Vitamin A cycle byproducts impede dark adaptation.

Impaired dark adaptation (DA), a defect in the ability to adjust to dimly lit settings, is a universal hallmark of aging. However, the mechanisms responsible for impaired DA are poorly understood. Vitamin A byproducts, such as vitamin A dimers, are small molecules that form in the retina during the vitamin A cycle. We show that later in life, in the human eye, these byproducts reach levels commensurate with those of vitamin A. In mice, selectively inhibiting the formation of these byproducts, with the investigational drug C20D3-vitamin A, results in faster DA. In contrast, acutely increasing these ocular byproducts through exogenous delivery leads to slower DA, with otherwise preserved retinal function and morphology. Our findings reveal that vitamin A cycle byproducts alone are sufficient to cause delays in DA and suggest that they may contribute to universal age-related DA impairment. Our data further indicate that the age-related decline in DA may be tractable to pharmacological intervention by C20D3-vitamin A.

Ameliorated Effects of (-)-Epigallocatechin Gallate Against Toxicity Induced by Vanadium in the Kidneys of Wistar Rats.

The aim of the study was to assess the protective effect of (-)-epigallocatechin gallate (EGCG), a flavonoid abundant in green tea, against ammonium metavanadate (AMV)-induced oxidative stress in male Wistar rats. Four groups of animals have been used, a control group and three test groups. In the first test group, AMV was intra-peritoneally (i.p) injected daily (5 mg/kg body weight for five consecutive days). The second test group of animals was also injected daily with EGCG (5 mg/kg body weight) during the same period. However, the third test group was i.p. injected with both AMV and EGCG (5 mg/kg body weight for five consecutive days). When given alone, AMV induced an oxidative stress evidenced by an increase of lipid peroxidation levels (expressed as TBARS concentration) in kidney. In these animals, activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were significantly decreased, suggesting significant reduction of the antioxidant defense system at the cell level. Kidney histological sections, showed glomerular hypertrophy and tubular dilatation. In AMV-treated animals receiving EGCG, the oxidative stress was much less pronounced and activities of antioxidant enzymes were kept close to control values. Histopathological changes were less prominent. Our results confirm that green tea and other sources of flavonoids might confer a strong protection against ammonium metavanadate-induced oxidative stress.

Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-ß-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

Retinol deprivation partially rescues the skeletal mineralization defects of Phex-deficient Hyp mice.

X-linked hypophosphatemic rickets (XLH) is a genetic disorder caused by mutational inactivation of the PHEX gene, encoding a transmembrane endopeptidase expressed in osteoblasts. Since several experiments involving Phex-deficient Hyp mice have demonstrated that an increased expression of Fgf23 in osteoblasts is causative for the renal phosphate loss characteristic of XLH, we performed genome-wide expression analysis to compare differentiated osteoblasts from wildtype and Hyp mice. Here we did not only observe the expected increase of Fgf23 expression in the latter ones, but also a differential expression of genes that are either induced by or involved in retinoic acid signaling, which led us to analyze whether dietary retinol deprivation would influence the phenotype of Hyp mice. Unexpectedly, feeding a retinol-free diet resulted in a partial rescue of the growth plate and bone mineralization defects in 6 weeks old Hyp mice. When we fed the same diet for 24 weeks the amount of non-mineralized bone matrix (osteoid) was reduced by more than 70%, although phosphate homeostasis was unaffected. In contrast, a dietary normalization of serum phosphate levels in Hyp mice reduced the osteoid amount by less than 30%, thereby demonstrating a previously unknown impact of retinol on the cell-autonomous mineralization defect of Phex-deficient osteoblasts.

FATP1 inhibits 11-cis retinol formation via interaction with the visual cycle retinoid isomerase RPE65 and lecithin:retinol acyltransferase.

The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.

Retinol inhibits the invasion of retinoic acid-resistant colon cancer cells in vitro and decreases matrix metalloproteinase mRNA, protein, and activity levels.

Retinol inhibits the growth of all-trans-retinoic acid (ATRA)-resistant human colon cancer cell lines through a retinoic acid receptor (RAR)-independent mechanism. The objectives of the current study were to determine if retinol inhibited the invasion of ATRA-resistant colon cancer cells independent of RAR and the effects of retinol on matrix metalloproteinases (MMPs). Retinol inhibited the migration and invasion of two ATRA-resistant colon cancer cell lines, HCT-116 and SW620, in a dose-dependent manner. To determine if transcription, particularly RAR-mediated transcription, or translation of new genes was required for retinol to inhibit cell invasion, cells were treated with retinol and cycloheximide, actinomycin D, or an RAR pan-antagonist. Treatment of cells with retinol and cycloheximide, actinomycin D, or an RAR pan-antagonist did not block the ability of retinol to inhibit cell invasion. In addition, retinol decreased MMP-1 mRNA levels in both cell lines, MMP-2 mRNA levels in the SW620 cell line, and MMP-7 and -9 mRNA levels in the HCT-116 cell line. Retinol also decreased the activity of MMP-2 and -9 and MMP-9 protein levels while increasing tissue inhibitor of MMP-1 media levels. In conclusion, retinol reduces the metastatic potential of ATRA-resistant colon cancer cells via a novel RAR-independent mechanism that may involve decreased MMP mRNA levels and activity.

Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A.

Administration of 50 microg of bisphenol A (BPA) for the first 5 days after birth resulted in a decrease in the percentage of moving sperm, and an increase in the incidence of malformed sperm, in the epididymides of mice at 10 weeks of age, although no marked changes were found in the testicular histology between BPA-treated and vehicle-treated control mice. The deteriorating effects of 50 microg of BPA were ameliorated by the concurrent administration of 100 IU of retinol acetate (RA). Neonatal treatment with 0.5 microg of BPA for 5 days resulted in an increase in the incidence of malformed sperm, whereas the BPA effect became more severe in mice nursed by mothers fed a vitamin A-deficient (VAD) diet only a few days before and after parturition. On the other hand, neonatal treatment with 20 microg of estrogen for the first 5 days after birth resulted in an increase in the number of estrogen receptor alpha (ERalpha)-positive cells in the epithelium of the vas deferens, whereas only a few epithelial cells showed weak ERalpha-positive signals in the vehicle-treated control mice at 18 days after birth. This increase, however, was suppressed by the concurrent administration of RA. Although five daily treatments with 50 microg BPA led to no significant increase in the number of ERalpha-positive cells, it may have been due to the weak estrogenic activity of BPA, as discussed. These findings clearly showed that in mice, neonatal exposure to a relatively large dose of BPA causes damage to the motility and morphology of sperm, but the BPA effect is, to some extent, inhibited by a supplement of VA, and enhanced under a VAD condition.

Molecular logic of 11-cis-retinoid biosynthesis in a cone-dominated species.

The biochemical pathway to visual chromophore biosynthesis in rod-dominated animals involves minimally a two component system in which all-trans-retinyl esters, generated by the action of lecithin retinol acyltransferase (LRAT) on vitamin A, are processed into 11-cis-retinol by isomerohydrolase. Possible differences in retinoid metabolism in cone-dominated animals have been noted in the literature, so it was of interest to explore whether these differences are tangential or fundamental. Central to this issue is whether cone-dominated animals use an isomerohydrolase (IMH)-based mechanism in the predominant pathway to 11-cis-retinoids. Here, it is shown that all-trans-retinyl esters (tREs) are the direct precursors of 11-cis-retinol formation in chicken retinyl pigment epithelium/retina preparations. This conclusion is based on at least three avenues of evidence. First, reagents that block tRE synthesis from vitamin A also block 11-cis-retinol synthesis. Second, pulse-chase experiments also establish that tREs are the precursors to 11-cis-retinol. Finally, 11-cis-retinyl-bromoacetate, a known affinity-labeling agent of isomerohydrolase, also blocks chromophore biosynthesis in the cone system.

N-benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-alpha,beta-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene.

Lignostilbene-alpha,beta-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and beta-carotene 15,15'-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 microM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 microM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.

Vitamin A depletion induced by cigarette smoke is associated with the development of emphysema in rats.

We showed previously that vitamin A deficiency per se causes emphysema. Benzo(a)pyrene, a constituent in cigarette smoke, induces vitamin A depletion when administered to rats; therefore, we tested the hypothesis that cigarette smoke induces vitamin A depletion, which is associated with the development of emphysema. Male weanling rats were fed a purified AIN-93G diet and divided into two groups. The experimental group was exposed to cigarette smoke from 20 nonfiltered commercial cigarettes/d for 5 d/wk, whereas the control group was exposed to air. After 6 wk, tissues were collected for histological and biochemical analyses. Retinol levels were measured in serum, lung and liver. The trachea, lung and liver were examined for histological changes. Vitamin A levels decreased significantly in serum, lung and liver of smoke-treated rats. Histological examination revealed the presence of interstitial pneumonitis along with severe emphysema. There was a significant inverse relationship between vitamin A concentration in the lung and the severity of emphysema (r = -0.69 and P < 0.03). Detachment or hyperplasia (and metaplasia) of the tracheal epithelium and liver vacuole formation also were evident in the smoke-treated rats. The results of this research indicate that exposure to cigarette smoke induces vitamin A depletion in rats, which is associated with the development of emphysema.

In vitro antimalarial activity of retinoids and the influence of selective retinoic acid receptor antagonists.

Retinol (vitamin A alcohol) may have a beneficial role in the host response to malaria in humans and previously published data have suggested that it has a direct inhibitory effect on the growth of Plasmodium falciparum in vitro. To further investigate the role of retinoids as potential antimalarial agents, we assessed the effect of all-trans-retinoic acid (RA), 9-cis-RA and 13-cis-RA, as well as retinol itself and its ester, retinyl palmitate, on 3H-hypoxanthine uptake by the laboratory-adapted strains of P. falciparum 3D7 and K1. In addition, we examined the influence of three specific RA receptor antagonists, ER 27191, Ro 415253 and AGN 194301, on retinoid-induced growth inhibition of 3D7. All-trans-RA, 9-cis-RA and 13-cis-RA in concentrations ranging from 1 x 10(-4) to 5 x 10(-10) M each had antimalarial activity, but at IC50 values (5.9 x 10(-5) to 7.9 x 10(-5) M) that were less than those of retinol (2.5 x 10(-5) to 3.2 x 10(-5) M). Retinyl palmitate had minimal effect on 3H-hypoxanthine uptake. Each of the three specific antagonists inhibited growth of 3D7 (IC50 range 1.2 x 10(-5) to 3.0 x 10(-5) M) but, in isobolographic analysis, were antagonistic to retinol (dose factor potentiation, DFP 0.46-0.79) and, in the case of Ro 415253, to all-trans-RA (DFP=0.39). Although we did not attempt to quantify losses of retinoids from the system, these data suggest that retinol has greater antimalarial activity than its RA metabolites and especially retinyl palmitate. The specific RA receptor antagonists showed paradoxical antimalarial activity but consistently antagonised the effect of retinol and all-trans-RA in isobolographic experiments. We conclude that RA metabolites may be less suitable than retinol per se as antimalarial agents and that P. falciparum might possess or acquire a RA receptor-like moiety.

Retinyl esters are the substrate for isomerohydrolase.

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.

Chlormethiazole treatment prevents reduced hepatic vitamin A levels in ethanol-fed rats.

BACKGROUND: Chronic ethanol intake results in decreased hepatic vitamin A levels through both enhanced degradation of vitamin A via a cytochrome P450 enzyme (CYP)-dependent process and increased mobilization of vitamin A from the liver into the circulation. This study investigated whether treatment with chlormethiazole, a CYP inhibitor, restores vitamin A in the livers of ethanol-fed rats. METHODS: Ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole (10 and 100 mg/kg body weight) for 1 month. Liver and plasma levels of retinol and retinyl palmitate were analyzed by high-performance liquid chromatography. Expressions of hepatic lecithin:retinol acyltransferase (LRAT) and cellular retinol-binding protein were analyzed with reverse transcription-polymerase chain reaction. Hepatic retinol esterification by LRAT was examined by using incubations of the microsomal fractions of livers with exogenous sources of retinol. RESULTS: Ethanol-feeding in rats for a month resulted in lower hepatic levels of retinol and retinyl palmitate than those found in controls and the occurrence of several polar retinoid metabolites. In contrast, treatment with chlormethiazole at two different doses in ethanol-fed rats completely blocked the formation of hepatic retinoid polar metabolites and restored hepatic levels of retinol and hepatic retinyl palmitate in a dose-dependent manner. Furthermore, increased plasma concentrations of retinyl palmitate in rats fed with ethanol, which indicate increased mobilization of vitamin A, were partially inhibited by chlormethiazole treatment. However, neither ethanol nor chlormethiazole treatment altered the expression and activity of LRAT in the liver of rats. Hepatic expression of cellular retinol-binding protein increased significantly in ethanol-fed rats with or without chlormethiazole treatment compared with control rats. CONCLUSIONS: These data suggest that chlormethiazole can restore both hepatic retinol and retinyl ester concentrations to normal levels in ethanol-fed rats through blocking enhanced both degradation of vitamin A and mobilization of vitamin A from the liver into the circulation.

Retinol/ethanol drug interaction during acute alcohol intoxication in mice involves inhibition of retinol metabolism to retinoic acid by alcohol dehydrogenase.

Substantial evidence indicates that one consequence of alcohol intoxication is a reduction in retinoic acid (RA) levels. Studies on the mechanism have shown that chronic ethanol consumption induces P450 enzymes that increase RA degradation, thus accounting for much but not all of the observed decrease in RA. A reduction in RA synthesis may also be involved as ethanol competitively inhibits retinol oxidation catalyzed by alcohol dehydrogenase (ADH) in vitro. This may be important during acute ethanol intoxication and may contribute to adverse retinol/ethanol drug interactions. Here we have examined mice for the effect of either acute ethanol intoxication or Adh1 gene disruption on RA synthesis and degradation. RA produced following a dose of retinol (50 mg/kg) was reduced 87% by pretreatment with an intoxicating dose of ethanol (3.5 g/kg). RA produced in Adh1-null mutant mice following a 50-mg/kg dose of retinol was reduced 82% relative to wild-type mice, thus similar to wild-type mice pretreated with ethanol. Reduced RA production was associated with increased retinol levels in both ethanol-treated wild-type mice and Adh1-null mutant mice, indicating reduced clearance of the retinol dose. RA degradation following a dose of RA (10 mg/kg) was increased only 42% by ethanol pretreatment (3.5 g/kg) and only 26% in Adh1-null mutant mice relative to wild-type mice. These findings demonstrate that the reduced RA levels observed during acute retinol/ethanol drug interaction are due primarily to a decrease in ADH-catalyzed RA synthesis and secondarily to an increase in RA degradation.

Prevention of vitamin A teratogenesis by phytol or phytanic acid results from reduced metabolism of retinol to the teratogenic metabolite, all-trans-retinoic acid.

Previous studies in our laboratory showed a synergistic interaction of synthetic ligands selective for the retinoid receptors RAR and RXR in regard to teratogenic effects produced in mice (M. M. Elmazar et al., 2001, TOXICOL: Appl. Pharmacol. 170, 2-9). In the present study the influence of phytol and phytanic acid (a RXR-selective ligand) on the teratogenicity of retinol and the RAR-selective ligand all-trans-retinoic acid was investigated by coadministration experiments on day 8.25 of gestation in NMRI mice. Phytol and phytanic acid, noneffective when administered alone, did not potentiate the teratogenicity induced by retinol or all-trans-retinoic acid. On the contrary, phytol and phytanic acid greatly reduced retinol-induced teratogenic effects (ear anotia, tail defects, exencephaly). The effect of phytol on all-trans-retinoic acid teratogenesis was limited (only resorptions and tail defects were reduced). Pharmacokinetic studies in nonpregnant animals revealed that phytol coadministration with retinol reduced plasma levels of retinol and retinyl esters, and drastically reduced the levels of the teratogenic retinol metabolite, all-trans-retinoic acid. Phytanic acid also reduced the oxidative metabolism and teratogenic effects of retinol. These results indicate that phytol and phytanic acid did not synergize with retinol and all-trans-retinoic acid in our mouse teratogenesis model. Instead, phytol and phytanic acid effectively blocked the teratogenic effects of retinol by drastically reducing the metabolic production of all-trans-retinoic acid. Phytol and phytanic acid may be useful for the prevention of vitamin A teratogenicity.

Oxidative stress-independent depletion of epidermal vitamin A by UVA.

In hairless mice, epidermal vitamin A (retinol and retinyl esters) is strongly decreased following a single exposure to UVB. Here, using the same mouse model, we studied the effects of UVA on epidermal vitamin A content, lipid peroxidation, and CRBP-I expression, as well as the putative prevention of vitamin A depletion or lipid peroxidation by topical alpha-tocopherol. An acute exposure to UVA completely depleted epidermal vitamin A with EC50 of 0.25 and 0.5 J per cm2 for retinyl esters and retinol, respectively; these values were 0.1 J per cm2 for both retinoids under UVB exposure. CRBP-I expression was increased 2-fold 8 h following UVA exposure (10 J per cm2), and this increase persisted for at least 16 h. A single UVA exposure induced a concentration-dependent epidermal lipid peroxidation (EC50 = 3.5 J per cm2) giving rise to 55.4 +/- 4.2 nmol lipid peroxides per g at 20 J per cm2, whereas UVB, up to 1 J per cm2, did not increase the basal concentration of 6.7 +/- 0.9 nmol lipid peroxides per g. On the other hand, topical menadione induced a concentration-dependent lipid peroxidation, but did not affect vitamin A content. Pretreatment with alpha-tocopherol (i) did not inhibit UV-induced vitamin A depletion, (ii) completely inhibited the increased lipid peroxidation induced by UVA or menadione, and (iii) accelerated reconstitution of epidermal vitamin A after UVB but not UVA induced depletion. Thus acute UVA induced both epidermal vitamin A depletion and lipid peroxidation, UVB induced only vitamin A depletion, and menadione induced only a lipid peroxidation; topical alpha-tocopherol prevented lipid peroxidation but not vitamin A depletion. These observations indicate (i) that CRBP-I neither provides protection to UVB- and UVA-induced epidermal vitamin A depletion, nor interferes significantly with reconstitution, and (ii) that the UV-induced vitamin A depletion and lipid peroxidation in mouse epidermis are unrelated processes. UV light does not destroy epidermal vitamin A through an oxidative stress but probably by a photochemical reaction in which UV radiations at about 325 nm give the corresponding activation energy.

Inhibition of retinol oxidation by ethanol in the rat liver and colon.

BACKGROUND: Epidemiological evidence has been presented for an increased risk of development of colon cancer after chronic alcohol abuse. Alcohol is degraded by cytosolic alcohol dehydrogenases that also are capable of retinol oxidation. Inhibition of retinol oxidation to retinoic acid has been shown to occur in parallel with profound impairment of intracellular retinoid signal transduction and loss of cell differentiation control. AIMS: In the present study, the change in cytosolic retinol oxidation and retinoic acid formation by ethanol concentrations that occur in body tissues in humans after social drinking was measured in cells from the liver, and small and large intestine of the rat. RESULTS: The specific catalytic efficiency V(max)/K(m) (ml/min/g) of cytosolic retinol oxidation in the large intestine (28.9) was found to be distinctly higher than that in the liver (3.4), while the efficiency in the small intestine was negligible (0.20). In the presence of increasing ethanol concentrations (9, 17, and 34 mM), V(max)/K(m) for retinol oxidation decreased in a dose dependent manner to 7.8% of the initial value in the large intestine and to 12% in the liver. The V(max)/K(m) of retinoic acid formation in the liver cytosol decreased to 15%. CONCLUSIONS: Our data demonstrate impairment of hepatic and intestinal cytosolic retinol oxidation and retinoic acid formation by ethanol at concentrations in body tissues after social drinking in humans. The results suggest that the increased risk of developing colorectal neoplasias after alcohol abuse may, at least in part, be caused by impaired retinoid signal transduction.

Effect of cellular retinol-binding protein on retinol oxidation by human class IV retinol/alcohol dehydrogenase and inhibition by ethanol.

All-trans retinoic acid (atRA) is a powerful morphogen synthesized in a variety of tissues. Oxidation of all-trans retinol to all-trans retinal determines the overall rate of atRA biosynthesis. This reaction is catalyzed by multiple dehydrogenases in vitro. In the cells, most all-trans retinol is bound to cellular retinol binding protein (CRBP). Whether retinoic acid is produced from the free or CRBP-bound retinol in vivo is not known. The current study investigated whether human medium-chain alcohol/retinol dehydrogenases (ADH) can oxidize the CRBP-bound retinol. The results of this study suggest that retinol bound to CRBP cannot be channeled to the active site of ADH. Thus, the contribution of ADH isozymes to retinoic acid biosynthesis will depend on the amount of free retinol in each cell. Physiological levels of ethanol will substantially inhibit the oxidation of free retinol by human ADHs: class I, alpha alpha and beta 2 beta 2; class II, pi pi; and class IV, sigma sigma.

Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants.

During late gestation, the lungs of rats contain retinyl esters, but their concentration decreases considerably at the time of birth. The regulation of the acquisition and utilization of these stored retinoids remains poorly understood, although it has been hypothesized that they are involved in surfactant production and alveolar septal formation. Previous investigations demonstrated that exogenous retinoic acid increases elastin production in cultured neonatal lung fibroblasts and increases the number of alveoli when it is administered to neonatal rats. It has been hypothesized that these pulmonary stores of retinyl esters may regulate the perinatal expression of various genes in the lung, including elastin. To test this hypothesis, inhibitors of retinoid metabolism were used to reduce the flux of retinyl esters to retinoic acid, and the effects of this maneuver on elastin gene expression were analyzed. Inhibitors of alcohol and aldehyde dehydrogenases and of retinyl ester hydrolases decreased the steady-state level of tropoelastin mRNA without reducing alpha 1(I) procollagen mRNA. The magnitude of the effects of the inhibitors was retinol dependent and was significantly reduced in lung tissue that was obtained from vitamin A-deficient fetuses. These findings suggest that the late gestational pulmonary stores of retinoids may increase elastin gene expression during the fetal and early postnatal life in the rat.