Light-induced Oxidative Stress Research Articles

Light-Induced Oxidative Stress in Choroidal Endothelial Cells in Mice

Although light-induced oxidative stress in the retina has been extensively reported, little information regarding light-induced oxidative stress in choroidal endothelial cells (CECs) is available. In the current study, light-induced DNA oxidation and the activation of nuclear factor-kappaB (NF-kappaB), a major oxidative responsive transcription factor, were investigated in mouse CECs. Mice were exposed to green light. Light-induced DNA oxidation in CECs was detected by in situ 8-hydroxy-2-deoxyguanosine (8-oxo-dG) immunolabeling. CECs were isolated from the retinal pigment epithelium (RPE)/choroid by using immunomagnetic beads. The isolated CECs were immunochemically characterized by the expression of endothelial markers, CD31, and P1H12. The quality of total RNA from CECs was assessed by a bioanalyzer and RT-PCR. NF-kappaB activation in situ and in isolated CECs was investigated. After a 3-hour exposure to light, the immunoreactivity to anti-8-oxo-dG antibody or anti-NF-kappaB p65 antibody in CECs in situ was significantly increased when compared with unexposed mice. Isolated CECs expressed CD31 and P1H12. The 28S/18S rRNA ratio of RNA isolated from CECs was 1.5:1. CD31 and von Willebrand Factor (vWF) transcripts were predominantly expressed in the RNA from isolated CECs. IkappaBalpha was more heavily phosphorylated in light-exposed than untreated CECs. IkappaBalpha expression levels were increased fivefold in isolated CECs after exposure to light compared to unexposed control subjects. Exposure to light induces oxidative stress in CECs in vivo. A method for CEC isolation from the mouse RPE/choroid with preservation of RNA quality has been developed. The results of this study may facilitate the ability to identify CEC-specific genes and gene products that respond to photo-oxidative stress.

A Proposed Mechanism for the Inhibitory Effects of Oxidative Stress on Rubisco Assembly and Its Subunit Expression

In Chlamydomonas reinhardtii, a light-induced oxidative stress shifts the glutathione pool toward its oxidized form, resulting in a translational arrest of the large subunit (LSU) of Rubisco. We show here that the translational arrest of LSU is tightly coordinated with cessation of Rubisco assembly, and both processes take place after a threshold level of reactive oxygen species is reached. As a result, the small subunit is also eliminated by rapid degradation. We previously showed that the amino terminus of the LSU could bind RNA in a sequence-independent manner, as it shares a structural similarity with the RNA recognition motif. This domain becomes exposed only under oxidizing conditions, thus restricting the RNA-binding activity. Here we show that in vitro, thiol groups of both subunits become oxidized in the presence of oxidized glutathione. The structural changes are mediated by oxidized glutathione, whereas only very high concentrations of H2O2 confer similar results in vitro. Changes in the redox state of the LSU thiol groups are also observed in vivo, in response to a physiological light shock caused by transfer of cells from low light to high light. We propose that during a photooxidative stress, oxidation of thiol groups occurs already in nascent LSU chains, perhaps hindering their association with chaperones. As a result, their RNA recognition motif domain becomes exposed and will bind any RNA in its vicinity, including its own transcript. Due to this binding the ribosome stalls, preventing the assembly of additional ribosomes on the transcript. Polysome analysis using Suc gradients indeed shows that the rbcL RNA is associated with the polysomal fraction at all times but shifts toward fractions that contain smaller polysomes and monosomes during oxidative stress. Thus, translational arrest of the LSU most likely occurs at a postinitiation stage.

Dietary Lutein/Zeaxanthin Decreases Ultraviolet B-Induced Epidermal Hyperproliferation and Acute Inflammation in Hairless Mice

Lutein and zeaxanthin are carotenoids found in green leafy vegetables with interesting antioxidant properties. They are present in high concentrations in the fovea centralis of the human retina and their role in the prevention of age-related macula degeneration has been reported. We have investigated the effect of orally administered lutein and zeaxanthin in the cutaneous response to ultraviolet B irradiation. Female hairless SKh-1 mice receiving 0.4% and 0.04% lutein plus zeaxanthin-enriched diet for 2 wk were exposed to single doses of ultraviolet B radiation. Skin biopsies were taken at 24 and 48 h after irradiation and analyzed for the presence of apoptotic cells, proliferating cells, and expression of proliferating cell nuclear antigen. Our results show a clear ultraviolet-induced dose-dependent inflammatory response. Orally administered 0.4% lutein and zeaxanthin decreased significantly the edematous cutaneous response (p<0.01) as determined by the reduction of the UVB-induced increase of ear bifold thickening. Additionally, dietary carotenoids were efficient in reducing the ultraviolet B-induced increases in the percentage of proliferating cell nuclear antigen (p<0.05), bromodeoxyuridine (p<0.05), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive cells (p<0.01). These data demonstrate that oral supplementation of lutein and zeaxanthin diminishes the effects of ultraviolet B irradiation by reducing acute inflammatory responses and ultraviolet-induced hyperproliferative rebound.

Ginkgo biloba extracts and cancer: a research area in its infancy.

Recent studies conducted with various molecular, cellular and whole animal models have revealed that leaf extracts of Ginkgo biloba may have anticancer (chemopreventive) properties that are related to their antioxidant, anti-angiogenic and gene-regulatory actions. The antioxidant and associated anti-lipoperoxidative effects of Ginkgo extracts appear to involve both their flavonoid and terpenoid constituents. The anti-angiogenic activity of the extracts may involve their antioxidant activity and their ability to inhibit both inducible and endothelial forms of nitric oxide synthase. With regard to gene expression, a Ginkgo extract and one of its terpenoid constituents, ginkgolide B, inhibited the proliferation of a highly aggressive human breast cancer cell line and xenografts of this cell line in nude mice. cDNA microarray analyses have shown that exposure of human breast cancer cells to a Ginkgo extract altered the expression of genes that are involved in the regulation of cell proliferation, cell differentiation or apoptosis, and that exposure of human bladder cancer cells to a Ginkgo extract produced an adaptive transcriptional response that augments antioxidant status and inhibits DNA damage. In humans, Ginkgo extracts inhibit the formation of radiation-induced (chromosome-damaging) clastogenic factors and ultraviolet light-induced oxidative stress - effects that may also be associated with anticancer activity. Flavonoid and terpenoid constituents of Ginkgo extracts may act in a complementary manner to inhibit several carcinogenesis-related processes, and therefore the total extracts may be required for producing optimal effects.

Developmental changes of superoxide dismutase, peroxidase and catalase isoenzyme profiles in leaves of Impatiens flanaganiae Hemsl. associated with variations in light intensity

Changes in isoenzyme profiles of antioxidative enzymes, superoxide dismutase, peroxidase and catalase, in leaves of Impatiens flanaganiae Hemsl. subjected to different light conditions were investigated. Unbranched plantlets of I. flanaganiae propagated from tubers were exposed to three light regimes: 55 μmol m− 2 sec− 1, 280 μmol m− 2 sec− 1 and 30 μmol m− 2 sec− 1 (variants I, II and III respectively) for a period of seven weeks. Isoenzymes of superoxide dismutase, peroxidase and catalase and their differential responses to light variations were studied in plant extracts prepared from leaves of all three variants at weekly intervals. The activity of most SOD isoenzymes from plants subjected to low light intensity (variants I and II) remained relatively constant except for one Cu/Zn-SOD that increased gradually during the course of development. Little or no change in catalase activity was observed during the initial stage development of plants from these variants. A profound enhancement of catalase level was detected only from the fifth week onward, especially in variant III. The induction of some SODs and CAT in plants from these variants could be related to possible oxidative stress associated with plant aging. In contrast to CAT, the activity of some of the peroxidase isoenzymes in variant III decreased from the second week onward and was lower than in the plants from the other two variants during the initial stages of development. Plants from variants I and III also showed a steady increase in activity of most isoperoxidases (low and high mobility) with time, which suggests their indirect involvement in the regulation of plant growth and development. A rapid increase in the activity of superoxide dismutase and catalase during the experimental period was observed in plants exposed to relatively high light intensity (variant II). The activities of both Mn, and some of the Cu/Zn- containing SODs increased sharply from the second week onward under high light intensity as compared to the other two variants subjected to low light intensity. The Mn-SOD activity remained high throughout the experimental period, while the light-induced Cu/Zn- SODs showed a reduction in activity after the third week. This light-induced response could be an attempt to protect plants from the harmful radicals generated due to photo-oxidative stress under high light. The low activity of some isoperoxidase observed under these conditions is considered to be compensated by high catalase activity as the active oxygen species, hydrogen peroxide, is removed by catalase and/or peroxidase. These metal containing enzymes seem to respond differentially to variations in light and could be suitable as a marker for the evaluation of light-induced oxidative stress in Impatiens flanaganiae plants.

Protective effects of quercetin on ultraviolet A light-induced oxidative stress in the blood of rat.

The oxidative effects of ultraviolet A (UVA) light (320-400 nm) and the antioxidant effects of quercetin were examined in rat blood. For this purpose, rats were divided into three groups: control, ultraviolet (UV) and ultraviolet + quercetin (UV + Q). The UV and UV + Q groups were irradiated for 4 h a day with UVA light (1.25 mW cm(2)) during periods of 3, 6 and 9 days. Quercetin (50 mg kg(-1) body wt.) was administered intraperitoneally in the UV + Q group rats before irradiation periods. Blood was taken 3, 6 and 9 days post-treatment. Plasma malondialdehyde (MDA) levels significantly increased after 9 days of daily exposure to UVA. Whole blood glutathione (GSH) levels significantly declined after 3-9 days of irradiation. Glutathione peroxidase activity on days 6 and 9 and glutathione reductase activities on days 3, 6 and 9 post-irradiation were diminished significantly. Superoxide dismutase and catalase activities decreased significantly 3-9 days post-irradiation. The administration of quercetin before the 9-day period of irradiation significantly reduced the increase in plasma MDA value. Whole blood GSH levels significantly decreased with the administration of quercetin on all days. Quercetin significantly increased antioxidant enzymes diminished by UVA irradiation. Exposure of rats to UVA light leads to oxidative stress, reflected by increased MDA and reduced antioxidant enzyme levels. The administration of quercetin appears to be a useful approach to reduce the damage produced by UVA radiation.

C-Jun N-terminal Kinase Specifically Phosphorylates p66ShcA at Serine 36 in Response to Ultraviolet Irradiation

Mice lacking expression of the p66 isoform of the ShcA adaptor protein (p66(ShcA)) are less susceptible to oxidative stress and have an extended life span. Specifically, phosphorylation of p66(ShcA) at serine 36 is critical for the cell death response elicited by oxidative damage. We sought to identify the kinase(s) responsible for this phosphorylation. Utilizing the SH-SY5Y human neuroblastoma cell model, it is demonstrated that p66(ShcA) is phosphorylated on serine/threonine residues in response to UV irradiation. Both c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases are activated by UV irradiation, and we show that both are capable of phosphorylating serine 36 of p66(ShcA) in vitro. However, treatment of cells with a multiple lineage kinase inhibitor, CEP-1347, that blocks UV-induced JNK activation, but not p38, phosphatidylinositol 3-kinase, or MEK1 inhibitors, prevented p66(ShcA) phosphorylation in SH-SY5Y cells. Consistent with this finding, transfected activated JNK1, but not the kinase-dead JNK1, leads to phosphorylation of serine 36 of p66(ShcA) in Chinese hamster ovary cells. In conclusion, JNKs are the kinases that phosphorylate serine 36 of p66(ShcA) in response to UV irradiation in SH-SY5Y cells, and blocking p66(ShcA) phosphorylation by intervening in the JNK pathway may prevent cellular damage due to light-induced oxidative stress.

A method for the assessment of light-induced oxidative stress in embryos of fucoid algae via confocal laserscan microscopy

A method was developed for measurement of active oxygen production in embryonic stages of the brown seaweed Fucus spiralis, using the label CM-DCFH-DA. Active oxygen species convert the label into the green fluorescent CM-DCF (exc/em 488/530 nm) that is detected via confocal laserscan microscopy and quantitative image analysis. Loading of the label did not harm the embryos; loading efficiency was age-independent, and the esterases needed for conversion to CM-DCFH were not inhibited by the effective UV dose (2 W m−2) applied in the experiments. After correction for daily variation of the laser power, and calibration with DCF standard solutions, this automated analysis of confocal images rendered active oxygen concentrations in fucoid embryos (μM DCF). An experiment was designed for the assessment of active oxygen production following irradiance stress in the light-sensitive embryos. Dim-light-acclimated, 1-, 2- and 4-day-old embryos were transferred for 60 min to defined high-light conditions (4π-irradiance 300 μmol photons m−2 s−1), optionally without UV radiation, including UVA, or including UVA plus UVB. PSII yield measurements (PAM fluorometer) were carried out in order to assess the degree of photoinhibition under these light conditions. The imposed light stress initially caused a rapid decrease of the PSII yields (ΦP). With increasing embryo age, minimum ΦP values attained under light stress remained higher. Consequently, electron transport rates (ETR) would increase with embryo age, i.e., with the development of their photosynthetic apparatus. Active oxygen production increased with ETR, and when UVB was included, relatively greater amounts of active oxygen were produced. A slow, second-phase decrease of ΦP under light stress that was proportional to active oxygen production indicated that some photooxidative damage was caused, in particular during UVB exposure. Recovery from light stress was a rapid process in the absence of UVB; in such cases ΦP was almost restored to the initial values within 60 min. The relative state of recovery of ΦP was correlated with both the effective UV dose and active oxygen production rate (DCF). Recovery was slowest in embryos exposed for 60 min to an experimental UVB dose, which was representative of a situation at low tide, on a sunny day. The results suggest that active oxygen may cause an in situ inhibition of growth of the earliest life stages of F. spiralis.

Reduction of ultraviolet light-induced oxidative stress by amino acid-based iron chelators

The generation of free radicals by ultraviolet (UV) light accelerates skin aging, which is known as photoaging. Cutaneous iron catalyzes the generation of free radicals. We designed novel antioxidants that suppressed the iron-catalyzed free radical generation and the ensuing UV-induced damage by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(2-hydroxybenzyl)amino acids, were prepared by condensation of amino acids such as glycine and L-serine with salicylaldehyde and followed by catalytic reduction. The compounds formed a 2:1 complex to iron ion. These amino acid derivatives inhibited the iron-induced hydroxyl radical generation (the Fenton reaction). The compounds also suppressed UV-induced lipid peroxidation in murine dermal fibroblast homogenates. In addition, N-(2-hydroxybenzyl)- L-serine showed protective activity against UV-induced cytotoxicity in murine dermal fibroblasts. Desferrioxamine, a strong iron sequestering compound, was effective in inhibiting the Fenton reaction and the lipid peroxidation, but it was ineffective in protecting against UV-induced cytotoxicity. The results suggest that UV-induced oxidative stress can be reduced by these amino acid derivatives.

Modulation of UVA light-induced oxidative stress by β-carotene, lutein and astaxanthin in cultured fibroblasts

The ability of β-carotene, lutein or astaxanthin to protect against UVA-induced oxidative stress in rat kidney fibroblasts (NRK) was assessed. Activities of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), and changes in thiobarbituric acid reactive substances (TBARS) were measured as indices of oxidative stress. Exposure to UVA light at a dose intensity of 5.6 mW/cm 2 for 4 h resulted in a significant decrease in CAT and SOD activities and a significant increase in TBARS. No cytotoxicity, as indicated by lactate dehydrogenase (LDH) release, was observed. β-Carotene (1 μM), lutein (1 μM) and astaxanthin (10 nM) protect against UVA light-induced oxidative stress in vitro with astaxanthin exhibiting superior protective properties.