg have produced behavioral alterations.

The functional teratogenic potential of 13-cis retinoic acid has been studied in considerably less detail and only following administration on GD 11-13. Following treatment at this time, 100 mg/kg of isotretinoin produces effects on multiple behavioral endpoints and lower doses are suggested to be effective.

Therefore, as summarized in Table 3, the structure-activity relationships across retinoid compounds when multiple behavioral alterations are used as the marker of abnormal development are: (a) following exposure on GD 8-10, retinol appears to be 1/5 as potent as all-trans retinoic acid in disrupting behavioral function (26 mg/kg retinol: 5 mg/kg all-trans retinoic acid), (b) following exposure on GD 11-13, retinol appears to have 1/10th the potency of all-trans retinoic acid (26 mg/kg retinol: 2.5 mg/kg all-trans retinoic acid), and (c) following exposure on GD 11-13, isotretinoin appears to be 1/40th the potency of all-trans retinoic acid.

Table 3 presents the structure-activity relationships for potency in inducing abnormal development defined by different endpoints following differing gestational periods of exposure. Evaluation of the data in this table reveals a striking degree of consistency for the structure-activity relationships between retinol and all-trans retinoic acid across gestational stages and diverse endpoints. This systematic replication of a four- to six-fold potency differential between retinol and all-trans retinoic acid suggests a commonality in the active agent for disruption of developmental processes relevant to CNS teratogenesis, postnatal death, growth, and behavioral functioning. It appears to be both valid and parsimonious, therefore, to accept all-trans retinoic acid and certain of its metabolites as the agent of developmental toxicity.

Generally speaking, lower doses of retinol and isotretinoin which induce postnatal behavioral alterations appear to have reduced potency relative to all-trans retinoic acid. It is proposed that lower doses of retinol or isotretinoin may produce less metabolic conversion and/or placental transfer of active all-trans metabolites to the embryo. Clearly more research is needed to determine these relationships with greater resolution of dose-response effects and certainly a great deal more research is needed to explain pharmacokinetic characteristics. Some insight into this issue may be gained by an examination of the similarity of dose-response relationships across endpoints for each retinoid.

DOSE-RESPONSE CONTINUUM FOR THE EXPRESSION OF RETINOID-INDUCED

ENDPOINTS OF ABNORMAL DEVELOPMENT

Early vitamin A studies explored dose-response and period- response relationships for the expressed purpose of establish- ing principles governing the relationships between prenatal exposure and developmental insult as defined by gross malformations versus functional deficits. The principle that a repro- ductive casualty continuum describes dose-response relationships when all endpoints of abnormal development are evaluated arose largely from this work (see historical reviews in refs. 19,52,55). This dose-response continuum is supported by five primary relationships evident in Table 3. First, as shown in Table 3, the general trend for postnatal growth and behavioral endpoints to be altered by lower doses than malformation or postnatal death is present for retinol, all-trans retinoic acid, and isotretinoin. Second, GD 9-13 appear to be more sensitive than earlier or later stages for the induction of effects on postnatal endpoints. Third, the data further suggest that within this window, GD l l -13 of embryogenesis are particularly sensitive to the induction of effects on postnatal survival, growth, and behavior as exemplified by

RETINOID TERATOGENICITY OF THE CNS 201

the lower effective doses during this period. Fourth, following exposure on GD 11-13, postnatal growth appears to be effected by doses less than what are necessary to reduce viability. Fifth, behavioral function appears to be effected by all compounds at doses lower than what are necessary to produce effects on growth.

Beyond these fundamental dose-response relationships, one must ask whether or not proportionality is maintained in the expression of the dose-response continuum across retinoid compounds. Although data are limited in the floor definition for lowest effective behavioral doses in several cases, it nevertheless seems of interest to address this issue. As shown in Tables 1 and 2, available data suggest that the range from effective doses for producing behavioral insult to that for inducing frank CNS teratogenesis (defined by °70 increase) during GD 8-10 is for retinol, 26-40 mg/kg (54070) and for all- trails retinoic acid, 5-10 mg/kg (100070). Using figures from GD 10-12 or 11-13, the range for retinol is from 26 to 80 mg/ kg (208%), for all-trans retinoic acid, 2.5 to 20 mg/kg (700070), and for isotretinoin, 100 to more than 150 mg/kg (50070). Although derived with considerable restriction, the dose- response gradient appears to be steeper for all-trans retinoic acid than for the other two compounds.

The previous examinations of structure-activity relationships showed that retinol and isotretinoin are less potent developmental toxicants than is all-trans retinoic acid, and that relative potency of retinol and isotretinoin appear to he reduced at lower doses. Coupled with the suggestion that the dose-response range across endpoints is also narrowed for retinol and isotretinoin relative to all-trans retinoic acid, it is proposed that a threshold dose of these compounds must be present for conversion of the compounds to adequate amounts of all-trans retinoic acid to take place and /o r become available to relevant embryonic target tissues. This dose-dependent metabolic and/or distributional threshold may be high enough to obscure a more marked definition of the dose-response

continuum across endpoints of abnormal development for retinol and isotretinoin. Since all-trans retinoic acid needs no or only minimal conversion to other active metabolites, smaller amounts are readily available to embryonic tissues, thus allow- ing a wider range of doses to define the dose-response continuum across endpoints.

CONCLUSIONS

This review began with the posing of three questions: (a) Do the rules that govern structure-activity relationships for one malformation apply to that of an embryologically distinct malformation? The answer appears to be a clear "Yes" for both retinol and isotretinoin; Co) Do these rules generalize to the expression of functional teratogenesis in animals who are free of detectable malformations? For retinol, the answer is a clear "Yes" for postnatal viability, growth, and function. There is one caveat, however, that the relative potency may be reduced at the lowest doses used to examine behavior. For isotretinoin, structure-activity relationships appear to be dose- dependent and consequently to change across endpoints. (c) Are dose-response and period-response relationships between anatomic and functional endpoints similar across retinoids? The answer is a clear "Yes", however, exact proportionality is not maintained.

These results demonstrate systematic relationships for the induction of CNS teratogcnesis, postnatal death, reduced postnatal growth, and behavioral dyfunction. The relationships have an orderly nature when viewed in light of shared active agents for developmental disruption, all-trans retinoic acid and metabolites. The information summarized in this review constitutes further validation of the principle that all four endpoints of abnormal development share similar controlling variables, represent a continuum of expression of developmental insult, and merit equal consideration in risk esti- mation.

REFERENCES

1. Adams, J. Ultrasonic vocalizations as diagnostic tools in studies of developmental toxicity: An investigation of the effects of hypervitaminosis A. Neurotoxicol. Teratol. 4:299-304; 1982.

2. Adams, J.; Lammer, E. J. Relationship between dysmorphology and neuropsychological function in children exposed to isotretinoin "in utero." In: Fujii, T.; Boer, G. J., eds. Functional neuro- teratology of short term exposure to drugs. Tokyo; Teikyo Uni- versity Press; 159-170, 1991.

3. Adams, J.; Lammer, E. J.; Holmes, L, B. A syndrome of cogni- tive dysfunctions following human embryonic exposure to isotretinoin. Teratol. 43:497; 1991.

4. Agnish, N, (personal communications). Cited in anonymous. Recommendations for isotretinoin use in women of childbearing potential. The Public Affairs Committee and the Council of the Teratology Society. Teratol. 44:1-6; 1991,

5. Allen, J. G.; Bloxham, D. P. The pharmacology and pharmacokinetics of the retinoids. Pharmacol. Ther. 40:1-27; 1989.

6. Anonymous. Accutane Package Insert. Nut/ey, N J: Hoffman- LaRoche, 1983.

7. Anonymous. Position paper by the Teratology Society: Vitamin A during pregnancy, Public Affairs Committee of the Teratology Society. Teratol. 35:267-269; 1987.

8. Anonymous. Recommendations for isotretinoin use in women of clfildbearing potential. The Public Affairs Committee and the Council of the Teratology Society. Teratol. 44:1-6; 1991.

9. Butcher, R. E.; Brurmer, R. L.; Roth, T.; Kimmel, C. A. A learning impairment associated with maternal hypervitaminosis-A in rats. Life Science 2:141-145; 1972.

10. Cohlan, S. Q. Excessive intake of vitamin A as a canse of congenital anomalies in the rat. Science. 117:535-536; 1953.

11. Cohian, S. Q. Congenital anomalies in the rat produced by excessive intake of vitamin A during pregnancy. Pediatrics 13:556- 567; 1954.

12. Coyle, I. R.; Singer, G. The interaction of post-weaning housing conditions and prenatal drug effects on behavior. Psycho- pharmacologia 41: 237-244; 1975.

13. Creech Kraft, J.; Kochhar, D. M.; Scott, W. J.; Nan, H. Low teratogenicity of 13-cis retinoic acid (isotretinoin) in the mouse corresponds to low embryo concentrations during organogenesis: Comparison to the all-trans isomer. Toxicol. Appl Pharmacol. 87:474-482; 1987.

14. Creech Kraft, J.; Lofberg, B.; Chahoud, I.; Bochert, G.; Nau, H. Teratogenicity and placental transfer of all-trans-, 13-cis, 4- Oxo-all-trans-, and 4-Oxo-13-cis-retinoic acid after administration of a low oral dose during organogenesis in mice. Tox. Ap- plied Pharm. 100:162-176; 1989.

15. Durston, A. J.; Timmermans, J. P. M.; Hage, W. J.; Kendriks, H. F. J.; de Vries, N. J.; Heideveid, M.; Nieuwkoop, P. D. Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature. 340:140--144; 1989.

16. Geelen, J. A. Hypervitaminosis A induced teratogenesis. CRC Crit. Rev. Toxicol. 6:351-375; 1979.

17. Giroud, P. A.; Martinet, M. Hypervitaminoses A et anomalies chez le foetus de rat. Rev. Intern. Vitaminol. 26:10-18; 1955.

18. Giroud, P. A.; Martinet, M. In; Kalter, H. Teratology of the

202 A D A M S

central nervous system. Chicago, IL: University of Chicago Press, Chap. 3; 1968.

19. Hutchings, D. E. Behavioral teratology: A new frontier in neurobehavioral research. In: Johnson, E. D.; Hutchings, D. M., eds. Handbook of experimental pharmacology: Teratogenesis and re- productive toxicology, VUl. 65. Berlin: Springer-Verlag; 1983.

20. Hutchings, D. E. Gaston, J. The effects of vitamin A excess administered during the mid-fetal period on learning and development in rat offspring. Dev. Psychobiol. 7:225-233; 1974.

21. Hutchings, D. E.; Gibbon, J.; Kaufman, M. A. Maternal vitamin A excess during the early fetal period: Effects on learning and development in the offspring. Dev. Psychobiol. 6:445-457; 1973.

22. Jensh, R. P.; Kochhar, D. M.; Till, M. K.; Eskessen, M. B. Postnatal behavioral sequelae of prenatal exposure to 13-cis retinoic acid. Teratol. 41:621-622; 1990.

23. Jensh, R. P.; Kochhar, D. M.; Till, M. K.; Eskessen, M. B. Effects of prenatal exposure of isotretinoin (13-cis retinoic acid) on neonatal vocalization. Teratol. 43:497; 1991.

24. Kalter, H. Teratology of the central nervous system. Chicago, IL: University of Chicago Press; Chap 3; 1968.

25. King, S.; Creech Kraft, J.; Wildi, E.; Merker, H. J.; Persaud, T. V. N.; Nan, H.; Neubert, D. Influence of 13-cis and ail-trans retinoic acid on rat embryonic development in vitro: Correlation with isomerisation and drug transfer to the embryo. Arch. Tox- icol. 63:185-192; 1989.

26. Kochhar, D. M. Teratogenic activity of retinoic acid. Acta Path. et Microbiol Scandinav 70:398-404; 1967.

27. Kochhar, D. M.; Johnson, E. M. Morphological and autoradio- graphic studies of cleft palate induced in rat embryos by maternal hypervitaminosis A. J. Embryol. Exp. Morph. 14:223-238; 1965.

28. Kochhar, D. M.; Penner, J. D. Developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicity. Teratol. 36:67-76; 1987.

29. Kochhar, D. M.; Penner, J. D.; Satre, M. A. Derivation of retinoic acid metabolites from a teratogenic dose retinol (vitamin A) in mice. Tox. Appl. Pharm. 96:429--441; 1988.

30. Kochhar, D. M.; Penner, J. D.; Tellone, C. I. Comparative teratogenic activities of two retinoids: effects on palate and limb development. Teratogen. Carcinogen. Mutagen. 4:377-387; 1984.

31. Lammer, E. J.; Chen, D. T.; Hoar, R. M.; Agnish, N. D.; Benke, P. J.; Braun, J. T.; Curry, C. J.; Ferhnoff, P. M.; Grix, A. W.; Lott, I. T. et al. Retinoic acid embryopathy. New Eng J. Med. 313:837-841; 1985.

32. Lammer, E. L.; Hayes, A. M.; Schunior, A,; Holmes, L. B. Unusually high risk for adverse outcomes of pregnancy following fetal isotretinoin exposure. Am. J. Hum. Genet. 43:A58; 1988.

33. Langman, J.; Welch, G. W. Effect of vitamin A on development of the central nervous system. J. Comp. Neural. 128:1-16; 1966.

34. Lorente, C. A.; Miller, S. A. The effect of hypervitaminosis A on rat palatal development. Teratol. 18:277-284; 1978.

35. Madden, M.; Ong, D. E.; Chytil, F. Retinoid-binding protein distribution in the developing mammalian nervous system. Devel- opment 109:75-80; 1990.

36. Malakhovskii, V. G. Behavioral disturbances in rats receiving teratogenic agents antenatally. Bull. Exp. Biol. Med. 68:1230- 1232; 1969.

37. Malakhovskii, V. G. Antenatal effect of pyrimethamine and vitamin A on behavior of the rat progeny. Bull. Exp. Biol. Med. 71; 254-256; 1971.

38. Marcus, R.; Coulston, A. M. Fat soluble vitamins. In: Gilman, A. G.; Rail, T. W.; Nies, A. S.; Taylor, P., eds. Goodman and Gilman's The pharmacological basis of therapeutics. Elmsford, NY: Pergamon Press: 1553-1563; 1990.

39. Mooney, M. P.; Hoyenga, K. T.; Hoyenga, K. B.; Morton, J. R. C. Prenatal hypervitaminosis A and postnatal behavioral development in the rat. Neurobehav. Toxicol. Teratol. 3:1-4; 1981.

40. Morriss, G. M. Morphogenesis of the malformations induced in rat embryos by maternal hypervitaminosis A. J. Anat. 113:241- 250; 1972.

41. Murakami, U.; Kameyama, Y. Malformations of the mouse fetus caused by hypervitaminosis A of the mother during pregnancy. Arch. Environ. Health 10:732; 1965.

42. Nanda, R. Effect of vitamin A on the potentiality of rat palatal processes to fuse in vivo and in vitro. Cleft Palate 11:123-133; 1974.

43. Nanda, R.; Romeo, D. Effect of intraamniotic administration of vitamin A on rat fetuses. Teratol. 16:35-40; 1977.

44. Nolen, G. A. Variations in teratogenic response to hypervitaminosis A in three strains of the albino rat. Fd Cosmet. Toxicol. 7: 209-214; 1969.

45. Nulen, G. A. The effects of various levels of dietary protein on retinoic acid-induced teratogenicity in rats. Teratol. 5:143-152; 1972.

46. Nolen, G. A. The effects of prenatal retinoic acid on the viability and behavior of the offspring. Neurotoxicol. Teratol. 8:643-654; 1986.

47. Pratt, R. M.; Goulding, E. H.; Abbott, B. D. Retinoic acid inhib- its migration of cranial neural crest cells in the cultured mouse embryo. J. Craniofacial Gen. Dev. Bio. 7:205-217; 1987.

48. Roberts, A. B.; Sporn, M. B. Cellular biology and biochemistry of the retinoids. In: Sporn, M. B.; Roberts, A. B.; Goodman, D. S., eds., The retinoids, Vol. 2. New York: Academic Press; 1984: 109-296.

49. Saillenfait, A. M.; Vannier, B. Methodological proposal in behavioural teratogenicity testing: Assessment of propoxyphene, chlorpromazine, and vitamin A as positive controls. Teratol. 37: 185-199; 1988.

50. Shenefelt, R. E. Morphogenesis of malformations in hamsters caused by retinoic acid: Relation to dose and stage at treatment. Teratol. 5:103; 1972.

51. Vorhees, C. V. Some behavioral effects of maternal hypervitaminosis A in rats. Teratol. 10:269-274; 1974.

52. Vorhees, C. V. Principles of behavioral teratology. In: Riley, E. P.; Vorhees, C. V., eds., Handbook of behavioral teratology. New York: Plenum Press; 1986:23-48.

53. Vorhees, C. V.; Brnnner, R. L.; Butcher, R. E. Psychotropic drugs as behavioral teratogens. Science. 205:1220-1225; 1979.

54. Vorhees, C. V.; Brunner, R. L.; McDaniel, C. R.; Butcher, R. E. The relationship of gestational age to vitamin A induced postnatal dysfunction. Teratol. 17:271-276; 1978.

55. Vorhees, C. V,; Butcher, R. E. Behavioural teratogenicity. In: Snell, K., ed. Developmental toxicology. New York: Praeger; 1982: 249-298.

56. Vorhees, C. V.; MoUnow, E. Behavioral teratogenesis: Long- term influences on behavior from early exposure to environmen- tal agents. In: Osofsky, J. D., ed. Handbook of infant development, 2nd ed. New York, NY: Wiley; 1986.

57. Willhite, C. C.; Hill, R. M.; Irving, D. W. Isotretinoin-induced craniofacial malformations in humans and hamsters. J. Craniofa- cial Gen. Dev. Bio. 2:193-209; 1986,

58. Wolf, G. Recent progress in vitamin A research: Nuclear retinoic acid receptors and their interaction with gene elements. J. Nutr. Biochem. 1: 284-289; 1990.

59. Wolf, G. The intracellular vitamin A-binding proteins: An over- view of their functions. Nutrition Rev. 49:1-12; 1991.

Documents

Structure-activity and dose-response relationships in the neural and behavioral teratogenesis of retinoids