CAROTENOID PIGMENT ADMINISTRATION and DELAY IN DEVELOPMENT OF UV-B-INDUCED TUMORS

—The results of two studies are reported. In the first study, the carotenoid pigments, β-carotene. canthaxanthin and phytoene were administered to mice after one UV-B-induced skin tumor had developed, to see if carotenoid administration would delay the development of subsequent tumors. Canthaxanthin significantly delayed the development of subsequent tumors. In the second study, the same pigments were administered starting 10 weeks before, and for 24 weeks after, exposure to a large single fluence (8 × 10⁴J/m²) of UV-B radiation, to determine the effect of the pigments on tumor development. Phytoene significantly prevented the development of tumors to this fluence of radiation.     

PHYTOENE AS A PROTECTIVE AGENT AGAINST SUNBURN (>280nm) RADIATION IN GUINEA PIGS

Abstract— Phytoene, the colorless triene precursor of β-carotene (17 mg/g body weight) or a placebo was given to guinea pigs by daily intraperitoneal injection for 14 days, after which the animals were exposed to radiation (> 280 nm). It was found that the animals receiving phytoene developed significantly less erythema to the radiation than did the animals receiving placebo.     

CAROTENOIDS QUENCH EVOLUTION OF EXCITED SPECIES IN EPIDERMIS EXPOSED TO UV-B (290-320 nm) LIGHT

Abstract— Reactions involving singlet oxygen and other free radicals have been identified in epidermis containing either exogenous or endogenous photosensitizers. soaked in a singlet oxygen/free radical trap, and then exposed to visible or UV-A (320-400 nm) light. Such reactions can be quenched by the presence of the carotenoid pigments β-carotene and canthaxanthin which accumulate in epidermis after oral administration. We report here that the carotenoid pigments β-carotene. canthaxanthin and phytoene accumulating in epidermis can also quench to some degree those photochemical reactions involving singlet oxygen and free radicals that occur when epidermis is exposed to the sunburn spectrum of light (UV-B. 290–320 nm).     

PHOTOELECTRON QUANTUM YIELD SPECTRUM AND PHOTOELECTRON MICROSCOPY OF β-CAROTENE

Abstract—The absolute photoelectron quantum yield spectrum for β-carotene in the wavelength range 180–230 nm is reported. β-Carotene is very photoemissive over this wavelength region with photoelectron quantum yields on the order of 2 × 10^--³ electrons per incident photon at 180 nm, 4 × 10^--⁴ at 210 nm, and 3 × 10^--⁵ at 230 nm. At wavelengths longer than 240 nm, β-carotene photoemission dropped off monotonically with increasing wavelength. The photoelectron quantum yield spectrum of β-carotene is very similar to that of chlorophyll a . A photoelectron micrograph of β-carotene deposited on a thin layer of the fatty acid arachidic acid demonstrates the marked photoemission contrast between β-carotene and membrane lipid. Photoelectron micrographs of samples with β-carotene and Chl a in the same field show that prolonged (1 h) illumination in vacuo causes β-carotene to markedly fade while leaving the Chl a photoemissive. This differential bleaching of β-carotene may allow it to be distinguished from Chl in high magnification photoelectron micrographs of photosynthetic membranes.     

ACTION SPECTRA FOR BIOSYNTHESIS OF CHLOROPHYLLS a AND b AND β-CAROTENE

Abstract— Action spectra for the formation of chlorophyll b and β-carotene were determined with etiolated wheat leaves and compared with the action spectrum for the formation of chlorophyll a determined for the same samples. The action spectra were measured with etiolated leaves which had been pre-illuminated for 10 min and incubated in the dark for 4 h to eliminate induction of pigments. The action spectra for chlorophyll b and for β-carotene accorded with the action spectrum for chlorophyll a and with the absorption spectrum of protochlorophyllide in intact etiolated leaves. It is postulated from this result with chlorophyll b that this pigment is formed from protochlorophyllide through chlorophyll a or some intermediates to chlorophyll a. Complexing between chlorophylls and β-carotene and proteins is postulated to interpret the action spectrum for β-carotene. It is assumed that the low concentration of chlorophylls formed photochemically limits the rate of complexing, and that consumption of β-carotene for the complexing induces formation of new β-carotene.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES—II. THE TRIPLET STATES OF β-CAROTENE AND LYCOPENE EXCITED BY PULSE RADIOLYSIS

Abstract— –Pulse radiolysis has been used to excite the triplet states of β-carotene (τ# 9μ sec) and lycopene (τ= 8μsec) in hexane solution, both in the presence and absence of naphthalene as a triplet sensitiser. The absorption spectra of both triplets have been measured in the range 430–550 nm and have thus been extended into the region of the corresponding singlet absorptions. The overlap of the triplet and singlet spectra is discussed in relation to in vivo studies. Extinction coefficients of 1.3±0.1 × 10⁵ l/mole cm for β-carotene triplet 515 nm and 3.9±0.2 × 10⁵ l/mole cm for lycopene triplet at 525 nm were obtained. Isomerisation of the all- trans polyenes used was detected and preliminary measurements indicate that the yield of isomerisation was greater than the triplet yield. The rate of triplet energy transfer from naphthalene to β-carotene was estimated to be 1.5 × 10¹⁰ l/mole sec. The corresponding value for lycopene was 1.4× 10¹⁰ l/mole sec. The measured efficient quenching of triplet β-carotene by oxygen may occur by an energy transfer mechanism, leading to the formation of singlet oxygen (¹Δ_g. This would suggest that the triplet energy level of β-carotene lies between 121 and 94 kJ mole^-1.     

DIETARY FAT MODULATES IMMUNORESPONSIVENESS IN UV-IRRADIATED MICE

Previous studies have shown that a high level of dietary lipid (corn oil) exacerbates UV-carcinogenic expression in hairless mice. Furthermore, it was demonstrated that this effect occurs at the postinitiation, or promotion, stage of UV-carcinogenesis-a stage believed to be modulated immunologically. Thus, we sought to examine the influence of dietary lipid on specific immune parameters at various times within a UV-carcinogenic protocol, with the purpose of detecting potential relationships to UV carcinogenesis. Hairless mice were fed either a high- (12%, wt/wt, corn oil) or low-fat (0.75%, wt/wt, corn oil) diet for 2 weeks prior to start of the UV or experimental protocols. Animals were sensitized to dinitrochlorobenzene (DNCB) hapten and delayed-type hypersensitivity (DTH) was assessed. Delayed-type hypersensitivity was significantly suppressed (P= 0.01) in the high-fat group, even before UV irradiation. Although both groups exhibited UV-induced suppression of this response, the high-fat group was totally suppressed after 3 weeks of UV, whereas the low-fat group exhibited reactivity through week 8. The splenic T-lymphocyte (Thy 1.2⁺) population had declined by about 50% at the time of UV termination (11 weeks). Dietary lipid exerted no apparent influence upon this T-cell population. However, after 6 weeks of UV, I-J⁺ cells (a marker shown to be acquired adaptively by suppressor T lymphocytes) began to increase. By week 15 (4 weeks post-UV) I-J⁺ cells had increased by about 65% in the high-fat group, twice the % increase that occurred in the low-fat group. When UV-induced tumors were transplanted to recipient animals receiving various periods (0, 6, 11 weeks) of UV irradiation, no significant differences in median tumor rejection times between the two dietary groups occurred at 0 or 6 weeks. After 11 weeks of UV, the low-fat group exhibited a tumor rejection time that was comparable to that of nonirradiated animals, i.e. 21 days. However, median tumor rejection time for the high-fat group was greater than 63 days, significantly (P= 0.01) longer than that of the low-fat group. Thus, suppression of tumor rejection by high fat occurred at a time when high fat had been shown to exacerbate carcinogenic expression and when I-J⁺ cells had markedly increased. These data demonstrate that level of dietary lipid modulates immunoresponsiveness in UV-irradiated animals and is compatible with the thesis that immune suppression may account for the exacerbation of carcinogenic expression elicited by high dietary fat.     

PHOTOTROPISM IN PHYCOMYCES MUTANTS LACKING β-CAROTENE

Abstract. β-carotene and riboflavin are considered as the major candidates for the photoreceptor for physiological responses to blue light in Phycomyces and a number of other organisms. Mutants of Phycomyces blocked in all six steps of the biosynthesis of β-carotene from phytoene contain no detectable β-carotene (less than 4 times 10^-5 of wild-type amount) but exhibit phototropic responses identical to wild-type. Moreover, wild-type Phycomyces , while abundant in trans -β-carotene, contains no detectable cis -β-carotene, sometimes proposed as a photoreceptor candidate on the basis of the close similarity of the cis -species absorption spectrum in the near UV region to many action spectra for blue-light responses. These results indicate that β-carotene cannot be the photoreceptor for phototropism in Phycomyces.     

UPTAKE AND BIOCONVERSION OF α-TOCOPHERYL ACETATE TO α-TOCOPHEROL IN SKIN OF HAIRLESS MICE

Abstract— The photoprotective effect of topically applied α-tocopheryl acetate (vitamin E acetate), a stable derivative of α-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (α-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied α-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of α-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical α-tocopheryl acetate treatments. Finally, free α-tocopherol levels in skin also were significantly increased (P < 0.00 1) by topical applications of α-tocopheryl acetate and skin levels of free α-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments than in non-UV-Birradiated animals. These results suggest that UV-B irradiation enhances both the absorption of α-tocopheryl acetate and its bioconversion to free α-tocopherol.     

Lipid lowering effects of high linoleate and high alpha-linolenate diets in rats and mice. Consequence of long-term feedings.

Diets high in linoleate (safflower oil) or high in alpha-linolenate (perilla oil) were fed to rats for 11 months, and the effects of the diets on plasma and tissue lipids were compared. The plasma levels of total cholesterol (Cho), phospholipids (PL) and triacylglycerol (TG) were significantly lower in the high alpha-linolenate group than in the high linoleate group, the differences being more than 30% in the levels of total Cho and TG. The diets had differential effects on the lipid contents of major tissues: the TG level in muscle was higher but both the TG level in depot fat and the PL level in muscle were lower in the high alpha-linolenate group than in the high linoleate group. In order to clarify whether or not the hypolipidemic effect of the high alpha-linolenate diet was due to changes in the distribution of lipids among tissues, whole body lipids were estimated in mice fed these diets for 5 months. The whole body Cho content was significantly lower, by 28%, in the high alpha-linolenate group compared with the high linoleate group, but the total lipid content, PL and neutral lipids were similar between the groups. Our results indicate that the high alpha-linolenate diet has a more potent cholesterol lowering effect in plasma and body tissue than the high linoleate diet; interestingly, whole body TG levels are similar but tissue distributions of TG are different between the two dietary groups.     

PHOTOSENSITIZED BLEACHING OF β-CAROTENE WITH LIGHT AT 632·8 nm FROM A CONTINUOUS-WAVE GAS LASER

Abstract— β-Carotene was not affected when irradiated with a monochromatic beam of light (632·8 nm) from a continuous-wave gas laser. However, β -carotene undenvent numerous changes when irradiated in the presence of the photosensitizing dye toluidine blue. Initially there was a considerable decrease in the absorbance of the β-carotene, accompanied by small shifts of the absorption maxima to shorter wavelengths as well as the formation of two new peaks at 400 and 375 nm. Ultimately a complete bleaching of the solution was observed. By the use of column and thin layer chromatography up to 13 different compounds including cis-isomers, epoxides and possibly hydroxy compounds were shown to be formed by the photosensitization of the β-carotene. These reactions were found to be oxygen-dependent.     

重复性"饥饿/再投喂"对大鼠脂代谢及主动脉影响的研究

为探讨经过重复性"饥饿/再投喂"方式处理后大鼠摄取高脂食物时有关体质量控制与能量平衡调节的瘦素(leptin)和神经肽Y(NPY)的表达变化,设计"重复性饥饿1 d/再投喂基础饲料1 d,持续6周"的饲喂方式处理动物模型,6周后改为每天喂以高脂饲料(高脂模型组),另设高脂对照组和基础对照组,喂养至12周,测定了血脂水平...     

INTERACTION OF HUMAN SERUM LOW DENSITY LIPOPROTEINS WITH PORPHYRINS: A SPECTROSCOPIC AND PHOTOCHEMICAL STUDY

The incorporation of proto-, uro- and hematoporphyrin in low density lipoproteins (LDL) of human blood has been studied by equilibrium dialysis, fluorescence and absorption spectroscopy. The lipoproteins may efficiently compete with albumin in the binding of protoporphyrin to human blood proteins in patients suffering from protoporphyria. It can be concluded that hydrophobic porphyrins bind to blood proteins.
The complexation of hydrophobic porphyrins in LDL is responsible not only for efficient photodynamic effect at the lipoprotein level, but also for photoinduced lipid peroxidation and for consumption of β-carotene incorporated into LDL which are one of their natural carriers. The water-soluble uroporphyrin, although an efficient photosensitizer for the LDL apoprotein photoinactivation, is much less efficient for lipid peroxidation and β-carotene bleaching. The 353 nm laser flash photolysis shows that porphyrin triplet states are not affected by the physiological β-carotene content of LDL but are fully accessible to oxygen.     

Production of β-carotene from beet molasses by Blakeslea trispora in stirred-tank and bubble column reactors

The effect of aeration rate and agitation speed on β-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentor and optimization of the production of the pigment in a bubble column reactor were investigated. In addition, a central composite design was employed to determine the maximum β-carotene concentration at optimum values for the process variables (aeration rate, sugar concentration, linoleic acid, kerosene). By image analysis of the morphology of the fungus, a quantitative characterization of the hyphae and zygospores formed was obtained. The hyphae were differentiated to intacthyphae, vacuolated hyphae, evacuated cells and degenerated hyphae. An increased proportion of zygospores was correlated to high β-carotene production. In the stirred-tank fermentor, the highest concentration of the carotenoid pigment (92.0 mg/L) was obtained at an aeration rate of 1.5 vvm and agitation speed of 60 rpm. In the bubble column reactor, the aeration rate and concentration of sugars, linoleic acid, kerosene, and antioxidant significantly affected the production of β-carotene. In all cases, the fit of the model was found to be good. Aeration rate, sugar concentration, linoleic acid, and kerosene had a strong positive linear effect on β-carotene concentration. Moreover, the concentration of the pigment was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. Maximum β-carotene concentration (360.2 mg/L) was obtained in culture grown in molasses solution containing 5% (w/v) sugar supplemented with linoleic acid (37.59 g/L), kerosene (39.11 g/L), and antioxidant (1.0 g/L).     

THE PHOTOSENSITIZED OXIDATION OF β-CAROTENE

Abstract— –Products of oxidation of β-carotene, photosensitized by hypericin in acetone, appear to include mutatochrome, aurochrome, and a number of other compounds absorbing in the violet and near u.v. regions of the spectrum. The 5, 6-monoepoxide is not formed in appreciable quantity, though it is the principal product of oxidation of carotene by perphthalate. Mutatochrome, lutein, and zeaxanthin are photooxidized more slowly than β-carotene, and the products do not appear to include 5, 6-epoxides. β-carotene-5, 6-monoepoxide is oxidized to what is probably luteochrome. Quantum yields appear to be low. We found no evidence to support a photochemical contribution to the oxidation of zeaxanthin to violaxanthin, a process known to accompany photosynthesis.     

INFLUENCE OF DIETARY OMEGA-6, -3 FATTY ACID SOURCES ON THE INITIATION AND PROMOTION STAGES OF PHOTOCARCINOGENESIS

To determine the segment along the carcinogenic continuum at which dietary lipid exerts its principal effect, six groups of 35 Skh-HR-1 hairless mice were placed on defined isocaloric diets containing either 0.75%, 12% corn oil or 12% menhaden oil as sources of omega-6 or omega-3 fatty acids, respectively. All animals received an 11 week course of UV-radiation from fluorescent sunlamps. Upon termination of UV, diets of some groups were crossed-over to either low fat, high fat, omega-6 or omega-3 fatty acid sources. The first tumor appeared at week 14. Life-table analysis of the tumor incidence curves and Wilcoxon tests of tumor multiplicity provided evidence that high corn oil diets significantly (P < 0.01) enhance carcinogenic expression; that tumor enhancement by the omega-6 fatty acid source occurs during the post-initiation, or promotion, stage; that replacement with a low corn oil diet after UV-initiation will negate the exacerbating effect of high corn oil; and that an omega-3 fatty acid source inhibits UV-carcinogenesis even at high dietary levels, although not during the post-initiation stage.     

TARGETED MUTAGENESIS OF M13 DNA INDUCED BY PHOTOSENSITIZED REACTION

Abstract— Photoacoustic spectroscopy has been used to observe a qualitative depth profile in vitro in mouse skin. Because the photoacoustic response is a vector quantity, it can be used to qualitatively determine the depth of a chromophore in skin. The spectrum of an induced layer of β-carotene in albino mouse skin was separated from the spectrum of hemoglobin. Dyes were then used to determine more precisely the position of the β-carotene in the epidermis. The spectrum of melanin was also separated from that of the underlying hemoglobin in pigmented mouse skin.     

β-Carotene removal from soybean oil with smectite clay using central composite design

In this study removal of β-carotene from soybean oil by adsorption on acid activated smectite clay from Serbia was investigated and a factorial 2³ experimental design was applied. The effects of relevant factors, such as temperature, solid-to-liquid ratio and time, on removal of β-carotene were investigated. In order to check these factors and their effect on the removal of β-carotene, we have established a model of this technique following a methodological strategy using experiments design. The mathematical model is established using a central composite design. The model describes the changes of the measured responses of β-carotene removal efficiency according to the temperature, solid-to-liquid ratio and time. The graphical representation of this model in the space of the variables enabled us to define the optimum conditions of these parameters. The optimum conditions to obtain the maximum removal of β-carotene from soybean oil were a temperature of 80°C, a solid-to-liquid ratio of 1: 25 and a time of 1255 s. Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating a high goodness of fit of the model used and the success of response surface methodology for optimizing adsorption β-carotene of acid activated smectite clay from soybean oil. The article is published in the original.     

ULTRAVIOLET HYPERSENSITIVITY OF COCKAYNE SYNDROME LYMPHOBLASTOID LINES—THE EFFECTS OF EXOGENOUS β-NICOTINAMIDE ADENINE DINUCLEOTIDE

Four Cockayne Syndrome (CS) lymphoblastoid lines were tested for the lethal effects of UV radiation (254 nm) with or without addition of exogenous β-nicotinamide adenine dinucleotide (β-NAD ⁺) to their culture medium. Two of them exhibited a small but significantly increased resistance to UV radiation when β-NAD⁺ was added to the culture. However, their UV sensitivity after β-NAD ⁺ addition was still much greater than that of normal control lines. Normal control lymphoblastoid lines and those from complementation group A and group C of xeroderma pigmentosum (XP) did not reveal any differences in post-UV sensitivity after the addition of exogenous β-NAD⁺. Thus the abnormal response to the lethal effects of UV radiation of CS lymphoblastoid lines could not be rectified by β-NAD⁺ addition. However, β-NAD⁺ does appear to play some partial role in reducing the high UV sensitivity of some CS lymphoblastoid lines.     

The β-carotene dilemma: ESR study with coordinated peroxyl radicals

The apparently unpredictable behaviour of β-carotene in the supplementation of the diet of smokers is discussed in the light of the reactions of peroxyl radicals with β-carotene in the absence of oxygen. The decay of tert-butylperoxyl radicals in the presence of β-carotene was studied at ambient temperature in non-polar solvents by ESR spectroscopy. The primary reaction in the absence of oxygen is interpreted as a spin-trapping effect of a peroxyl radical by β-carotene producing an intermediate labile free radical, which disappears after recombination with a second tert-butylperoxyl radical. The result is the transformation of β-carotene to a diamagnetic compound with two peroxy bonds. In the presence of chelating transition metals with unpaired d-electrons as electron donors the peroxy group of the oxidized β-carotene can be split to alkoxyl free radicals. The primary attack of tert-butylperoxyl radicals is completely inhibited in the presence of vitamin E followed by production of free aryloxy radicals and the presence of oxygen has no significant effect on this reaction. Spin-trapping of peroxyl radicals by the double bond of vitamin A leads to its oxidation in the absence of vitamin E. Transition metal ions such as Co, Cr, Fe, and Mn, known to be present in the aerosol of cigarette smoke, homolyse the peroxyl bonds of peroxidised β-carotene, which results in cell damage.