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Concluding Remarks W e began to apply model-based compartmental analysisto whole-body retinoldynamics in 198 I. Based on conventional wisdom at that time,45we postulated a five-compartment startingmodel. W e then conducted an in vivo turnover study in rats with marginal vitamin A status; short- and long-term tracer and tracce data were collectedfor plasma, liver,kidneys, and the rest of the carcass.Data wcrc not compatible, however, with the initialmodel; wc used S A A M / C O N S A M to arrive at a "simplest" model with 1 1 physiologicalcompartmcnts.i Some parameters were well defined (i.e.,statisticaluncertaintieswcrc low); others, for example, those describing liver vitamin A dynamics, were not. This study not only generated some new and thought-provoking concepts about retinolmetabolism, but it suggested improvements in experimental design and hypotheses to bc tested in subscqucnt and ongoing cxpcriments. It is clear that the application of model-based compartmental analysis will continue to add to our understanding of the complex dynamics of whole-body retinol metabolism.

Acknowledgments Work in the laboratory ofM. H. Green was supported by U.S. Department of Agriculture Competitive Research Grants 81-CRCR-1-0702 and 88-37200-3537. We acknowledge Barbara A. Underwood who introduced us to the exciting field of vitamin A dynamics through a collaborative research project. 45B. A. Underwood, J. D. Loerch, and K. C. Lewis, J. Nutr. 109, 796 (1979).

[33] Q u a n t i f i c a t i o n o f E m b r y o n i c R e t i n o i c A c i d D e r i v e d from Maternally Administered Retinol By DEVENDRA M. KOCHHAR

Introduction Hypervitaminosis A during pregnancy is probably teratogenic in humans as it certainly is in animals. ~-3 It is generally assumed that the human embryo is safe as long as the mother does not overdose herself, but S. Q. Cohlan, Science117, 535 (1953). 2 F. W. Rosa, A. L. Wilk, and F. O. Kelsey, Teratology33, 355 (1986). 3Anonymous, Teratology35, 269 (1987).

METHODS IN ENZYMOLOGY, VOL 190

Copyright @ 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.

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the question is, What constitutes a vitamin A "overdose"? From a survey of malformed human babies whose mothers ingested isotretinoin [Accutane (Hoffmann-La Roche), 13-cis-refinoic acid] a derivative of retinol, it turned out that even small quantifies of the drug resulted in teratogenesis.2,4, 5

Several questions can be addressed in experimental animals which may yield not only initial estimates of the risk associated with vitamin A-induced teratogenesis but also some information on mechanisms. These include the following: What is the identity of the chemical form(s) of vitamin A [retinol, retinal, retinyl esters, retinoic acid (RA), or metabolites] that poses the greatest teratogenic threat to the embryo? Is there a threshold dose of retinol beyond which teratogenic outcome becomes certain? At the threshold dose, is there a relationship of the levels of retinol (or of its teratogenic metabolites) in maternal circulation and the embryonic target tissues? Are there species differences in transplacental pharmacokinetics of retinol and other retinoids? Using retinol as an example, the following describes an experimental approach and methodologies suitable to address these questions. The aim is to quantitate teratogenicity in mice after exposure to a stepwise increase in an oral dose of retinol in order to correlate this response with the patterns of formation and distribution of the parent compound and its individual metabolites in the maternal and embryonic compartments. Some requirements for such an experiment should be noted. The degree and the type of teratogenic effects vary with the stage of embryonic development; this is so even if the dose of the test chemical is held constant. Hence, the onset of pregnancy should be timed with some precision so as to obtain consistency in the teratogenic response. Another requirement is the selection of one or more distinctive end point(s) from among a variety of embryonic defects produced by highly embryotoxic agents such as retinoids. It is preferable that the end point selected for measuring the teratogenic response should be a specific and a direct effect of the chemical rather than a nonspecific pharmacologic change in the dam. Therefore, care should be taken to ensure that the treatment does not result in overt maternal toxicity. Although indirect effects are of considerable importance in safety evaluations, they may not be so useful as the direct effects in understanding the mechanisms of retinoid action. For the same reason the selected defect should be such that it can be unambiguously and rapidly ascertained in a large number of exposed fetuses without 4 E. J. Lammer, D. T. Chen, R. M. Hoar, N. D. Agnish, P. J. Benke, J. T. Braun, C. J. Curry, P. M. Fernohoff, A. W. Gifixte, L T. Lott, L M. Richard, and S. C. Sun, N. Engl. J. Med. 313, 837 (1985). 5 j. C. Kraft, H. Nan, E. Lammer, and A. Olney, N. Engl. J. Med. 321, 262 (1989).

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the need for elaborate sample processing or extensive anatomic expertise. These are some of the criteria we have used in planning the following experimental protocols. Dosing of Animals Four doses of retinol are chosen for examination: 0, 10, 100, and 200 mg/kg maternal body weight. Retinol (all-trans-retinol, Sigma Chemical Co., St. Louis, MO) is dissolved in absolute ethanol (10 mg/ml) from which dilutions are made fresh in soybean oil on the day of use. The volume of vehicle is held constant at 5 ml/kg body weight.6 Thus, a mouse weighing 40 g receives 0.2 ml of ethanol/oil vehicle containing either 0, 0.4, 4, or 8 mg retinol. Precautions against exposure of retinoid preparations to light, air, and heat are observed. Teratology Five pregnant females are dosed orally for each time point, using an animal feeding needle (Perfektum, 18 gauge X 2 inches, from Popper & Sons, Inc., New Hyde Park, NY) attached to a l-ml syringe. For the teratology study, dams are intubated once on the morning (10 am) or evening (10 p r o ) o f Day 10 of gestation (designated as Days 10 and 10.5, respectively; day of vaginal plug is designated Day 0 of gestation), or on the morning of Day 11. The dose and treatment regimens are based on previous studies where the teratologic outcome is known for a number of diverse retinoids. 7-10 A single 100 mg/kg dose of an active retinoid affects virtually every mouse embryo exposed on Days 10 or 11 of gestation, and these embryos exhibit pronounced reduction of limb bones and are born with a cleft palate. If exposed earlier in gestation, on Days 8 or 9, the embryos frequently die and are resorbed or survive with a pattern of malformations quite different and anatomically more complex than that obtained with the chosen regimen. Embryos at more advanced developmental stages (Days 13 and 14) become increasingly resistant to the teratological effects of retinoids. The dams are sacrificed by cervical dislocation 1 day before delivery, on Day 18, to prevent loss of litters through cannibalism. The fetuses are 6A. Kisflerand W. B. Howard,this volume[44]-[46]. D. M. Koehharand J. D. Penner, Teratology36, 67 (1987). s B. Zimmerman,D. Tsambaos,and H. Sturje, Teratog. Carcinog. Mutagen. 5, 415 (1985). 9D. M. Koehhar,J. C. Kraft, and H. Nau, in "Pharmacokineticsin Teratogenesis"(H. Nau and W. J. Scott,eds.), p. 173.CRC Press,BocaRaton, Florida, 1987. ~oW. S. Webster,M. C. Johnston,E. J. l_ammer,and K. K. Sulik,J. Craniofacial Genet. Dev. Biol. 6, 211 (1986).

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FlO. 1. Cleft palate (left) in a Day 18 mouse fetus is compared with the complete secondary palate of a normal fetus. The specimens were fixed in Bouin's fixative and the mandible removedto confirm the palatal abnormality. weighed and examined fresh under a dissection microscope or fixed and prepared for subsequent examination. TM A typical example of cleft palate in a Day 18 mouse fetus after fixation in Bouin's solution is shown (Fig. 1). For examples of limb reduction defects, see Kistler and Howard.H Dose-dependent frequencies of malformed fetuses are expressed as the percentage of total number of live fetuses obtained after exposure on either Day 10, 10.5, or 11 (Fig. 2). All fetuses exposed to the l0 mg/kg dose of retinol survive without any malformations. About one-quarter of fetuses are affected at the 100 mg/kg dose; the frequencies of cleft palate among the survivors varies from 7% after exposure on Day l0 to 28% after Day I l; some of these affected fetuses also show a mild degree of limb bone reductions. The highest dose of retinol (200 mg/kg) results in virtually 100% incidence of not only cleft palate but also severe shortening of limb bones (phocomelia). Of importance is that even at the highest dose the embryonic mortality (resorbed or dead fetuses) is only 8%, which is only slightly greater than that observed in control litters. ~2 Pharmacodynamics Our aim is to correlate maternal/fetal levels of retinol derivatives with teratogenesis. Each pregnant female yields only a single blood sample at it A. Kisflerand W. B. Howard,this volume [44]-[46]. t2 D. M. Kochhar, J. D. Penner, and M. A. Satre, Toxico[. Appl. Pharmacol. 96, 429 (1988).

[33]

RETINOIC ACID IN THE EMBRYO A

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Day of Gestation FIG. 2. Teratologic effects of an oral dose of retinol (A, 10 mg/kg; B, 100 mg/kg; C, 200 mg/kg) given to pregnant ICR mice on Other Day I0, 10.5, or 11 of gestation. The fetuses were examined for anomalies on Day 18 of gestation. The frequency of occurrence of deft palate and limb reduction defects is given as the percentage of the total surviving fetal populations. No teratologic effects are associated with the 10 mg/kg dose, whereas the 100 mg/kg dose is mildly teratogenic when given on Days 10.5 and 11. The 200 mg/kg dose is strongly teratogenic, resulting in virtually 100% frequencies of cleft palate and limb defects.

the time of removal of her litter. Although serial samples can be taken from an individual animal, controls must be included to assess the resultant effects of maternal stress or hemodynamic change, either of which may influence not only teratogenesis but also the pharmacokinetic parameters themselves. Maternal blood, embryo, and placental samples are obtained at designated time intervals. The blood is drawn from the inferior vena cava of an ether-anesthetized animal into a heparinized syringe, after which the animal is sacrificed by cervical dislocation and its implantation sites removed, t3 Each embryo is removed from its placenta, divested of embryonic membranes, and examined for its proper developmental stage designation before processing for the extraction of retinoids. Each mouse litter consists of an average of 12 embryos, and a Day 11 embryo weighs on an average about 30 nag (wet weight) and contains about 40/tg protein. A sample size of about 200 mg wet weight of embryos is generally adequate 13M. A. Satre and D. M. Kochhar, Dev. Biol. 133, 529 (1989).

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PHARMACOKINETICS

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FIG. 3. Retinol concentrations in the embryo and in maternal circulation during the first 24 hr after intubation of Day 11 pregnant mice with nonteratogenic (10 mg/kg, A) or teratogenic (100 mg/kg, B; 200 mg/k& C) doses of retinol. Although the magnitude of increase in retinol concentration is roughly proportional to the dose in either compartment, the higher levels persist longer in the embryo than in the maternal circulation.

for measurement; therefore, each fitter provides an average o f two replicate samples. The sample collection, processing, extraction, and quantification o f retinoids are essentially similar to protocols reported elsewhere, m~3 An internal standard, Ro 13-7410 (Hoffmann-La Roche, Inc., Nufley, NJ), is added to the ethanolic extracts o f the sample prior to analysis by high-per-

[33]

RETINOIC ACID IN TIlE EMBRYO

323

formance liquid chromatography (HPLC). Retinoid concentrations are calculated from the integrated peak areas with reference to standard curves obtained by analyzing known amounts of authentic retinoids. We have found that in pregnant, untreated mice the circulating retinol levels normally decline by midgestation to about one-fourth the level of nonpregnant animals in our ICR colony. 12,~3Intubation of the animals on Day 11 of gestation with the subteratogenic 10 mg/kg dose raises slightly the retinol levels in circulation without unduly influencing the embryonic level (Fig. 3A). The presence of RA isomers is detectable after this dose, but their amounts are similar to the levels normally encountered in untreated control animals. Intubation of the dams with the higher doses results in prompt and proportional increase in plasma levels. The peak is reached within 2 - 3 hr with either the 100 mg/kg or the 200 mg/kg dose followed by a gradual decline to near normal levels by 24 hr (Fig. 3B,C). Transfer to the embryo occurs fairly rapidly, and the peak is attained simultaneously with the plasma. The peak concentrations in embryo after 100 and 200 mg/kg doses are, respectively, 1 and 2/tg/g wet weight (Table I). The notable feature is that these high retinol levels in the embryo persist for a prolonged period of

TABLE I PEAK CONCENTRATIONS (Cmax) OF RETINOL AND DERIVATIVES FOLLOWINGA SINGLE, ORAL DOSE OF RETINOL TO DAY I 1 PREGNANT MICE

Retinoid Retinol

Retinol Maternal dose plasma (mg/kg) (ng/ml + S.E.) 0 10

100 aIl-trans-Refinoic acid

4-Oxoretinoic acid

200 0 10 100 200 0 l0

100 13-eis-Retinoie acid

200 0 10 100 200

53 4- 15 235 4- 12 1790_+410 3560 + 610 < 10 < 10 412 ",- 56 1200 -+ 220 0 0 148-+20 990 4- 60 < l0