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.     

CAROTENOID DOSE LEVEL and PROTECTION AGAINST UV-B INDUCED SKIN TUMORS

Abstract— Two groups of workers have reported that low levels of carotenoids (0.07 and 0.01%) were effective in preventing tumor development in experimental animals. To see whether these low concentrations were effective in preventing the development of UV-B induced skin tumors, groups of hairless mice were fed beta-carotene beadlets or canthaxanthin beadlets or the equivalent weight of placebo beadlets at these two final concentrations of carotenoid, and also at a final concentration of 2%, the amount found effective by us in past experiments. The mice received their pigments either for 4 days (the group receiving 0.01%), one month (the group receiving 0.07%) or for 12 weeks (the group receiving 2%) before irradiation started, in order to duplicate the conditions used in the previously reported experiments, and continued to receive their respective diets during the irradiation and observation period. All groups received a total dose of 10.8 J/cm² of UV-B radiation. At 2 and 0.07%, beta-carotene and canthaxanthin each conferred significant protection against skin tumor development. However, at 0.01 %, neither carotenoid offered protection against tumor development.     

INFLUENCE OF PHOTON FLUX DENSITY IN THE 400–700 nm WAVEBAND ON INHIBITION OF PHOTOSYNTHESIS BY UV-B (280–320 nm) IRRADIATION IN SOYBEAN LEAVES: SEPARATION OF INDIRECT AND IMMEDIATE EFFECTS

Abstract— Visible radiation can substantially influence the degree to which plant photosynthesis is inhibited by UV-B radiation. This study was designed to separate the immediate effects of visible radiation on UV-B photosynthetic inhibition from the indirect influence of visible irradiation on morphological and physiological properties of leaves during leaf development. Soybean plants were pretreated in growth chambers with either high or low visible irradiance (750 and 70 μmol m^-2s^-1 quantum flux in the 400–700 nm waveband, respectively) during the development of leaves used subsequently for UV irradiation. Test leaves still attached to the plant were exposed to 5 h of polychromatic UV-B irradiation and the photosynthetic capacity (net CO₂ exchange) was determined before and after the UV irradiation. During the UV irradiation, plants from both pretreatment groups received either high or low visible flux. Development of leaves in the high visible flux pretreatment conditions resulted in thicker leaves, higher chlorophyll a/b ratios, more UV-absorbing pigments, and reduced sensitivity to the UV-B irradiation. However, higher visible flux during the UV-B irradiation resulted in greater depression of photosynthesis by the UV-B irradiation. The relative magnitude of photosynthetic depression under these treatment combinations was the same when photosynthesis was measured under either light-limited or light-saturated conditions.     

UNDERSTANDING PHOTOSYNTHESIS, PIGMENT AND GROWTH RESPONSES INDUCED BY UV-B AND UV-A IRRADIANCES

Abstract—Plant response to UV-B (0.290–0.320 μm) irradiation in controlled environments has been difficult to assess, possibly because plants also respond to UV-A (0.320–0.400 μm) and visible radiation. Photosynthetic dysfunction is often reported, but effects on photosynthetic pigments have been equivocal. Because UV-A/blue radiation is involved in pigment synthesis, the experimental UV-A irradiation was controlled and this study was conducted under high ambient photosynthetic photon flux (mid-day PPF > 1400 pmol m ^–2 s^–1). Two biologically effective UV-B irradiances (10.7 and 14.1 kJ m^-2 day^-I) were utilized and the UV-A irradiances were matched in controls (˜5 and 9 kJ m^-2 day^-1). Normal and two mutant pigment isolines (chlorophyll-deficient, flavonoid-deficient) of soybean cultivar Clark were utilized for comparisons. Many pigmedgrowth variables exhibited a statistical interaction between spectral quality and quantity. UV-A/blue photoregulation was demonstrated in the UV-A controls. The pigmentlgrowth pattern observed at the lower UV-B irradiance was interpreted as a photosystem II response similar to shade adaptation, suggesting phytochrome involvement in UV-B irradiation responses. On the other hand, two variables most commonly observed to manifest UV-B-induced effects—decreased photosynthesis and increased leaf flavonoid content—exhibited no interactions due to UV exposure or spectral quality. In general, the observed response patterns indicated either moderation of UV-B-induced responses by UV-A/blue radiation, or coaction between them, and provides an explanation for the common failure to demonstrate fluence-related responses in UV-B experiments.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis

Butylated hydroxytoluene (BHT) and certain carotenoid pigments have been found to inhibit photocarcinogenesis in animal models. In addition, BHT protects against UV-B-induced erythema and UV-B induction of ornithine decarboxylase. Studies on the photoprotective mechanism(s) of BHT suggested that changes in the physico-chemical properties of the keratin of the stratum corneum layer of skin occurred, leading to increases in UV absorption of that tissue. These changes might be exerted via the anti-radical action of BHT that retards oxidation and prevents cross-linking of the keratin chains, resulting in a diminution of UV-B radiation reaching potential target sites. The carotenoids beta-carotene, canthaxanthin and phytoene also inhibit UV-B carcinogenesis. beta-Carotene and canthaxanthin are excellent quenchers of singlet oxygen, and all three pigments can quench free radicals. beta-Carotene and canthaxanthin have been shown to quench singlet oxygen/free radical reactions in the skin of porphyric mice, and these two pigments as well as phytoene have been found to quench excited species formed on irradiation of mouse skin by UV-B.     

IMPACT OF UV-B RADIATION UPON ESTUARINE MICROCOSMS

Abstract— Twelve flow-through estuarine microcosms were exposed daily to four different levels of UV-B radiation (290–320. nm)(1.57 ± 10², 6.43 ± 10³, 6.86 ± 10³ and 7.61 ± 10³ J·m_-2d⁻¹) in addition to a natural level of visible solar radiation (380-800. nm). The parameters studied over a four week period were phytoplankton community composition, plankton biomass (ash-free dry weight), chlorophyll a concentration and primary productivity (radiocarbon uptake). With increased exposure to UV-B radiation there was an obvious alteration of the community composition. Daily exposure to enhanced levels of UV-B radiation also depressed the biomass, the chlorophyll a concentration and the radiocarbon uptake of samples from the ecosystems.     

ETUDE PAR SPECTROSCOPIE D''ECLAIRS DU TRANSFERT D''ENERGIE CHLOROPHYLLE-CAROTENOIDE

Abstract— Energy transfer from chlorophyll A in its lowest triplet state to carotenoid pigments is demonstrated by rapid flash photolysis experiments.
Two systems are used; the first consists of chlorophyll A and p carotene in organic solvents: in diluted solutions, energy transfer is diffusion controlled. The second consists of chlorophyll A and lutein incorporated into digitonin micelles suspended in water; with this system a very rapid energy transfer is observed (< 0.4 × 10^--⁶ sec).
Energy transfer results in a carotenoid metastable state, which is supposed to be a triplet state; for lutein its half-life is 8·9 × 10^--⁶ sec, and it has an absorption peak at 518 nm. Depopulation of lutein ground state, around 450 nm, can be observed, as well as the reactivity of oxygen towards the metastable state.
Most of these results were obtained with a Q -switch ruby laser as exciting source (6943 Å). A 4350 Å flash can also be obtained by two successive non linear effects. Using this flash for exciting chlorophyll A alone, a strong signal is detected, due to its triplet state. By exciting directly B carotene or lutein, it is not possible to detect any metastable state with our technique.     

UV-B RADIATION EFFECTS ON PHOTOSYNTHESIS, GROWTH and CANNABINOID PRODUCTION OF TWO Cannabis sativa CHEMOTYPES

The effects of UV-B radiation on photosynthesis, growth and cannabinoid production of two greenhouse-grown C. sativa chemotypes (drug and fiber) were assessed. Terminal meristems of vegetative and reproductive tissues were irradiated for 40 days at a daily dose of 0, 6.7 or 13.4 kJ m^-2 biologically effective UV-B radiation. Infrared gas analysis was used to measure the physiological response of mature leaves, whereas gas-liquid chromatography was used to determine the concentration of cannabinoids in leaf and floral tissue.
There were no significant physiological or morphological differences among UV-B treatments in either drug- or fiber-type plants. The concentration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), but not of other cannabinoids, in both leaf and floral tissues increased with UV-B dose in drug-type plants. None of the cannabinoids in fiber-type plants were affected by UV-B radiation.
The increased levels of Δ⁹-THC in leaves after irradiation may account for the physiological and morphological tolerance to UV-B radiation in the drug-type plants. However, fiber plants showed no comparable change in the level of cannabidiol (a cannabinoid with UV-B absorptive characteristics similar to Δ⁹ THC). Thus the contribution of cannabinoids as selective UV-B filters in C. sativa is equivocal.     

PHOTOREACTIVATION OF Halobacterium halobium: ACTION SPECTRUM AND ROLE OF PIGMENTATION

Abstract— An action spectrum for photoreactivation was measured with Halobacterium halobium R₁m₁ to prove a role of carotenoid pigments in photoreactivation of the bacteria. The action spectrum obtained showed a main peak at 435 nm and a minor peak at about 325 nm. The action spectrum was similar to that of Streptomyces pigment (Eker et al. , 1981) suggesting that the chromophore of the photoreactivating enzyme in Halobacterium halobium is 8-OH-5-deazaflavin. The minor peak may be due to photochemical cleavage of a pyrimidine6–4 hetero adduct. The result indicates that carotenoid pigments do not play a positive role in enhancing photoreactivation. This was confirmed also by comparing the efficiency of photoreactivation at 465 nm among three strains of Halobacterium halobium having different carotenoid pigments; R₁m₁. R₁ and W5002–1.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

EFFECTS OF UV IRRADIATION ON A LIVING SKIN EQUIVALENT

Abstract— The Living Skin Equivalent (LSE™) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280–400 nm) or UV-A (320–400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R₅₀ or UV-A₅₀) in different lots of LSE were 0.053 ± 0.021 J/cm² (n = 29) and 11.6 ± 4.9 J/cm² (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-α, tumor necrosis factor-α and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of U V radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.     

Comparison of changes in metal toxicity following exposure of water with high dissolved organic carbon content to solar, UV-B and UV-A radiation

This study examines the effects of natural solar radiation on the metal-binding capacity of dissolved organic matter (DOM). Newington Bog water (35.5 mg L⁻¹ dissolved organic carbon [DOC]) was irradiated for 20 days under UV-B lamps in the laboratory and under natural solar radiation. In the presence of irradiated DOM, IC₅₀ (contaminant concentration required to reduce algal growth by 50%) was significantly decreased with UV-B treatment for four metals: Pb, 64%; Cu, 63%; Ni, 35% and Cd, 40%. Solar radiation also significantly decreased IC₅₀ of Pb (58%) and Cu (49%), DOC concentration (11%), DOM fluorescence (DOMFL, 33%) and DOC-specific UV absorbance. Further experiments on Raisin River water (20.7 mg DOC L⁻¹) exposed to 20 days of artificial UVA and UV-B radiation produced significant decreases in IC₅₀ for Cu (48%) with UV-A and for Pb (43%) with UV-B. DOC concentration was decreased 20% by UV-B and 24% by UV-A. DOMFL decreased 51.5% in the first 5 days of UV-A exposure, an effect that was not observed with the UV-B treatment. The UV-A treatment decreased UV absorbance more at longer wavelengths and over a broader wavelength band than did the UV-B treatment. Change in toxicity with UV irradiation was inconsistent among the metals tested in this study, indicating that some organic metal-binding ligands were more quickly removed or altered than others. The DOM remaining after irradiation appears to be qualitatively different from the unirradiated DOM. The much greater irradiance of UV-A makes its contribution to the removal and/or alteration of DOM at least as important as the influence of higher energy UV-B.     

SKIN DAMAGE IN ADULT AMPHIBIANS AFTER CHRONIC EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The effects of repeated UV exposure on the skin of the European crested newt, Triturus cristatus carnifex , have been investigated. The animals were irradiated 3 times per week with a Westing-house FS40T12 fluorescent sun lamp (wavelength spectrum 275–350 nm). Two groups of animals received the same total fluence of 1.3 × 10⁵ J/m² in single fluences of either 1570 J/m² (group A) or 9430 J/m² (group C), and one group received a total fluence of 2.6 × 10⁵ J/m² in single fluences of 4710 J/m² (group B). All the animals were killed 7 months after the first UV exposure, but at different intervals after the last exposure. Striking epidermal hyperplasia was found in the newts irradiated at the lower fluence rate (group A). In the animals given the higher total fluence (group B), the most prominent skin changes were dermal fibrosis and irregular thinning and thickening of the epidermis. No significant skin changes were found in group C., in which if there had been UV lesions, they had been repaired during the 5 month interval between the last irradiation and the killing of the animals. No skin tumors developed in any experimental group.     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

EFFECTS OF UV-B IRRADIATION ON SOROCARP DEVELOPMENT OF Dictyostelium discoideum

Abstract— The effect of UV-B irradiation on the development of Dictyostelium discoideum from amoebae to mature sorocarps was studied. Radiation with wavelength ≤ 310 nm was very efficient in retarding and inhibiting the development especially when the organisms were exposed during the first 12 h. At a wavelength of 280 nm an irradiation of 1 h at an irradiance of < 0.2 W m^-2 was sufficient to completely inhibit sorocarp development. The fluence-dependence shows as well that the development of D. discoideum is a very sensitive system to indicate UV-B irradiation. Furthermore, since the sorocarp development is concluded within 48 h it can serve as a fast bioassay for hazardous levels of increased UV-B irradiation which have been predicted as a result of the ozone reduction in the stratosphere due to the manmade production and emission of chlorofluoromethanes.     

Distribution of Chlorophyll- and Bacteriochlorophyll-derived Photosensitizers in Human Blood Plasma

Chlorophyll a and, in particular, bacteriochlorophyll a derivatives are promising candidates for photosensitizers in photodynamic therapy. The distribution of 21 (bacterio)chlorophyll derivatives among human blood plasma fractions was studied by iodixanol gradient ultracentrifugation and in situ absorption spectroscopy. Modifications of the natural pigments involved the central metal (Mg²⁺, Zn²⁺, Pd²⁺, none), the isocyclic ring (closed, open and taurinated), substituents at C-3 (vinyl, acetyl, 1-hydroxyethyl) and C-17³ (phytyl ester, free acid). Cellular blood components bound only a small fraction of the pigments. Distribution among low-density lipoproteins (LDL), high-density lipoproteins (HDL) and high-density proteins (HDP) of the plasma was influenced as follows: (1) application in Cremophor^® EL slightly altered pigment distribution by lipoprotein modification, (2) only very polar pigments with multiple hydrophilic substituents showed substantial HDP binding, (3) the presence of the esterifying alcohol at C-17³ caused enrichment in LDL, this was more pronounced with bacteriochlorophylls than with chlorophylls, (4) substituents at C-3 had only little influence on the distribution, (5) Zn²⁺-complexes were enriched in HDL compared to Mg²⁺ and Pd²⁺ complexes, indicating specific binding of the former. Equilibration of pigments among the different fractions was largely complete within 3 h.     

Increased UV-B Radiation Affects the Viability, Reactive Oxygen Species Accumulation and Antioxidant Enzyme Activities in Maize (Zea mays L.) Pollen

The increase in UV-B radiation reaching the earth's surface has prompted extensive studies on the effects of UV-B on plants. However, most of these studies have not addressed the close characteristics related to future survival of plant populations. The purpose of this study was to investigate the effects of UV-B radiation on reactive oxygen species (ROS) accumulation and antioxidant defense system in relation to germination, tube length and viability of maize pollen. Our results indicate that increased UV-B radiation decreased the pollen germination rate and tube length in vitro and also its fertilization ability in the field. Production of O₂^•− and H₂O₂ increased by UV-B radiation treatment, and their continuous accumulation resulted in lipid peroxidization. The activities of superoxide dismutase, catalase, peroxidase and DPPH-radical scavenging were decreased by increased UV-B radiation. The increased ROS and lipid peroxidization, and decreased activities of the antioxidants may be attributed to the effects of UV-B radiation on pollen germination, tube growth and fertilization ability.     

PROTECTION OF CHLOROPHYLL a BY CAROTENOID FROM PHOTODYNAMIC DECOMPOSITION

Abstract— The order of inhibition of the photooxidation of chlorophyll a in ethanol and ethanol-benzene is as follows: β-carotene, α-tocopherol, benzoquinone, DABCO, menadione, cholesterol and KI. The quenching of singlet oxygen by β-carotene occurs by a collisional quenching mechanism with a diffusion-controlled rate of 1.7 × 10¹⁰ M ^-1 s^-1. Photodecomposition of Chi a is faster in ethanol-D₂O than in ethanol-H₂O. Photoirradiation (660 nm) of the peridinin-Chl a -protein complex, a photosynthetic light-harvesting pigment isolated from marine dinoflagellates, did not show any photo-decomposition of its Chi a in H₂O or D₂O, even after an extended period (12 h) of irradiation. However, the carotenoid, peridinin, in the photosynthetic antenna pigment was photobleached (ca. 10%) during the irradiation. We conclude that the singlet oxygen formed as a result of the Chi photosensitization is immediately quenched by the low-lying triplet state of four peridinin molecules (per Chl a ) bound within the same protein crevice. The carotenoid thus effectively protects Chl a from photodynamic damage, providing a direct proof for the protective role of carotenoids in the photosynthetic pigment complex.