Mitogenic signaling mediated by oxidants in retinol treated sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 μM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.     

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol-RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.     

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.     

Increased Receptor for Advanced Glycation Endproducts Immunocontent in the Cerebral Cortex of Vitamin A-Treated Rats

Vitamin A, beyond its biological role, is an alternative choice in treating some life threatening pathologies, for instance leukemia and immunodeficiency. On the other hand, vitamin A therapy at moderate to high doses has caused concern among public health researchers due to the toxicological aspect resulting from such habit. It has been described hepatotoxicity, cognitive disturbances and increased mortality rates among subjects ingesting increased levels of vitamin A daily. Then, based on the previously reported data, we investigated here receptor for advanced glycation endproducts (RAGE) immunocontent and oxidative damage levels in cerebral cortex of vitamin A-treated rats at clinical doses (1,000–9,000 IU/kg day⁻¹). RAGE immunocontent, as well as oxidative damage levels, were observed increased in cerebral cortex of vitamin A-treated rats. Whether increased RAGE levels exert negative effects during vitamin A supplementation it remains to be investigated, but it is very likely that deleterious consequences may arise from such alteration.     

Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells

Summary Epidermal mucous metaplasia of cultured skin can be induced by treatment with excess retinol for several days (Fell 1957). In the induction of mucous metaplasia, retinol primarily affects the dermal cells and retinol-pretreated dermis can alter epidermal differentiation towards secretory epithelium (Obinata et al. 1987). In this work, we found that mucous metaplasia could be induced by culturing 13-day-old chick embryonic tarsometatarsal skin in medium containing retinol (20 M) for only 8–24 h, followed by culture in a chemically defined medium (BGJb) without retinol or serum for 6 days. The application of cycloheximide together with retinol during the first 8 h of culture inhibited epidermal mucous metaplasia during subsequent culture for 6 days in BGJb, indicating that induction of a signal(s) in the dermis by excess retinol requires protein synthesis. However, the presence of 20 nM hydrocortisone (Takata et al. 1981) throughout the culture period did not inhibit retinol-induced epidermal mucous metaplasia of the epidermis. This indicates that a brief treatment of the skin with excess retinol determines the direction of epithelial differentiation toward secretory epithelium; this is a simpler in vitro system for the induction of epidermal mucous metaplasia than those established before. Offprint requests to: A. Obinata     

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.     

Dopaminergic and adrenergic toxicities on SK-N-MC human neuroblastoma cells are mediated through G protein signaling and oxidative stress

Dopamine and norepinephrine are neurotransmitters which participate in various regulatory functions of the human brain. These functions are lost in neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease. In this study, we used SK-N-MC neuroblastoma cells to investigate the cytotoxicities of high concentrations of dopamine and norepinephrine on neuronal cells. Dopamine, norepinephrine, as well as their corresponding synthetic agonists (SKF38393 and isoproterenol, respectively) triggered SK-N-MC cell death when applied at 50–100 μM persistently for 2 days. This catecholamine-induced cell death appears to be neuronal specific, as demonstrated by their inabilities of triggering apoptosis of A549 lung carcinoma cells and Cos-7 kidney fibroblasts. By pretreating SK-N-MC cells with target-specific inhibitors before administration of catecholamine, components of G protein signaling (i.e. G _s /cAMP/PKA), monoamine oxidases, nitric oxide synthase, c-Jun N-terminal kinase and oxidative stress were found to be involved in this dopamine/norepinephrine-induced cytotoxicity, which subsequently led to caspase-dependent and -independent apoptotic responses as well as DNA degradation. In contrast, agonists of G _i -coupled dopamine receptors and adrenergic receptors (quinpirole and UK14,304, respectively) were incapable of triggering apoptosis of SK-N-MC cells. Our results suggest that both G protein (G _s )-mediated signaling cascade and oxidative stress participate in the dopamine/norepinephrine-induced neuronal apoptosis. Anthony Chan and Ng Contributed equally to this work.     

Serum α- and γ-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, β-carotene, α-carotene, cryptoxanthin, lutein, lycopene, α-tocopherol, and γ-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk’s Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P<0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 μg/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P<0.05) higher for Seven Seas dolphins (16.4 μg/ml) than for Hawk’s Cay (13.0 μg/ml) and free-ranging dolphins (12.5 μg/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and β-carotene) while the captive animals did not. Additional carotenoids (lycopene, α-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells

Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.     

Serum levels of antioxidant vitamins, copper, zinc and magnesium in children with chronic rhinosinusitis

Reactive oxygen species including hydroxyl radicals, superoxide anions and hydrogen peroxide which are produced by activated granulocytes play an essential role in many biochemical processes and diseases. Oxidant-mediated tissue damage may be important in the development of chronic sinusitis. The aim of this study was to investigate the serum levels of antioxidant vitamins and elements in 24 children (14 boys and 10 girls, age range: 7-12 years, mean age: 9.2 years) with chronic rhinosinusitis, compared to 20 age and sex matched healthy children. Blood samples were collected in the morning before breakfast and prior to any medication. Vitamin A, E and C levels were determined using reagent kits for high performance liquid chromatography. Cu, Zn and Mg levels were analyzed by atomic absorption spectrometry. Vitamin E, vitamin C, Cu and Zn levels were significantly lower in the patients group than in the control group. However, vitamin A and Mg levels did not differ. In conclusion, serum levels of antioxidant vitamins and elements may be important in the pathogenesis and treatment of chronic rhinosinusitis in children.     

Role of ascorbate in lung cellular toxicity mediated by light-exposed parenteral nutrition solution

Abstract

Neonatal lung injury has been induced experimentally by infusion of multivitamin-containing light-exposed parenteral nutrition (PN) solutions. The objective was to explore the role of ascorbate in toxic effects of light-exposed PN on primary cultured foetal rat lung epithelial cells. Hydroperoxides were measured in 3% amino acid solutions at baseline, immediately after addition of either multivitamins or ascorbate alone (400 μg/mL) and again after a 24-h period of exposure to (or protection from) ambient light. Cellular toxicity was assessed by [C¹⁴]adenine release. Multivitamins or ascorbate alone increased hydroperoxides in PN, which was attenuated by light protection. Light-exposed PN containing multivitamins was more toxic to cells than baseline or light-protected PN. Exposure to ascorbate at concentrations both lower (< 5 μg/mL) and higher (> 1000 μg/mL) than normally contained in PN-induced oxidant-mediated cell death, as indicated by protective effects of hydroperoxide and hydroxyl radical scavengers. This study concludes that ascorbate generates toxic amounts of peroxide in PN solutions. The types and physiological importance of hydroperoxides induced by pro-oxidant effects of ascorbate require further evaluation in vivo.     

Effects of retinol and dexamethasone on cytokine-mediated control of metalloproteinases and their inhibitors by human articular chondrocytes and synovial cells in culture

Human articular chondrocytes in culture produced large amounts of specific mammalian collagenase, gelatinase and proteoglycanase when exposed to dialysed supernatant medium derived from cultured human blood mononuclear cells (mononuclear cell factor) or to conditioned medium, partially purified by fractionation with ammonium sulphate (60–90% fraction), from cultures of human synovial tissue (synovial factor). Human chondrocytes and synovial cells also released into culture medium an inhibitor of collagenase of apparent molecular weight about 30 000, which appeared to be similar to the tissue inhibitor of metalloproteinases synthesised by tissues in culture. The amounts of free collagenase inhibitor were reduced in culture media from chondrocytes or synovial cells exposed to mononuclear cell factor or synovial factor. While retinol inhibited the production of collagenase brought about by mononuclear cell factor or synovial factor, it restored the levels of inhibitor, which were reduced in the presence of mononuclear cell factor or synovial factor. Dexamethasone markedly reduced the production of collagenase by synovial cells, while only partially inhibiting factor-stimulated collagenase production by chondrocytes. Addition of puromycin as an inhibitor of protein synthesis reduced the amounts of both collagenase and inhibitor to control or undetectable levels.     

Decreased plasma and tissue levels of vitamin C in a rat model of aging: implications for antioxidative defense

Aging is an independent risk factor for the development of cardiovascular and many other diseases. The aging process is known to be associated with increased oxidative stress, possibly related to an age-inherent loss of antioxidant capacity. Vitamin C is a major naturally occurring antioxidant. Thus, we investigated its role in a rat model of aging. Vitamin C in plasma and tissues as well as malondialdehyde in the heart were measured in young (6 months old) and old (27-30 months old) F1 (F344 x BN) healthy male rats fed a normal diet. In old rats, vitamin C plasma levels were found to be decreased (p<0.02) as compared with young animals. Furthermore, there was a tissue-specific distribution: in the heart, liver, kidney, lungs, and skeletal muscle, vitamin C decreased with age (p<0.005 to p<0.05), while no significant differences could be observed in the aortic wall and in the brain. Organs of the digestive tract rather showed an increase of vitamin C levels with age. Oxidative stress, determined representatively in the heart by measuring malondialdehyde tissue levels, exhibited an age-dependent increase (p<0.05). A distinct pattern of specific tissue distribution of vitamin C suggests a differential age-associated regulation. As vitamin C decreased concomitantly to an increase in cardiac lipid peroxidation, its supplementation may be useful to prevent age-related oxidative stress and tissue aging.     

Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition

Cyclosporine A (CsA), a widely used immunosuppressant shows cytotoxic effects by either inducing apoptosis or redirecting the cell towards non-apoptotic cell death. However, there still remains a lacuna in understanding the mechanism of CsA induced non-apoptotic cell death. In the present study we investigated calcineurin dependent or independent cytotoxic effects of CsA, a calcineurin inhibitor, in cervical cancerous SiHa cells. Decreased cell viability and massive cytoplasmic vacuolations were observed in CsA treated SiHa cells, having increased calcineurin activity. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR), accompanied by a decrease in cyclophilin B (ER resident PPIase), preceded the formation of the vacuoles. These vacuoles stained positive for many ER resident markers confirming their ER origin; but the absence of autophagosomal marker, LC3II, ruled out autophagy. Extensively vacuolated cells eventually undergo cell death which lacked the typical apoptotic features, but showed significant decrease in AIP (ALG2 interacting protein) as seen in paraptosis. ER-vacuolation was prevented by cycloheximide and salubrinal thereby indicating requirement of active protein synthesis. Inhibiting calcineurin activity by either Tacrolimus (FK506) or by knockdown of calcineurin B subunit did not result in either ER-stress or cellular vacuolation. However, knockdown of cyclophilin B by siRNA resulted in increased expression of Bip and IRE1α, together with cytoplasmic vacuolation. In conclusion, we report that persistent ER stress due to cyclophilin B inhibition in CsA treated cervical cancer cells caused cellular vacuolation which culminated in a non-apoptotic cell death response similar to paraptosis. Additionally, the paraptotic effects of CsA are independent of calcineurin inhibition.     

Influence of high vitamin E dosages on retinol and carotinoid concentration in body tissues and eggs of laying hens

The aim of the study was to contribute to the discussion of overdosing vitamin E in laying hens. A total of 45 laying hens, divided into 5 groups were fed diets supplemented with either 0; 100; 1000; 10 000 or 20 000 mg dl‐α‐tocopheryl acetate/kg diet over a period of 10 weeks. Concentrations of vitamins A and E were measured in plasma, various tissues and egg yolk. Furthermore egg yolk colour and some carotinoids were measured in egg yolks. None of the vitamin E doses significantly influenced performance of the hens. As expected, vitamin E concentration in plasma, all tissue samples and egg yolk was significantly increased with increasing tocopherol content in the diet. The egg yolk showed the highest vitamin E concentration, followed by liver and muscles. Feeding 1000 mg α‐tocopheryl acetate per kg diet resulted in an increase of vitamin A concentration in the liver. Very high doses (10 000 and 20 000 mg/kg diet) significantly decreased retinol concentration in the liver and egg yolk, as well as carotinoid concentration in the egg yolk. The lower carotinoid concentration in egg yolk resulted in a decreased intensity of egg yolk colour. A prooxidative and/or competitive effect of very high doses of vitamin E with other fat soluble substances has been discussed.     

SIN-1 cytotoxicity to PC12 cells is mediated by thiol-sensitive short-lived substances generated through SIN-1 decomposition in culture medium

As a generator of peroxynitrite (ONOO⁻), 3-morpholinosydnonimine (SIN-1) is widely used in the study of oxidative/nitrosative stress in cultured cells, although controversy exists regarding active species responsible for cytotoxicity. In this study, we report that unstable thiol-sensitive substances, generated from the reaction of SIN-1 with components in culture medium, play a crucial role in SIN-1 cytotoxicity in PC12 cells. Exposure of cells to culture medium obtained after almost complete SIN-1 decomposition at 37 °C for 2 h demonstrated almost the same degree of cytotoxicity as did fresh SIN-1. The cytotoxicity of SIN-1-decomposed medium largely depended on serum, decayed with time, and could be completely abolished by the addition of thiols. Degradation of synthetic ONOO⁻ in the culture medium did not reproduce the unstable cytotoxicity. The presence of superoxide dismutase (SOD) during SIN-1 decomposition prevented the formation of the cytotoxic substances, whereas SOD had no protection against the cytotoxicity itself, suggesting a crucial role of simultaneously generated superoxide and nitric oxide in the formation of the toxicants, but not in their cytotoxic action. The cytotoxicity of fresh SIN-1 is dramatically suppressed in a basal medium (Hanks balanced salt), suggesting that the cytotoxicity of fresh SIN-1 also requires components of culture medium. These results suggest that SIN-1 cytotoxicity in PC12 cells is mediated via the generation of cytotoxic substances in the medium during its decomposition.