Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, β-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10–15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low μM concentrations of vitamin E, vitamin C, or carnosic acid but not with β-carotene or lycopene. Indeed, in the presence of 0.5–1.0 μM β-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5–2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, β-carotene or lycopene (0.5–1.0 μM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and β-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Effects of beta-carotene,vitamin C and E on antioxidant status in hyperlipidemic smokers

Smoking can accelerate the consumption of the stored antioxidant vitamins and increase the oxidative stress in the hyperlipidemic patients. The study investigated the effects of combined beta-carotene, vitamin C, and vitamin E on plasma antioxidant levels, erythrocyte antioxidative enzyme activities, and LDL lipid peroxides. Male hyperlipidemic smokers (35-78 years old) were randomly divided into two antioxidant supplemented groups: intervention 1 (I1, n = 22) (15 mg beta-carotene/day, 500 mg vitamin C/day, and 400 mg alpha-tocopherol equivalent/day) and intervention 2 (I2, n = 20) (30 mg beta-carotene/day, 1000 mg vitamin C/day, and 800 mg alpha-tocopherol equivalent/day). After 6-week supplementation, plasma beta-carotene, vitamin C, vitamin E, and erythrocyte glutathione levels increased significantly by 200%, 98%, 129%, and 39%, respectively, in the I1 group, and by 209%, 216%, 197%, and 32%, respectively, in the I2 group. Plasma Fe(+2) concentrations and Fe(+2)/Fe(+3) decreased significantly in both groups. Except erythrocyte glutathione peroxidase activity in the I1 group, erythrocyte catalase, glutathione peroxidase, and superoxide dismutase activities increased significantly in both groups. Lipid peroxides in LDL decreased significantly by 56% and 72% in the I1 and I2 groups, respectively. However, the levels of plasma iron, erythrocyte glutathione, and LDL lipid peroxides, and the activities of erythrocyte antioxidative enzymes did not differ between two groups. In conclusion, combined antioxidant supplements increased plasma antioxidant levels and antioxidative enzyme activities, and lowered LDL lipid peroxides in male hyperlipidemic smokers. Higher dosage of the supplements did not have an additive effect.     

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.     

Production of antioxidant vitamins, beta-carotene, vitamin C, and vitamin E, by two-step culture of Euglena gracilis Z

Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as beta-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of beta-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C. (c) 1997 John Wiley & Sons, Inc.     

cis-trans isomers of lycopene and beta-carotene in human serum and tissues.

Since cis or trans isomers of carotenoids may have different biological reactivities, the isomeric composition of lycopene and beta-carotene was measured in serum and seven human tissues. In addition to all-trans lycopene, at least three cis-isomers (9-, 13-, and 15-cis) were present, accounting for more than 50% of total lycopene. 13- and 15-cis-beta-carotene, however, were present at only 5% of the all-trans isomer. In addition, 9-cis-beta-carotene was present in tissue samples but not in serum. There were interindividual differences in carotenoid levels of the different tissue types, but liver, adrenal gland, and testes always contained significantly higher amounts of the carotenoids than kidney, ovary, and fat; carotenoids in brain stem tissue were below the detection limit. beta-Carotene was the major carotenoid in liver, adrenal gland, kidney, ovary, and fat, whereas lycopene was the predominant carotenoid in testes.     

Concentrations of vitamin A, beta-carotene and vitamin E in individual bovine follicles of different quality

The degree of atresia of the follicle had no influence on the intrafollicular concentrations of beta-carotene, vitamin E and cholesterol. This might result from the passive transfer of these substances from blood to follicular fluid bound to high density lipoproteins. However, concentrations of vitamin A in follicular fluid were significantly (P less than 0.001) influenced by follicle quality, with highest concentrations (0.32 microgram/ml) in non-atretic follicles and lowest values (0.15 microgram/ml) in greatly atretic follicles. The higher concentrations of vitamin A in healthy follicles might be due to a local conversion of beta-carotene into vitamin A in follicular structures. By influencing hormone and protein synthesis, vitamin A may have a potential for local modulation of follicular development and therefore be one of the factors controlling recruitment, selection and growth of the dominant follicle in cattle.     

Comparing beta-carotene,vitamin E and nitric oxide as membrane antioxidants

Singlet oxygen initiates lipid peroxidation via a nonfree radical mechanism by reacting directly with unsaturated lipids to form lipid hydroperoxides (LOOHs). These LOOHs can initiate free radical chain reactions leading to membrane leakage and cell death. Here we compare the ability and mechanism by which three small-molecule membrane antioxidants (beta-carotene, alpha-tocopherol and nitric oxide) inhibit lipid peroxidation in membranes. We demonstrate that beta-carotene provides protection against singlet oxygen-mediated lipid peroxidation, but does not slow free radical-mediated lipid peroxidation. Alpha-Tocopherol does not protect cells from singlet oxygen, but does inhibit free radical formation in cell membranes. Nitric oxide provides no direct protection against singlet oxygen exposure, but is an exceptional chain-breaking antioxidant as evident from its ability to blunt oxygen consumption during free radical-mediated lipid peroxidation. These three small-molecule antioxidants appear to have complementary mechanisms for the protection of cell membranes from detrimental oxidations.     

Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.     

Effects of vitamin E and beta-carotene on sperm competitiveness

Sperm are particularly prone to oxidative damage because they generate reactive oxygen species (ROS), have a high polyunsaturated fat content and a reduced capacity to repair DNA damage. The dietary compounds vitamin E and beta-carotene are argued to have antioxidant properties that help to counter the damaging effects of excess ROS. Here in, we tested the post-copulatory consequences for male crickets (Teleogryllus oceanicus) of dietary intake of these two candidate antioxidants. During competitive fertilisation trials, vitamin E, but not beta-carotene, singularly enhanced sperm competitiveness. However, the diet combining a high vitamin E dose and beta-carotene produced males with the most competitive ejaculates, possibly due to the known ability of beta-carotene to recycle vitamin E. Our results provide support for the idea that these two common dietary compounds have interactive antioxidant properties in vivo, by affecting the outcomes of male reproductive success under competitive conditions.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

Synthesis and regulation of C1 inhibitor in human skin fibroblasts

Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.     

Characterization of acid phosphatase isoenzymes in human skin fibroblasts.

The multiple acid phosphatase isoenzymes of cultivated skin fibroblasts were investigated in an effort to further clarify the basis of the observed molecular heterogeneity. Treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment with neuraminidase did not alter the migration of isoenzymes present prior to treatment, but additional isoenzymes were detectable after treatment. Variation of enzymes, permitting prediction of net charge and molecular size differences. Isoelectric focusing between pH 5.0 and pH 8.0 demonstrated three isoenzymes of acid phosphatase in the total cell homogenate and in the lysosomal fraction, two of which appeared to have similar isoelectric points.     

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.     

Enhancement of the UVA induction of haem oxygenase-1 expression by beta-carotene in human skin fibroblasts.

beta-Carotene has often been discussed as a means to reduce the risk of skin photodamage. We studied the antioxidative potential of beta-carotene in human skin fibroblasts exposed to ultraviolet A light. Surprisingly, we found a pro-oxidative effect of beta-carotene. Using the induction of haem oxygenase-1 as a marker for oxidative stress, we found a strong enhancement of gene expression by beta-carotene in ultraviolet A-irradiated cells. This effect was clearly suppressed by concomitant addition of vitamin E but only moderately by vitamin C. The results show that beta-carotene has pro-oxidative properties in human skin fibroblasts exposed to ultraviolet-A light.     

Effect of vitamin C and vitamin E on prostaglandin synthesis by fibroblasts and squamous carcinoma cells.

Dietary levels of vitamins C and E have been associated with cancer prevention and to a lesser extent with therapeutic enhancement of cancer treatment. Inhibition of prostaglandins (PGs) by pharmacological agents has been demonstrated to enhance immunocompetence, and to suppress growth of tumors in animals and humans. We report here on the effect of vitamins C and E on PGE2 production by human gingival fibroblasts and SCC-25 oral squamous carcinoma cells. The results indicate: 1. vitamins C and E exert a dose-dependent effect on arachidonic acid (AA) release and PGE2 synthesis; 2. vitamin E has a biphasic effect which is stimulatory at 1 and 10 microM and inhibitory at 100 microM; 3. vitamin E is considerably more potent than vitamin C in its inhibitory effect on AA and PGE2 in both cell types; 4. a combination of the two vitamins has a consistent dose-dependent inhibitory effect on AA and PGE2; 5. vitamin C stimulates PGE2 synthesis from exogenous AA in fibroblasts, and inhibits it in SCC-25 cells. The in vivo significance of these findings requires further investigation.     

Metabolic engineering of beta-carotene and lycopene content in tomato fruit

Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A). Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (beta-Lcy). We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the beta-Lcy gene in a fruit-specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta-carotene content. The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio. Fruits from AS transformants show up to 50% inhibition of beta-Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.     

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.     

Inhibition of immunoglobulin E production in allergic model mice by supplementation with vitamin E and beta-carotene

A diet containing different amounts of vitamin E (alpha-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of beta-carotene was supplemented to the basal diet containing vitamin E as alpha-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with beta-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus beta-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and beta-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and beta-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.