Temporal distribution of retinoic acid and cellular retinoic acid-binding protein (CRABP) in the fetal hamster

Temporal distribution of retinoic acid and cellular retinoic acid-binding protein (CRABP) in the fetal hamster

EXPERIMENTAL AND MOLECULAR PATHOLOGY 55, 38-54 (191) Temporal Distribution of Retinoic Acid and Cellular Retinoic Acid-Binding Protein (CRABP) i...

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EXPERIMENTAL

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MOLECULAR

PATHOLOGY

55,

38-54 (191)

Temporal Distribution of Retinoic Acid and Cellular Retinoic Acid-Binding Protein (CRABP) in the Fetal Hamster’ AKIKO

HATORI, 2,3 AKIYO SHIGEMATSU,~ ANNA M. MCCORMICK,’ AND RAGHUBIR P. SHARMA~,’ CALVIN C. WILLHITE,**~

‘Toxicology Program, Utah State University, Logan, Utah 84322; 41nstitute of Whole Body Metabolism, Shiroi, Inba, Chiba, Japan; and ‘National Institute of Aging, NIH, Bethesda, Maryland 20892 Received November 7, 1990, and in revised form April 8, 1991 The temporal relationship between the distribution of retinoic acid, a known human and rodent teratogen, and that of cellular retinoic acid-binding protein (CRABP) was investigated from Day 11 to Day 14 of hamster prenatal development. The 11 ,12-3H, and 1S-‘4C forms of all-trans-retinoic acid were used for quantitative distribution studies and autoradiography, respectively, and were evaluated 15 min after a single intravenous injection. Radioactivity was detected in all fetal tissues examined (brain, liver, heart, spinal cord, limb, and skin), and at Day 14, -66% of the total radioactivity was present as parent all-trans-retinoic acid. High concentrations of total radioactivity were observed by autoradiography in the midbrain and hindbrain (mesencephalon, metencephalon, and myelencephalon) and spinal cord, but not in the forebrain. At the earliest time studied, limb buds showed relatively high concentrations of radioactivity. Levels of radioactivity were also high in portions of the developing face, nose, and tongue. Immunohistochemical analyses indicated that the amount of CRABP in Day 14 tissues was the highest in spinal cord followed by limb and skin; heart and liver contained only relatively small amounts of this protein. From Day 1I to Day 14, the amount of CRABP, as measured by high-performance size-exclusion liquid chromatography, in the whole body decreased as gestation progressed. Microscopic immunohistochemical localization of CRABP found the highest concentration in the ventral midbrain and in the ventral and lateral sides of the hindbrain and spinal cord; CRABP was also abundant in tongue, limb, and skin. The distribution of CRABP-positive cells in the central nervous system was similar to the distribution of retinoic acid. The data presented here indicate that fetal CRABP appears to play a role in differential accumulation of retinoic acid in certain structures of the developing hamster, The patterns of tissue retinoid and CRABP distribution observed here are consistent with the patterns of congenital malformations induced by prenatal retinoid exposure. 0 1~ Academic PKSS. IIK.

INTRODUCTION Vitamin A (retinol) is required for normal embryonic development and may be involved in the control of germ layer differentiation and organogenesis (Takahashi et al., 1975). Retinoic acid, an endogenous oxidized metabolite of retinol, shares with retinol the properties of the maintenance of normal growth and differentiation of tissues derived from mesoderm and ectoderm. Retinoic acid and certain other retinoids are, however, teratogens in animals and humans (Willhite, 1990; ’ Presented in part at the 40th Annual Meeting of the American Society for Pharmacology and Experimental Therapeutics, Salt Lake City, UT (Pharmacologist 31, 139, 1989). 3 Current address: Institute of Whole Body Metabolism, Shiroi, Inba, Chiba 270-14, Japan. 6 Permanent address: Department of Health Services, State of California, 700 Heinz Street, Berkeley, CA 94710. ’ To whom correspondence should be addressed. 38 OOl4-4800/91 $3.00 Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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Willhite et al., 1989). Cellular retinol-binding proteins (CRBP types I and II) and cellular retinoic acid-binding protein (CRABP) have binding specificity for retinol and retinoic acid, respectively. The CRABP apparently mediates the transfer of retinoic acid from the cytoplasm to the nucleus (Chytil and Ong, 1984), or perhaps controls local concentrations of free endogenous retinoic acid. Tissue distribution of CRABP differs from that of CRBP; both CRBP and CRABP have been detected in all fetal rat organs examined (brain, eye, skin, liver, lung, kidney, intestine, heart, and skeletal muscle) with the exception of serum. The CRABP is suppressed during the development of lung, liver, kidney, and intestine (Ong and Chytil, 1975, 1976; Ong et al., 1982). Besides brain, eye, and skin, CRABP is present in adult ovary, testis, uterus, bladder, prostate, trachea, and mammary gland of rats and mice (Sani and Corbett, 1977; Ong and Chytil, 1975); in epididymis, pituitary, and thymus of rats (Ong et al., 1982); and in bovine retina (Saari et al., 1982). Ong and Chytil (1976) suggested that the widespread distribution of CRABP in fetal tissue compared with adult tissue provided some evidence for an important role for retinoic acid in embryogenesis. Recent studies have suggested that retinoic acid is a natural morphogen involved in generation of the digit pattern in the chick limb bud, where local retinoid concentrations determine the morphology of the apical ectodermal ridge (Thaller and Eichele, 1987); CRABP was abundant in a concentration gradient in chick limb buds (Tickle et al., 1985) and has been detected in the embryonic chick central nervous system. The expression of CRABP in the brain changes with development, where, for example, by stages 38-43, CRABP cannot be detected in chick brain (Momoi et al., 1989b). How the interaction between teratogenic retinoids, CRABP, and the family of retinoid nuclear receptors (Doll6 ef al., 1989; Willhite et al., 1989) leads to pathologic changes in the embryonic germ layers and their derivatives is not at all clear, and the existence of more than one type of CRABP (Bailey and Siu, 1988) complicates the matter. As hamsters exhibit malformations not unlike those observed in retinoid-damaged human fetuses and neonates (Willhite, 1990), the objective of the present study was to investigate the temporal and quantitative relationships between the distribution of CRABP and the distribution of radiolabeled all-trans-retinoic acid during morphogenesis of the hamster. The results are discussed in light of previous retinoid placental permeability and metabolic fate studies and identification of retinoid-binding proteins and receptors in embryonic and fetal tissues. MATERIALS

AND METHODS

Chemicals. all-tram-[ 11, 12-3H,]retinoic acid (47.8 Ci/mmole, 98.8% purity) was obtained from NEN Research Products (Boston, MA). All-trans[carboxyl-‘4C]retinoic acid ([‘4C]retinoic acid, 13.7 mCi/mmole, >95%) was supplied by the Institute of Whole Body Metabolism (Chiba, Japan). Figure 1 illustrates the location of label in two forms of retinoic acid used in the study. These two forms of retinoic acid were necessary because only the 3H-labeled form was available in the high specific activity required for distribution studies, and the i4C-labeled form was needed for autoradiographic evaluations. Nonlabeled alltrans-retinoic acid (98%) and 13-cis-retinoic acid (99%) were purchased from Kodak Chemical Co. (Rochester, NY). All-trans-4-oxo-retinoic acid was a gift from Hoffman-LaRoche, Inc. (Nutley, NJ). Radioactive and all nonlabeled reti-

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(W FIG. 1. Chemical structures of all-rruns-retinoic acid indicating the location of label. The alltruns-[ 10,l I-‘HZ]-retinoic acid (A) was used in tissue distribution and metabolic fate studies and the aN-trans-[15-‘4C]retinoic acid was used for autoradiographic localization.

noids were handled and administered under a yellow light with as little exposure to air as possible as previously described in detail (Willhite and Book, 1990). Animals. Virgin male and female Syrian Golden hamsters [Lak:LVG(SYR)], 100-140 g body wt, were purchased from the Charles River Breeding Laboratories, Inc. (Wilmington, MA). All animals were allowed free access to tap water and laboratory stock diet (Wayne F6 Lab Blox, No. 8664-00) and were housed separately in polycarbonate cages with pine shavings for bedding. The light cycle was maintained at 14-hr light/lo-hr dark. Animals were housed in an AAALACaccredited facility (Laboratory Animal Research Center, Utah State Univ.). Hamsters were mated and the day following the evening of breeding was considered Day 1 of gestation (Willhite and Book, 1990). Radiolabeled all-trans-retinoic acid, dissolved in a small volume of ethanol (8.3 &i/O.05 bg to 4.8 pCi/O. 1 mg per 100 g body wt) was injected intravenously into the sublingual vein of pregnant hamsters on Days 11-14 of gestation (0.1 ml/100 g) under ketamine anesthesia. Animals were sacrificed under excess CO,. Quantification of tissue radioactivity. Tissues were minced and samples were weighed (5&100 mg) and placed into liquid scintillation vials. The samples were digested with 70% aqueous perchloric acid, decolorized with hydrogen peroxide, and mixed with scintillation cocktail (ScintiVerse, Fisher Scientific, Pittsburgh, PA), and the radioactivity was counted in a Packard Tri-Carb 2660 liquid scintillation spectrometer (Packard Instruments, Downers Grove, IL) equipped with an automatic external standard. Analysis and identification of parent retinoid and radioactive metabolites. Day 14 fetal tissues and maternal liver were collected 15 min after a single iv injection of 3H- or 14C-labeled all-trans-retinoic acid and homogenized on ice in equal volumes (w/v) of ice-cold Tris-HCl-buffered saline (pH 7.6). The homogenates and plasma samples were extracted with 2 vol of ice-cold ethanol and then centrifuged at 12,OOOg for 15 min. The supernatant was removed and filtered through a 0.45~pm microfilter at 1OOOg for 5 min. The ethanol-extracted samples were analyzed by HPLC using a Spectra-Physics LC8800/8100 liquid chromatograph equipped with a Spherisorb ODS 5-km column (Universal Scientific, Atlanta, GA) and a Spherex Cl8 5 X 4.6cm precolumn (Phenomonex, Ranch0 Palos Verdes, CA). The chromatography conditions were similar to those described by Kochhar et al. (1988). The eluent was collected in 0.5-ml fractions and the radioactivity was measured. The radioactivity of each metabolite was calculated against the total radioactivity of the original ethanol extract; parent retinoid and metabolites were

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identified by chromatographic elution and/or retention time of authentic 13-c& retinoic acid and all-truns-4-oxo-retinoic acid. Purity of all radiolabeled and nonlabeled dosing solutions was quantified using the same HPLC system; the 14C dosing solution comprising 93.1% all-truns-retinoic acid, 5.5% 13-cis-retinoic acid, and 1.4% all-truns-4-oxo-retinoic acid. The tritiated solution constituted 92.8% all-truns-retinoic acid and 7.2% 13-cis-retinoic acid, and the ulltruns-4-oxo-retinoic acid was less than the analytical limit of detection (2 fmole/ ml). Quantitative analysis of CRABP. For quantification of CRABP, highperformance size-exclusion liquid chromatography (HPLC) as modified from Rainier et al. (1983) and described elsewhere (Howard et al., 1990) was used. Briefly, whole fetus or selected isolated tissues were homogenized and 105,OOOg supernatant was incubated with high specific activity [3H,]retinoic acid, with or without a 200-fold molar excess of unlabeled retinoic acid. The label associated with the CRABP was isolated by size-exclusion HPLC and the radioactivity was quantified (Howard et al., 1990). Protein in cytosol was measured by Pierce protein assay reagent (Pierce, Rockford, IL). Autoradiography. [14C]Retinoic acid (4.8 @i/O.1 mg/lOO g maternal body wt) was administered via a single iv bolus on Days 11-14 of pregnancy. Fifteen minutes later, animals were sacrificed and the gravid uteri were rapidly removed. Fetuses were frozen by dry ice in a gel of carboxymethyl cellulose. Sagittal and cross sections, 16-Frn thick, were made with a microtome in a cryostat and collected on an adherent tape (Scotch 810, 3M Co., St. Paul, MN). After freezedrying, the sections were placed in contact with X-ray film (Kodak XAR-5; Rochester, NY) and exposed for 3 months. Films were developed by routine processing, and the distribution of radioactivity was observed and representative sections were photographed. Localization of CRABP in fetal tissues. The presence of CRABP in hamster fetuses at different stages of development was investigated using an immunohistochemical method. Fetuses from 11 to 14 days of gestation were obtained via laparotomy immediately after CO, asphyxiation. Fetuses were dissected free from the placenta and associated membranes and were then fixed in 10% neutralbuffered formalin (Howard et al., 1989a). After fixation, the fetuses were mounted in paraffin blocks using routine histological procedures, and 5-pm sections were obtained with a microtome. Sections from the central sagittal plane and brain and myelencephalon and limb cross sections from each day of prenatal development were studied. For immunohistochemistry, anti-bovine testis CRABP immunoglobulin prepared in rabbits was employed. For the preparation of this antibody, the CRABP was conjugated with keyhole-limpet hemocyanin using glutaraldehyde and injected in rabbits subcutaneously three times at 6-week intervals; the serum was obtained 1 week after the last injection, and IgG was purified using a Perllex 35s Protein A column (NEN-DuPont, Wilmington, DE). A commercial peroxidaseantiperoxidase immunochemical staining kit (Dako Japan Co., Kyoto) was used according to the following protocol. The sections were deparaffinized in xylene (twice for 5 min each) and hydrated in an ethanol series (absolute, 90%, and 70% ethanol, respectively, for 2 min each) and finally washed in distilled water and 0.05 M Tris buffer (pH 7.6). The sections were then incubated with 3% H202 for 5 min to inactivate nonspecific tissue peroxidases. Following a buffer wash (20

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min) the sections were incubated at room temperature with dilute normal swine serum (NSS, blocking, 20 min), the serum was removed, and anti-CRABP antibody (suitably diluted after initial trials) was added. The sections were incubated at 4°C in humidified chambers overnight. After this incubation, the sections were again washed in buffer (20 min) and reincubated with swine anti-rabbit IgG (20 min) at room temperature and washed. Finally, the rabbit peroxidaseantiperoxidase complex was added to the sections (20 min), and after buffer washing, the sections were stained with diaminobenzidine (20 mg/lOO ml, containing 0.05 ml of 5% H,O,, freshly prepared). The sections were thoroughly rinsed in running tap water, lightly stained with an aqueous hematoxylin solution, and washed again. Following this staining procedure, the sections were dehydrated using graded series of ethanol and xylene and mounted using the Eukitt mounting reagent (0. Kindler, Germany). The sections were then evaluated under a light microscope. Appropriate blanks using a preimmune rabbit serum (instead of anti-CRABP antibody) and omitting the NSS, anti-CRABP or second antibody were employed on duplicate sections. RESULTS Preliminary studies were carried out to optimize the dose of retinoic acid and the time of sampling after retinoic acid injection. When retinoic acid was injected at 0.5 to 250 t&kg, the amount of total radioactivity in fetal and maternal tissues was linearly related to the administered dose (data not shown). In order to minimize nonspecific target tissue binding, the lowest dose was employed in subsequent distribution studies. Similarly, when the tissues were sampled at 7, 15, and 30 min after the injection, the total radioactivity in the conceptus increased between the first two periods but later decreased or plateaued, and a sampling period of 15 min was hence selected. The distribution of total radioactivity (representing parent retinoic acid and its metabolies) on different days of gestation was observed 15 min after the injection of [3H,]retinoic acid (Table I). In each instance, the concentration of total radioactivity was highest in maternal liver followed by that in maternal plasma. Circulating retinoid was primarily associated with the plasma, consistent with the known binding of retinoic acid with albumin. The amount of radioactivity in the whole fetal body generally decreased from Day 11 to Day 14; however, radioactivity in maternal tissues was consistent over the experimental period. In fetal tissues, a level of radioactivity was observed in the spinal cord and limb higher than the average for other fetal organs. Radiolabeled all-truns-retinoic acid and related metabolites in fetus, maternal liver, and maternal plasma were measured 15 min after an intravenous injection of 1 mg/kg afl-trans-[‘4C]retinoic acid or 0.5 pg/kg of all-truns-[3H,]retinoic acid on Day 14. Table II shows that 64 and 61% of the radioactivity in fetuses sampled at 15 min after maternal dosing remained as all-trans-[i4C]and ulltruns-[3H,]retinoic acid. Relatively smaller amounts of the 13-c&isomer were detected in the fetus, with only very small amounts of the 4-0~0 metabolite. The data in maternal liver and plasma provide evidence that all-truns-retinoic acid was isomerized or oxidized more rapidly in these tissues than in the fetus. To determine the amount of CRABP in various fetal tissues, high-performance size-exclusion chromatographic analyses were performed on cytosolic supernatants. Figure 2 shows the radioactive elution of cytosol incubated with ufl-

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TABLE I Distribution of Total Residual Radioactivity 15 min after Intravenous Injection of nll-rrans-[3H]Retinoic Acid on Different Days of Gestation in Hamsters” dpm x 10m3/g tissue on gestation day

Organ or tissue

Day 11

Day 12

Day 13

Day 14

Fetalb Whole body Brain Liver Heart Spinal cord Limb Skin

101 2 16 ND ND ND ND ND ND

972 110 2 117 2 35 ‘124 2 128 r ND

11 9 9 6 8 4

94 87 92 75 125 120 105

2 t 2 2 t 2 ”

4 1 24 14 13 10 20

81 f 94 2 992 63 2 123 2 105 * 93 +

30 55 269 18 31

185 248 1,057 228 175

” k + * *

1 43 19 61 24

Maternal’ Whole blood Plasma Liver Placenta Uterus

187 254 1,203 133 178

* t + 2 +

6 12 289 4 26

186 278 1,257 159 182

* ” + 2 +

184 275 1,143 187 156

2 2 * k ?

6 27 12 14 9 7 3 18 34 155 37 24

Note. ND, not determined due to inadequate tissue mass. a Administered dose, 8.3 &i/O.05 ug retinoic acid100 g body wt. b Mean * SD of three fetuses of two to five litters studied. c Mean lr SD of two dams, except on Day 14 when five dams were sampled.

trans-[3H,]retinoic acid (2 FM, 20 ~1). The second peak, which coincided with the position of myoglobin (M, 17,000), was nearly abolished by incubating with a 200-fold molar excess of unlabeled retinoic acid. This finding confirmed the presence of a specific binding protein for retinoic acid in these cytosolic fractions. Figure 3 shows the amount of CRABP as measured by the HPLC in various tissues of the Day 14 fetal hamster. The spinal cord possessed the highest amount of CRABP, followed by the limb and skin, but the heart and liver contained only TABLE II Radioactive Retinoids and Metabolites of all-frans-[“‘ClType of radiolabel and sample

or a/l-trans-[3H]Retinoic

Acid

retinoic acid

13-cis-retinoic acid

Fetus Maternal liver

63.9” 52.2

25.5 41.8

0 5.4

3H Fetusb Maternal livep Maternal plasma

61.4 + 4.7 20.5 k 0.4 37.6

34.8 f 4.8 67.7 + 9.4 45.7

3.8 k 0.2 7.7 k 4.2 16.1

all-trans-

4-oxo-all-trans-

retinoic acid

‘T

a Values represent the percentage of radioactivity in the corresponding peak in relation to total radioactivity in a sample at 15 min after a single iv injection on Day 14. The all-rrans-retinoic acid, 13-cis-retinoic acid, and 4-oxo-all-trans-retinoic acid were contirmed using standard samples and ultraviolet detection. Each value represents a composite of one litter. b Means ? SD of two dams.

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FIG. 2. High-performance size-exclusion liquid chromatographic analysis of spinal cord CRABP in Day 14 hamster fetus. The cytosol was incubated with all-rruns-[3H,]retinoic acid in the absence (m) or presence (0) of a 200-fold molar excess of unlabeled all-rruns-retinoic acid. The arrow shows the elution volume of myoglobin.

relatively small amounts of this protein. The brain had only a small amount of CRABP (1.4 pmole/mg protein) compared with the spinal cord (12.7 pmole/mg protein). The total amount of CRABP in fetuses on different days of gestation is shown in Fig. 4. From Day 11 to Day 14 of gestation, CRABP (pmole/mg cytosol protein) in whole fetuses steadily decreased. Sagittal and autoradiographic cross sections after al&runs-[ 14C]retinoic acid distribution are shown in Fig. 5. High concentrations of total residual radioactivity were observed in the mesencephalon, metencephalon, myelencephalon, and spi-

Brain

SC.

Limb

Skin

Heart

Lung

Liver

Intes.

FIG. 3. Quantitative analysis of cellular retinoic acid-binding protein in tissues of Day 14 hamster fetuses. Abbreviations: SC., spinal cord; Intes., intestine. Values are given as means 2 SD (n = 2).

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4. Quantitative analysis of cellular retinoic acid-binding protein in fetuses from Day 11 to Day 14 of gestation. Values are given as the mean 2 SD (n = 2). FIG.

nal cord but not in the forebrain (telencephalon and diencephalon). Limbs also showed relatively high concentrations of total residual radioactivity, especially on Day 11. On other days, the radioactivity was distributed at the distal peripheral areas of the limb rather than being uniformly distributed. High levels of radioactivity were also observed in parts of the face, particularly in those structures derived from the cranial neural crest. Rabbit anti-rat testis CRABP antibody was employed in evaluating the tissuespecific localization of CRABP in fetal tissues after several days gestation. Using appropriate controls, the specific binding of this antibody in cells was noticed, and the distribution of CRABP was highly specific. In many cases, only a few cells were stained, whereas many surrounding cells failed to show any detectable antiCRABP binding. Representative results at various stages indicating the anatomical localization of CRABP are indicated in Fig. 6. In the midsagittal section of the Day 11 fetus, the highest concentration of CRABP was observed in the ventral midbrain, hindbrain, and spinal cord. The highest CRABP concentrations were found in the ventral and lateral myelencephalon and spinal cord and these were particularly evident in cross sections. Some cells were also stained with brown color in the myelencephalon and spinal cord, but the central and dorsal areas of these structures were not stained. In sagittal sections, the cells were stained near a line in the middle of the spinal cord. This stain line was emphasized day by day according to development, and by Day 13, CRABP concentrations in the central portions (near the spinal canal) of the spinal cord were higher than those in the ventral regions. On Day 11, the derivatives of the branchial arches (which develop into the tongue and mandible) were also stained and by Day 13, when the tongue was distinguishable from the mandible, cells were stained clearly in the dorsal tongue. In the limb, brown staining cells were dispersed throughout; however, in the skin, the epidermal cells that were located within one to two cell layers from the surface were stained.

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FIG. 5. Autoradiograms of (A) cross and (B) sagittal sections of fetal hamsters showing the distribution of radioactivity 15 min after intravenous injection of all-trans-[‘4C]retinoic acid on different days of gestation. Abbreviations: te, telencephalon; di, diencephalon, me, mesencephalon; my, myelencephalon; s.c., spinal cord; h, heart; Ii, liver; III, lung; k, kidney; to, tongue; lim, limb; in, intestine.

DISCUSSION The placental permeability of all-trans-retinoic acid and its metabolites in hamsters, mice, and humans has been previously documented. Howard et al. (1989b) found the distribution of radiolabel in the hamster conceptus approaching that in maternal plasma on Day 12 of gestation at 96 hr after a single oral teratogenic dose (IO.5 mg/kg) of all-trans-retinoic acid. Kochhar (1976) reported that the totai

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me my

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me my

FIG. 5-Continued

radioactivity after 3.85 mg/kg of all-truns-retinoic acid increased linearly in mouse embryo for 6 hr. In the maternal liver and in the placenta, however, the concentration of total radioactivity began to decline much earlier (Howard et al., 1989b). The presence of all-truns-retinoic acid and its oxidized metabolites at later periods was demonstrated in the placenta and embryo after larger doses (100 mg and 200 mg/kg) of retinol to pregnant mice (Kochhar et al., 1988). An active teratogenic metabolite of all-truns-retinoic acid, all-truns-Coxo-retinoic acid, also accumulated in the mouse embryo (Satre et al., 1989); however, the peak concentration was less than 30% of that in the maternal plasma. Creech-Kraft and associates (1989b) measured 1.2,2.8, and 0.003 &g of 13-cis, all-tram, and the 13-cis-4-0~0 isomers of retinoic acid in a human abortus collected 72 hr after the last of 20 consecutive oral doses (40 mg/day) of 13-cis-retinoic acid. Of the fetal tissues studied in the present experiments, the highest concentrations of total radioactivity were observed in the spinal cord. Dencker et al. (1987) reported the distribution of [14C]retinoic acid by autoradiography 4 hr after injection of pregnant mice between Day 9 and Day 18 of gestation. Accumulation of

FIG. 6. Immunohistochemical localization of CRABP in the central nervous system of the fetal hamster. (A) The mesencephalon in a sagittal section of 11-day hamster fetus immunostained with anti-CRABP IgG and (B) a duplicate of the same organ stained with the preimmune serum (respective control). Note the CRABP-containing cells in localized ventral region of the mesencephalon in A. (C) Cross section of the spinal cord from an 1 l-day hamster fetus. CRABP-positive cells were localized primarily in the ventral and lateral peripheral areas of the spinal cord at this age; however, they were not observed in a similar zone at later periods. (D) Sagittal section of the spinal cord from a 12-day hamster fetus. Brown-stained cells were noted primarily in the zone surrounding the spinal canal. Also note CRABP-positive cells in the cutaneous region. (E) In the limb bud (left) of a 13-day hamster fetus, cells containing CRABP were primarily in the few peripheral cutaneous layers of this organ; they were not detected in a similar region in the branchial arch (right). Both limb bud and branchial arch contained CRABP-positive cells in deeper layers at an early fetal age (data not shown). Arrows indicate the development of brown color in cells, indicating the presence of CRABP. All sections (A-E) were counterstained with hematoxylin. x 112. 48

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G--Continued

radioactivity in the neuroepithelium and the developing central nervous system occurred in early but not in late fetal development (after Day 13) in the mouse. Of special interest in the current study was the chemical form of the radioactivity localized in the embryo. At 15 min after iv injection of all-rrans-retinoic acid to the mother more than 66% of the radioactivity in the fetus was in the parent form, whereas in the maternal liver and plasma much of the radioactivity was observed in the form of metabolites (Table II); the metabolites found in the embryo were consistent with those reported earlier. Howard ef al. (1989a) reported 13-cis-retinoic acid and 4-oxo-all-rrans-retinoic acid as the principal metabolites in

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maternal hamster plasma after a single oral administration of all-truns-retinoic et al. (1989a) quantified the 4-oxo-allacid (10.5 mg/kg). Creech-Kraft truns-retinoic acid and the 13-cis-retinoic acid in fetal mouse tissues after administration of all-truns-retinoic acid to the dam. In the current study, 25-30% of the radioactivity in hamster fetuses was present as 13-cis-retinoic acid and up to 4% of the radioactivity was present as all-truns-4-oxo-retinoic acid. It should be noted that the parent retinoid and these metabolites are all teratogenically active (Willhite et al., 1989). The differences in the ratio of parent chemical to metabolite in maternal liver are perhaps due to a large difference in the doses employed. A more precise and localized distribution of radioactivity 15 min after [14C]retinoic acid was observed using autoradiography. The highest concentration was observed in the spinal cord, midbrain, and hindbrain but not in the forebrain; limb, nose, and tongue showed high concentrations of radioactivity. Dencker et al. (1987) reported localization of radioactivity associated with all-truns-retinoic acid in the fetal mouse maxillary region, lateral and medial nasal processes, brain, and spinal cord, and in the limb. The present results are consistent with those of Dencker and co-workers. The fetal hamster heart and lung showed low concentrations of radioactivity by autoradiographic measures, similar to the observation that radioactivity in the heart was consistently lower than other tissues as determined by scintillation counting (Table I). Autoradiogrphy showed that distribution of radioactivity in the brain was not uniform. The radioactivity was specifically concentrated in mesencephalon, metencephalon, and myelencephalon. These results may explain why the amount of radioactivity in the whole brain as determined by liquid scintillation counting was lower than that in the spinal cord (Table I). In Day 14 hamster fetuses, high concentrations of CRABP were detected in the spinal cord (12.68 pmole/mg), followed by limb (8.85 pmole/mg) and skin (6.18 pmole/mg). Ong and Chytil (1976) reported that CRABP was detected in all fetal

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rat organs examined (brain, eye, skin, liver, lung, kidney, intestine, heart, and skeletal muscle) but no quantitative data were presented. Maden and Summerbell (1986) detected CRABP in chick limb bud and suggested that this protein was present at approximately similar levels (4.42 to 6.66 pmole/mg of cytosolic protein) at all development stages tested (stages 20 to 35). Although CRABP was not detected in adult rat liver (Ong et al., 1982), CRABP was observed in fetal liver in the present experiments, however, only in very small amounts. There are limited quantitative data for CRABP regarding age-related embryonic and fetal development; the present study illustrated the reduction in CRABP in whole fetuses between Day 11 and Day 14. These data are consistent with those of Momoi et al. (1989b) who observed the developmental change of CRABP in chick brain by immunoblot analysis wherein the highest level of CRABP expression occurred during developmental stages 22-28 and gradually declined thereafter. Using the immunohistochemical technique, the highest concentration of CRABP was observed here in the ventral and lateral myelencephalon and the spinal cord on Day Il. The ventral marginal layer of the spinal cord provides an enveloping zone into which the processes of nerve cells grow over the course of their morphogenesis (Arey, 1966). The area also correlated with the “floor plate” of the neural tube, where a zone of polarizing activity with the potential of endogenous retinoid synthesis has been recently described (Wagner et al., 1990). The cells located in the spinal cord mantle layer also contained CRABP. The concentrations of CRABP in the marginal layer (floor plate) decreased but those in the mantle layer increased over time. Momoi et al. (1989a) reported CRABPpositive ventral areas in the murine neural tube located in the mantle layer with differentiated neurons. The expression of CRABP in specific subpopulations of neural cells in developing mouse embryo was recently reported (Vaessen et al., 1989). Maden et al. (1989) also reported similar specific distribution of CRABP in chick embryo. The present results also agree well with those of Perez-Castro et al. (1989) and DollC et al. (1989), who reported the distribution of CRABP in mouse embryo and the developing limb bud, respectively, employing in situ hybridization of CRABP-specific mRNA. A transient expression of CRABP in the central nervous system of developing mouse embryo has been suggested (Momoi et al., 1990). As mentioned above, the label associated with retinoic acid accumulated in the spinal cord, localized in the brain, limb, and skin, and occurred at relatively lower levels in the intrathoracic organs of the fetal hamster. Similarly, CRABP concentrated in the fetal spinal cord, limb, skin, brain, and intestine. In the heart, lung, and liver, CRABP was detected only in minimal amounts. The results indicated a temporal correlation between the distribution of these acidic retinoids and CRABP in various tissues, except fetal liver. The central nervous system, craniofacial structures, and limb, where both retinoid concentrations and CRABP were detected at high levels, constituted those structures which exhibited malformations when teratogenic doses of all-trans-retinoic acid or its acidic metabolites were administered to pregnant hamsters (Willhite, 1990). Since retinoic acid and/or its metabolites accumulated in maternal and fetal hepatic tissue (Tables I and II), yet CRABP levels remained extremely low in fetal liver, these observations suggest the hypothesis that fetal liver might contain other proteins that bind and accumulate these retinoids. The fetal radioactivity derived from retinoic acid decreased gradually from Day 11 to 14 of gestation but the amount of CRABP

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decreased more rapidly. This relationship does not appear to be linear at the whole fetus or tissue level. It should be pointed out that the localization of CRABP by immunochemistry will not necessarily distinguish between different forms of this protein. Two distinct retinoid-binding proteins, CRABP I and II, have been observed in the neonatal rat (Bailey and Siu, 1988). The antibody used here was prepared against bovine testis CRABP, presumably CRABP I, since CRABP II was not observed in adult tissues, except the skin (Kitamoto et al., 1989; Giguere et al., 1990). Bailey and Siu (1988) suggested that antibodies against mouse fetal CRABP I did not react with CRABP II despite an extensive sequence homology between the two proteins. Subsequently Maden et al. (1990) reported that antibodies against rat CRABP distinguished between CRABP I and II from the rat but not when the two proteins were derived from the chick. The fetal hamster CRABP structureactivity data where CRABP I binds both all-trans-retinoic acid and 13-cis-retinoic acid (Howard et al., 1990), taken together with the fact that CRABP I was the isoform found in adult tissues used to raise the antibody employed in the present study, suggest that the fetal CRABP isoform studied here was analogous to rat CRABP I, but determination of the amino acid sequence is needed for rigorous identification of the fetal hamster CRABP molecules evaluated in this study. The distribution of CRABP-positive cells was similar to the distribution of radioactivity associated with the parental all-trans-retinoic acid. In autoradiograms, the high radioactivity from retinoic acid was also observed in the ventral and lateral myelencephalon and the spinal cord on Day 11. On Days 13 and 14, high concentrations of total residual radioactivity were observed in the mid-spinal cord, just as was the distribution of CRABP. Shenefelt (1972) observed limb and digit malformations, cleft palate, and hypoplastic mandible after maternal oral treatment with all-truns-retinoic acid on Day 11, 12, or 13 of hamster gestation and retinoic acid induced malformations of the hamster spine and spinal cord (rachischisis, myeloschisis, myelocystocele) (Tibbles and Wiley, 1988) that are also readily compared with the results of the present study. From these data we conclude that the distribution of exogenous retinoic acid has a temporal relationship with the distribution of CRABP (likely CRABP I), at least in the central nervous system, in portions of the developing face, tongue, skin, and limb. ACKNOWLEDGMENTS This study was supported in part by Reproductive Hazards in the Workplace, Home, Community, and Environment Research Grant (15-130) from the March of Dimes Birth Defects Foundation. The authors are grateful to Dr. Peter Sorter, Hoffman-LaRoche, Inc.. for the gift of all-trans4-oxo-retinoic acid and to Dr. B. Simizu for helpful suggestions in immunocytochemistry. This work was published as Utah State University Agricultural Experiment Station Journal Paper No. 4017.

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