Photoprotective potential of lycopene,beta-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, beta-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 microM concentrations of vitamin E, vitamin C, or carnosic acid but not with beta-carotene or lycopene. Indeed, in the presence of 0.5-1.0 microM beta-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, beta-carotene or lycopene (0.5-1.0 microM) 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 beta-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.     

Protection of Heme Proteins by Vitamin E, Selenium, and β-Carotene Against Oxidative Damage in Rat Heart, Kidney, Lung and Spleen

Effects of the combination of vitamin E, selenium, and β-carotene on oxidative damage to rat heart, kidney, lung, and spleen were studied by measurement of the production of oxidized heme proteins (OHP) during spontaneous and prooxidant-induced oxidation. Male SD rats were fed with a vitamin E and selenium deficient diet or a diet supplemented with vitamin E, selenium, and β-carotene, Homogenates of heart, kidney, lung, and spleen were incubated at 37°C with and without the presence of bromotrichloromethane (CBrCl₃). The diet supplemented with antioxidants showed a strong protective effect against oxidative damage to heme proteins during the early stages of both spontaneous and CBrCl₃-induced oxidation in contrast to the antioxidant deficient diet. Synergism of multiple antioxygenic nutrients against oxidative damage to various animal tissues is discussed.     

Prooxidant activity of β-carotene under 100% oxygen pressure in rat liver microsomes

The effects of the partial pressure of oxygen (pO₂ on antioxidant efficiency of β-carotene in inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced lipid peroxidation are investigated in rat liver microsomal membranes. The rate of peroxyl radicals generated by thermolysis of AAPH at 37°C is markedly higher at 150 than 760 mm Hg pO₂. At 150 mm Hg pO₂ β-carotene acts as an antioxidant, inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced Malondialdehyde (MDA) formation. At 760 mm Hg pO₂, it loses its antioxidant activity and shows a prooxidant effect, increasing lipid peroxidation products, -Tocopherol prevents the prooxidant effect of β-carotene in a dose-dependent manner. Our data provide the first evidence of a prooxidant effect of β-carotene under 100% oxygen pressure in a biological membrane model and point out the existence of cooperative interactions between β-carotene and -tocopherol.     

Kinetics of Photobleaching of β-Carotene in Chloroform and Formation of Transient Carotenoid Species Absorbing in the Near Infrared

Upon laser flash photolysis of β-carotene in chloroform instantaneous bleaching of β-carotene and concomitant formation of near infrared absorbing species are observed. One species, absorbing with maximum at 920 nm, is formed during the laser pulse (10 ns) and is practically gone in one millisecond, the decay showing a bi-exponential behaviour. The second species, absorbing with maximum at 1000 nm, is formed from the species absorbing at 920 nm by first order kinetics with a rate constant of 4.9·10⁴ s^-1 at 20°C. This second species decays by second order kinetics and is gone within a few milliseconds. An additional slow bleaching of β-carotene and formation of the species absorbing at 920 nm is observed. This slow bleaching/formation of transient absorption is probably due to processes involving free radicals generated during the instantaneous bleaching. The species absorbing at 920 nm is suggested to be either (i) a free radical adduct formed from β-carotene and chloroform or (ii) β-carotene after abstraction of a hydrogen atom. The species absorbing at 1000 nm is most likely the radical cation. Formation and decay of the near infrared absorbing species and bleaching of β-carotene are independent of whether oxygen is present or absent in the solutions.     

The effect of α-tocopherol transfer protein gene disruption on Trypanosoma congolense infection in mice

At present 15 to 20 million people are estimated to be infected with pathogenic trypanosome parasites worldwide, mainly in developing countries. There are a number of factors that affect the severity of trypanosomiasis, including the nutritional status of the host. However, the relationship between micronutrient levels and trypanosomiasis outcome has yet to be reported in detail. Here, we demonstrate that the inhibition of α-tocopherol transfer protein, a determinant of the vitamin E concentration in host circulation, confers resistance to Trypanosoma congolense infection, evidently owing to oxidative damage to parasite DNA. These results suggest that transient inhibition of α-tocopherol transfer gene activity could possibly be exploited as a strategy for both the prevention and the treatment of trypanosomiasis.     

β-Carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources

Variations in the β-carotene, vitamin C and vitamin E content of D. tertiolecta have been shown to result from the nitrogen source used in the culture medium. Differences of 101%, 38% and 69% have been found in β-carotene, ascorbic acid and tocopherol content in mg/g of dry matter, respectively, and differences of 147%, 63% and 37% occurred in β-carotene, vitamin C and E concentrations in mg/litre of culture, respectively. Considering the β-carotene, vitamin C and vitamin E content in mg/g of chlorophyll a, maximum variations occurred in β-carotene content, with differences of 145% among the different nitrogen sources. Maximum β-carotene and vitamin C values were found in urea cultures, whereas urea cultures showed the minimum values for vitamin E.     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Effects of triple antioxidant combination (vitamin E, vitamin C and alpha-lipoic acid) with insulin on lipid and cholesterol levels and fatty acid composition of brain tissue in experimental diabetic and non-diabetic rats

The aim of this research was to examine the effects of a triple antioxidant combination (vitamins E (VE) and C (VC) plus alpha-lipoic acid (LA)) on the total lipid and cholesterol levels and the fatty acid composition of brain tissues in experimental diabetic and non-diabetic rats. VE and LA were injected intraperitoneally (50 mg/kg) four times per week and VC was provided as a supplement dissolved in the drinking water (50 mg/kg). In addition, rats in the diabetes 1 and D+VELAVC groups were given daily by subcutaneous insulin injections (8 IU/kg), but no insulin was given to rats in the diabetes 2 group. The results indicate that the brain lipid levels in the D+VELAVC, diabetes 1 and diabetes 2 groups were higher than in the control group (P<0.01). Total lipid was also higher in the non-diabetic rats treated with LA and VC. Total cholesterol was higher in the diabetes 1 and diabetes 2 groups (P<0.05) than in controls. Cholesterol levels were similar in the D+VELAVC and LA groups but lower in the VC, VE and VELAVC groups of non-diabetic rats (P<0.05 and P<0.01). In respect of fatty acid composition, palmitic acid levels were lower in the diabetes 2 and non-diabetic VE groups than the control group (P<0.05), but higher in the non-diabetic LA group (P<0.05). Oleic acid (18:1 n-9) levels were lower in the diabetic and non-diabetic groups than the control group (P<0.01), but higher in the non-diabetic LA group. Arachidonic acid (20:4 n-6) levels were similar in the diabetes 1, D+VELAVC and control groups (P>0.05) but higher in the non-diabetic VE, VC, LA and VEVCLA groups (P<0.05) and lower in the diabetes 2 group (P<0.05). Docosahexaenoic acid (22:6 n-3) was elevated in the diabetes 2 and VEVCLA groups (P<0.01, P<0.05). In conclusion, the current study confirmed that treatment with a triple combination of VE, VC and LA protects the arachidonic acid level in the brains of diabetic and non-diabetic rats.     

Separative preparation of the four stereoisomers of β-methylphenylalanine with N-carbamoyl amino acid amidohydrolases

The selective preparation of the four stereoisomers of β-methylphenylalanine (Mphe) from mixtures of the four stereoisomers of N-carbamoyl-β-methylphenylalanine (NCMphe) with N-carbamoyl amino acid amidohydrolases (carbamoylases) was developed. -Carbamoylase specifically hydrolyzed threo- -NCMphe with a little side activity toward erythro- -NCMphe, thus threo- -Mphe was produced with high optical purity from a mixture of the four stereoisomers of NCMphe. -Carbamoylase specifically produced threo- -Mphe from a mixture of the four stereoisomers of NCMphe. The erythro- -Mphe was obtained from erythro- -NCMphe which was prepared through diastereomer resolution by separative crystallization of benzoyl Mphe with a little side activity of -carbamoylase toward erythro- -NCMphe and the remaining erythro- -NCMphe was chemically hydrolyzed to erythro- -Mphe.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Pro-oxidative effect of beta-carotene and the interaction with flavonoids on UVA-induced DNA strand breaks in mouse fibroblast C3H10T1/2 cells

It has been suggested that β-carotene itself is unstable under certain conditions and that a combination of antioxidants may prevent the pro-oxidative effects of β-carotene. Thus, the present study aimed to investigate the interaction of β-carotene with three flavonoids—naringin, rutin and quercetin—on DNA damage induced by ultraviolet A (UVA) in C3H10T1/2 cells, a mouse embryo fibroblast. The cells were preincubated with β-carotene and/or flavonoid for 1 h followed by UVA irradiation, and DNA damage was measured using comet assay. We showed that β-carotene at 20 μM enhanced DNA damage (by 35%; P<.05) induced by UVA (7.6 kJ/m²), whereas naringin, rutin and quercetin significantly decreased UVA-induced DNA damage. When each flavonoid was combined with β-carotene during preincubation, UVA-induced cellular DNA damage was significantly suppressed and the effects were in the order of naringin≥rutin>quercetin. The flavonoids decreased UVA-induced oxidation of preincorporated β-carotene in the same order. Using electron spin resonance spectroscopy, we showed that the ability of these flavonoids to quench singlet oxygen was consistent with protection against DNA damage and β-carotene oxidation. All three flavonoids had some absorption at the UVA range (320–380 nm), but the effects were opposite to those on DNA damage and β-carotene oxidation. Taken together, this cell culture study demonstrates an interaction between flavonoids and β-carotene in UVA-induced DNA damage, and the results suggest that a combination of β-carotene with naringin, rutin or quercetin may increase the safety of β-carotene.     

Modification of subunit interaction in membrane-bound acid β-glucosidase from Gaucher disease

The radiation inactivation method has been used to determine the molecular mass of membrane-bound acid β-glucosidase (EC 3.2.1.21) in situ, in normal human spleen and in that of two patients with type I Gaucher disease: the molecular mass in Gaucher spleen is about double (125 000 ± 8900) of that found in the normal spleen (67 000 ± 7700) which is compatible with the existence of subunit coupling in the muted acid β-glucosidase. From the results, we conclude that subunit interaction is altered in mutant acid β-glucosidase and that this may be due to a direct effect of the mutation.     

Kinetics of Parallel Electron Transfer from β-Carotene to Phenoxyl Radical and Adduct Formation Between Phenoxyl Radical and β-Carotene

Phenoxyl radicals generated by laser flash photolysis were found to react with β-carotene with concomitant β-carotene bleaching in two parallel reactions with similar rates: (i) formation of a β-carotene adduct with a (pseudo) first order rate constant of 1-1.5 ± 10⁴ s^-1 with absorption maximum around 800 nm, and (ii) formation of a β-carotene radical cation with a (pseudo) first order rate constant of 2-3 ± 10⁴ s^-1 with absorption maximum around 920 nm. Both β-carotene radicals decay on a similar time scale and have virtually disappeared after 100 ms, the β-carotene adduct by a second order process. Oxygen had no effect on β-carotene bleaching or radical formation and decay. The reduction of phenoxyl radicals by β-carotene may prove important for an understanding of how β-carotene acts as an antioxidant.     

Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal caco-2 cells

Oxidative stress has been linked to the development of various chronic diseases. Vegetables and fruits, which contain polyphenols, were shown to have protective effects. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenol abundant in tea, has been shown to have antioxidant activities in cell-free conditions and this study focused on the effect of cellular EGCG. Using an intestinal cell model to examine the oxidative stress induced by hydroxyl radicals, we report here that physiological concentrations (0.1-1 microM) of EGCG have dose- and incubation duration-dependent cell-associated lipid antioxidant activity (measuring malondialdehyde production). Vitamin E and vitamin C at 10-40 microM also showed cell-associated lipid antioxidant activities under shorter incubation durations. When EGCG was included in the incubation with vitamin E or C, more antioxidant activities were consistently observed than when vitamins were added alone. Catechin (widely present in fruits and vegetables) at 1 microM also significantly increased the antioxidant activity of vitamins E and C. Previous studies examining cell-associated activity of EGCG mainly focused on the 10-100 microM concentration range. Our results suggest that although the physiological level (0.1-1 microM) of dietary catechins is much lower than that of vitamins, they further contribute to the total antioxidant capacity even in the presence of vitamins.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Differential inhibition by α- and β-tocopherol of human erythroleukemia cell adhesion: role of integrins

The effect of α- and β-tocopherol on human erythroleukemia cell (HEL) adhesion induced by phorbol 12-myristate 13-acetate (PMA) has been studied. Adhesion induced by PMA stimulation was prevented by 44.5% by physiological concentrations of α-tocopherol. Under the same experimental conditions, β-tocopherol, an analogue of α-tocopherol, produced 11% inhibition of adhesion. Cell response gradually increased from 0 to 24 h of α-tocopherol treatment. Only a slight time dependency of β-tocopherol inhibition was observed. Another human erythroleukemia cell line (K562) and the human monocyte tumor cell line U937 showed 5.0 and 11.2% inhibition, respectively. Similar to α-tocopherol, the protein kinase C inhibitor, Calphostin C, and the MAPK inhibitor, PD98059, prevented PMA-induced cell adhesion. An inhibition of ERK-1 phosphorylation was observed for α-tocopherol only in HEL, implying that MAP kinase pathway is involved in this cell line. Fluorescence-activated cell sorting (FACS), by using various integrin-specific monoclonal antibodies, has shown that α (1–6), β1, and αv integrins are less expressed at the cell surface after α-tocopherol treatment. Beta-tocopherol treatment was less effective.