Testing of retinoids for teratogenicity in vivo

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RETINOID TERATOGENICITY i n Vivo

433

As an alternative method of quantifying inhibition of chondrogenesis, the number of chondrogenic foci can be determined.6 Some retinoids require bioactivation to the active form. 1,4,7In preliminary experiments we were able to activate some retinoids by coincubation of limb bud cells with small amounts (< 1 gl/ml) of S9 liver fraction without cofactors. However, a metabolic activation system for routine use has not yet been established. 60. P. Flint and T. C. Orton, Toxicol. Appl. Pharmacol. 76, 383 (1984). 7 D. M. Kochhar, J. D. Penner, and L. M. Minutella, DrugMetab. Dispos. 17, 618 (1989).

[46] T e s t i n g

of Retinoids for Teratogenicity

in Vivo

By ANDREAS KISTLERand W. BRIAN HOWARD Introduction Retinoids are powerful teratogens that induce specific malformation patterns in animals ~,2 as well as in humans. 3 Representative malformed rodent fetuses at term are shown in Fig. 1.4 Their mothers were treated with retinoids daily from Days 6 through 15 of gestation. It should be noted that some investigators consider plug day as Day 0 whereas others consider it as Day 1 of gestation; we consider plug day as Day 0 of gestation. In rats retinoic acid has a short half-life of elimination5 and induces stage-specific malformations depending on the day of treatment6 (Fig. 2). Thus, the malformations noted at term are unique for each day of treatment. A susceptible period regarding embryolethality and malformations of the head (including exencephaly, encephalocele, open eyes, cleft palate, and facial and cranial abnormalities) occurs between Days 8 and l0 of gestation (Fig. 2). Day 9 embryos are at the most susceptible stage of development (Table I). Therefore, for testing the teratogenic potential of a large number of new retinoids in vivo, we suggest treating rats once on Day J J. A. G. Geelen, Crit. Rev. Toxicol. 6, 351 (1979). 2 j. j. Kamm, J. Am. Acad. Dermatol. 6, 652 (1982). 3 E. J. Lammer, D. T. Claen, R. M. Hoar, N. D. Agnish, P. J. Benke, J. T. Braun, C. J. Curry, P. M. Fernhoff, A. W. Grix, I. T. Tott, J. M, Richard, and S. C. Sun, N. Engl. J. Med. 313, 837 (1985). 4 A. Kisfler, Arch. Toxicol. 60, 403 (1987). 5 A. B. Roberts and H. F. De Luca, Biochem. J. 102, 600 (1967). 6 A. Kistler, Teratology 23, 25 (1981).

METHODS IN ENZYMOLOGY, VOL. 190

Copy~ht © 1990 by Academic Prem, Inc. All rights of reproduction in any form reserved.

434

TOXICOLOGY

[46]

FIo. 1, Representative,severe malformations induced by retinoids in rodents. Dams were treated from Days 6 through 15 of gestation. Details regarding the various retinoids and the doses used have been reported.4Fetuses were obtained by cesarean section on Days 18 and 20 of gestation for mice and rats, respectively.(a-c) Mouse, (d-f) rat. The followingmalformations were observed: (a) exencephaly, protruding tongue, and open eyes; (b) protruding tongue and malformations of head and limbs; (c) protruding tongue, open eyes, and malformations of head, limbs, and tail; (d) exencephalyand malformations of head, limbs, and tail; (e) open eyes and malformations of head, limbs, and tail. (f) Control.

9 o f gestation. After cesarean section on Day 20 o f gestation, the fetuses can be examined for only externally visible abnormalities including cleft palate. This procedure enables the investigator to detect the major malformations induced by retinoids at this early susceptible stage o f development without further laborious techniques such as skeletal examination after alizarin red staining ([44], this volume). Caution Most retinoids are teratogenic, some at such diminutive doses as 1 #g/ kg. Therefore, they should be handled with utmost care, especially by women. Wearing a dust mask, working under a hood, and avoiding skin contact are recommended.

[46]

RETINOID TERATOGENICITY In Vivo

435

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Fro. 2. Resorption rate and incidence of specific malformations induced by retinoic acid after a tingle treatment at specific days of gestation. Pregnant rats were treated orally with 120 mg/kg body weight at the day indicated. After cesarean section on Day 20 of gestation, fetuses were examined for external malformations, processed for visualization of the skeleton with alizarin red, and examined for skeletal malformations.

Method of Dose Preparation Retinoids are light-sensitive, especially in solution. Stock solutions/suspensions should be kept only for a few days, protected from light and stored under argon or N2 gas. Use high shear mixing during formulation in rapeseed off (or other vegetable off) and keep the suspension homogeneous during dosing with the use of a magnetic stirrer. The application volume is 5 ml/kg. Retinoids have a broad teratogenic activity range from 0.001 to over 100 mg/kg depending on the specific retinoid. 4 Therefore, for the selection of doses for exploratory studies we suggest logarithmic dose steps (i.e., l, 3, l0 mg/kg) or even steps by one order of magnitude (i.e., 1, 10, 100 mg/kg). T r e a t m e n t of Animals and Recording of Reproduction and Malformation Data Mated female rats on gestational Day 9 (or 8 and 9 or 8-10) are treated orally by gavage with the respective retinoid dose. Controls receive the vehicle only. On day 20 of gestation the dams are sacrificed, fetuses removed by cesarean section, and the uteri examined for the number of fetuses and sites of implantations and resorptions. The fetuses are weighed and examined for external malformations and cleft palate. The palate can

436

TOXICOLOGY

[46]

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[47]

RETINOIDS DURING ORGANOGENESIS WITH TERATOGENICITY

437

be examined through the opened mouth of the freshly dissected fetuses. A more reliable method is the examination of the palate of formalin-fixed heads after dissection of the lower jaw. Comments Mice are an alternative species for teratogenicity testing. Mice have a similar susceptibility of retinoids as rats: An advantage is that less drug is required. For the treatment, Days 8 and 9 of gestation are recommended, days which are very susceptible to retinoids. 7 An important advantage of a single treatment is that, in general, the drug is well tolerated by the dams, s in contrast to repeated dosing which induces hypervitaminosis A (weight loss, desquamation of the skin, hair loss, and alterations of the skeletal system including bone fractures). 9 7 D. M. Kochhar, Acta Pathol. Microbiol. Scand. 70, 398 (1967). s K. Teelmann, Pharmacol. Ther. 40, 29 (1989). 9 A. Kistler, H. Sterz, and IC Teelmann, Arch. Toxicol. 56, 117 (1984).

[47] C o r r e l a t i o n o f T r a n s p l a c e n t a l a n d M a t e r n a l Pharrnacokinetics of Retinoids during Organogenesis with Teratogenicity B y HEINZ NAU

Introduction Retinoids are teratogenic in humans as well as in all animal species which have been investigated. 1'2 In humans, isotretinoin3 (13-cis-retinoic acid) and etretinate4 are established teratogens; of recent concern have also been excessive doses of vitamin A 5 (retinol or esters), although a causal

l j. j. Kamm, J. Am. Acad. Dermatol. 6, 652 (1982). B. W. Howard and C. C. Willhite, J. Toxicol. Toxin Rev. 5, 55 (1986). 3 E. J. Lammer, D. T. Chen, R. M. Hoar, N. D. Agnish, P. J. Benke, J. T. Braun, C. J. Curry, P. M. Fernhoff, A. W. Grix, I. T. Lott, J. M. Richard, and S. C. Sun, N. Engl. J. Med. 313, 837 (1985). 4 R. Happle, H. Traupe, Y. Bounameaux, and T. Fisch, Dtsch. Med. Wochenschr. 109, 1476 (1984). F. W. Rosa, A. L. Wilk, and F. O. Kelsey, Teratology33, 355 (1986).

METHODS IN ENZYMOLKK3Y,VOL. 190

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