Microinjections of cultured rat conceptuses: studies with 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide.

4-Oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and all-trans-retinoyl-beta-glucuronide were intraamniotically microinjected in rat embryos on day 10 of gestation and cultured until day 11.5. A comparison of the concentration-effect relationships showed that the dysmorphogenic effects produced by these metabolites were qualitatively similar to those of parent all-trans-retinoic acid. Compared with all-trans-retinoic acid (300 ng/ml), the dysmorphogenic effects were elicited by a 2-fold higher concentration of 4-oxo-all-trans-retinoic acid, an approximately 10-fold higher concentration of 4-oxo-13-cis-retinoic acid and a 16-fold higher concentration of all-trans-retinoyl-beta-glucuronide. A surplus of uridine 5'-diphospho-glucuronic acid, microinjected together with 300 ng/ml all-trans-retinoic acid, decreased the observed embryo-toxicity of all-trans-retinoic acid, suggesting the possibility of glucuronidation in tissues of the conceptus per se. The results of the study provide further support for the hypothesis that 4-oxo-all-trans-retinoic acid and all-trans-retinoic acid are, in contrast to the corresponding cis-isomers and glucuronides, ultimate dysmorphogenic retinoids.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

Multiple retinol and retinal dehydrogenases catalyze all-trans-retinoic acid biosynthesis in astrocytes

All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ～40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.     

Comparison of the effects of retinol and retinoic acid on postimplantation rat embryos in vitro

Rat embryos were explanted on day 8 or 9 of pregnancy and cultured for up to 48 hours in serum containing added retinol (vitamin A), retinoic acid (vitamin A acid), or absolute ethanol. They were examined morphologically and their protein content determined. Retinoic acid was more teratogenic and growth-retarding than retinol. Electron microscopy of embryos cultured for 30 minutes or one hour revealed that both forms of vitamin A brought about similar ultrastructural effects on the embryonic cells; however, the abnormally large intracellular lipid droplets observed in a previous study following exposure to retinol in vitro and retinyl palmitate in vivo were not observed in embryos exposed to retinoic acid. It is possible that the differential teratogenicity may be due to the inability of the embryonic cells to convert and store retinoic acid in a less teratogenic form.     

Biliary metabolites of all-trans-retinoic acid in the rat

Biliary metabolites from physiological doses of all-trans-[10-3H]retinoic acid were examined in normal and vitamin A-deficient rats. The bile from normal and vitamin A-deficient rats contained approximately 60% of the administered dose following a 24-h collection period. However, vitamin A-deficient rats show a 6-h delay in the excretion of radioactivity compared to normal rats. Retinoyl-beta-glucuronide excretion was particularly sensitive to the vitamin A status of the rats. In normal rats, retinoyl-beta-glucuronide reached a maximum concentration of 235 pmol/ml of bile 2 h following the dose and then rapidly declined. Vitamin A-deficient rats show a relatively constant concentration of this metabolite (100-150 pmol/ml of bile) over a 10-h collection period. Retinoic acid excretion was low in both normal and deficient rats. The concentration of retinotaurine, a recently identified biliary metabolite, was approximately equal to retinoyl-beta-glucuronide in normal rats and appeared in the bile 2 h later than the glucuronide.     

Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein

The interactions of retinol with rat cellular retinol-binding protein (CRBP) and with rat serum retinol-binding protein (RBP) were studied. The equilibrium dissociation constants of the two retinol-protein complexes (Kd) were found to be 13 x 10(-9) and 20 x 10(-9) M for CRBP and for RBP, respectively. The kinetic parameters governing the interactions of retinol with the two binding proteins were also studied. It was found that although the equilibrium dissociation constants of the two retinol-protein complexes were similar, retinol interacted with CRBP 3-5-fold faster than with RBP; the rate constants for dissociation of retinol from CRBP and from RBP (koff) were 0.57 and 0.18 min-1, respectively. The rate constants for association of retinol with the two proteins (kon) were calculated from the expression: Kd = koff/kon. The kon's for retinol associating with CRBP and with RBP were found to be 4.4 x 10(7) and 0.9 x 10(7) M-1 min-1, respectively. The data suggest that the initial events of uptake of retinol by cells are not rate-limiting for this process and that the rate of uptake is probably determined by the rate of metabolism of this ligand. The data indicate further that the distribution of retinol between RBP in blood and CRBP in cytosol is at equilibrium and that intracellular levels of retinol are regulated by the levels of CRBP.     

Hyperglycemia, hypoxia and their combination exert oxidative stress and changes in antioxidant gene expression: studies on cultured rat embryos

INTRODUCTION: Hyperglycemia and hypoxia are well‐known teratogens that may affect many animal species, including man. We hypothesize that a combination of hypoxia and hyperglycemia will increase embryonic damage produced by either factor individually. We investigated the interrelationship between hyperglycemia and hypoxia and their effects on genes involved in the balance of embryonic redox status. METHODS: Rat embryos (10.5‐day‐old) were cultured for 28 hr in culture medium with about 6 mg/ml of glucose and 20% oxygen (hyperglycemia), with 10% oxygen (hypoxia) and 2.4 g/ml glucose (normal) or a combination of both 6 mg/ml glucose and 10% oxygen. Antioxidant capacity was determined by activity and gene expression of antioxidant enzymes: Cu/Zn SOD, Mn‐SOD, CAT, and GSH‐px using real time PCR. RESULTS: Hyperglycemia, hypoxia, or their combination, decreased embryonic growth and induced a high rate (62–78%) of anomalies mainly of the nervous system, heart, and limbs. CAT mRNA and GSH‐px mRNA were decreased in the malformed embryos exposed to hyperglycemia, to hypoxia or their combination. CAT mRNA was also decreased in the nonmalformed embryos subjected to hyperglycemia and hypoxia. Cu/Zn SOD mRNA was increased in all experimental embryos whether malformed or not, whereas Mn‐SOD was drastically decreased. Total SOD and CAT like activity were changed very little in the experimental embryos compared to controls. CONCLUSIONS: Both hyperglycemia, hypoxia, and their combination reduce embryonic growth and development, induce embryonic anomalies, and modify the expression of the principle antioxidant genes. However, hypoxia does not seem to enhance the damaging effects of hyperglycemia except its effects of embryonic growth. Birth Defects Res (Part B) 92:231–239, 2011. © 2011 Wiley‐Liss, Inc.     

The protective effects of L-ascorbic acid and DL-alpha-tocopherol on cultured rat embryos treated with xanthine/xanthine oxidase

Abnormalities of the neural suture were observed in cultured rat embryos exposed to oxygen radicals generated by xanthine and xanthine oxidase. The distribution of the severity of these abnormalities was altered by the addition of L-ascorbic acid (AA) or DL-alpha-tocopherol (AT). The antioxidant effect of AA and AT were probably responsible for the protection of the embryos from the damaging effects of oxygen radicals.     

Early effects of retinol and retinoic acid on protein synthesis in retinol deficient rat testes

When an [35S] labeled mixture of methionine and cysteine was injected intratesticularly into retinol-deficient rats, two hours later more than 980 cytosolic proteins were detected by computer aided two dimensional gel electrophoresis. Furthermore, two hours after oral refeeding retinyl acetate as the source of retinol to retinol deficient rats, synthesis of 286 proteins was inhibited and that of 101 proteins was activated. Refeeding with retinoic acid leads in two hours to even higher inhibition of protein synthesis and the labeling patterns of proteins are not identical when compared to retinol refed rats. The results indicate that retinol or retinoic acid quickly influence expression of many proteins and suggest that retinol action in the testes is not identical to that of retinoic acid.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Effects of 9-cis- and all-trans-retinoic acids on blood glucose homeostasis in the fiddler crab, Uca pugilator

9-cis-Retinoic acid (9CRA) and all-trans-retinoic acid (ATRA) are known to be involved in the regulation of glucose homeostasis in vertebrates by inducing insulin release and expression of glucose transporter proteins. In view of the fact that both 9CRA and ATRA are endogenous to the fiddler crab, Uca pugilator, that a retinoid X receptor exists in this fiddler crab and that activities of insulin-like and insulin-like growth factor-like peptides have been reported for crustaceans, we investigated whether 9CRA and ATRA also play a role in glucose homeostasis in U. pugilator. Neither 9CRA nor ATRA was found to produce hypoglycemic effects at a dose of 10 microg/g live mass. However, 9CRA, but not ATRA, induced hyperglycemia. Such 9CRA-induced hyperglycemia was apparently mediated by the eyestalk hormone CHH since injection of 9CRA into eyestalk-ablated crabs did not result in hyperglycemia. ATRA was found to have an inhibitory effect on the recovery of blood glucose concentration following ATRA administration. Discussion on the possible mechanisms for the actions of 9CRA and ATRA was presented.     

Pathways of retinol and retinoic acid metabolism in the rat

1. The metabolism of retinoic acid and retinyl acetate labelled with (14)C in various positions was studied after intravenous injection of physiological amounts of these compounds into retinol-deficient rats. 2. Analysis of the resultant radio-activity in the urine, carbon dioxide and faeces led to a postulation of the existence of three major pathways for the metabolism of these two compounds. 3. Evidence is presented that retinoic acid and retinol are metabolized by either the same or at least similar pathways and that retinol becomes oxidized to the carboxyl state before any degradation of the isoprenoid side chain occurs. 4. It is not possible to decide from these data whether retinoic acid is an intermediate in the retinol pathway. 5. Possible sites for the regulation of retinol metabolism are discussed.     

Formation of retinoic acid from retinol in the rat

1. The formation in vivo of retinoic acid from microgram quantities of intrajugularly administered [15-(14)C]retinol was demonstrated in the rat. 2. Endogenously formed retinoic acid (about 0.1mug./rat) was found in liver, and to a much smaller extent in intestine, 12hr. after retinol administration. 3. Excretion of some of the endogenously formed retinoic acid occurred in the bile of bile-duct-cannulated rats. 4. Excretion of unaltered retinoic acid in the urine of intact rats did not occur even after the intrajugular administration of preformed retinoic acid.     

Tracer studies with 13C-labeled carbohydrates in cultured plant cells. Retrobiosynthetic analysis of chelidonic acid biosynthesis

The biosynthesis of chelidonic acid was studied in cell suspension cultures of Leucojum aestivum. Cell cultures were supplied with [U-13C]glucose, [l-13C]glucose or [U-13Cs]ribose/ribulose in standard medium containing unlabeled glucose. 13C labeling patterns of amino acids obtained by hydrolysis of biomass were determined by NMR spectroscopy and compared to the labeling pattern of chelidonic acid. The data document the incorporation of a contiguous 4-carbon fragment derived from the pentose phosphate pool into chelidonic acid. This suggests a biosynthetic pathway involving the condensation of phosphoenolpyruvate with a pentose phosphate followed by dehydration, dehydrogenation, ring closure and decarboxylation conducive to the loss of C-5 of the pentose precursor.     

The embryolethality and teratogenicity of acrolein in cultured rat embryos

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.     

Photoaffinity labeling of human IRBP with all-trans-retinoic acid

Interphotoreceptor retinoid-binding protein (IRBP), found only in photosensitive tissues, is a large approximately 135-kDa glycoprotein that contains a fourfold repeat structure. IRBP may function as a buffer and prevent retinoid toxicity and retinoid degeneration. Here we asked (i) whether each repeat of IRBP possesses the capability of photo-crosslinking all-trans-retinoic acid (RA), (ii) within Repeat 1 whether a single retinoic acid-binding domain exists, and (iii) whether protease and CNBr digestion of Repeat 1 bound RA indicate the exact location of the binding site. 3H-RA cross-linked to all four repeats, consistent with the current model of multiple binding sites in IRBP. Acetone precipitation was effective in removing unbound 3H-RA. LysC and tryptic digestion of the RA-Repeat 1 detected 18- and 5-kDa bands, respectively. CNBr digestion showed two bands about 9 and 11 kDa in size. Our data suggests a single binding site near positions 151-160 in the center of Repeat 1.     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

Effect of exogenous gibberellic acid,abscisic acid,and benzylaminopurine on epicotyl dormancy of cultured herbaceous peony embryos

Epicotyl dormancy was broken in cultured peony (Paeonia lactiflora Pall.) embryos after topical application of agarose gels containing gibberellic acid, with optimum growth at 1.5 mM gibberellic acid. Addition of 100 M abscisic acid to the medium resulted in complete inhibition of gibberellic acid-stimulated promotion of dormant epicotyls. Epicotyl dormancy was also broken in embryos by culture on media containing 1 or 10 M benzylaminopurine. A highly significant increase in leaf number occurred when embryos were both cultured on medium containing benzylaminopurine and treated topically with gibberellic acid. Anatomical and morphological studies indicated that the increase in shoot growth was due to the development and growth of 1) buds formed at the cotyledonary node, 2) axillary buds, and 3) adventitious meristems originating from subepidermal parenchymatous tissue.Abbreviations ABA abscisic acid - BA N⁶-benzylaminopurine - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - LS Linsmaier and Skoog